Polish Journal of Microbiology

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In the presented study, lactic acid bacteria strains of intestinal and plant origin were assessed for their ... of the bacteria was added to MRS agar medium, in the quantity sufficient to obtain the ..... R.S. Henderson, Mycotoxins and animal foods.
POLSKIE TOWARZYSTWO MIKROBIOLOGÓW POLISH SOCIETY OF MICROBIOLOGISTS

Polish Journal of Microbiology formerly

Acta Microbiologica Polonica

2005 POLSKIE TOWARZYSTWO MIKROBIOLOGÓW

EDITORS K.I. Wolska (Editor in Chief) J. Dziadek, A. Kraczkiewicz-Dowjat, A. Skorupska, H. Dahm E.K. Jagusztyn-Krynicka (Scientific Secretary)

EDITORIAL BOARD President: Zdzis³aw Markiewicz (Warsaw, Poland) Ryszard Chróst (Warsaw, Poland), Waleria Hryniewicz (Warsaw, Poland), Miros³aw Kañtoch (Warsaw, Poland), Donovan Kelly (Warwick, UK), Tadeusz Lachowicz (Wroc³aw, Poland), Wanda Ma³ek (Lublin, Poland), Andrzej Piekarowicz (Warsaw, Poland), Anna Podhajska (Gdañsk, Poland), Gerhard Pulverer (Cologne, Germany), Geoffrey Schild (Potters, Bar, UK), Torkel Wadström (Lund, Sweden), Jadwiga Wild (Madison, USA), Miros³awa W³odarczyk (Warsaw, Poland)

EDITORIAL OFFICE Miecznikowa 1, 02-096 Warsaw, Poland tel. 48 (22) 55 41 302, Tuesday and Thursday from 10 A.M. – till 2 P.M. only fax 48 (22) 55 41 402 e-mail izabelaw@ biol.uw.edu.pl

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QUARTERLY OF POLISH SOCIETY OF MICROBIOLOGISTS, PUBLISHED WITH THE FINANCIAL SUPORT OF THE STATE COMMITTEE OF SCIENTIFIC RESEARCH

The individual sections of the State Committee for Scientific Research have credited Polish Journal of Microbiology with the following points: P04 – 5, P05 – 5, P06 – 6, T09 – 6, T12 – 6

POLISH SOCIETY OF MICROBIOLOGISTS 00-725 Warsaw, Che³mska 30/34

Front cover: Long chain of Aspergillus sp. spores at the ends of the phialides (courtesy of Jaros³aw Wiœniewski, M.Sc. and Magdalena Sobolewska Ph.D)

Typesetting and print: Publishing House Letter Quality Warsaw, Brylowska 35/38, tel. 631 45 18, 607 217 879 Circulation: 500 + 15

Polish Journal of Microbiology formerly Acta Microbiologica Polonica

2005, Vol. 54, No 4

CONTENTS ORIGINAL PAPERS

Differentiated pattern of protein composition of crystalline inclusions of newly isolated Bacillus thuringiensis strains from Silesia in Poland

GUZ K., KUCIÑSKA J., LONC E., DOROSZKIEWICZ W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

Influence of N,N-bis(3-aminopropyl)dodecylamine on the mycelium growth and the cell wall composition of resistance and sensitive strains belonging to the genus Aspergillus

KOZIRÓG A., KUBERSKI S., ¯AKOWSKA Z., BRYCKI B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

The elimination of ochratoxin A by lactic acid bacteria strains

PIOTROWSKA M., ¯AKOWSKA Z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

Selection of potentially probiotic Lactobacillus strains towards their inhibitory activity against poultry enteropathogenic bacteria

KIZERWETTER-ŒWIDA M., BINEK M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

The effect of acid adaptation conditions on heat resistance of Escherichia coli O157: H7

TOSUN H., GÖNÜL Ô.A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Co-infection of hamsters with toxin A or toxin B-deficient Clostridium difficile strains

SZCZÊSNY A., MARTIROSIAN G., COHEN S., SILVA Jr. J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

The susceptibility of anaerobic bacteria isolated from periodontal diseases to photodynamic inactivation with fotolon (Chlorin e6)

DRULIS-KAWA Z., BEDNARKIEWICZ A., BUGLA-P£OSKONSKA G., STRÊK W., DOROSZKIEWICZ W. . . . . . . . . . . . . . 305

Susceptibility testing and resistance phenotypes detection in bacterial pathogens using the VITEK 2 system

STEFANIUK E., MRÓWKA A., HRYNIEWICZ W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Bacteriological urinalysis in patients after renal transplantation

£AZIÑSKA B., CISZEK M., ROKOSZ A., SAWICKA-GRZELAK A., P¥CZEK L. £UCZAK M. . . . . . . . . . . . . . . . . . . . . . . . . 317

The action of photosensitizers and serum in a bactericidal process. II. the effects of dyes: hypericin, eosin Y and saphranine O

JANKOWSKI A., JANKOWSKI S., MIROÑCZYK A., NIEDBACH J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Effect of intensity of feeding on the intestinal microflora of pigs

REKIEL A., GAJEWSKA J., TOPOL K., SAWOSZ E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

Identification of Aeromonas culicicola by 16S rDNA RFLP

KAZNOWSKI A., KONECKA E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Analysis of the murein of a Listeria monocytogenes EGD mutant lacking functional penicillin binding protein 5 (PBP5)

KORSAK D., POPOWSKA M., MARKIEWICZ Z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

INSTRUCTIONS TO AUTHORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

ERRATA Exposure to Moulds in Flats and the Prevalence of Allergic Diseases – Preliminary Study BEATA GUTAROWSKA, MARTA WISZNIEWSKA, JOLANTA WALUSIAK, MA£GORZATA PIOTROWSKA, CEZARY PA£CZYÑSKI AND ZOFIA ¯AKOWSKA

Polish Journal of Microbiology Vol. 54, Suppl. p 13 – 20, 2005 on page 13: The name of the second author should appear as shown above.

Polish Journal of Microbiology 2005, Vol. 54, No 4, 263– 269

Differentiated Pattern of Protein Composition of Crystalline Inclusions of Newly Isolated Bacillus thuringiensis Strains from Silesia in Poland KATARZYNA GUZ1, JOLANTA KUCIÑSKA2, EL¯BIETA LONC2 and W£ODZIMIERZ DOROSZKIEWICZ1 1 Department

of Microbiology, Institute of Genetics and Microbiology, Department of Parasitology, Institute of Genetics and Microbiology University of Wroc³aw, 63 Przybyszewskiego street, 51-148 Wroc³aw, Poland 2

Received 11 June 2005, received in revised form 21 October 2005, accepted 24 October 2005 Abstract Protein profiles of crystal delta-endotoxins were determined in twenty nine Bacillus thuringiensis strains-soil and phylloplane isolates – from Poland. Electrophoretic analysis revealed quantatively and qualitatively different patterns of delta-endotoxin crystal preparations of these B. thuringiensis strains. The crystalline parasporal inclusions of B. thuringiensis isolates were composed of two, three, four or five proteins. Molecular weights of these polypeptides varied from 23.4 kDa to 142 kDa. There is lack of correlation between serovars of B. thuringiensis strains, the morphology of crystals and the number and size of proteins in parasporal inclusions. K e y w o r d s: B. thuringiensis, delta-endotoxins, crystal proteins

Introduction Parasporal inclusions produced by gram-positive bacilli Bacillus thuringiensis and B. sphaericus are the subject of intense research because of their entomopathogenic properties which are widely used in biological control of many plant pests and vectors of pathogens, e.g. mosquitoes and black-flies transmitting first of all malaria and other tropical diseases (Enwistle et al., 1993). They are synthesized after stage II of sporulation (t2-24) and accumulate in the mother cell as crystals which can account for up to 25% of dry weight of the sporulating cells. Those crystalline inclusions, composed of one or a few proteins are named also delta-endotoxins, Cry proteins or Insecticidal Crystal Protein (ICP). Höfte and Whiteley (1989) proposed a system of nomenclature and classification of the delta-endotoxins according to their insecticidal properties and molecular relationships. Generally, Cry proteins are active against Lepidopteran (CryI of 130– 140 kDa), both Lepidopteran and Dipteran (CryII of 71 kDa), Coleopteran (CryIII of 66– 77 kDa) and Dipteran (CryIV of 125– 145 kDa and 68 kDa) larvae. Recent extensive screening programs have revealed numerous strains producing delta-endotoxins which do not fall into these categories. The increasingly rapid isolation and characterization of B. thuringiensis proteins with new pesticidal properties and a variety of sequences have resulted in a new nomenclature of Cry proteins based on hierarchical clustering using amino acid sequence identity (Crickmore et al., 1998). In most cases, the insecticidal specifity, if any, has not been determined yet. The present study was undertaken in order to establish protein patterns of newly isolated B. thuringiensis strains displaying different toxic activities in previous tests (Lonc et al., 2001a, b, 2003). 1

e-mail: [email protected], tel. (071) 324 72 27, fax: (071) 325 21 31

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Experimental Materials and Methods Bacterial strains. The studies encompassed 29 strains from the collection of the Institute of Genetics and Microbiology at the Wroc³aw University (Poland). Fourteen isolates originated from the phylloplanes and soil of the Lower Silesia, mainly from the Wroc³aw area. These strains (Table I) were described previously by Doroszkiewicz and Lonc (1999). The rest of fifteen strains of B. thuringiensis (Table II) were isolated from leaves and soil of the Upper Silesia regions. The standard biochemical tests were performed for Bacillus thuringiensis subspecies according to Lecadet et al. (1999). Stock bacterial culture were maintained on brain-heart infusion agar (Difco) slants and stored at 8°C.

Table I Bacillus thuringiensis soil and phylloplane isolates from the Lower Silesia, Poland (Doroszkiewicz and Lonc 1999) Symbol of B. thuringiensis strain*

Biochemical group/ Serovar

B. thuringiensis subspecies

OpAc1, OpPa1, OpPs1, OpQ3, KpC1, KpF3

I / H31, H18a18c2, H423

japonensis1, yoso2, jinghongiensis3

OpS1, OpF3, OpF4, KsS1, KsAc1

III / H2

finitimus

OpQ1

IV / H19

tochigiensis

OpF1, OpF2

V / H3a3c

alesti

Morphology of crystal** bp r c

* the tree names and strains origins were used to designate isolates: K = Karkonosze National Park, O = Osola (sampling sites for leaf and soil samples); sample source: p = phylloplane, s = soil; A c = Acer pseudoplatanus, C = Corylus avellana, F = Fagus silvatica, Pa = Picea abies, Ps = Pinus silvestris, Q = Quercus robur, S = Sorbus aucuparia; the numbers reflect the number of successive isolates ** bp = bipiramidal, r = rectangular, c = cuboidal

Table II B. thuringiensis soil and leaves isolates from the Upper Silesia, Poland Symbol of B. thuringiensis strains*

Biochemical group/ Serovar

ŒpPs1, ŒpCp5, BpQ11, BpQ18

I/H31, 4, 5, H72, H43, H446

kurstaki1, aizawai2, keynae3, sumiyoshiensis4, fukuokaensis5, higo6

bp

ŒpQ7, BsPs1

II/H12 , H25 , H30

thompsoni , coreanensis , medellin

bp

BsC1, BsC9

III/H1010, H3211

darmstandiensis10, cameroun11

BpTx5, BpAc4

IV/H17

tohokuensis

BpAc2, BsC16

V/H5

galleriae

s

BpTx1

VI/H24

neoleonensis

s

BsC6

VII/H812, H35,13 H3814

ostriniae12, seoulensis13, oswaldocruzi14

bp

ŒpPt3

VIII/H3a 3c

alesti

bp

7

8

9

B. thuringiensis subspecies

7

8

9

Morphology of crystal**

s bp

* the tree names and strains origins were used to designate isolates: Œ = Œwierklaniec (forest), B = Brynek (park) (sampling sites for leaf and soil samples), samples source: p = phylloplane, s = soil, Ac = Acer pseudoplatanus, Ps = Pinus silvestris, Q = Quercus robur, Pt = Populus tremula, Cp = Carpinus betulus, C = Cartaegus monogyna, Tx = Taxus baccata, the numbers reflect the number of successive isolates. ** bp = bipiramidal, s-spherical

Media. The sporulation medium (Kaelin et al., 1994) contained: glucose – 10 g, Casamino acids – 7.5 g, KH2PO4 – 6.8 g, MgS04 × 7H2O – 123 mg, MnSO 4 × 4H2O – 2.23 mg, ZnSO2 × 7H2O – 14 mg, Fe2(SO4)3 – 20 mg, H2O dist. – 1L, pH 7.5 was used. Separation and purification of parasporal crystal of B. thuringiensis strains. The strains were grown on sporulation medium at 28°C for 5 days with shaking, giving a spore content of about 10 8 ml–1. Crystals and spores of each cultures were washed twice in cold distilled water by centrifugation (30 min at 4 000 rpm) and resuspended in 50 mM Tris-HCl buffer, pH 7.5, containing 10 mM KCl, as previously described by Lonc et al. (1997). Crystals were purified on discontinuous sucrose gradients (67, 72, 79 and 87%) at 15 000 rpm for 30 min. Bands were collected and washed three time in distilled water. Samples of purified crystals were solubilized in 50 mM NaHCO 3 (pH 10.0) for 1h at 37°C (Gill et al., 1987). SDS-PAGE. The protein composition of parasporal bodies was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was conducted essentially as described by Laemmli (1970) with a 3% stacking gel and 10% running gel. Solubilized crystal proteins were mixed with denaturation buffer (1 : 1), heated for 2 min at 100°C and then loaded onto the gel. After elecrophoresis gels were stained with Coomassie blue. The molecular masses of the parasporal body proteins were estimated by comparison with a series of protein size standards (WIDE M 4038, Sigma).

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Protein composition of. B. thuringiensis crystalline inclusions

265

Results The protein content of purified parasporal crystals from twenty nine isolates of B. thuringiensis, analysed by SDS-PAGE, is shown in Figure 1 and Figure 2. The protein profiles of crystalline inclusions are quantatively and qualitatively different (Tables III and IV). The crystalline parasporal inclusions of B. thuringiensis isolates were composed of two, three, four or five proteins. Molecular weight of these polypeptides varied from 23.4 kDa to 142 kDa. Strains OpA c1, OpPa1, OpPs1, OpQ3, KpC1 and KpF3 belonging to B. thuringiensis japonensis, yoso and jinghongiensis group produced crystals composed of two or three poplypeptides of 34.1 to 142 kDa. Crystals of OpS1, OpF3, OpF4, KsS1 and KsAc1 isolates of B. thuringiensis finitimus contained two, three or four proteins; their molecular weight varied from 34.7 to 139 kDa.

Fig. 1. Proteinogram of parasporal crystals isolated from B. thuringiensis strains originating from the Lower Silesia area OpAc1 (line 1), OpP a1 (line 2), OpP s1 (line 3), OpQ3 (line 4), KpC1 (line 5), KpF3 (line 6), OpS1 (line 7), OpF3 (line 8), OpF4 (line 9), KsS1 (line 10), KsA c1 (line 11), OpQ1 (line 12), OpF1 (line 13), OpF2 (line 14); MM-molecular masses of standards WIDE M 4038 (Sigma).

Fig. 2. Electrophoregram of crystalline inclusions of B. thuringiensis isolates from the Upper Silesia regions BpTx5 (line 1), ŒpC p5 (line 2), BpQ11 (line 3), BsB9 (line 4), BsC1 (line 5), BsC6 (line 6), BsC16 (line 7), BpA c2 (line 8), ŒpPs1 (line 9), BpQ18 (line 10), BpTx1 (line 11), BsPs1 (line 12), ŒpQ7 (line 13), ŒpPt3 (line 14), BpA c4 (line 15); MM-molecular masses of standard WIDE M 4038 (Sigma).

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Guz K et al. Table III Molecular weight of delta-endotoxins in parasporal inclusions of B. thuringiensis isolates from the Lower Silesia Symbol of Num- Bacillus thuringiensis ber strain 1.

Molecular weight of delta-endotoxins (kDa)

Subspecies / Biochemical type

OpAc1

B. thuringiensis japonensis, yoso, jinghongiensis / I

136.3; 80; 63.6

2.

OpQ3

B. thuringiensis japonensis, yoso, jinghongiensis / I

136.3; 68

3.

OpPa1

B. thuringiensis japonensis, yoso, jinghongiensis / I

136.3; 64.4

4.

OpPs1

B. thuringiensis japonensis, yoso, jinghongiensis / I

133.6; 62.9

5.

KpC1

B. thuringiensis japonensis, yoso, jinghongiensis / I

142; 86.2; 34.1

6.

KpF3

B. thuringiensis japonensis, yoso, jinghongiensis / I

136.3; 66.2

7.

OpS1

B. thuringiensis finitimus / III

130.1; 66.2

8.

OpF3, OpF4

B. thuringiensis finitimus / III

139.2; 82; 64.4; 34.7

9.

KsS1

B. thuringiensis finitimus / III

133.6; 86.2; 60.5; 45

10.

KsA c1

B. thuringiensis finitimus / III

133.6; 61.3; 44.4

11.

OpQ1

12.

OpF1, OpF2

B. thuringiensis tochigiensis / IV

147.9; 100.2; 63.6

B. thuringiensis alesti / V

136.3; 80; 63.6; 80

Table IV Molecular weight of delta-endotoxins in parasporal inclusions of B. thuringiwnsis isolates from the Upper Silesia, Poland Number

Symbol of Bacillus thuringiensis strain

Subspecies/biochemical type

Molecular weight of delta-endotoxins (kDa)

1

ŒpCp5

B. thuringiensis kurstaki, B. thuringiensis aizawai, B. thuringiensis fukuokaensis, B. thuringiensis higo/ I

88; 65,5; 32,8; 23,4

2

ŒpPs1, BpQ18

B. thuringiensis kurstaki, B. thuringiensis aizawai, B. thuringiensis fukuokaensis, B. thuringiensis higo/ I

142; 102; 74; 66; 64

BpQ11

B. thuringiensis kurstaki, B. thuringiensis aizawai, B. thuringiensis fukuokaensi, B. thuringiensis higo/ I

136; 74; 65

3 4

ŒpQ7, BsPs1

B. thuringiensis thompsoni, B. thuringiensis coreanensis, B. thuringiensis medellin/ II

142; 102; 72; 64

5

BsC1, BsC9

B. thuringiensis darmstandiensis, B. thuringiensis cameroun/ III

138; 65,5

6

BpTx5

B. thuringiensis tohokuensis/ IV

132; 74; 65,5; 24,6

7

BpAc4

B. thuringiensis tohokuensis/ IV

142; 72; 66; 24

8

BsC16

B. thuringiensis galleriae/ V

142; 64

9

BpAc2

B. thuringiensis galleriae/ V

142; 98; 64

10

BpTx1

B. thuringiensis neoleonensis/ VI

74; 66; 64

BsC6

B. thuringiensis ostriniae, B. thuringiensis seoulensis, B. thuringiensis oswaldocruzi/ VII

138; 102; 74; 65,5

ŒpPt3

B. thuringiensis alesti/ VIII

142; 102; 72; 64

11 12

An identical protein pattern (34.7, 64.4, 82.0 and 139.2 kDa) was characteristic of phylloplane strains OpF3 and OpF4 (B. thuringiensis alesti). Two other strains of B. thuringiensis alesti: OpF1 and OpF2 had crystals composed of only 63.1, 80,0 and 136.3 kDa. Different protein profiles had parasporal inclusions of B. thuringiensis alesti ŒpPt3 isolated from the Upper Silesia region. Four proteins with estimated molecular masses of ca 142, 102, 72 and 64 kDa were detected as the main components of these crystals. The same protein profile of crystals had two other strains of B. thuringiensis ŒpQ7 and BsPs1 belonging to B. thuringiensis thompsoni, coreanensis and medellin group. Four strains with biochemical characters of B. thuringiensis kurstaki, aizawai, fukuokaenensis and higo isolated from phylloplane from the Upper Silesia area produced crystals which contained three, four or five delta-endotoxins of 23.4 to 142 kDa. Identical protein profiles of inclusions had two strains BsC1 and BsC9 belonging to B. thuringiensis darmstandiensis and cameroun

4

Protein composition of. B. thuringiensis crystalline inclusions

267

group. Their crystals had two polypeptides of 138 and 65.5 kDa. Both strains were isolated from soil of the same area. B. thuringiensis strains of two subspecies: B. thuringiensis tohokuensis and B. thuringiensis galleriae had delta-endotoxins that can vary in number and in size from around 24 to142 kDa. Different protein patterns of crystals were stated in strains BpT x5 and BpAc4 belonging to B. thuringiensis tohokuensis. Molecular weight of four delta-endotoxins included 132, 74, 65.5 and 24.6 kDa in BpTx5 strain and 142, 72, 66 and 24 kDa in BpAc4 strain. Crystals of BpAc2 and BsC16 isolates belonging to B. thuringiensis galleriae were composed of two or three proteins of 64 to 142 kDa. The unique protein pattern of crystalline inclusions was characteristic for three strains of different serovars of B. thuringiensis: BpTx1, BsC6 and OpQ1. Crystals of OpQ1 isolate belonging to B. thuringiensis tochigiensis contained three delta-endotoxins of 63.6, 100,2 and 147,9 kDa. Also proteins of 64, 66 and 74 kDa were present in parasporal inclusion of BpT x1 isolate of B. thuringiensis neoleonensis. Four delta-endotoxins of 138, 102, 74 and 65.5 kDa were found in soil isolate BsC6 belonging to B. thuringiensis ostriniae, seoulensis and oswaldocruzi group. It can be seen that in 29 analyzed B. thuringiensis strains both large (from 130.1 to 147.9 kDa) and/or medium (from 60.5 to 102 kDa) delta-endotoxins occurred. A great diversity of delta-endotoxin profiles was observed by SDS-PAGE methods. Some profiles indicated components lower than 60 kDa, and others showed multiple components or components ranging between 138 and 64 kDa. Components higher than 142 kDa appeared much less frequently and were detected in the crystals of OpQ1 isolate. Small proteins of 23.4– 45.0 kDa were found in eight isolates, namely KpC1, OpF3, OpF4, KsS1, KsA c1, ŒpCp5, BpTx5 and BpAc4 originating from different sample sources and areas. These components were isolated from strains belonging to various serovars: B. thuringiensis kustaki, B. thuringiensis finitimus, B. thuringiensis japonensis and B. thuringiensis tohokuensis. Analysis of the protein composition of crystalline inclusions of newly isolated B. thuringiensis strains from Poland indicate that two or more subgroup of the same serotype may possess several crystals with different protein profiles of delta-endotoxins. Discussion The previous entomopathogenic activity of B. thuringiensis strains from the Wroc³aw collection encouraged us to attempt a closer characteristics of their parasporal inclusions. The inclusions are a potential source of new, insecticidal proteins which in the future may join the world’s collection of delta-endotoxins, or even become directly useful in biological control of pest insects or vectors of infectious and invasive diseases. It is in the line with COST 862 programm “Bacterial toxins for insects control” (http:// cost.cordis.lu/src/cso_informatio.cfm). Our investigation indicated that crystals of twenty nine B. thuringiensis isolates possessed quantatively and qualitatively different patterns of delta-endotoxins. In addition there is no correlation between serovar B. thuringiensis strain and type of crystalline inclusion produced. Several isolates producing crystals of the same morphology belong to different serovars. Often the typical bipiramidal parasporal inclusions were synthesised by many various B. thuringiensis strains, including B. thuringiensis japonensis, B. thuringiensis kurstaki, B. thuringiensis thompsoni, B. thuringiensis tohokunensis, B. thuringiensis ostriniae, and B. thuringiensis alesti. Other B. thuringiensis isolates formed spherical, rectangular or cuboidal crystalline inclusions. Also the same crystals of various serovars of B. thuringiensis strains were morphologically indistinguishable. These phenomenon were observed also by many authors (Benintende et al., 1997; Lecadet et al., 1999; Martin et al., 1989; Ohba et al., 1986; Ohba et al., 1989; Ohba et al., 1992; Wassano et al., 1997). The presence of bipiramidal or cuboidal parasporal inclusions may indicate Lepidoptera-specific B. thuringiensis strains. However, isolates belonging to the anti-Lepidoperan strains of B. thuringiensis darmstandiensis, B. thuringiensis galleriae and B. thuringiensis finitimus produced another type of crystals, particularly spherical and rectangular. This result may indicate that amino acid composition of Cry proteins and the number of protoxins in the crystal determine the shape as well as insecticidal properties. Electrophoretic analysis revealed great differentiation of protein patterns of the purified delta-endotoxins from B. thuringiensis isolates. Most of them appear to belong to Cry1 class, associated with effects against pest moths. It is the largest group of medium (ca. 81 kDa) and large-sized (130– 142 kDa) proteins. There are about 135 Cry proteins (45%) among the 300 delta-endotoxins described to date (http:// www.biols.susx.ac.uk/home/ Neil_Crickmore). In our investigations the crystals of 27 out of 29 B. thuringiensis isolates possessed the large-sized proteins of 130.1 to 147.9 kDa. These molecular weight are also characteristic of numerous anti-Lepidopteran toxins of Cry1 category, eg. Cry1A (Lee et al., 1991), Cry1B

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(Brizzard et al., 1988; Kuo et al., 2000), Cry1C (Honee et al., 1988), Cry1F (Chambers et al., 1991), Cry1I (Choi et al., 2000; Tailor et al., 1992) or Cry1K (Koo et al., 1995). Several of them were found in parasporal inclusions of B. thuringiensis berliner, B. thuringiensis kurstaki, B. thuringiensis entomodiscus, B. thuringiensis alesti, B. thuringiensis aizawai, B. thuringiensis morrisoni and B. thuringiensis wuhanensis (Brizzard et al., 1988; Chambers et al., 1991; Choi et al., 2000; Entwistle et al., 1993; Kuo et al., 2000; Lee et al., 1991). However, the proteins of 130– 140 kDa can be similar in size to the Cry4 Diptera-specific proteins (Höfte et al., 1989) and the Cry3 Coleoptera-specific proteins (Ceron et al., 1995; Wasano et al., 1997). Whereas the medium-sized proteins (64 to 102 kDa) were identified in all the presently tested strains. The molecular masses of these proteins can correspond to various classes of known Cry proteins, eg. Cry2 specificity against Lepidoptera and Dipteran larvae (Espinasse et al., 2002; Honguy et al., 2000; Nichols et al., 1989), Cry3 and Cry5 toxicity against Coleopteran or Coleopteran and Lepidopteran larvae (Crickmore et al., 1998; Espinasse et al., 2002; Lopez-Meza et al., 1996; Shin et al., 1995) or Cry10 and Cry11 subclasses activity against Dipteran larvae (Delécluse et al., 1995; Saitoh et al., 1998; Wirth et al., 2000). The size of delta-endotoxins of 70 to 105 kDa may indicate also the proteins of Cry4 class (Kawalek et al., 1995; Tabashnik, 1992). A few crystalline inclusions of eight B. thuringiensis isolates classified to four biochemical groups, including B. thuringiensis tohokuensis, B. thuringiensis kurstaki, B. thuringiensis finitimus and B. thuringiensis japonensis were composed of the small components of polypeptides of 23.4 to 45 kDa. The size of these proteins may indicate the Cyt proteins (25–30 kDa) (Cheong et al., 1997; Gill et al., 1987; Georghiou et al., 1997; Ragni et al., 1996; Thomas et al., 1983) or anti-coleopteran delta-endotoxins of Cry3 and Cry35 subclasses (39–55 kDa) (Ellis et al., 2002; Entwistle et al., 1993; Kim et al., 2000). In the nearest future the immunological relationships of separated components of the crystals from B. thuringiensis isolates will be analysed by Western blot. Distribution of cry genes of B. thuringiensis strains isolated from Silesia also will be determined by PCR method. This investigation will complete the data on Cry proteins present in parasporal inclusions of B. thuringiensis strains originating from Poland. This way the genetic relationships of genes encoding ICPs will be compared with the data presented by Œwiêcicka et al. (2005). Literature B e n i n t e n d e G.B., J.E. L o p e z - M e z a, J.G. C o z z i, C.F. P i c c i n e t t i and J.E. I b a r r a. 1997. Characterization of INTA 51–3, a new atypical strain of Bacillus thuringiensis from Argentina. Appl. Environ. Microbiol. 41: 396–401. B r i z z a r d B.L. and H.R. W h i t e l e y. 1988. Nucleotide sequence of an additional crystal protein gene cloned from Bacillus thuringiensis subsp. kurstaki. Nucleic Acids Res. 16: 2723–2723. C e r o n J., A. O r t i z, R. Q u i n t e r o, L. G u e r e c a and A. B r a v o. 1995. specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection. Appl. Environ. Microbiol. 61: 3826–3831. C h a m b e r s J.A., A. J e l e n, M.P. G i l b e r t, C.S. J a n y, T.B. J o h n s o n and C. G a w r o n - B u r k e. 1991. Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai. J. Bacteriol. 173: 3966–3976 C h e o n g H. and S.S. G i l l. 1997. Cloning and characterization of a cytotytic and mosquitocidal *-endotoxin from Bacillus thuringiensis subsp. jegathesan. Appl. Environ. Microbiol. 63: 3254–3260. C h o i S.K., B.S. S h i n, E.M. K o n g, H.M. R h o and S.H. P a r k. 2000. Cloning of a new Bacillus thuringiensis cry1I-type crystal protein gene. Curr. Microbiol. 41: 65–69. C r i c k m o r e N., D.R. Z e i g l e r, J. F e i t e l s o n, E. S c h n e p f, J. Va n R i e, D. L e r e c l u s, J. B a u m and D.H. D e a n. 1998. Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins. Microbiol. Mol. Biol. Rev. 62: 807–821. D e l é c l u s e A., M.-L. R o s s o and A. R a g n i. 1995. Cloning and Expression of a Novel Toxin Gene from Bacillus thuringiensis subsp. jegathesan Encoding a Highly Mosquitocidal Protein. Appl. Environ. Microbiol. 61: 4230–4235. D o r o s z k i e w i c z W. and E. L o n c. 1999. Biodiversity of Bacillus thuringiensis Strains in the Phylloplane and Soil of Lower Silesia Region (Poland). Acta Microbiol. Pol. 48: 355–361. E l l i s R.T., B.A. S t o c k h o f f, L. S t a m p, H.E. S c h n e p f, G.E. S c h w a b, M. K n u t h, J. R u s s e l l, G.A. C a r d i n e a u and K.E. N a r v a. 2002. Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera. Appl. Environ. Microbiol. 68: 1137–1145. E n w i s t l e P.F., J.S. C o r y, M.J. B a i l e y and S. H i g g s. 1993. Bacillus thuringiensis, An environmental biopesticide: Theory and practice. John Wiley and Sons, Chichester, New York. E s p i n a s s e S., M. G o h a r, J. C h a f a u x, C. B u i s s o n, S. P e r c h a t and V. S a n c h i s. 2002. Correspondenne of high levels of beta-exotoxin I and the presence of cry1B in Bacillus thuringiensis. Appl. Environ. Microbiol. 68: 4182–4186 G i l l S.S. and B.A. F e d e r i c i. 1987. Cytolityc activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14. Appl. Environ. Microb. 53: 1251–1256 G e o r g h i o u G.P. and M.C. W i r t h. 1997. Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subs. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Appl. Envirom. Microbiol. 63: 1095–1101.

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H o n e e G., T.P.M. v a n d e r S a l m and B. V i s s e r. 1988. Nucleotide sequence of crystal protein gene isolated from B. thuringiensis subspecies entomocidus 60.5 coding for a toxin highly active against Spodoptera species. Nucleic Acids. Res. 16: 6240–6240. H o n g y u Z., Y. Z i n i u and D. W a n g x i. 2000. Composition and ecological distribution of Cry proteins and their genotypes of Bacillus thuringiensis isolates from Warehouses in China. J. Invert. Pathol. 79: 191–197. H ö f t e H. and H.R. W h i t e l e y. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53: 242–255. K a e l i n P., P. M o r e l and F. G a d a n i. 1994. isolation of Bacillus thuringiensis from stored tabacco and Lasioderma serricorne (F.). Appl. Environ. Microbiol. 60: 19–25 K a w a l e k M.D., S. B e n j a m i n, H.I. L e e and S.S. G i l l. 1995. Isolation and identification of novel toxins from Malaysia, Bacillus thuringiensis subsp. jegathesan. Appl. Environ. Microbiol. 61: 2965–2969 K i m H.-S., S. Y a m a s h i t a, T. A k a o, H. S a i t o h, K. H i g u c h i, Y.S. P a r k, E. M i z u k i and M. O h a b a. 2000. In vitro cytotoxicity of non-Cyt inclusion proteins of a Bacillus thuringiensis isolate against human cells, including cancer cells. Lett. Appl. Microbiol. 89: 16–23. K o o B.T., S.H. P a r k, S.K. C h o i, B.S. S h i n, J.I. K i m and J.H. Y u. 1995. Cloning of a novel crystal protein gene cry1K from Bacillus thuringiensis subsp. morrisoni. FEMS Microbiol. Lett. 134: 159–164. K u o W.-S., J.-H. L i n, C.-C. T z e n g, S.-S. K a o and K.-F. C h a k. 2000. Cloning of two new cry genes from Bacillus thuringiensis subsp. wuhanensis strain. Curr. Microbiol. 40: 227–232. L a e m m l i U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. L e e C.S. and A.I. A r o n s o n. 1991. Cloning and analysis of delta-endotoxin genes from Bacillus thuringiensis subsp. alesti. J. Bacteriol. 173: 6635–6638. L e c a d e t M.M., E. F r a c h o n, V.C. D u m a n o i r, H. R i p o u t e a u, S. H a m o n, P. L a u r e n t and I. T h i e r y. 1999. Updating the H-antigen classification of Bacillus thuringiensis. J. Appl. Microbiol. 86: 660–672. L o n c E., W. D o r o s z k i e w i c z, M.J. K l o w d e n, K. R y d z a n i c z and A. G a ³ g a n. 2001a. Entomopathogenic activities of environmental isolates of Bacillus thuringiensis against dipteran larvae. J. Vector Ecology 26: 15–20. L o n c E., J. K u c i ñ s k a and K. R y d z a n i c z. 2001b. Toxicity isolates of Bacillus thuringiensis from Wroc³aw against larvae of Aedes aegypti (in Polish). Wiad. Parazytol. 47: 297–303. L o n c E., J. K u c i ñ s k a and K. R y d z a n i c z. 2003. Comparative Delta-endotoxins of Bacillus thuringiensis against Mosquito Vectors (Aedes aegypti and Culex pipiens). Acta Microbiol. Pol. 52: 293–300. L o n c E., M.-M. L e c a d e t, T.M. L a c h o w i c z and E. P a n e k. 1997. Description of Bacillus thuringiensis wratislaviensis (H-47), a new serotype originating from Wroclaw (Poland), and other Bt isolates from the same area. Lett. Appl. Microbiol. 24: 467–473. L o p e z - M e z a J.E. and J.E. I b a r r a. 1996. Characterization of a novel strain of Bacillus thuringiensis. Appl. Environ. Microbiol. 62: 1306–1310. M a r t i n P.A. and R.S. T r a v e r s. 1989. Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl Environ. Mircobiol. 55: 2437–2442. N i c h o l s C.N., W. A h m a d and D.J. E l l a r. 1989. Evidence for two different types of insecticidal P2 toxins with dual specifity in Bacillus thuringiensis subspecies. J. Bacteriol. 171: 5141–5147. O h b a M., H. I w a h a n a, S. A s a n o, N. S u z u k i, R. S a t o and H. H o r i. 1992. A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvacidal activity specific for scarabaeid beetles. Lett. Appl. Microbiol. 14: 54–57. O h b a M. and K. A i z a w a. 1989. New flagellar (H) antigenic subfactors in Bacillus thuringiensis H serotype 3 with description of two new subspecies, Bacillus thuringiensis subsp. sumiyoshiensis (H3a: 3d) and Bacillus thuringiensis subsp. fukuokaenensis (H3a: 3d: 3e). J. Invert. Pathol. 54: 208–212. O h b a M. and K. A i z a w a. 1986. Insect toxicity of Bacillus thuringiensis isolated from soil of Japan. J. Invert. Pathol. 47: 12–20. R a g n i A., I. T h i e r y and A. D e l e c l u s e. 1996. Characterization of six highly mosquitocidal Bacillus thuringiensis strains that do not belong to H-14 serotype. Curr. Microbiol. 32: 48–54. S a i t o h H., K. H i g u c h i, E. M i z u k i, S.H. H w a n g and M. O h b a. 1998. characterization of mosquito larvacidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain. J. Appl. Microbiol. 84: 883–888. S h i n B.S., S.H. P a r k, S.K. C h o i, B.T. K o o, S.T. L e e and J.I. K i m. 1995. Distribution of cry-V type insecticidal protein genes in Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus. Appl. Environ. Microbiol. 61: 2402–2407. Œ w i ê c i c k a I. and I. M a h i l l o n. 2005. The clonal structure of Bacillus thuringiensis isolates from north-east Poland does not correlate with their cry gene diversity. Environ. Microbiol. 7: 34–39. T a b a s h n i k B.E. 1992. Evaluation of synergism among Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 58: 3343–3346. T a i l o r R., J. T i p p e t t, G. G i b b, S. P e l l s, D. P i k e, L. J o r d a n and S. E l y. 1992. Identification and characterization of a novel Bacillus thuringiensis delta-endotoxin entomocidal to coleopteran and lepidopteran larvae. Mol. Microbiol. 6: 1211–1217. T h o m a s W.E. and D.J. E l l a r. 1983. Bacillus thuringiensisvar israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo. J. Cell Sci. 60: 181–197. W a s a n o N., H. S a i t o and M. O h b a. 1997. A high homology exists in N-terminal amino acid sequences of delta-endotoxins between Lepidoptera-specific and Coleoptera-specific Bacillus thuringiensis strains. Lett. Appl. Microbiol. 24: 438–440. W i r t h M.C., B.A. F e d e r i c i and W.E. W a l t o n. 2000. Cyt 1A from Bacillus thuringiensis synergize activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae). Appl. Environ. Microbiol. 66: 1093–1097.

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Polish Journal of Microbiology 2005, Vol. 54, No 4, 271– 278

Influence of N,N-bis(3-aminopropyl)dodecylamine on the Mycelium Growth and the Cell Wall Composition of Resistance and Sensitive Strains Belonging to the Genus Aspergillus ANNA KOZIRÓG*1, S£AWOMIR KUBERSKI2, ZOFIA ¯AKOWSKA1 and BOGUMI£ BRYCKI3 1 Institute

of Fermentation Technology and Microbiology, Technical University of £ódŸ, Poland of Process and Environmental Engineering, Technical University of £ódŸ, Poland 3 Department of Chemistry, Adam Mickiewicz University, Poznañ, Poland

2 Faculty

Received 27 June 2005, received in revised form 26 September 2005, accepted 28 September 2005 Abstract Resistance causes of moulds to N,N-bis(3-aminopropyl)dodecylamine (APDA) for selected species of Aspergillus niger and Aspergillus flavus was examined. Control (sensitive) strains and resistant strains, cultured at 0.05% triamine, were used in the experiments. The non-resistant strains did not have growth capacity in this amount of ADPA while the resistant strains were characterised by a smaller biomass increase. Individual stages of the development of the mycelium occurred later than those in the control samples. The participation of the cell wall in the mycelium biomass of the resistant strains was higher by 7.5%. The glucan content in the wall dry mass was lower by 11% than that in the sensitive strains. A 41% increase in the lipid content was recorded in the cell wall of resistant Aspergillus flavus. A 21% protein increase occurred in the wall of Aspergillus niger comparing to the control strain. Infrared spectrophotometric analysis of the cell wall did not reveal the presence of triamine. Most absorption bands disappeared in the wall of Aspergillus flavus while no additional absorption bands were registered in Aspergillus niger; some bands were only stronger than those in the control sample. The resistant strains were characterised by a smaller ergosterol content, the main constituent of cell membranes. Spectrophotometric analysis of the mycelium did not reveal significant qualitative changes; only quantitative changes were observed. It was noticed that the resistance reaction did not occur with the same intensity in both species studied. The resistant strain of Aspergillus niger was characterised by a slightly more intensive absorption within its entire spectrum range in comparison to control strain. In case of Aspergillus flavus the absorption was higher for control strain. K e y w o r d s: moulds, resistance to disinfectants, N,N-bis(3-aminopropyl)dodecylamine, cell wall, ergosterol

Introduction Various anti-microbial agents are used in the pharmaceutical, cosmetic, paper and food industries as well as in medicine. Many products used so far have become less effective as a result of spontaneous cellular mutation or phenotypic adaptation to harmful environment conditions. Most studies on the mechanisms of microbial resistance or inactivation have until recently concentrated on bacteria; fungi, however, characterised by high adaptability to physical and climatic environment conditions that allows them to colonise new substrates and to invade the human environment, should also be examined to identify their resistance. A number of basic targets for antifungal agents are distinguished in a single cell (Groll et al., 1998; Wills et al., 2000). The cell wall, responsible for the cell shape, and the first contact point with the biocidal agent, is one of them. Chitin, glucan, mannan (these polysaccharides constitute 80– 90% of the cell wall dry mass) as well as proteins (ca 20%) and lipids (1– 3%) occur in the cell wall in most species (Farkaš, 1990; Fiema, 1994). It may be destroyed by inhibiting the synthesis of these structural components in the cell or by degrading the existing constituents of the wall. The cell membrane, consisting of phospholipids, sphingolipids, sterols and proteins, is another target for disinfectant agents. Ergosterol, also called provitamin D2, is the main sterol in the cell of moulds. It plays * Corresponding author: Anna Koziróg, Institute of Fermentation Technology and Microbiology, Technical University of £ódŸ, Wólczañska 171/173, 90-924 £ódŸ, Poland, e-mail: [email protected]

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a very important role in the membrane of the cytoplasmatic wall in fungi as it is responsible for its appropriate functioning, including substance transport. The mechanisms of the action of antifungal disinfectants and biocidal agents as well as the mechanisms of fungal resistance have been examined only in few studies and still little is known about them (Strzelczyk, 2001). The aim of the present study was to explain the presumable mechanism of the resistance of selected mould species belonging to the genus Aspergillus to N,N-bis(3-aminopropyl)dodecylamine (APDA). Researches were concentrated on mycelium growth dynamics assessment, cell wall composition, and ergosterol content of resistant and sensitive (control) strains. Moreover, based on infrared spectrophotometric analysis, the article gives the answer if triamine presence results in new bonds creation in biological material. The compound is a component of cleaning agents and disinfectants used in hospitals, the food industry and the cosmetic industry. It is used, for instance, in Australia, Germany or Ireland in agents such as Lonzabac, Gigasept or in bactericidal hand soap, KM 801 (Dibo and Brasch, 2001; web pages1). N,N-bis(3-aminopropyl)dodecylamine (APDA) belongs to the group of triamines, and is a derivative of fatty amines with two free amino groups and a 12-carbon aliphatic chain. Experimental Materials and Methods Fungal strains and culture conditions. Strains of Aspergillus niger £ 0439 and Aspergillus flavus £ 0422, deposited in the Collection of Pure Cultures at the Institute of Fermentation Technology and Microbiology, Technical University of £ódŸ, LOCK 105, as well as their varieties resistant to triamine, obtained in successive passages (the authors’ unpublished studies), were the biological material. Culture medium: nutrient solution M 0, pH = 6.2; containing 3% (w/v) of glucose, 0.3% (w/v) (NH 4)2SO4; 0.1% (w/v) KH2PO4; 0.5% (w/v) MgSO4 × 7H2O; 0.5% (w/v) yeast extract. Culture conditions: temperature – 28°C, time – 7 days. Resistant strains were cultured on the M0 solution to which 0.05% (w/w) triamine was added (89 ml culture medium + 10 ml APDA). Non-resistant control strains were cultured on M0 without APDA (99 ml culture medium). Samples prepared as described above were treated with 1ml conidial suspension of sensitive or control strains (107 spores/ml). Mycelium analysis comprised estimation of participation of the cell wall dry mass in the mycelium, chemical composition of the cell wall, spectrophotometric analysis of the mycelium and the walls. The results given below are the arithmetic mean of three series of experiments. Growth curves. After 1, 2, 3, 4, 5, 7, 8, 10, 12 days of culture, the samples were taken out from incubator, and washed with distilled water on the filter to separate mycelium from nutrient solution. Next, the mycelium was dried to constant mass, first at 30°C, next at 105°C. Cell wall isolation. The mycelium was hydrolysed with 1% (w/v) dodecyl sulphate solution in volume of 100 ml for each 10 g of wet mycelium biomass (Kisser et al., 1980). Then the mycelium was separated from the post-reaction mixture and washed with distilled water to remove cell protein structures. In order to check total flush, the absorbance of final filtrate was measured at wavelength 8 = 260 nm. Cell walls were dehydrated with cooled absolute alcohol and dried to constant mass. The results were given as percentage of cell wall mass to total dry mass of mycelium. Glucan and chitin determination. The cell wall was hydrolysed using 3 M HCl to obtain cell wall monomers (Rokem et al., 1986). The total content of reducing sugars was determined in 1ml of the obtained hydrolysate using the colorimetric method with 3,5-Dinitrosalicyl acid (DNS) using spectrophotometer at wavelength 8 = 540 nm. Acid was removed from the remaining hydrolysate using a Dowex1 anion exchanger. Determination consist in colour reaction between reducing sugars, DNS, and sodium-potassium tartrate. This method is characterised by high sensitivity – 0.1 mg of sugar at 1 ml. A Dowex50Wx4 cation exchanger was used to elute glucosamine from the sample studied. The eluate was concentrated to a defined volume under reduced pressure. The concentration of reducing sugars (glucan), expressed as the glucose content, was determined in this sample using the colorimetric method with DNS. The amount of glucosamine (chitin) was determined from the difference between the total number of reducing sugars and the amount of glucose obtained in the eluate. The results are given in mg per 1g of the cell wall dry mass. Determination of the total amount of proteins. The Kjeldahl method was used to determine the protein content. As described in the method, nitric organic substances are converted into ammonium salts, and ammonia is distilled to the standard acid solution. The results are given in mg per 1g of the cell wall dry mass. Lipid determination. The cell wall was suspended in distilled water and a mixture of chloroform and methane (1:2) was added (Kiejts, 1975). The extraction was conducted twice, a chloroform layer was separated, diluted with a mixture of chloroform and water (1:1), and shaken in a separatory funnel. The chloroform layer was transferred to a weighing bottle and evaporated to dryness. The results are given in mg per 1g of the cell wall dry mass. Spectrophotometric analysis in the infrared. Mycelia and cell walls of both strains were dried to constant mass and extracted using CCl4 after a 7-day culture. The extract was analysed using infrared spectroscopy (FTIR). Spectra were registered (FTIR Jasco 610) in the range 5000 – 400 cm –1 from the resolution 2 cm–1 in KSR-5 trays, 1.02 mm thick. The measured absorption value in the ranges characteristic of the compounds studied was linearly proportional to their concentrations. 1

Australian Government Department of Health And Ageing NICNAS web page: http://www.nicnas.gov.au/PUBLICATIONS/CAR/NEW/NA/NASUMMR/NA0100SR/na180.asp; Kitchenmaster NI Ltd web page: http://www.kitchenmaster-ni.com/safetyinfosheets/KM801.DOC

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Ergosterol determination. The content of this component in the mycelium dry mass was determined after day 5 and day 7 of the culture using the spectrophotometric method (Gutarowska and ¯akowska, 2002). The preliminary lipid extraction from the mycelium was conducted using methanol, and the samples were centrifuged. The unsaponifiable fraction was separated. The entire sample was boiled under a reflux condenser, and the cooled sample was extracted with hexane twice. The hexane fraction was evaporated to dryness. The dry residue obtained was dissolved in methanol and examined using spectrophotometry at wavelength 8 = 282.6nm. The results are given in mg of ergosterol per 1g of the mycelium dry mass. Statistic. Anova test were used in order to verify if obtained data are statistically essential. Test depends on variation analysis. Verification was performed in Origin, data analysis and technical graphics software.

Results Development of the mycelium. The results of the comparative analysis of the development of resistant strains, cultured in the presence of 0.05% of N,N-bis(3-aminopropyl)dodecylamine, and that of non-resistant strains growing in control conditions, are discussed in this part. The non-resistant strains did not show growth capacity at this amount of ADPA. The control strain was characterised by a greater biomass increase in comparison with the resistant strain, cultured in the presence of triamine, already in the the first day. The greatest differences are noticeable throughout first and second day, and also between fifth and seventh day of the experiment. The addition of triamine to the resistant strain culture suppressed its growth, so the growth phases were prolonged (Fig. 1).

Fig. 1. Growth of Aspergillus strains sensitive and resistant to triamine

The control strain entered the stationary phase on the seventh day, and the amount of its biomass began to decrease from tenth day. The stationary phase for the resistant strain did not begin until eighth day. The growth curves for the Aspergillus flavus strain were similar to those of Aspergillus niger; the biomass yield, however, was smaller. The greatest biomass differences between the resistant strain, cultured in the presence of 0.05% triamine, and the control (sensitive) strain occurred between day 1 and day 2, as well as on day 4 and day 7. The slope of the growth curve of the resistant strain is slightly greater. The vegetative growth phase of this strain was prolonged, and the stationary phase began around day 8, what is two days later than in control culture. Cell wall in the sensitive strains and the resistant strains. The cell wall content in the mycelium dry mass of the sensitive strains and the resistant strains is shown in Figure 2. For the resistant strains of both species belonging to the genus Aspergillus, the cell wall content in the mycelium dry mass in the presence of

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Cell wall content in the mycelium [%]

50 49 30 20 10 0

Aspergillus niger

Aspergillus flavus

control strain (sensitive) (K) resistant strain, cultured with 0.05% triamine (R + 0.05%) Fig. 2. Participation of the cell wall in the mycelium after 7 days of culture

0.05% triamine exceeded 50%. The non-resistant strains, growing in control conditions, were characterised by the cell wall content in mycelium dry mass lower by ca 7.5% than that in the resistant strains, cultured in the presence of triamine. Results are statistically essential at accepted significance level of 0.01. Structural polysaccharides, including glucan and chitin, are the greatest group of cell wall constituents. In the case of both resistant strains, the glucan content lower by ca 11% in comparison with that in the control strains cultured without triamine was recorded (Fig. 3). Results of variation analysis in Anova test did not show significant differences of chitin content between resistant and control strains. Determined values of F statistic for genus Aspergillus were lower than critical value at accepted significance level of 0.05. The share of lipids in relation to the cell wall mass was 92.5 mg/g d.m. in the sensitive strain of Aspergillus niger. Its content increased by ca 12% in the resistant strain. An increase in the amount of lipids by 41.3% was noticed, from 78.4 mg/g d.m. to 110.8 mg/g d.m., in the strain of Aspergillus flavus cultured in the presence of triamine. The protein content ranged between 219.2– 336.6 mg/g of the cell wall dry mass in the sensitive strains examined. A very slight divergence of protein contend was observed between resistant and control strains of Aspergillus flavus. Results differences are not statistically significant. However, increase in the amount of proteins, equal 21.2%, was observed in the resistant strain of Aspergillus niger in comparison with the sensitive (control) strain. The spectra obtained in spectroscopic analysis show the correlation of absorption as a function of the wave number value. No significant spectrum differences between the control sample and the resistant

Cell wall constitution [mg/g cell wall dry mass]

500 400 300 glucal chitin

200

lipids protein

100 0

K

R + 0.05% Aspergillus niger

K

R + 0.05% Aspergillus flavus

K-control strain (sensitive), cultured without triamine R + 0.05% – resistant strains, cultured with 0.05% triamine Fig. 3. Cell wall constituents of the sensitive and resistant strains after 7 days of culture

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sample were noticed for the cell wall of the strain of Aspergillus niger (Fig. 4). Additional absorption bands were not registered; the absorption zone, however, in the range of the wave number 3050– 2800 cm –1 was slightly stronger for the cell wall of resistant strain. The bands in this range are characteristic of vibrations stretching C-H bonds. Enhanced absorption was also observed in the range 1570– 1528 cm –1 that corresponds to N-O bonds. Spectrum differences were observed in the case of the cell wall of the strain of Aspergillus flavus (Fig. 5). Most bands disappear on the IR spectra of the resistant strain in comparison with the control sample. Slight peaks are visible below 3000 cm–1, in the range 1700– 1800 cm –1, and in the area of 1100 cm –1, which are successively characteristic for vibrations stretching C-H bonds, C = O bonds of carbonyl groups, and C-C bonds.

Fig. 4. Spectrum of cell wall of Aspergillus niger sensitive and resistant strains

Fig. 5. Spectrum of cell wall of Aspergillus flavus sensitive and resistant strains

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Ergosterol [µg/g d.m. of the mycelium]

1000

800

600

400

200

0

5

7 5 Culture time [days] Aspergillus niger

7 Aspergillus flavus

control strains (sensitive) (K) resistant strains, cultured with 0.05% triamine (R + 0,05%) Fig. 6. Ergosterol level in the mycelia of Aspergillus strains sensitive and resistant to triamine

The presence of triamine itself, however, in the cell wall was not observed for both strains of the genus Aspergillus. The individuals observed on the basis of spectra described above may be products of triamine decomposition or of metabolic processes. Ergosterol in the mycelium of the sensitive strains and the resistant strains. The examination of obtained data shows that the synthesis of ergosterol in the resistant strain of Aspergillus niger, cultured in the presence of 0.05% triamine, was inhibited in comparison with the control (sensitive) strain, not exposed to APDA (Fig. 6). After 5 days of culture in the presence of APDA, the ergosterol content was lower in the resistant strain by 33.6% in comparison to the control strain. Further growth of this cell membrane constituent was noticed after 48h. The ergosterol content was also higher by 19.65% in the mycelium of the control strain than that of the resistant strain on seventh day. The sensitive strain of Aspergillus flavus was characterised by the ergosterol content lower by 10% than that in the resistant strain after 5 days of culture. Its growth by 161 mg/g d.m. of the mycelium in the control sample and only by 64 µg/g of d.m. of the mycelium in the resistant strain was noticed after 7 days. Because

Fig. 7. Spectrum of mycelium of Aspergillus niger sensitive and resistant strains

4

Influence of APDA on Aspergillus mycelium

277

Fig. 8. Spectrum of mycelium of Aspergillus flavus sensitive and resistant strains

of such a small increase of tested cell membrane component, a higher ergosterol content was observed for the control strain than for the resistant strain, cultured in the presence of APDA. Differences of ergosterol content between control and cultured mycelium in both growth phases are equal to 6–10%. With the accepted significance level of 0.01, these differences are statistically significant. Spectrophotometric analysis of the mycelium. To identify the differences between the strains of Aspergillus sensitive or resistant to triamine, the dry mass of their mycelia was analysed in the infrared. The examination of the FTIR spectra of the mycelia of both species in the samples with triamine did not show additional peaks in comparison with the control samples (Fig. 7 and 8). This could suggest that no additional functional groups occurred in the mycelia as a result of the presence of APDA. Qualitative changes were not recorded; quantitative changes, however, are noticeable, especially in the wave number ranges 1530– 1260 cm–1 and 700– 800 cm–1. A slightly stronger absorption in the entire spectrum range in comparison with the control sample was noticed in the mycelium of the resistant strain of Aspergillus niger. A reverse situation was recorded in the case of Aspergillus flavus. Discussion The cell wall of moulds belonging to the genus Aspergillus constitutes on the average 50% of the mycelium dry mass (Fiema 1994). The cell wall content reached 54% in the resistant mycelia of the genus Aspergillus, obtained as a result of ten-fold passages and cultured in the presence of 0.05% triamine. The sensitive strains were characterised by a smaller participation of the cell wall, equal to 46%. The growth dynamics of resistant strains was inhibited. According to the Bartnicki-Garcia’s classification of cell wall structures (Bartnicki-Garcia, 1968), moulds of the genus Aspergillus belong to the chitin-glucan category. The content of these structural polysaccharides in the walls of the studied strains is higher in comparison with that given by Rokem et al. (1986) and Fiema (1994). As shown by ¯akowska et al. (1997), the glucan content in the mycelium of sensitive strains of Aspergillus niger in the cell wall is 43%, and that of chitin – 12%. These values are higher for resistant strains, and equal to 55% and 20%, respectively. Similar results were also obtained by Kisser et al. (1980), who reports the glucan content in moulds cell wall is 60% and chitin content – ca 18%. Comparable content values of structural polymers were recorded in the examinations conducted using 0.05% triamine. In contrast to these studies, however, it was noticed that glucan synthesis was suppressed in the cell walls in the resistant strains belonging to the genus Aspergillus. The chitin content did not change almost at all. The resistant strain of Aspergillus niger, cultured in the presence of APDA, showed the protein content growth by 21.6%. In the case of Aspergillus flavus, an increase in the amount of lipids in the wall of the

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4

resistant strain by 41.3% in comparison with the sensitive strain was recorded. These results contradict those obtained by ¯akowska et al. (1997), who recorded a lower level of proteins and lipids for strains resistant to various compounds of molasses than that for sensitive ones. Divergent data may be caused by the use of different substrates or the specificity of the examined strains. Lipids constitute spare substances in the cell; they fill up spaces and merge the cell wall. The growth of their content could then be a defence reaction to the exposition to a harmful agent such as N,N-bis(3-aminopropyl)dodecylamine. Cell wall spectra of the strains examined in the infrared did not reveal the presence of triamine in this part of the cell. The presence of new functional groups that may derive from this chemical compound was not recorded. No qualitative changes were observed in the cell wall of the control and resistant strains of Aspergillus niger, and only quantitative changes were noticed. In the case of the resistant strain of Aspergillus flavus, the disappearance of most bands in comparison with the control sample was observed. The data obtained in the chemical and spectroscopic analyses of the examined cell walls show, that changes caused by triamine does not occur with the same intensity in both species. These changes probably enhance resistance, and are the defence reaction. The cell membrane, also containing ergosterol, is another target for antifungal agents in the mycelium. Richardson and Warnock (1995) report that some strains of moulds resistant to amphotericin B were characterised by a reduced ergosterol content. The ergosterol content recorded in the resistant strains of Aspergillus niger as well as of Aspergillus flavus, subjected to triamine, was also lower than that in the sensitive strains. These findings contradict the data given by Pawiroharsono et al. (1987), who reported that an increased ergosterol content in cytoplasmatic membrane protects the cell from unfavourable environmental conditions. Spectrophotometric analysis in the infrared of the mycelia of both sensitive strains and resistant strains did not reveal significant qualitative differences, only qualitative differences were recorded. The absence of any significant differences was also demonstrated in the analysis of the mycelium ultrastructure (the authors’ unpublished studies). These findings make it possible to claim that the resistant strains belonging to the genus Aspergillus adapted to new conditions, unfavourable for them. Their adaptability may be confirmed by the suppressed development of the mycelium of the resistant strains until eighth day, and then its intensification, which may indicate the enzymatic adaptation of the mycelium. The increase in the participation of the cell wall and the quantitative changes that took place in its composition are also indicative of the adaptation to unfavourable conditions. The results discussed do not comprise all the changes, which reduce the sensitivity to product studied. They confirm, however, the occurrence of moulds resistance in practice. They also indicate types of defence mechanisms developed by growing mycelium as a reaction to a harmful agent. Literature B a r t n i c k i - G a r c i a S. 1968. Cell wall chemistry, morphogenesis and taxonomy of fungi. Ann. Rev. Microbiol. 22: 87–108. D i b o M. and J. B r a s c h. 2001. Occupational allergic contact dermatitis from N,N-bis(3-aminopropyl)dodecylamine and dimethyldidecylammonium chloride in 2 hospital staff. Contact Dermatitis 45: 40. F a r k a š V. 1990. Fungal cell walls: their structure, biosynthesis and biotechnological aspects. Acta Biotechnol. 10: 225–238. F i e m a J. 1994. Research on polysaccharides in selected species of moulds. (in Polish) Biotechnologia 3: 45–53. G r o l l A.H., A.J. D e L u c c a and T.J. W a l s h. 1998. Emerging targets for the development of novel antifungal therapeutics. Trends in Microbiology 6: 117–124. G u t a r o w s k a B. and Z. ¯ a k o w s k a. 2002. Elaboration and application of mathematical model for estimation of mould contamination of some building materials based on ergosterol content determination. Inter. Biodeter. Biodegrad. 49: 299–305. K i e j t s M. 1975. Technique of lipipds, isolation, analysis and identification. (in Russian) p. 72–75. Mir Moskwa. K i s s e r M., C.P. K u b i c e k and M. R ü h r. 1980. Influence of manganes on morphology and cell wall composition of Aspergillus niger during citric acid fermentation. Arch. Microbiol. 128: 26–33. P a w i r o h a r s o n o S., B. N a j i, R. B o n a l y, F. T o n e t t i, C. C h a s s e b o e u f and J.P. R i c h t e r. 1987. Permeability and membrane sterol distribution in Saccharomyces uvarum and Kluyveromyces bulgaricus grown in presence of polyoxyalkylene glycol oleic acid condensates. Appl. Microbiol. Biotechnol. 27: 181–185. R i c h a r d s o n M.D. and D.W. W a r n o c k. 1995. Mycosis. Recognition and therapy. (in Polish) SPRINGER PWN. Warszawa. R o k e m J.S., D. K l e i n, H. T o d e r and E. Z o m e r. 1986. Degradation of fungal cell walls taking into consideration the polysaccharide composition. Enzyme Microb. Technol. 8: 588–592. S t r z e l c z y k A.B. 2001. Adaptation to fungicides of fungi damaging paper. Inter. Biodeter. Biodegr. 48: 255–262. W i l l s E., M.R. R e d i n b o, J.R. P e r f e c t and M. D e l P o e t a. 2000. New potential targets for antifungal development. Emer. Therap. Targets 4: 1–32. ¯ a k o w s k a Z., B. G a b a r a and D. K u s e w i c z. 1997. Cell wall analysis on Aspergillus niger strains characterized by different tolerance to toxic compounds of beet molasses. Acta Microbiol. Pol. 46: 27–36.

Polish Journal of Microbiology 2005, Vol. 54, No 4, 279– 286

The Elimination of Ochratoxin A by Lactic Acid Bacteria Strains MA£GORZATA PIOTROWSKA and ZOFIA ¯AKOWSKA

Institute of Fermentation Technology and Microbiology, Technical University of £ódŸ Wólczañska 171/173, 90-924 £ódŸ, Poland Received 3 June 2005, received in revised form 22 June 2005, accepted 15 July 2005 Abstract The aim of this study was to examine 29 strains of lactic acid bacteria of the Lactobacillus and Lactococcus genera, assessing their sensitivity to ochratoxin A and their ability to remove it from a liquid medium. It was demonstrated that most strains are insensitive to the presence of OTA at the quantity of 5 µg/disc. It was demonstrated that all strains caused a reduction of the toxin amount in the liquid medium. The highest decrease, exceeding 50% of the initial OTA content, was caused by the strains Lactobacillus acidophilus CH-5, L. rhamnosus GG, L. plantarum BS, L. brevis and L. sanfranciscensis. The example of three selected strains confirmed the negative effect of the toxin on the dynamics of bacterial growth. A sharp decrease of ochratoxin A was observed during the first 15 hours of culture growth. In the course of cultivation the amount of the toxin in the medium increased, indicating that the toxin elimination is partially reversible. A small quantity of ochratoxin A became bound to the bacterial biomass. K e y w o r d s: ochratoxin A, lactic acid bacteria, Lactobacillus, mycotoxin elimination

Introduction Most common contaminating agents of food raw materials and products include fungal metabolites: mycotoxins, and among them ochratoxin A (OTA), which most frequently occurs in the countries of Central Europe, also in Poland (Böhm, 1995). The toxin is produced by numerous fungal species belonging to the genera Aspergillus and Penicillum, which grow on plant materials stored in excessively humid conditions. It is heat resistant and is not destroyed during thermal processing, also contaminating final food products, e.g. bread (Alldrick, 1996). If domestic animals are fed with contaminated feed, ochratoxin A cumulates in their bodies and remains in food products of animal origin (Petzinger and Weidenbach, 2002). Ochratoxin A owes its toxicity to its chemical structure: a chlorine atom and a phenol group, as well as a molecule of isocumarin play the main role. Ochratoxin A is believed to be carcinogenic, genotoxic, teratogenic, immunosuppressive and nephrotoxic (Petzinger and Ziegler, 2000). In the case of plant raw materials processed by means of biotechnological tools and used as animal feeds, protection against mycotoxins consists mainly in providing proper cultivation, harvesting and storage conditions (Doyle et al., 1982; Northolt and Bullerman, 1982; Park, 1993). The use of chemical substances and physical processes is allowed to eliminate toxins from animal feeds, a number of requirements must be met, though, e.g. the nutritive and sensory value as well as the physical properties of the product have to be maintained, and the decontamination process has to be economically viable (Sinha, 1998). Detoxification of raw materials used in food processing poses a more serious problem. New opportunities have been created by biological methods, involving the elimination of mycotoxins by microorganisms. Reviews of literature on the subject of biodegradation have been presented by a number of authors (Bhatnagar et al., 1991; Bata and Lasztity, 1999; Karlovsky, 1999). The ability to eliminate ochratoxin A from the environment has been observed for bacteria: Acinetobacter calcoaceticus (Hwang and Draughon, 1994), Phenylobacterium inmobile (Wegst and Lingens, 1983) and Saccharomyces cerevisiae (Štyriak et al., 1998). Aspergillus fumigatus and Aspergillus niger are also capable of OTA degradation (Varga et al., 2000). Enzymatic hydrolysis of the peptide bond, resulting in formation of ochratoxin a and release of Corresponding author: tel. (42) 631 34 70, fax: (42) 636 59 76, e-mail: [email protected]

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phenyloalanine, is considered in literature as the basic toxicity reduction mechanism for ochratoxin A. Peptide bond hydrolysis is carried out by rumen microflora (Hult et al., 1976; Özpinar et al., 1999) and, to a lesser degree, in the alimentary tract of monogastric animals (Madhyastha et al., 1992; Li et al., 2000). Of all microorganisms capable of OTA elimination, lactic acid bacteria attract greatest interest. They have a positive effect on human health, they are safe, and they are applied as a biological agent in numerous biotechnological processes involving raw materials potentially contaminated with ochratoxin A (Wood, 1998). Lactic acid bacteria Lactococcus salivarius, Lactobacillus delbrueckii subsp. bulgaricus and Bifidobacterium bifidum cause a decrease in OTA level in milk (Škinjar et al., 1996). In the presented study, lactic acid bacteria strains of intestinal and plant origin were assessed for their sensitivity to the presence of ochratoxin A in the medium and their ability to eliminate ochratoxin A. The process of OTA elimination from the liquid culture medium during the subsequent phases of growth has been described on the example of the most effective strains. Experimental Materials and Methods Bacterial strains. 29 strains of lactic acid bacteria were used in the research: Lactobacillus rhamnosus GG ATCC 53105 received from Collegium Medicum, Jagiellonian University, Cracow, Lactobacillus acidophilus CH-5 and A92 from Prague Technical University, Department of Milk and Fat Technology, from Danisco Biolacta, Olsztyn: Lactobacillus acidophilus CH-2, H-1 and lnd 1, Lactobacillus delbrueckii subsp. bulgaricus strains J7, P7, 171/2, SL, Lactobacillus helveticus strains 8, D(a), 3035, CH-1, E8, Lactobacillus casei B, 150, 18H, 18cz strains, Lactococcus lactis subsp. lactis 168, Cz, 147, 8FD, 202, from National Collection of Agricultural and Industrial Microorganisms, Budapest: Lactobacillus plantarum strains B1074, B1149, from Collection of Industrial Microorganisms of Institute of Technology Fermentation and Microbiology £OCK 105: Lactobacillus plantarum £OCK 0862, Lactobacillus sanfranciscensis £OCK 0867 and Lactobacillus brevis £OCK 0845 The biomass was stored in 15% glycerol at –20°C. Before each experiment, a portion of bacterial biomass was defrosted and activated by a single passage in liquid MRS medium (MERCK) and cultivation for 24 hours at 37°C. Ochratoxin A standard. Ochratoxin A standard (SIGMA-Aldrich, St Louis, MO, USA) was stored as stock solution in absolute ethanol (200 ppm) at –20°C. Determination of sensitivity of lactic acid bacteria to the presence of ochratoxin A. The degree of sensitivity of the strains of lactic fermentation bacteria to the presence of ochratoxin A was assayed by disc diffusion method (Xiao et al., 1996). A suspension of the bacteria was added to MRS agar medium, in the quantity sufficient to obtain the final number of cells amounting to ca.106 cells in 1 cm3 of the medium. After thoroughly mixing the medium, it was poured into sterile Petri dishes, 15 cm 3 into each dish. Sterile paper discs, 10 mm in diameter, soaked with solutions prepared from standard ochratoxin A solution at the quantity of 20 µl were placed on the surface of the medium. The content of ochratoxin A on the disc amounted to 0.1; 0.5; 1, 5 and 10 µg. After 48 hours of incubation at the temperature of 37oC it was examined if zones inhibiting the growth of the investigated microorganisms appeared around the discs with specific OTA concentrations or not. The strains’ viability was also controlled by plating them on MRS medium. Screening of the strains capable to reduce the amount of ochratoxin A in model media. Liquid MRS medium (Merck) was used in the studies at the quantity of 10 cm3 supplemented with 0.05 cm 3 of ochratoxin A standard solution. The initial concentration of ochratoxin A in the medium was 1000 ppb. The media were inoculated with biomass suspended in physiological salt solution (5% vol.) derived from the logarithmic growth phase with density of 107 CFU/cm3. The bacterial cultures were conducted in static conditions at the temperature of 37oC, for 120 hours, then the cultures were centrifuged (3500 rpm, 10 min) and the amount of ochratoxin A was determined in the post-culture liquid. The decrease of the toxin in the medium in relation to the initial value was expressed in percentages. Assessment of changes in the amount of ochratoxin A during cultivation. Ochratoxin A standard solution was added to 50 ml of liquid MRS medium. The initial concentration of OTA was ca. 1000 ppb, and was precisely determined at time t = 0. Then inoculum was added, consisting of the tested microorganisms in the exponential growth phase, suspended in normal saline (107 cells ml–1). The culture was grown at 37 oC. The amount of ochratoxin A in the post-culture liquid and the biomass was determined after 5, 15, 24 and 40 hours of cultivation. The number of bacterial cells was calculated with standard plate method on MRS medium at the same time intervals. Ochratoxin A analysis. Immunoenzymatic quantitative method ELISA was applied to determine the concentration of ochratoxin A in the biomass and in the post-culture medium, after the biomass was centrifuged. The test used was Ridascreen®Ochratoxin A made by R-Biopharm, Darmstadt, Germany. The extraction procedure was applied in accordance with the producer’s instructions. The detection limit of this test is 80 ppt. Statistical Analysis. The results presented in this study are the average of three measurements. Variance analysis (one-way ANOVA test) was performed with the Microcal (TM) ORIGIN ver. 6.0 software (Northampton, USA).

Results The first stage of the study concerned a comparison of the sensitivity of lactic acid bacteria to the action of different concentrations of ochratoxin A. It was discovered that only one of all examined strains – Lactobacillus helveticus CH-1 was sensitive to ochratoxin A in the whole studied range of concentrations, i.e. from

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Ochratoxin A elimination by lactic acid bacteria Table I The sensitivity of lactic acid bacteria to the presence of ochratoxin A Species

Lactobacillus acidophilus

Lactobacillus delbrueckii subsp. bulgaricus

Lactobacillus helveticus

Lactobacillus casei

Lactococcus lactis, subsp. lactis

Lactobacillus plantarum Lactobacillus sanfranciscensis Lactobacillus brevis Lactobacillus rhamnosus

Strain

µg ochratoxin A/disc 0.1

0.5

1

5

10

CH-2 A92

– –

– –

– –

– –

+ +

H-1 Ind 1

– –

– –

– –

+ +

+ +

CH-5

–

–

–

–

–

J7 P7

– –

– –

– –

– –

+ +

1712 SL

– –

– –

– –

– –

+ +

8

–

–

–

–

+

D(a) CH-1

– +

– +

– +

– +

+ +

3035 E8 B 150

– – – –

– – – –

– – – –

– – – –

+ + + +

18H

–

–

–

–

+

18 cz 168 CZ 147 8FD

– – – – –

– – – – –

– – – – –

– – – – –

+ + + + +

202 B 1074 B 1149 BS BS

– – – – –

– – – – –

– – – – –

– – – – –

+ + + + +

BS GG

– –

– –

– –

– –

+ –

(+) the presence of inhibition zone, sensitive strain

0.1 µg to 10 µg (Table I). The remaining strains showed no sensitivity to this toxin in concentrations ranging from 0.1 to 5 µg, with the exception of the strains Lactobacillus acidophilus Ind1 and L. acidophilus H-1, whose growth was inhibited by OTA at the quantity of 5 µg. The highest dose of the mycotoxin used in the experiment, i.e. 10 µg proved to be inhibitory for the growth of most strains with the exception of L. acidophilus B, L. acidophilus CH-5 and Lactobacillus rhamnosus GG. Strains with the highest sensitivity to ochratoxin A, i.e. L. acidophilus A92, L. acidophilus Ind1 and L. helveticus CH-1 were excluded from further tests. The next experiment aimed to demonstrate whether the phenomenon of ochratoxin A elimination by lactic acid bacteria exists and which of the investigated microorganisms exhibits this feature most strongly. It was proved that the ability to reduce the amount of ochratoxin A is common among lactic acid bacteria but it is varied, depending on the species and the strain of bacteria. Taking into account the average values for the species, it is possible to distinguish those with the greatest ability to remove the toxin: Lactobacillus acidophilus, L. rhamnosus, L. sanfranciscensis and L. plantarum (Figure 1). The remaining species were characterized by removing the toxin in a much smaller quantity. However, the analysis of the values obtained for particular strains within species revealed considerable differentiation, indicating that the examined feature is strain-specific. Out of the 25 investigated strains 10 were capable of removing about a half of the amount of ochratoxin A from the liquid medium. The biggest decrease of the toxin was observed in the culture of intestinal lactobacilli L. acidophilus CH-5, Lactobacillus

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decreasing of ochratoxin A [%]

Piotrowska M., ¯akowska Z.

L.

100 80 60 40 20 0 a

o cid

ph

ilu

s L.

L.

v hel

del

br

eti

cus

ki uec

i su

. bsp

bu

lg.

..

L.

cas

ei L.

rh

n am

osu

s

L.

lac

tis L.

n pla

tar

L.

um

san

fra

sc nci

ens

is

L.

vi bre

s

species

Fig. 1. The elimination of ochratoxin A by different species of lactic acid bacteria

rhamnosus GG, 70.5% and 87.5% respectively (Table II). These strains were also least sensitive to the presence of ochratoxin A in the environment. A somewhat smaller decrease, about 50% reduction of the OTA amount, was observed in the strains of lactic acid bacteria of plant origin Lactobacillus plantarum, L. sanfranciscensis and L. brevis. These strains were classified into a group of organisms moderately resistant to ochratoxin A (Table I). A small degree of OTA elimination from the medium, amounting to approximately 10%, was demonstrated for 7 strains belonging to different species with the same sensitivity to the toxin as the ones mentioned earlier. The comparison of these data leads to the conclusion that there is no correlation between the sensitivity of the strains to the toxin and the ability to eliminate it. The first stage of the study aimed to demonstrate whether the phenomenon of ochratoxin A elimination by lactic acid bacteria exists and which of the investigated microorganisms exhibits this feature most strongly. It was proved that the ability to reduce the amount of ochratoxin A is common among lactic acid bacteria but it is varied, depending on the species and the strain of bacteria. Three strains – L. acidophilus CH-5, L. rhamnosus GG and L. plantarum £OCK 0862 – were selected for further tests, the purpose of which was to evaluate the impact of ochratoxin A on the increase of bacterial biomass and to observe changes in the amount of ochratoxin A during cultivation on liquid medium. The presence of 1000 ppm of ochratoxin A in the medium slowed down the multiplication rate of the lactic Table II The decrease of the amount of ochratoxin A in model medium Species Lactobacillus acidophilus

Lactobacillus delbrueckii subsp. bulgaricus

Lactobacillus helveticus

Lactobacillus casei

Strain

Decreasing of ochratoxin A [%]

CH-2

45.1 ± 0.72

168

16.8 ± 0.62

A92

50.2 ± 0.62

CZ

45.7 ± 0.26

CH-5

70.5 ± 0.98

J7

5.9 ± 0.62

Species

Lactococcus lactis

Strain

Decreasing of ochratoxin A [%]

147

21.0 ± 0.53

8FD

7.8 ± 0.61 59.6 ± 0.44

P7

34.3 ± 0.46

202

171 2

9.6 ± 0.26

B 1074

11.9 ± 0.75

SL

28.3 ± 0.53

B 1149

35.5 ± 0.46

8

67.1 ± 0.46

BS

56.2 ± 0.72

D(a)

11.9 ± 0.44

Lactobacillus sanfranciscensis

BS

52.0 ± 0.53

3035

17.0 ± 0.26

Lactobacillus brevis

BS

56.2 ± 0.72

E8

31.0 ± 0.36

Lactobacillus rhamnosus

GG

87.5 ± 0.66

B

11.5 ± 0.44

150

29.8 ± 0.30

18H

5.7 ± 0.44

18 cz

16.6 ± 0.46

Lactobacillus plantarum

4

283

Ochratoxin A elimination by lactic acid bacteria 9.5

log cell number

9.0 8.5 8.0 7.5 7.0

0

5

10

15

20 time [h]

25

30

35

40

Fig, 2. The effect of ochratoxin A on biomass yield of Lactobacillus acidophilus CH-5, (¨), toxin-free medium; (n), medium with toxin 9.5

log cell number

9.0 8.5 8.0 7.5 7.0

0

5

10

15

20 time [h]

25

30

35

40

Fig. 3. The effect of ochratoxin A on biomass yield of Lactobacillus rhamnosus GG, (o), toxin-free medium; (n), medium with toxin

acid bacteria (Figures 2, 3). Maximum biomass yield for L. acidophilus CH-5 (6.0× 108 CFU ml–1) was only reached after 20 hours, for the control – 5 hours earlier. The cell number was over three times higher in the toxin-free medium, amounting to 1.9 × 109 CFU ml–1 (9.3 Log value). The other strain, L. rhamnosus GG, proved to be less sensitive to the presence of ochratoxin A in the medium. Maximum yield in both toxinfree and contaminated medium was reached at the same time, i.e. after 15 hours of growth. The cell number in the medium with OTA was 2.0 × 108 CFU ml–1, only twice lower than in control (Figure 3). A similar negative effect of ochratoxin A on the increase of bacterial biomass was observed for L. platarum. The changes in the content of ochratoxin A in the post-culture medium are presented in Figure 4. The amount of OTA in the L. acidophilus CH-5 culture plunged dramatically during the first 5 hours, reaching the level of 145 ppb, which means that 85% of the initial OTA content was eliminated. After the next 10 hours, the amount of toxin dropped to 120 ppb. Starting from the 15th hour of incubation, the release of toxin to the medium restarted and lasted until the 40th hour of the process. The level of toxin in the medium finally reached 270 ppb, which means that 17% of the previously eliminated toxin (150 ppb) returned into the medium during the entire growth process. The difference in the OTA level between the 15th and 40th hour is of statistical relevance (P< 0.05). During the 40-hour growth of Lactobacillus acidophilus CH-5, ochratoxin A was eliminated in 72% (Figure 4). For the strain of Lactobacillus rhamnosus GG, a similar process of ochratoxin A reduction was observed. However, the first 5 hours were not as effective as in the

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Piotrowska M., ¯akowska Z. 1000

ochratoxin A [ppb]

800 600 400 200 0 0

10

20 time [h]

30

40

Fig. 4. Change dynamics of the amount of ochratoxin A in the cultures of lactic acid bacteria, (¡) , Lactobacillus rhamnosus GG; (●), L. acidophilus CH-5; (n), Lactobacillus plantarum £OCK 0862

case of the other strain, as only 50% of the initial toxin amount was eliminated. The lowest OTA concentration in the post-culture medium, 80 ppb, was observed during the 15th hour of growth, which means that 90% of toxin had been removed. After 15 hours of incubation, an increase (P < 0.05) in the toxin level occurred, and it reached 160 ppb in the 40th hour. Thus, between the 15 th and 40th hour of incubation, about 9% of previously eliminated toxin returned into the medium. During cultivation of the strain Lactobacillus plantarum BS the amount of ochratoxin A also dropped significantly in the medium in the first 5 hours of incubation, reaching 30 ppm. The next stages of cultivation, i.e. to the 15th hour resulted in a further decrease to the level of 200 ppm. Just as it occurred in the cultures of previously discussed strains, after the 15 th hour of incubation a release of a part of OTA to the medium was observed. Between the 15 th and the 24th hour of incubation 140 ppm returned to the environment, i.e. 17.5% of the previously eliminated toxin, prolonging the cultivation to 40 hours resulted in an increase of the amount of the toxin in the medium to 410 ppm, which is a level comparable to that obtained after 5 hours of cultivation. In the L. plantarum culture the amount of the toxin that returned to the medium was bigger than for the other strains. According to literature data concerning aflatoxin B 1 (El-Nezami et al., 1998), elimination of the toxin from the environment occurs through binding it to the bacterial biomass. To find out if this hypothesis is also correct for the examined toxin, the amount of OTA in the centrifuged bacterial biomass was determined (Table III). The values in the table are expressed in pg, they take account of the level of cell multiplication and relate to one colony forming unit (CFU). The content of ochratoxin A in the biomass extract of L. acidophilus CH-5 increased in the course of growth, reaching 0.167 mg in the 15th hour of incubation, which means that 0.00012 pg of toxin was bound by each CFU. The prolongation of the culture up to 40 hours resulted in a further increase in the amount of OTA up to 0.232 mg, i.e. 0.0011 pg CFU–1. In the case of the other strain, L. rhamnosus GG, a similar tendency was observed, but the amounts of toxin bound by the biomass were higher: 0.00023 pg CFU –1 in the 15th and 0.0021 pg CFU–1 in the 40th hour of growth. Upon analysis of the results presented in Table III, it can be assumed that ochratoxin A is bound by the cells of lactic acid bacteria. However, it is impossible to balance the amount of ochratoxin A, which suggests that apart from binding to the biomass there is another mechanism of removing ochratoxin A. Table III Ochratoxin A in bacterial biomass Time [h] 15 24 40

Ochratoxin A [pg/CFU] Lactobacillus acidophilus CH-5

Lactobacillus rhamnosus GG

0.00012 0.00047 0.0011

0.00023 0.00082 0.0021

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Ochratoxin A elimination by lactic acid bacteria

285

Discussion The scarce published studies suggest that bacteria, including lactic acid bacteria, are microorganisms resistant to this toxin, although the toxicity thresholds reported by different authors are varied. According to the studies of Xiao et al. (1996) on biological activity of OTA in relation to Bacillus brevis, 2 µg/disc is the lowest dose of the toxin able to create a zone inhibiting growth, which is less than in the case of the strains of lactic fermentation bacteria investigated in this study. The above-presented values demonstrate that ochratoxin A has a negative effect on the growth of lactic acid bacteria. The results are different from those presented by Ali-Vehmas et al. (1998), which showed that ochratoxin A at 20 ppm does not inhibit the growth of L. plantarum and L. casei. The results of this study demonstrate that the tested strains of lactic acid bacteria, L. acidophilus CH-5, the probiotic L. rhamnosus GG and lactobacilli of plant origin L. plantarum are capable of eliminating ochratoxin A from model media. The process is partly reversible and, upon culture prolongation, the part of toxin is released back into the medium after 40 hours of incubation. A part of the toxin becomes bound by the bacterial biomass, however, the remaining amount is eliminated in a different way. These results are consistent with the studies on aflatoxin B1, according to which the strain of L. rhamnosus GG is capable of eliminating 80% of toxin from the medium exclusively through physical binding by the cell wall components (El-Nezami et al., 1996; 1998; 2000). On the basis of the conducted studies it is possible to conclude that the application of selected strains of lactic acid bacteria in the production of fermented food and probiotic products may reduce the health risk related to the possible contamination of food products exposition to fungal toxins. These strains may protect directly humans eating several foods contaminated by toxins too. Literature A l l d r i c k A.J. 1996. The effects of processing on the occurrence of ochratoxin A in cereals. Food Additiv. Contamin. 13, suppl. 27–28. A l i - V e h m a s T., A. R i z z o, T. W e s t e r m a r c k and F. A t r o s h i. 1998. Measurement of antibacterial activities of T-2 toxin, deoxynivalenol, ochratoxin A, aflatoxin B 1 and fumonisin B1 using microtitration tray-based turbidimetric techniques. Vet. Med. 45: 453–458. B a t a Á. and R. L a s z t i t y. 1999. Detoxification of mycotoxin-contaminated food and feed by microorganisms. Trends Food Sci. Technol. 19: 223–228. B h a t n a g a r D., E.B. L i l l e h o j and J.W. B e n n e t t. 1991. Biological detoxification of mycotoxins. In: J.E. Smith and R.S. Henderson, Mycotoxins and animal foods. (pp. 815–826) London: CRC Press Inc. B ö h m J. 1995. Occurrence and noxiousness of mycotoxins in European foods. Pol. J. Food Nutr. Sci. 4/45: 2, 3–7. D o y l e M.P., R.S. A p p l e b a u m, R.E. B r a c k e t t and E.H. M a r t h. 1982. Physical, chemical and biological degradation of mycotoxins in foods and agricultural commodities. J. Food Protect. 45: 964–971. E l - N e z a m i H., P. K a n k a a n p ä ä, S. S a l m i n e n and J. A h o k a s. 1998. Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1. Food Chem. Toxicol. 36: 321–326. E l - N e z a m i H., H. M y k k ä n e n, P. K a n k a a n p ä ä, S. S a l m i n e n and J. A h o k a s. 2000. Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B1 from chicken duodenum. J. Food Protect. 63: 549–552. E l - N e z a m i H., S. S a l m i n e n and J. A h o k a s. 1996. Biological control of food carcinogens with use of Lactobacillus GG. Nutrit. Today. Supplement, 31: 41–42S. H u l t K., A. T e i l i n g and S. G a t e n b e c k. 1976. Degradation of ochratoxin A by a ruminant. Appl. Environ. Microbiol. 32: 443–444. H w a n g C.A. and F. D r a u g h o n. 1994. Degradation of ochratoxin A by Acinetobacter calcoceticus. J. Food Protect. 57: 410–414. K a r l o v s k y P. 1999. Biological detoxification of fungal toxins and its use in plant breeding, feed and food production. Nat. Toxins, 7: 1–23. L i S., R.R. M a r q u a r d t and A.A. F r o h l i c h. 2000. Identification of ochratoxins and some of their metabolites in bile and urine of rats. Food Chem. Toxicol. 38: 141–152. M a d h y a s a t h a M.S., R.R. M a r q u a r d t and A.A. F r o h l i c h. 1992. Hydrolysis of ochratoxin A by the microbial activity of digesta in the gastrointestinal tract of rats. Arch. Environ. Contamin. Toxicol. 23: 468–472. N o r t h o l t M.D. and L.B. B u l l e r m a n. 1982. Prevention of mould growth and toxin production through control of environmental conditions. J. Food Protect. 45: 519–526. Ö z p i n a r H., G. A u g o n y t e and W. D r o c h n e r. 1999. Inactivation of ochratoxin in ruminal fluid with variation of pH-value and fermentation parameters in an in vitro system. Environm. Toxicol. Pharmacol. 7: 1–9. P a r k D.L. 1993. Controlling aflatoxin in food and feed. Food Technol. 8: 92–96. P e t z i n g e r E. and A. W e i d e n b a c h. 2002. Mycotoxins in the food chain: the role of ochratoxins. Livestock Prod. Sci. 76: 245–250.

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P e t z i n g e r E. and K. Z i e g l e r. 2000. Ochratoxin A from a toxicological perspective. J. Vet. Pharmacol. Therap. 23: 91–98. S i n h a K.K. 1998. Detoxification of mycotoxins and food safety. pp.381–405. In: K.K Sinha and D. Bhatnagar (eds), Mycotoxins in agricultural and food safety. New York, Marcel Dekker Inc. Š t y r i a k I., E. È o n k o v á, E. R a z z a z i c and J. B ö h m. 1998. Inhibition of mycotoxins production and their biodegradation by lactobacilli and yeast’s. Proceedings of IV Conference “Mycotoxins in food and feed” Bydgoszcz, 101–108. Š k i n j a r M., J.L. R a š i c and V. S t o j i c i c. 1996. Lowering of ochratoxin A level in milk by yoghurt bacteria and Bifidobacteria. Folia Microbiol. 41: 26–28. V a r g a J., K. R i g o and J. T e r e n. 2000. Degradation of ochratoxin A by Aspergillus species. Int. J. Food Microbiol. 59: 1–7. W e g s t W. and F. L i n g e n s. 1983. Bacterial degradation of ochratoxin A. FEMS Lett. 17: 341– 344. W o o d B.J.B. ed. 1998. Microbiology of fermented foods. Blackie Academic and Profesional. X i a o H., S. M a d h y a s t h a, R.R. M a r q u a r d t, S. L i, J.K. Vo d e l a, A.A. F r o h l i c h and B.W. K e m p p a i n e n. 1996. Toxicity of ochratoxin A, its opened lactone form and several of its analogs: structure-activity relationships. Toxicol. Appl. Pharmacol. 137: 182–192.

Polish Journal of Microbiology 2005, Vol. 54, No 4, 287– 294

Selection of Potentially Probiotic Lactobacillus Strains Towards their Inhibitory Activity against Poultry Enteropathogenic Bacteria MAGDALENA KIZERWETTER-ŒWIDA and MARIAN BINEK

Division of Bacteriology and Molecular Biology, Department of Pre-Clinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Ciszewskiego 8, 02-786 Warsaw, Poland Received 1 March 2005, received in revised form 27 September 2005, accepted 29 September 2005 Abstract Lactobacilli were isolated from chicken gastrointestinal tract and examined for their potentially probiotic properties towards their inhibitory activity against poultry enteropathogenic bacteria. Biochemical tests, ITS-PCR and cell wall protein analysis were used to characterize the Lactobacillus isolates. The identification of isolated Lactobacillus strains based on phenotypic properties was not always satisfactory. ITS-PCR together with protein profile were found to be helpful in strain identification. Lactobacilli were tested for the inhibitory activity against selected strains of poultry enteropathogenic bacteria (Salmonella Enteritidis, Escherichia coli and Clostridium perfringens). Examined supernatants from Lactobacillus broth cultures demonstrated major antimicrobial activity against C. perfringens. Lower antimicrobial activity were observed against E. coli and Salmonella Enteritidis. The strongest inhibition effect were obtained using supernatant of Lactobacillus acidophilus strain 3D. Results received from this study confirmed that identification of Lactobacillus spp. is often tedious. Some isolates, which are in vitro antagonistic against enteropathogenic bacteria may be considered as potential candidates for poultry probiotics, especially in controlling necrotic enteritis caused by C. perfringens. K e y w o r d s: Lactobacillus spp., poultry, probiotics.

Introduction Lactobacilli are a heterogenous, non-sporing, rod-shapped and catalase-negative group of Gram-positive bacteria. They are inseparable components of natural microflora in the gastrointestinal tract of humans and animals and they have been widely used for medical and veterinary applications as well as in food fermentation processes. Bacteria of this group are commonly used as probiotics, which beneficially affect the animal host (Fuller, 1992; Gomes and Malcata, 1999). The general concept of probiotics administration is to improve the intestinal microbial balance which is assumed to provide better protection against various diseases (Fuller, 1992). They may be also beneficial in growth promotion, better feed utilization, diseases resistance, reduction of colonization and shedding of enteropathogenic bacteria. One of the most desirable features of probiotic bacteria is their ability to synthesize antimicrobial substances such as organic acids, hydrogen peroxiode and bacteriocins (Roy et al., 2000). Bacteriocins are polypeptides which have bactericidal or bacteriostatic effect, usually against bacteria closely related to the producer strain. Most bacteriocins produced by lactobacilli have narrow spectrum of activity, but some of them are active also against Gram-negative bacteria (Callewaert et al., 1999; Cuozzo et al., 2000; Jack et al., 1995). Bacterial strains to be used as probiotics in animal production could be therefore isolated from natural gastrointestinal microflora with the purpose of more specific application (La Ragione et al., 2004; Tsai et. al., 2005). However, testing their efficacy in vivo is expensive and time consuming. For that reason reliable in vitro methods are required for selecting new strains of probiotic bacteria to obtain profitable in vivo effects. Thus one of the important features of a new probiotic bacterial strains, which could be easily tested in vitro, is their activity against enteropathogenic bacteria (Tsai et. al., 2005). Conventional phenotypical identification of lactobacilli is based on their morphology and carbohydrate fermentation profiles. However, it is not always sufficient. There are closely related strains, which can not

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4

be easily distinguished by biochemical tests, since phenotypic properties may also depend on environmental conditions (Tynkkynen et al., 1999). Over the past years the significant progress has been made in the molecular taxonomy and identification of lactobacilli. Several molecular methods have been used for species identification and differentiation i.e., pulse field gel electrophoresis (PFGE) (Roy et al., 2000; Ventura and Zink, 2002; Tynkkynen et al., 1999; Zapparoli et. al., 1998), sequencing of rRNA genes (Kullen et al., 2000), protein profiling (Andrighetto et. al., 1998; Drake et al., 1996; Gevers et al., 2001), ribotyping (Tynkkynen et al., 1999), conventional PCR (Guarneri et al., 2001; Spano et. al., 2002), ITS-PCR (Drake et al., 1996), RAPD-PCR (Daud Khaled et al., 1997; Roy et al., 2000; Tynkkynen et al., 1999; Zapparoli et al., 1998), and REP-PCR (Gevers et al., 2001; Ventura and Zink, 2002). The objective of this study was to examine lactobacilli strains isolated from chicken gastrointestinal tract for the antagonistic activity against selected enteropothogenic bacteria. Isolates were identified using phenotyping and molecular biology methods and tested for activity against poultry enteropathogenic bacteria (Salmonella Enteritidis, Escherichia coli and Clostridium perfringens). Experimental Materials and Methods Bacteria and growth conditions. Lactobacilli were isolated from faeces and intestines of healthy chickens. Samples were inoculated into Rogosa medium (Merck). The plates were incubated at 37°C for 48 h under anaerobic conditions generated by BBLTM GasPak system (Becton Dickinson). All isolates growing on Rogosa agar were stained by Gram method for microscopic examination and tested for catalase production using 3% H2O2. Carbohydrate fermentation patterns were determined using the API 50 CHL test according to the instructions of the manufacturer. The results were analyzed by MiniApi analyser (BioMérieux). The following Lactobacillus spp. strains were obtained from the Collection of Industrial Microorganisms, Institute of Agriculture and Food Biotechnology in Warsaw: L. plantarum CIM 813, L. brevis CIM 369 and L. fermentum CIM 592. Salmonella Enteritidis (10 strains), Escherichia coli (10 strains) and Clostridium perfringens (11 strains) have been isolated from chicken organs in Laboratory of Division of Bacteriology and Molecular Biology, Faculty of Veterinary Medicine, Warsaw Agricultural University. Salmonella Enteritidis, E. coli and Clostridium perfringens were grown according to standard bacteriological procedures (Malicki et. al., 2004). All E. coli isolates were haemolytic and all C. perfringens isolates were shown in PCR to possess genes encoding alpha toxin production, therefore they were regarded as pathogenic isolates. Internal Transcribed Spacer PCR (ITS-PCR). For the genotypic characterization of isolated Lactobacillus spp. strains, ITS-PCR was applied according to the method described by Drake et al. (1996). ITS-PCR was carried out with primers G1 (5’-GAAGTCGTAACAAGG-3’) and L1 (5’-CAAGGCATCCACCGT-3’). Genomic DNA was extracted using InstaGeneTM Matrix (BioRad) from separate colonies on MRS (acc. to De Man, Rogosa and Sharpe) agar according to the manufacturer instruction. Each reaction mixture contained 0.2 mM of each dNTPs, 5 µL of PCR buffer, 1 µM of each primer, 2.5 mM MgCl2, 2.5 U Taq DNA Polymerase and water to the volume of 50 µL. All reagents for PCR were purchased from Fermentas. The following thermal cycling conditions were used: initial denaturation for 1 min at 94°C and 35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C, extension for 2 min at 72°C and final extension for 7 min at 72°C. The PCR amplifications were performed with a TC-312 Thermal Cycler (Techne). Volume of 15 µL of the products was analyzed by gel electrophoresis in 2% agarose gel. The molecular size of the obtained DNA fragments was estimated by comparison with GeneRuller TM 100bp DNA Ladder Plus (Fermentas). Each ITS-PCR gel was documented by VersaDoc apparatus (BioRad). Protein profiling. Extraction of cell wall proteins and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed following the procedure described by Henriksson et al. (1995). The isolates were incubated in MRS broth at 37°C anaerobically overnight. Volume of 1 mL of culture was centrifuged at 3500 g for 10 min. The pellet was washed twice with PBS, resuspended in 1 mL lysozyme buffer (lysozyme in PBS, 60 mg/mL) and incubated at 37°C for 3 h. Then 0.5 mL of lysis buffer (2% SDS, 0.01 M Tris, 0.01 M EDTA, pH 8.0) was added and incubated at 100°C for 2 min. Electophoresis was carried out using Mini-PROTEAN 3 Electrophoresis Cell (BioRad) on 10% polyacrylamide gels, using the Laemmli buffer system. Gels were stained with Coomasie blue. Detection of antibacterial activity – agar spot test. Gram-positive, catalase-negative rods were tested for inhibitory activity against randomly selected Salmonella Enteritidis clinical isolate in agar spot test. The agar spot test was performed on MRS agar plates (diameter 90 mm) as described previously by Schillinger and Lucke (1989). Isolated lactobacilli were spot inoculated on MRS agar plates and incubated at 37°C for 24 h under anaerobic atmosphere. The plates were then overlaid with 7 mL of soft agar (0.75% agar) containing 10 7 cells of Salmonella Enteritidis per mL and incubated aerobically at 37°C for additional 24 h. Formation of clear zones of growth inhibition around Lactobacillus colonies and their diameters were recorded. Inhibition was scored as positive if a diameter of clear zone around the colony was 5 mm or larger. Only lactobacilli, which showed inhibitory activity against Salmonella Enteritidis in agar spot test were selected for further analysis. Detection of antibacterial activity – agar well diffusion assay. The inhibitory activity of culture supernatants of selected Lactobacillus strains were tested against Salmonella Enteritidis, E. coli and C. perfringens by the agar well diffusion assay following the procedure of Schillinger and Lucke (1989). Selected Lactobacillus strains were grown in MRS broth for 24 h at 37°C under anaerobic conditions. Cells were removed by centrifugation (4000 g for 30 min at 4°C), the pH of the supernatant was adjusted to 6.0 with 10 M NaOH and supernatant was filtered through 0.45 µm-pore-size membrane (Millipore). The culture supernatants were concentrated five times using rotary

4

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Poultry enteropathogenic bacteria inhibition by Lactobacillus

evaporation according to Strompfová et. al. (2003) and Zhu et. al. (2000). Portions of 35 mL of Mueller-Hinton agar (BioMérieux) or TSN agar (BioMérieux) were autoclaved and cooled to about 48°C and then 100 µL of overnight cultures of eneropathogenic bacteria containing approximately 2 × 10 7 cells per mL were added. The inoculated medium was then poured into plates (diameter 90 mm) and wells 6 mm in diameter were cut. Aliquots (100 mL) of supernatants from different Lactobacillus isolates were dispensed into the wells and plates were incubated overnight at 37°C with appropriate atmosphere. The diameter of clear zones of growth inhibition around each well was measured, inhibition zones of 8 mm or more were scored as positive.

Results Bacteriological examination of chicken faeces and intestinal content provided to obtained sixteen isolates of Gram-positive, catalase-negative rods, which phenotypically correspond to the genus of Lactobacillus. According to the carbohydrate fermentation pattern analyzed with MiniApi system three strains were recognized as L. salivarius (9m, R1, 1a), three as L. brevis (E, 1/12, K1), two as L. acidophilus (3D, K2), one as L. fermentum (K13), one as L. plantarum (1/2s), four as Leuconostoc lactis (1D, 6d, K16, 2m) alternatively L. salivarius, one as Lactococcus lactis (Go³) alternatively L. brevis and one as Lactococcus raffinolactis (2/s) alternatively L. plantarum (Table I). Table I Identification of examined strains with API 50 CHL, ITS-PCR profile similarity and protein clusters Identification with API 50 CHL Strain

Identification /Other possibility

Percentage of identification

ITS-PCR profile similarity

Protein cluster

99.5

*

A

1D

Leuconostoc lactis / L. salivarius

3D

Lactobacillus acidophilus

99.9

*

A

6d

Leuconostoc lactis / L. salivarius

99.5

*

A

9m

Lactobacillus salivarius

98.9

*

A

R1

Lactobacillus salivarius

99.9

*

A

K13

Lactobacillus fermentum

97.3

*

A

K16

Leuconostoc lactis / L. salivarius

96.9

*

A

E

Lactobacillus brevis 3

98.8

Similar to L. brevis CIM 369

B

1/12

Lactobacillus brevis 3

98.5

Similar to L. brevis CIM 369

B

2m

Leuconostoc lactis / L. salivarius

99.5

*

B

½s

Lactobacillus plantarum 1

98.6

Similar to L. plantarum CIM 813

C

1a

Lactobacillus salivarius

98.9

*

D

2/s

Lactococcus raffinolactis / L. plantarum 1

82.3

**

D

Go³

Lactococcus lactis / L. brevis 1

84.2

**

D

K1

Lactobacillus brevis 1

49.7

unique

E

K2

Lactobacillus acidophilus 1

51.0

unique

F

* similarity between nine isolated strains (2m, 6d, 9m, R1, 1D, 1a, 3D and non scheduled in Fig. 1 K13 and K16) in ITS-PCR profile ** similarity between two isolated strains (2/s and non scheduled in Fig. 1, Go³) in ITS-PCR profile

The application of ITS-PCR allows to obtain bands from about 300 bp to 1500 bp (Fig. 1). Products below 300 bp were obtained from all isolates. Strain 1/2s showed very similar ITS-PCR pattern to L. plantarum CIM 813. Four additional bands, which have been observed for 1/2s were from 900 bp to above 1500 bp in size. Two isolates 1/12 and E produced similar ITS-PCR profiles to L. brevis CIM 369. Additional band, about 350 bp was seen only in L. brevis CIM 369. Other profiles did not correspond with strains from the Collection of Industrial Microorganisms, Institute of Agriculture and Food Biotechnology, used in this study. Nine strains (2m, 6d, 9m, R1, K13, K16, 1D, 1a, 3D and non scheduled in Fig. 1 K13 and K16) had very similar ITS-PCR profiles comprised of three or four bands and additional fourth band was detected only in two (1a, 3D) isolates. Two strains 2/s and non scheduled in Fig. 1 Go³ produced the same ITS-PCR pattern and two others (K1, K2) showed a unique ITS-PCR patterns (data not shown). Based on their cell wall protein profiles all examined strains were divided into six clusters. Representative cell wall protein profiles are shown in Fig. 2. Strains and protein profiles are presented in Table I. Each

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Kizerwelter-Œwida M., Binek M.

Fig. 1. Representative ITS-PCR products of isolated strains and reference Lactobacillus spp.

protein profile was represented by several bands, but some of them were cluster typical. Seven strains were included into cluster A (1D, 3D, 6d, 9m, R1, K13, K16) characterized by presence of distinct bands at approximately 25, 30, 37 and 48 kDa. Three strains were included into cluster B (E, 1/12, 2m) together with L. brevis CIM 369. L. brevis CIM 369 produced species specific bands at 38 kDa, which were identified also Table II Inhibitory activity of examined Lactobacillus spp. strains obtained in agar spot test against Salmonella Enteritidis Diameter of growth inhibition zone

Strain 1D 3D 6d 9m R1 K13 K16 E

Diameter of growth inhibition zone

Strain

6 mm 15 mm 10 mm 5 mm 10 mm 6 mm 10 mm 5 mm

1/12 2m ½s 1a 2/s Go³ K1 K2

6 mm 10 mm 10 mm 15 mm 10 mm 15 mm 8 mm 10 mm

Table III Inhibitory activity of Lactobacillus spp. culture supernatants against selected intestinal pathogens

Tested strains

Concentrated culture supernatant of Lactobacillus strains Number 3D 1/2s 1a R1 Go³ of Inhibition zone in mm tested strains 8–10 >10 8–10 >10 8–10 >10 8–10 >10 8–10 >10

Clostridium perfringens

11

3

8

E. coli

10

1

*2

Salmonella Enteritidis

10

1*3

*1

– 6 strains non inhibited;

*2

2

7

4

4*1

1

10

1

5

6

*2

0

6

*2

0

6*2

0

4*2

2

4

1*4

0

3*1

1

1*4

0

1*4

0

– 4 strains non inhibited;

9

*3

– 5 strains non inhibited;

*4

– 9 strains non inhibited

4

Poultry enteropathogenic bacteria inhibition by Lactobacillus

291

Fig. 2. Types of protein profiles for representative Lactobacillus strains M – protein weight marker (77.0 kDa; 50.0 kDa; 34.3 kDa; 28.8 kDa; 20.7 kDa); L. br. – L. brevis CIM 369; L. pl. – L. plantarum CIM 813.

Fig. 3. Agar well diffusion assay: A – C. perfringens; B – Salmonella Enteritidis

in three isolates from cluster B. Only one strain was included into cluster C (1/2s), which revealed very similar protein profile as L. plantarum CIM 813. Cluster D was characterized by three distinct bands at 25, 30 and 36 kDa, contained three isolates (1a, 2/s, Go³). Two isolates (K1, K2) produced a unique protein profiles and were included in separate clusters: E and F. Results obtained from agar spot test are presented in Table II and from agar well diffusion assay are presented in Table III. Examined supernatants demonstrated the strongest antimicrobial activity in agar well diffusion assay against C. perfringens. Lower activity were observed against E. coli and Salmonella Enteritidis. Culture supernatant from strain 3D showed the best inhibition properties and the diameter of zones of growth inhibition for two C. perfringens isolates were larger then 15 mm.

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Discussion Identification of sixteen isolated strains based on API 50 CHL system revealed that ten were Lactobacillus spp., four were Leuconostoc spp. and two were Lactococcus spp. Other properties such as cell morphology, protein profile and ITS-PCR pattern suggested that all isolates belong to the genus Lactobacillus. This finding confirmed that sugar fermentation profile should not be used for reliable identification of lactobacilli (Nigatu et. al., 2000; Reinheimer et. al., 1995). Percentage of identification (%id) obtained from MiniApi computer system was 96.9 or higher, with exception of two strains recognized as Lactococcus (2/s, Go³) with %id 82.3 and 84.2 respectively. Two other strains recognized as L. brevis and L. acidophilus (K1, K2) with %id respectively 49.7 and 51.0, were classified as separate clusters E and F, based on their protein profile. However, even satisfactory %id obtained for Leuconostoc might not be reliable because examined strains showed other properties common with genus Lactobacillus. Analysis of cell wall proteins has been used to differentiate numerous strains of lactobacilli. In particular Drake et. al. (1996) reported that extraction of cell wall proteins followed by SDS-polyacrylamide gel electrophoresis (PAGE) has been proved to be a reliable and rapid method for strains characterization. SDS-PAGE of cell wall proteins and ITS-PCR were found to be useful as complementary methods for strain identification (Gatti et. al., 1997; Du Toit et.al., 2003; Henriksson et. al., 1995; Mukai et. al. 2003). Protein cluster A contained seven isolates recognized with API 50 CH system as L. salivarius (2), L. fermentum (1), L. acidophilus (1), and also as Leuconostoc lactis (3). What is interesting, three isolates identified as Leuconostoc lactis, each with different carbohydrate fermentation pattern, were alternatively recognized as L. salivarius. Two other strains from cluster A identified phenotypically as L. fermentum (K13) and L. acidophilus (3D) produced protein profile characteristic for cluster A, therefore their phenotypical identification was not reliable. Two isolates from cluster B (E, 1/12) produced identical protein profile as L. brevis CIM 369, thus confirming their identification. Another strain 2m from cluster B recognized as Leuconostoc lactis might also belonging to L. brevis, because it produced protein bands specific for this species. In one strain from cluster C (1/2s) phenotypical identification as L. plantarum was confirmed by protein profile. Cluster D contained three isolates (1a, 2/s, Go³) recognized as L. salivarius, Lactococcus raffinolactis and Lactococcus lactis. Results of phenotypical identification seems to be not reliable, because for Lactococcus %id was not satisfactory, as was mentioned above. Results confirmed that identification of Lactobacillus spp. is tedious. Particularly L. acidophilus is difficult to identify, since L. acidophilus group representing six species was described (Johnson et. al., 1980). Gatti et. al. (1997) described different proteins specific for this genus (20, 31 and 55 kDa). Results obtained in this study proved the antimicrobial activity of the culture supernatants from isolated Lactobacillus strains. The activity might be due to antimicrobial substances, probably proteinaceous molecules, which are produced into the culture broth. Further studies focused on the nature of those antimicrobial substances produced by isolated Lactobacillus strains are in progress and will render more information about their characteristic. Bacteriocins produced by Lactobacillus spp. are mainly antagonistic against other members within this genus and against other Gram-positive bacteria (Sablon et. al., 2000). Previous studies on bacteriocin producing lactobacilli were conducted predominantly using another Lactobacillus spp. as an indicator strains (Callewaert et al., 1999; Cuozzo et al., 2000). The aim of our study was to isolate lactobacilli strongly antagonistic against enteropathogenic poultry microorganisms including C. perfringens and Gram negative bacteria i.e., Salmonella Enteritidis and E. coli. Those pathogens are frequently associated with poultry diseases and is of great significance in human health protection. Our objective was to isolate Lactobacillus spp. with desirable in vitro properties, which can be further analyzed and eventually used for developing a new probiotic for poultry. Strain 3D revealed inhibitory activity against C. perfringens and some E. coli and Salmonella Enteritidis and will be used in future in vivo studies in chickens. Demonstrated antibacterial activity may enable to use them in prevention or even therapy of necrotic enteritis caused by C. perfringens. Specific antagonistic activity of L. johnsonii FI9785 against C. perfringens in poultry was previously reported by La Ragione et al. (2004). They showed that single dose of L. johnsonii FI9785 suppressed in vivo colonization and persistance of C. perfringens, reduced E. coli colonization, but there were no significant effects against Salmonella Enteritidis colonization. The use of antibiotics in animal production should be seriously considered due to increased bacterial resistance and antibiotic residues in animal products. Particular attention was given to the subject when use

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of certain antibiotics was banned by the Council of the European Commission (1998). As a result, alternative methods such as probiotics have been widely investigated. Presented results indicate that potentially probiotic lactobacilli may be isolated from natural gut microflora of poultry. Acknowledgements. This work was supported by the Ministry of Science and Information Society Technologies (grant no. 2 P06K 020 26).

Literature A n d r i g h e t t o C., P. D e D e a, A. L o m b a r d i, E. N e v i a n i, L. R o s s e t t i and G. G i r a f f a. 1998. Molecular identification and cluster analysis of homofermentative thermophilic lactobacilli isolated from dairy products. Res. Microbiol. 149: 631–643. C a l l e w a e r t R., H. H o l o, B. D e v r e e s e, J. V a n B e e u m e n, I. N e s and L. D e V u y s t. 1999. Characterization and production of amylovorin L471, a bacteriocin purified from Lactobacillus amylovorus DCE 471 by a novel three-step method. Microbiology 145: 2559–2568. Council of the European Communities. 1998. Regulation EEC no. 2821/98. Off. J. Eur. Commun., L351/4. C u o z z o S.A., F. S e s m a, J.M. P a l a c i o s, A.P. d e R u i z H o l g a d o and R.R. R a y a. 2000. Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705. FEMS Microbiol. Lett. 185: 157–161. D a u d K h a l e d A.K., B.A. N e i l a n, A. H e n r i k s s o n and P.L. C o n w a y. 1997. Identification and phylogenetic analysis of Lactobacillus using mulitiplex RAPD-PCR. FEMS Microbiol. Lett. 153: 191–197. D r a k e M.A., C.L. S m a l l, K.D. S p e n c e and B.G. S w a n s o n. 1996. Differentiation of Lactobacillus helveticus strains using molecular typing methods. Food. Res. Int. 29: 451–455. D u T o i t M., L.M.T. D i c k s and W.H. H o l z a p f e l. 2003. Identification of heterofermentative lactobacilli isolated from pig feaces by numerical analysis of total soluble cell protein patterns and RAPD-PCR. Lett. Appl. Microbiol. 37: 12–16. F u l l e r R. 1992. Probiotics: The scientific basis. London, New York, Chapman & Hall. G a t t i M., E. F o r n a s a r i and E. N e v i a n i. 1997. Cell-wall protein profiles of diary thermophilic lactobacilli. Lett. Appl. Microbiol. 25: 345–348. G e v e r s D., G. H u y s and J. S w i n g s. 2001. Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol. Lett. 205: 31–36. G o m e s A.M.P. and F.X. M a l c a t a. 1999. Bifidobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trends Food Sci. Technol. 10: 139–157. G u a r n e r i T., L. R o s s e t t i and G. G i r a f f a. 2001. Rapid identification of Lactobacillus brevis using the polymerase chain reaction. Lett. Appl. Microbiol. 33: 377–381. H e n r i k s s o n A., L. A n d r é and P.L. C o n w a y. 1995. Distribution of lactobacilli in the porcine gastrointestinal tract. FEMS Microbiol. Ecol. 16: 55–60. J a c k R.W., J.R. T a g g and R. B i b e k. 1995. Bacteriocins of Gram-Positive Bacteria. Microbiol. Rev. 59; 171–200. J o h n s o n J.L., C.F. P h e l p s, C.S. C u m m i n s, J. L o n d o n and F. G a s s e r. 1980. Taxonomy of the Lactobacillus acidophilus group. Int. J. Syst. Bacteriol. 30: 53–68. K u l l e n M.J., R.B. S a n o z k y - D a w e s, D.C. C r o w e l l and T.R. K l a e n h a m m e r. 2000. Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex. J. Appl. Microbiol. 89: 511–516. L a R a g i o n e R.M., A. N a r b a d, M.J. G a s s o n and M.J. W o o d w a r d. 2004. In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry. Lett. Appl. Microbiol. 38: 197–205. M a l i c k i K. and M. B i n e k. 2004. Outline of the clinical veterinary bacteriology (In Polish). SGGW , Warszawa. M u k a i T., K. A r i h a r a, A. I k e d a, K. N o m u r a, F. S u z u k i and H. O h o r i. 2003. Lactobacillus kitasatonis sp. nov., from chicken intestine. Int. J. Syst. Evol. Microbiol. 53: 2055–2059. N i g a t u A., S. A h r n e and G. M o l i n. 2000. Temperature-dependent variation in API 50 CH fermentation profiles of Lactobacillus species. Curr. Microbiol. 41: 21–6. R e i n h e i m e r J.A., L. M o r e l l i, V. B o t t a z z i and V. S u a r e z. 1995. Phenotypic Variability among Cells of Lactobacillus helveticus ATCC 15807. Int. Diary J. 5: 97–103. R o y D., P. W a r d, D. V i n c e n t and F. M o n d o u. 2000. Molecular identification of Potentially Probiotic Lactobacilli. Curr. Microbiol. 40: 40–46. S a b l o n E., B. C o n t r e r a s and E. V a n d a m m e. 2000. Antimicrobial peptides of lactic acid bacteria: mode of action, genetics and biosynthesis. Adv. Biochem. Eng. Biotechnol. 68: 21–60. S c h i l l i n g e r U. and F.K. L u c k e. 1989. Antibacterial activity of Lactobacillus sake isolated from meat. Appl. Environ. Microbiol. 55: 1901–1906. S p a n o G., L. B e n e d u c e, D. T a r a n t i n o, G. Z a p p a r o l i and S. M a s s a. 2002, Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR. Lett. Appl. Microbiol. 35: 370–374. S t r o m p f o v á V., A. L a u k o v á and D. M u d r o ò o v á. 2003, Effect of bacteriocin-like substance produced by Enterococcus faecium EF55 on the composition of avian gastrointestinal microflora. Acta Vet. Brno 72: 559–564. T s a i C.C., H.Y. H s i h, H.H. C h i u, Y.Y. L a i, J.H. L i u, B. Yu and H.Y. T s e n. 2005, Antagonistic activity against Salmonella infection in vitro and in vivo for two Lactobacillus strains from swine and poultry. Int. J. Food Microbiol. 2: 185–194.

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T y n k k y n e n S., R. S a t o k a r i, M. S a a r e l a, T. M a t t i l a - S a n d h o l m and M. S a x e l i n. 1999. Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulse-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei Strains. Appl. Environ. Microbiol. 65: 3908–3914. V e n t u r a M. and R. Z i n k. 2002. Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis. FEMS Microbiol. Lett. 217: 141–154. Z a p p a r o l i G., S. T o r r i a n i and F. D e l l a g l i o. 1998. Differentiation of Lactobacillus sanfranciscensis strains by randomly amplified polymorphic DNA and pulsed-field gel electrophoresis. FEMS Microbiol Lett. 166: 325–332. Z h u M.W., W. L i n and D.Q. W u. 2000. Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7, J. Appl. Mcrobiol. 88: 877–886.

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The Effect of Acid Adaptation Conditions on Heat Resistance of Escherichia coli O157: H7 HAL¤L TOSUN1* and ÔAH¤KA AKTU˜ GÖNÜL2 1 Department

of Food Engineering, Faculty of Engineering, Celal Bayar University, Manisa-Turkey 2 Department of Food Engineering, Faculty of Engineering, Ege University, ¤zmir-Turkey Received 30 May 2005, received in revised form 12 September 2005, accepted 15 September 2005 Abstract The objective of this study was to determine the effect of acid adaptation conditions on heat resistance of E. coli O157: H7 932. E. coli O157: H7 was adapted to acid by exposing the cells to pH 4.5 (2h), pH 5.0 (1h), and pH 5.5 (1 h) in tryptic soy broth. D and z values of the acid adapted and control cultures at 54°C, 56°C, and 58°C were determined in E buffer. The heat resistance of E. coli O157: H7 increased significantly (p < 0.05) after acid adaptation at pH 4.5 or pH 5.0. E. coli O157: H7 adapted to acid at pH 4.5 for 2 h had the highest D values at all temperatures tested (20.3 – 10.7 – 3.3 min) while D values of culture adapted to acid at pH 5.0 for 1 h were 18.2, 7.9, and 2.6 min at 54°C, 56°C and 58°C, respectively. Heat resistance of culture adapted to acid at pH 5.5 for 1 h and the control culture was not significantly different (P < 0.05). Culture adapted to acid at pH 4.5 had the highest z value (5.10°C), whereas control culture had the lowest z value (4.33°C). This study showed that the magnitude of heat tolerance changed with the adaptation pH and at low adaptation pH, E. coli O157: H7 showed maximum heat resistance. Acid adaptation at pH 4.5 or 5.0 provides E. coli O157: H7 with cross-protection against heat treatments, and that this factor must be considered to estimate this pathogen’s thermal tolerance accurately. K e y w o r d s: E. coli O157:H7, acid adaptation, heat resistance.

Introduction E. coli O157: H7 is an important food-borne pathogen that causes the disease syndromes of hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura in humans (Griffin and Tauxe, 1991). It was identified in 1982 and outbreaks of food-borne illness due to E. coli O157:H7 have been reported with increasing frequency since that time (Bell, 2002). Undercooked ground beef has been implicated most often in outbreaks of food-borne illness caused by E. coli O157:H7, but more acidic foods such as unpasteurized apple juice (McCarty, 1996), apple cider (Besser et al., 1993), mayonnaise (Weagant et al., 1994) and yoghurt (Morgan et al., 1993) have also been implicated in outbreaks. The acid tolerance property may allow E. coli O157:H7 to survive in highly acidic foods. The extended survival of enteric pathogens in acidic foods and the increased tolerance of acid adapted cells to unfavourable growth conditions is well established (Arnold and Kaspar, 1995; Leyer et al., 1995; Semanchek and Golden, 1996). Briefly acid adaptation or acid tolerance is a phenomenon by which microorganisms show an increased resistance to environmental stress after the exposure to a moderate acid environment (Foster, 1991). Acid adaptation may also increase the heat resistance of pathogens. Leyer and Johnson (1993) showed that acid adaptation of Salmonella typhimurium resulted in increased thermal tolerance. Farber and Pagotto (1992) also reported that acid adapted Listeria monocytogenes enhanced thermal tolerance. Previously in our laboratory we have demonstrated that E. coli O157:H7 has the ability to survive at extremely low pH (pH 3.0) if first adapted to mild pH (pH 4.5– 5.5). Furthermore, it was also noted that the * Corresponding author: Department of Food Engineering, Faculty of Engineering, Celal Bayar University, Muradiye, ManisaTURKEY. Telephone: + 902362412144 (221). Fax: + 902362412143, e-mail: [email protected].

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extent of increased acid tolerance was affected by the adaptation pH. Maximum acid tolerance was observed in E. coli O157:H7 adapted to acid at pH 4.5, subsequently to pH 5.0 and pH 5.5 (Tosun, 2003). The objective of this study was to determine the effect of acid adaptation conditions on heat resistance of E. coli O157:H7. Experimental Materials and Methods Bacterial strain and media. E. coli O157:H7 932 strain (a clinical isolate) was provided by M. P. Doyle (Center for Food Safety and Quality Enhancement. Department of Food Science and Technology, The University of Georgia, Griffin, USA). E. coli O157:H7 was stored at + 4°C in tryptic soy agar (TSA, Oxoid) and subcultured every month. Culture was activated from stock culture after two successive transfers of the test organism in tryptic soy broth (TSB, Oxoid) at 37°C for 24 h. This activated culture was used in experimental studies. Acid adaptation of test organism. To prepare the acid adapted cells of E. coli O157:H7 the procedure described by Tosun (2003) was followed. Cells were grown overnight at 37°C in TSB at pH 7.0 and 40 millilitres of the cultures was taken and dispensed equally to four centrifuge tubes. Cultures were centrifuged at 5000 rpm. The supernatant was discarded, and the cell pellets were suspended in 10 ml of pH 4.5, 5.0 and 5.5 TSB (pH adjusted with 6N HCI) for acid adapted cells and other cell pellets were suspended in 10 ml of pH 7.0 TSB for nonadapted cells (control culture). Culture adapted to acid at pH 4.5 was incubated at 37°C for 2 h. Other acid adapted and control cultures were incubated at 37°C for 1 h. Calculation of D and z values. Acid adapted and control cultures were centrifuged at 5000 rpm. The supernatant was discarded and the cell pellets were washed once with 10 ml of E buffer (Vogel and Bonner, 1956) pH 7.0, centrifuged and resuspended in the same amount of E buffer. E buffer was prepared at 50x strength as follows. In distilled water (670 ml) were dissolved successively, MgSO 4 × 7H2O (10 g), citric acid × H20 (100 g), K2HPO4 × anhydrous (500 g), and NaNH4HPO4 × 4H2O (175 g), the final volume being about 1 liter. After 50-fold dilution with distilled water, the resulting single strength medium has pH of 7.00. 1 ml inoculum was taken from acid adapted and control culture and added to 100 ml of E buffer heated at 54°C, 56°C and 58°C in 250 ml Erlenmeyer flasks in a water bath. The surface of E buffer in the flask was 2 cm below the level of the water surface during heat treatment. Uninoculated sample was used for temperature control by using a thermometer. D values (time to inactivate 90% of the population) were calculated by plotting the log number of survivors against time for each heating temperature. The line of best fit for survivors plots was determined by linear regression analysis. Z values (change in heating temperature needed to change the D value by 90%) were estimated by plotting the log D values versus heating temperatures. Enumeration of E. coli O157:H7. Viable cells of acid adapted and control cells were enumerated immediately at every 4 or 10 minute of heating. Serial decimal dilutions in 0.1% peptone water were prepared. The viable populations of E. coli O157:H7 were than determined by plating 0.1 ml of the serially diluted samples on sorbitol MacConkey agar (SMAC, Oxoid) and incubated at 37°C fo2r 4 – 48 h. E. coli O157:H7 formed colourless colonies on SMAC agar. Statistical analysis. Experiments were run in duplicates. Data were analysed using the SPSS 9.0 statistical software (SPSS Inc., Chicago, IL, USA) for analysis of variance and Duncan’s multiple range test.

Results Thermal inactivation curves for acid adapted and control E. coli O157:H7 cells heated in E buffer at 54°C, 56°C and 58°C are shown in Figure 1. D values for acid adapted and control cultures are listed in Table I. E. coli O157:H7 adapted to acid at pH 4.5 for 2 h had the highest D values of all temperatures Table I D values and correlation coefficients of thermal inactivation curves for acid adapted and control culture of E. coli O157:H7 Temperature 54°C

56°C

58°C

Cell type acid adapted (pH 4.5) acid adapted (pH 5.0) acid adapted (pH 5.5) control culture (pH 7.0) acid adapted (pH 4.5) acid adapted (pH 5.0) acid adapted (pH 5.5) control culture (pH 7.0) acid adapted (pH 4.5) acid adapted (pH 5.0) acid adapted (pH 5.5) control culture (pH 7.0)

Correlation coefficient

D value

0.86 0.80 0.84 0.86 0.96 0.99 0.97 0.94 0.93 0.98 0.99 0.98

20.3 18.2 14.0 15.9 10.7 7.90 5.58 7.41 3.3 2.6 2.3 1.9

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(a)

log cfu/ml

6 5 4 3 2 1 0

0

20

40 60 Heating time (min)

80

100

8

(b)

log cfu/ml

6 4 2 0

0

10

20 30 Heating time (min)

40

50

8

(c)

log cfu/ml

6 4 2 0

0

4

8 Heating time (min)

12

16

Fig. 1. Thermal inactivation curves for E. coli O157:H7 adapted to acid at various pH. pH 4.5 (u), pH 5.0 (n), pH 5.5 (▲), control culture (●); heated at 54°C (a), 56°C (b) and 58°C (c) in E buffer

tested. There was no statistically significant difference between the D values of culture adapted to acid at pH 5.5 and control culture (P < 0.05). However D values of culture adapted to acid at pH 5.0 for 1 h were higher than those of culture adapted to acid at pH 5.5 or those of control culture (P< 0.05). Table II Z values and correlation coefficients of thermal death time curves for acid adapted and control culture of E. coli O157:H7 Cell type

Correlation coefficient

Z value (°C)

Acid adapted (pH 4.5) Acid adapted (pH 5.0) Acid adapted (pH 5.5) Control culture (pH 7.0)

0.97 0.99 0.99 0.97

5.10 4.71 5.09 4.33

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Regression statistics and z values are listed in Table II. E. coli O157:H7 adapted to acid at pH 4.5 for 2 h had the highest z value (5.10°C), whereas control culture had the lowest z value (4.33°C). Acid adaptation of E. coli O157: H7 at pH 4.5 or 5.0 significantly increased the heat resistance of E. coli O157: H7 in all temperatures tested (P< 0.05). However heat resistance of E. coli O157: H7 adapted to acid at pH 5.5 for 1 h was not significantly different from that of the control (P< 0.05). Discussion Microbial adaptation responses to one stress can lead to cross protection against another stress. It has been long recognised that pH can affect microbial thermal resistance. Increased thermal tolerance resulting from bacterial responses to exposure to an acidic environment has been demonstrated in L. monocytogenes (Farber and Pagotto, 1992). Leyer and Johnson (1993) also reported that acid adapted S. typhimurium cells had higher heat tolerance than nonadapted counterparts. Cheville et al. (1996) reported that exposure of E. coli O157: H7 to an environment induced acid shock resulting in expression of new genes, regulated by the alternative sigma factor that is encoded by the rpoS locus. Perhaps, RpoS regulated proteins cross-protection against heat. Ryu and Beuchat (1998) and Mazzotta (2001) reported that acid adaptation significantly increased the heat resistance of E. coli O157: H7. In these studies acid tolerance was determined for only one pH value, however in our current study heat tolerance of E. coli O157: H7 adapted to acid at different pH value was determined. In this study we showed that the magnitude of heat tolerance changed with the adaptation pH and lower the adaptation pH the greater the heat tolerance. At each heating temperature the D values of cells adapted to acid at pH 4.5 or 5.0 were significantly higher than D values of control cells. The level of heat tolerance in E. coli O157: H7 was influenced by adaptation pH. Splittstoesser et al. (1996) reported that D values of E. coli O157: H7 was 2.5 min when heated in apple cider (pH 4.5) at 58°C. D58°C value of our test strain adapted to acid at pH 4.5 observed in our study was considerably higher than the value of 2.5 min reported by these researches. Doyle and Schoeni (1984) reported that D values for E. coli O157: H7 (strain 932) in ground beef were 39.83 min at 54.4°C and 4.5 min at 57.2°C. Our study shows that D54°C values of strain 932 were 20.3, 18.2, 14.0 and 15 min according to adaptation pH. Our test strain has lower D values because TSB contains no fat to protect cells which might occur in ground beef. In summary, maximum heat resistance was observed when E. coli O157: H7 was adapted to acid at pH 4.5 for 2 h. These findings may have important implications in the food industry because most of fermented foods have pH value near 4.5. In addition, hot water and organic acid sprays are frequently used for the decontamination of beef carcasses in the meat industry. Increased tolerance of acid adapted E. coli O157: H7 cells to heat could have practical implications when establishing mild thermal processing schemes to eliminate the organism from fermented foods. Acknowledgements: The authors gratefully acknowledge Celal Bayar University’s Scientific Research Project Fund for providing financial support.

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Polish Journal of Microbiology 2005, Vol. 54, No 4, 301– 304

Co-Infection of Hamsters with Toxin A or Toxin B-Deficient Clostridium difficile Strains ADAM SZCZÊSNY 1,2, GAYANE MARTIROSIAN1,3*, STUART COHEN2 and JOSEPH SILVA Jr.2 1 Department

of Medical Microbiology Medical University of Silesia, Katowice, Poland of Internal Medicine, Division of Infectious and Immunologic Diseases University of California, Davis Medical Center, Sacramento, CA, USA 3 Department of Histology and Embryology Center of Biostructure Research, Medical University of Warsaw, Warsaw, Poland 2 Department

Received 22 June 2005, received in revised form 21 November 2005, accepted 24 November 2005 Abstract Male Syrian hamsters (Mesocricetus auratus) were used to study interactions between different toxin deficient strains of C. difficile. After sensitization with clindamycin, hamsters were intragastrically co-infected with the appropriate dilutions corresponding to 100, 1000 and 10,000 cells of four (toxin A or B-deficient) C. difficile strains (8864, P-829, W-38 and W-74). In addition, a group of hamsters was infected with C. difficile VPI 10463, a reference toxigenic strain. Colonization and mortality was observed within 48 hours in the group of hamsters infected with the reference toxigenic strain. No clinical disease was observed in the groups of hamsters co-infected with the toxin A or B-deficient strains. Re-infection of these hamsters (co-infected with toxin deficient isolates) with C. difficile VPI 10463 resulted in clinical disease and death suggesting that these strains do not confer protection against infection with a toxigenic strain. Macroscopic and microscopic observations of the cecum of re-infected hamsters demonstrated uniformly multiple large hemorrhagic areas without pseudomembranes. Hamsters infected with as few as 100 – 500 cells of the toxigenic strain – VPI 10463 alone demonstrated pseudomembranes and multiple hemorrhages. These results suggest that even though the toxin deficient strains did not prevent re-infection with a toxigenic strain of C. difficile, they may play a role in the histopathologic changes after re-infections in the hamster model. Further studies with a larger number of hamsters and C. difficile strains of various molecular profiles are required to better understand the interaction between these strains. K e y w o r d s: Clostridium difficile, toxin deficiency, hamsters

Introduction Clostridium difficile causes antibiotic-associated diarrhea (CDAD), colitis and pseudomembranous colitis (PMC) in humans through the actions of toxins (mainly A and B). C. difficile, a spore-forming anaerobic bacteria, is the predominant causative agent of nosocomial infectious diarrhea , and the most common cause of hospital acquired diarrhea in the USA (Martirosian et al., 1995). CDAD has become a major clinical problem with the increased use of antibiotics such as clindamycin, penicillins, cephalosporins and others. Cases of C. difficile associated diarrhea have also been reported after cancer chemotherapy. Antibiotic treatment is problematic in patients with CDAD. Incidences of C. difficile – associated diseases are described even after treatment of patients with vancomycin or metronidazole (Szczêsny et al., 2002). The later is preferred in cases of CDAD or PMC since vancomycin therapy is associated with colonization of patients with vancomycin-resistant enterococci. Toxins A and B are considered the major virulence factors in C. difficile. Genes for toxins A and B (tcdA and tcdB) are located on a large 19.6 kb chromosomal fragment known as the pathogenicity locus (PaLoc) which is a prerequisite for virulence (Poxton et al., 2001). The PaLoc also includes three other genes (tcdC, * Corresponding author address: Department of Microbiology, Medical University of Silesia, 18 Medyków str., 40-572 Katowice, Poland, phone/fax: (48-32) 252 60 75, e-mail: [email protected]; [email protected]

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tcdD, tcdE). Molecular changes in the PaLoc of C. difficile strains correlate with the virulence. Toxigenic strains of C. difficile produce both toxins and non-toxigenic strains lack both toxins and genes. Recently mutant strains producing only one toxin are described (toxin A-negative/toxin B-positive and toxin A-positive/toxin B-negative) (Rupnik et al., 2005; Cohen et al., 1998). One of these strains, 8864 (tcdA–/tcdB+) is known as a causative agent of diarrhea in humans and animals (Borriello et al., 1992). Nevertheless limited work has been done on characterization of a possible virulence factors in such clinical isolates of C. difficile. The hamster provides the most useful model to study the pathophysiology of CDAD (Sambol et al., 2001). It is the closest model of CDAD to that observed in humans. Fatal colitis in the hamster model occurs 3– 5 days after administration of one dose of an antibiotic and toxigenic C. difficile strain. In this preliminary study, we used hamster model to investigate interactions between different mutant strains of C. difficile given intragastrically as a mixture. Experimental Materials and Methods Strains of Clostridium difficile. Table I, lists the strains of C. difficile and their phenotypic characteristics. All strains were cultured on Brucella Blood agar (Anaerobic Systems, San Jose, CA). Plates were incubated anaerobically at 37°C for 48 hours. Strains were identified by routine laboratory methods (Martirosian et al., 2004). One colony of each strains was inoculated into Brain Heart Infusion broth – BHI (Difco) and incubated anaerobically at 37°C overnight. Cultures were centrifuged at 3000 rpm for 10 min to pellet the cells. Cells were re-suspended in sterile Phosphate Buffer Saline (PBS). Dilutions of each C. difficile strain corresponding to 100, 1000 and 10,000 cells were prepared in PBS. The appropriate dilutions of four C. difficile strains (8864, P-829, W-38 and W-74) were mixed and used as the inoculum. C. difficile VPI 10463, a reference toxigenic strain was used as the control for clinical disease. Dilutions of the reference strains were done as described above. Each strain was tested for susceptibility to clindamycin and erythromycin by the E-test method (AB Biodisk, Sweden). For detection of both toxins (A and B) of C. difficile strains, Tox A/B ELISA test (TechLab, USA) and PCR for toxin A and B genes (primer pairs YT-28 & YT-29 and YT-17 & YT-18) was performed as previously described. Additionally TCD toxin A test (Becton Dickinson, USA) for detection of toxin A was also performed (Kuhl et al., 1993; Martirosian et al., 2004). Toxin B was detected in vitro by cytotoxicity and neutralization assays on McCoy cell line. PCR ribotyping was performed to compare all five C. difficile strains. For PCR – ribotyping primers for amplification of 16S-23S rRNA intergenic spacer were used as previously described (Martirosian et al., 1995).

Table I Molecular profile of Clostridium difficile strains Strain VPI 10463

1

Characteristics

Source

tcdA +/tcdB +

ATCC

8864

tcdA –/tcdB +

ATCC

P-829

tcdA +/tcdB –

UCDMC1

W-38

tcdA +/tcdB –

UCDMC1

W-74

tcdA –/tcdB +

UCDMC1

These strains are part of the C. difficile collection at the University of California, Davis Medical Center, and were isolates from cases of CDAD in Seattle, WA (Cohen et al., 1998).

Hamsters. Twenty four male Syrian hamsters 6 – 7 weeks old (Mesocricetus auratus) were obtained from Charles River. Hamsters were housed in groups of 4 in isolator cages with air filters fitted in their lids. They were observed during a one week period and fecal pellets were screened for the presence of C. difficile by direct application of a small amount of material on a selective medium Cycloserine Cefoxitine Fructose Agar (CCFA) supplemented with sodium taurocholate. Twenty hamsters were sensitized on day 0 with 3 mg/ml (subcutaneously – SC) of clindamycin (Cleocin phosphate, Pharmacia). Two days after receiving the antibiotic, hamsters were infected respectively with: the mixture of C. difficile toxin deficient strains (groups 3, 4, 5), VPI 10463 (group 6). Hamsters were infected by intragastric intubation (gavage) with the appropriate feeding needles. Gavage was performed following light anesthesia with Sevoflurane. As controls, a group of hamsters (group 1) received saline (SC) instead of clindamycin and 0.5 ml of saline was administered intragastrically. In addition, the clindamycin control group (group 2) received clindamycin (SC) and 0.5 ml of saline intragastrically. All hamsters were weighed on day 0 and every day there after. Hamsters were observed and scored twice daily for the following symptoms: lethargy, weight loss, onset to wet tail and time to death, for a two-week period. Every second day during this two weeks period fecal pellets from each hamster was cultured for the presence of C. difficile on selective CCFA plates with sodium taurocholate, as described above. After a two-weeks period, all animals in groups 3, 4 and 5 were re-infected with C. difficile VPI 10463. Table II shows the animal groups and treatment.

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Co-infection of hamsters with toxin A or toxin B-deficient C. difficile Table II Groups of hamsters and treatment Group 1

Saline s.c. (0.2 ml) + 0,1 ml saline by gavage (Control group)

Group 2

Clindamycin (3 mg s.c. in 0.2 ml) + 0.1 ml saline by gavage (Clindamycin control)

Group 3

Clindamycin s.c. + 0.1 ml mixture of Clostridium difficile strain (10,000 cells)

Group 4

Clindamycin s.c. + 0.1 ml mixture of Clostridium difficile strain (1000 cells)

Group 5

Clindamycin s.c. + 0.1 ml mixture of Clostridium difficile strain (100 cells)

Group 6

Clindamycin s.c. + 0.1 ml VPI 10643 Clostridium difficile strain (100 – 500 cells)

Results and Discussion Characterization of C. difficile strains. All 5 C. difficile strains demonstrated similar colony morphology and characteristic C. difficile biochemical reactions. Results of antibiotic susceptibility testing by E-tests are presented in Table III. E-tests demonstrated susceptibility to clindamycin and erythromycin in all C. difficile strains. Only one strain W-74 demonstrated higher MIC (2.0 µg/ml) of clindamycin and erythromycin, compared with 4 other C. difficile strains (range 0.094–1.0 µg/ml). Toxigenicity and PCR-ribotyping of C. difficile strains. All five C. difficile strains used in this experiment demonstrated positive results in Tox A/B ELISA designed for detection of both toxins A and B. The culture filtrates of each C. difficile strains were analyzed for cytotoxicity using fibroblasts cell line. Strains VPI 10463 and 8864 demonstrated cytopathic effect on tissue culture, and this effect was neutralized by the specific anti-C. difficile serum (TechLab, USA). The culture filtrates of strains P-829, W-38, W-74 showed no cytopathic activity on cells. Toxin A activity using the TOX A ELISA kit was positive for all strains except strain 8864. The results for PCR of toxin A and B gene and PCR ribotyping of C. difficile are shown in Table III. Table III Characterization of C. difficile strains C. difficile Clindamycin/Erithromycin Tox A/B strains MICs (µg/ml) ELISA VPI 10643

0.38

0.50

+

TCD Tox A

Cytotoxicity PCR for toxin PCR riboand neutralization genes (A/B) typing

+

+

+/+

C

8864

0.50

0.75

+

–

+

–/+

A

P-829

0.094

0.38

+

+

–

+/–

B

W-38

0.60

1.0

+

+

–

+/–

D

W-74

2.0

2.0

+

+

–

+/–

E

Hamsters. Clinical disease was not observed in the groups of hamsters co-infected with the toxin deficient strains (groups 3, 4, 5) or in the control groups (1 and 2). No C. difficile growth was obtained from fecal samples taken from hamsters of groups 1, 2, 3, 4 and 5. In the case of group 6 (strain VPI 10463) we observed colonization and mortality of all animals within 48 hours after infection. All the hamsters showed pseudomembranes and hemorrhages in the colonic mucosa, both, macroscopically and microscopically (histology slides). C. difficile was cultured from fecal contents of all hamsters in group 6. Toxigenicity, antibiotic susceptibility testing and PCR-ribotyping were consistent with the characteristics of the reference strain VPI 10463 (Table III). After the two weeks period we re-infected (by intragastric intubation – gavage) 12 hamsters (group 3, 4 and 5) which had previously received the mixture of toxin deficient C. difficile strains, with strain VPI 10463 (100– 500 cells). Within two days, all hamsters showed clinical symptoms of CDAD and died. C. difficile was cultured on selective CCFA plates from the fecal contents of all animals. All characteristics of the strain isolated were identical to strain VPI 10463. Macroscopic and microscopic studies of colons demonstrated multiple large hemorrhagic areas without pseudomembranes. Several reports indicate that previous colonization with nontoxigenic C. difficile may be protective against colonization with a toxigenic strain (Wilson and Sheagren, 1983). Attempts have been made to treat patients with a nontoxigenic strain to prevent further relapses with a toxigenic strain. Even though some

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results have been promising, it is not clear whether this approach will be useful in clinical practice. This preliminary study was undertaken to determine if previous infection with toxin A or B-deficient strains confers protection in hamsters against infection with a toxigenic strain. No colonization or clinical symptoms were observed when hamsters were co-infected with C. difficile toxin deficient strains (3 toxin B-deficient and one toxin A-deficient strain). Co-infection with as high as 10,000 cfu of these isolates did not result in clinical symptoms. Re-infection with C. difficile VPI 10463 resulted in death within 48 hours. These results suggest that previous infection with toxin deficient isolates does not result in protection against colonization and disease development by a toxigenic strain. Why a mixture of these toxin deficient isolates does not cause clinical symptoms in the hamster remains to be investigated. It is possible that adherence plays a major role in this case, and that other genetic characteristics of the deficient strains related to adherence mechanisms and colonization may play a role. In summary, one implication derived from this study is that there may be other pathogenic factors that are co-linked to the pathogenicity locus which are important in colonization and development of clinical colitis. Thus, strains having the ability to produce only one toxin may have other pathogenic factors that are eliminated or not expressed. These can include production of anti-chemotactic factors, proteinases, alterations in adhesion, etc. The studies of these factors are very important because biotherapy still holds promise for either preventing C. difficile colitis or treatment of patients with active disease and/or relapses. The mutant isolates studied do not appear to offer any significant protection against toxigenic C. difficile strains in the hamster model. However, a larger study with more animals and combination of strains fully characterized may be provide useful information. Acknowledgements. We would like to thank Dr Yajarayma Tang-Fredman for critical comments and advices.

Literature B o r r i e l l o S.P., B.W. W r e n, S. H y d e, S.V. S e d d o n, P. S i b b o n s, M.M. K r i s h n a, S. T a b a q c h a l i, S. M a n e k and A.B. P r i c e. 1992. Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile. Infect. Immun. 60: 4192–4199. C o h e n S., Y. T a n g, B. H a n s e n and J.Jr. S i l v a. 1998. Isolation of a toxin B-deficient mutant strain of C. difficile in a case of recurrent C. difficile – associated diarrhea. Clin. Infect. Dis. 26: 410–412. K u h l S.L., Y.J. T a n g, L. N a w a r r o, P.H. G u m e r l o c k and J.Jr. S i l v a. 1993. Diagnosis and monitoring of C. difficile infections with the polymerase chain reaction. Clin. Infect. Dis. 16: 234–238. M a r t i r o s i a n G., S. K u i p e r s, A. v a n B e l k u m, H. V e r b r o u g h and F. M e i s e l - M i k o l a j c z y k. 1995. PCR ribotyping and arbitrarily primed PCR for typing of C. difficile from Polish maternity hospital. J. Clin. Microbiol. 33: 2016–2021. M a r t i r o s i a n G., A. S z c z ê s n y, S.H. C o h e n and J.Jr. S i l v a. 2004. Isolation of non-toxigenic strains of C. difficile from cases of diarrhea among patients hospitalized in hematology/oncology ward. Pol. J. Microbiol. 53: 197–200. P o x t o n I.R., J. M c C o u b r e y and G. B l a i r. 2001 The pathogenicity of C. difficile. Clin. Microbiol. Infect. 7: 421–427. R u p n i k M., B. D u p u y, N.F. F a i r w e a t h e r, D.N. G e r d i n g, S. J o h n s o n, I. J u s t, D.M. L y e r l y, M.R. P o p o f f, J.I. R o o d, A.L. S o n e n s h e i n, M. T h e l e s t a m, B.W. W r e n, T.D. W i l k i n s and C. v o n E i c h e l - S t r e i b e r. 2005. Revised nomenclature of C. difficile toxins and associated genes. J. Med. Microbiol. 54: 113–117. S a m b o l S.P., J.K. T a n g, M.M. M e r r i g a n, S. J o h n s o n and D.N. G e r d i n g. 2001. Infection of hamsters with epidemiologically important strains of C. difficile. J. Infect. Dis. 183: 1760–1766. S z c z ê s n y A., A. K a ñ s k i and G. M a r t i r o s i a n. 2002. Incidence of pseumembranous colitis after vancomycin-treated MRSA infection. Clin. Microbiol. Infect. 8: 58–59. W i l s o n K.H. and J.N. S h e a g r e n. 1983. Antagonism of toxigenic C. difficile by nontoxigenic C. difficile. J. Infect. Dis. 147: 733–736.

Polish Journal of Microbiology 2005, Vol. 54, No 4, 305– 310

The Susceptibility of Anaerobic Bacteria Isolated from Periodontal Diseases to Photodynamic Inactivation with Fotolon (Chlorin e6) ZUZANNA DRULIS-KAWA1,2*, ARTUR BEDNARKIEWICZ3, GABRIELA BUGLA-P£OSKONSKA1, WIES£AW STRÊK3 and W£ODZIMIERZ DOROSZKIEWICZ1 1 Institute

of Genetics and Microbiology, University of Wroc³aw, Przybyszewskiego 63/77, 51-148 Wroc³aw, Poland 2 Lower Silesian Centre of Paediatrics in Wroc³aw, Kasprowicza 64/66, 51-147 Wroc³aw, Poland 3 Institute of Low Temperatures and Structure Research, Polish Academy of Sciences, Okólna 2, 50-422 Wroc³aw, Poland Received 27 June 2005, received in revised form 24 October 2005, accepted 25 October 2005 Abstract Photodynamic inactivation (PDI) may be a very promising alternative method for the antimicrobial treatment of periodontitis. Several studies have demonstrated the sensitivity of subgingival flora to PDI using toluidine blue, methylene blue, and chlorin e6 derivatives. In the present study we report the activity of the Fotolon sensitizer, composed of chlorin e6 and polyvinylpyrrolidone (PVP), against anaerobic bacteria isolated from periodontal diseases. Over 99.9% reduction in colony forming units in 20 Gram-positive and 30 Gram-negative clinical anaerobic strains was obtained. K e y w o r d s: photodynamic inactivation, Fotolon (chlorin e6), anaerobic bacteria

Introduction Periodontal diseases result from the accumulation of bacterial plaque on the tooth surface. The occurrence of anaerobic bacteria such as Porphyromonas gingivalis, Prevotella sp., Treponema sp., Veillonella sp., Bacteroides sp., Capnocytophaga sp., and Actinomyces sp. in periodontal pockets results from the accumulation of metabolic products. Later, inflammation begins together with posterior destruction which leads to periodontal disease. The common treatments of periodontitits involve mechanical removal of the biofilm (plaque) and antimicrobial chemotherapy (Sbordone et al., 1990; Slot, 1979; Wirkström et al., 1993). However, the overuse of antibiotics favours the natural selection of drug-resistant oral pathogens (Kleinfelder et al., 1999). The alternative method of periodontal disease treatment could be photodynamic inactivation (PDI). To be efficient, PDI requires a proper dose of light delivered to the appriopriate amount of photo sensitizer (PS) accumulated inside the bacteria or on their cell walls. The (laser) light illumination leads to the formation of reactive singlet oxygen or/and redox reactions (Wainwright, 1998). The effects of PDI on microorganisms depend on the structure of the PS and the incubation time of the drug with bacteria cells. PDI can cause damage to the cell wall and increase cytoplasmic membrane permeability and nucleic acid strand breakage (Wainwright, 1998; Jacob and Hamann, 1975; Menezes et al., 1990). The photodynamic eradication of oral and wound pathogens (localised infection) seems to be a very promising technique (Bertoloni et al., 1990; Embleton et al., 2002; Hamblin et al., 2002; Hamblin et al., 2003; Merchat et al., 1996; Soncin et al., 2002; Soukos et al., 1996). Several studies have demonstrated the sensitivity of subgingival flora to PDI using cationic charged photosensitizers: toluidine blue (Dortbudak et al., 2001; Kömerik et al., 2003; Sarkar and Wilson, 1993; Wilson, 1994), methylene blue (Chan and Chern-Hsiung, 2003), and poly-L-lysine- chlorin e6 conjugates (Rovaldi et al., 2000; Soukos et al., 1998). * Corresponding author. Mailing address: Dr. Zuzanna Drulis-Kawa, Institute of Genetics and Microbiology, University of Wroc³aw, Przybyszewskiego 63/77, 51-148 Wroc³aw, Poland, Tel./fax: +48-71-325-21-51, E-mail: [email protected]

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Drulis-Kawa Z. et al.

4

In the present study we demonstrate that Fotolon (chlorin e6), having been successfully used in diagnosing tumours and in treating surface tumours as well as mucous and open organs (Parkhots et al., 2003), could also be applied in the photodynamic inactivation of local oral infections. Experimental Materials and Methods Microorganisms. In our study, 50 anaerobic clinical strains isolated from periodontal infections were used. These were: Prevotella oralis (n = 8), Prevotella denticola (n = 7), Leptotrichia buccalis (n = 4), Veillonella sp. (n = 11), Actinomyces sp. (n = 4), Clostridium sp. (n = 4), Eubacterium sp. (n = 7), Peptostreptococcus sp. (n = 4), and Propionibacterium sp. (n = 1). The microbiological investigations were performed at the Bacteriology Department of the Lower Silesian Center of Paediatrics in Wroc³aw. The samples were taken from the periodontal pockets of patients. The growth of anaerobic bacteria was carried out on Schaedler Agar + 5% Sheep Blood plates in an anaerostat Genbox System (bioMerieux, France) at 35°C for 7 days. The bacteria were identified on the basis of microscopic morphology in Gram-stained preparations and their metabolic properties using ID32 A tests (bioMérieux). Photosensitizer. Fotolon (Haemato-Polska, Wroc³aw, Poland) is a complex of the trisodium salt chlorin e6 and polyvinylpyrrolidone (PVP). It is a water-soluble, lyophilic, dry substance of green-black colour. Chlorin in suspension tends to be unstable and it aggregates quickly when applied without PVP, leading to a decrease in bioavailability. PVP in an aqueous medium forms hydrophobic cavities into which various molecules can be built (Parkhots et al., 2003). The admixture of PVP to chlorin e6 increases quantum yield and fluorescence lifetime compared with chlorin e6 in 0.9% sodium chloride alone, indicating some changes in the local environment and reduced aggregation of chlorin e6. Nevertheless, absorption spectra measured with freshly prepared Fotolon solutions and those several days/weeks old showed the Soret band shifting towards red (data not shown), decreasing in intensity and increasing in its full width at half-maximum in old solutions. This is a clear indication of polimerisation. To keep Fotolon’s bioavailability high, a PS solution in 0.9% sodium chloride was prepared just before the experiments. The manufacturer of Fotolon recommends intravenous application of 2.5 – 3 mg of PS per kg of body weight. The calculated PS dose is dissolved ex tempore in 100 ml of 0.9% sodium chloride and administered by i.v. infusion over 30 minutes. The initial solution used for cancer treatment is around 1.7 mM of chlorin e6 and is still safe (Parkhots et al., 2003). Photobleaching monitoring. Photobleaching of the Fotolon solution was experimentally monitored by means of quantitative absorption and fluorescence spectra measurements while illuminating the solution with a laser diode. Ten miligrams of Fotolon was dissolved in 1 ml of 0.9% sodium chloride, then dilutions were made to obtain concentrations of ce6 = 31 mg ml–1 and c4xe6 = 125 mg ml–1. Four ml of the Fotolon solution was then placed in an absorption-emission 10.00 mm quartz cuvette in a cuvette holder dedicated to absorption and emission spectra. Absorption and fluorescence were measured with an OceanOptics SD2000 two-channel miniature spectrophotometer. The absorption spectra in the 450 – 800 nm range were measured with an LS-1 tungsten-halogen miniature lamp after subtraction of dark current and proper baseline correction. The fluorescence was monitored by the second channel of the spectrophotometer under 405 nm excitation with an LED (Light Emitting Diode from Roithner-LaserTechnik, Austria). The excitation light (651 nm), perfectly matching the absorption spectrum of Fotolon in 0.9% sodium chloride, was delivered through a fibre to the second transparent surface of the cuvette. A Coherent FieldMaster with an LS-10 head shadowed with a 0.5 cm2 hole was used to measure the light flux (power density), which was fixed at 500 mW cm–2 at the cuvette surface for the photostability experiments. Light source. A pigtailed laser diode (LaserSecura, Wroclaw, Poland) was used as an excitation source emitting at 651 nm, exactly matching the Q absorption band maximum of chlorin e6 (Fotolon) in 0.9% sodium chloride. The microtitration plate was fixed about 4 cm above the tip of the laser fibre. A light power meter (Coherent, Field Master) equipped with a measuring head (Coherent, LM-10) shielded with a 0.5 cm 2 diaphragm was used to obtain a light flux of 250 mW cm–2 by diode current changes. Sixty seconds of illumination was used, giving a total light dose of 15 J cm–2. Photostability experiments have shown that this light dose does not completely photobleach the chlorin e6 solution and, in fact, a higher (e.g. ≥ 30 J cm–2) light dose could be used for further enhancement of PDI. The manufacturer of Fotolon recommends delivering a minimum of 300 J cm–2 at a power density of 200 – 300 mW cm –2 for efficient photodynamic therapy of cancer. The power density used (250 ± 10 mW cm –2) is high enough to increase the local temperature; however, no cfu decreases were noted compared with untreated control groups. Experimental procedure. After 48 h of incubation (stationary phase) on Schaedler Agar + 5% Sheep Blood plates in anaerobic conditions, the bacteria were diluted in 0.9% sodium chloride to the optical density of McFarland No 0.5. One hundred µl of bacterial culture at an approximate concentration of 107 cells ml–1 was placed in the wells of a flat-bottom 96-well microtitration plate in triplicate. Five µl of chlorin e6 solution was added to each well to a final chlorin e6 concentration of 50 µg ml–1 (84 µM). Before the 60-sec illumination, the bacteria were incubated for 5 min in the dark at room temperature. To determine the number of colony-forming units (cfu), aliquots of 10 µl were taken from each well, serially diluted in 0.9% sodium chloride, and placed on Schaedler Agar + 5% Sheep Blood. The anaerobic bacteria were cultured in an anaerostat Genbox System at 35°C for 3 days in the dark. The respective controls were bacteria strains (i) treated with neither PS nor light, (ii) exposed to light in the absence of PS, and (iii) treated with PS but not illuminated. To determine the optimal experimental conditions, preliminary tests were carried out with various concentrations of e6: (i) 50 µg ml–1 (84µìM) with 30 and 60 s of illumination; (ii) 25 µg ml–1 (42 µM) with 30 and 60 s of illumination; (iii) 2.5 µg ml–1 (4.2 µM) with 30 and 60 s of illumination; and (iv) 50 µg ml–1 , 25 µg ml–1, and 2.5 µg ml–1 with 60 s of illumination and 5 min of incubation. Two clinical isolates were taken for preliminary tests: the Prevotella oralis strain 9 as a Gram-negative and Actinomyces naeslundii strain 52 as a Gram-positive representative were used. Statistical analysis. Statistical analysis was performed using the t-test for independent samples (Statistica 5.0 StatSoft, Polska).

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Results

normalised intensity [a.u.]

Since PDI requires short incubation and illumination times with limited light flux in order not to sensitise or kill eukaryotic cells, it is potentially useful to increase the PS concentration to obtain PDI-induced cfu reduction. However, applying high doses of photosensitizer in PDI experiments may reduce PDI efficiency due to self-shielding. Increasing the light flux is another way to increase the efficiency of PDI. In cancer treatment with Fotolon the suggested light flux is no less than 200 – 300 mW cm–2 (Parkhots, 2003). To assess the self-shielding effect experimentally we carried out Fotolon photobleaching studies. Figure 1 presents the photobleaching of the Fotolon 0.9% NaCl solution. Absorption was monitored as intensity of Q-band at 653nm and normalised to the absorption coefficient for the not irradiated sample. The emission was monitored as luminescence intensity of Q-band at 660 nm when excited with 405 nm by Light Emitting Diode and normalized to luminescence intensity for the not irradiated sample. Saturation of the absorption curve starts at 30 J cm-2, indicating that delivering more than 30 J cm-2 will not have any therapeutic effect due to the lack of active PS molecules (Fig. 1). It can also be noticed that increasing the PS concentration reduces the photobleaching rate, and about 60 J cm-2 was needed to complete photobleaching of the PS.

abs cc abs e6e6

1.0

lum cce6 lum e6 abs cc abs 4xe6 4xe6

0.8

lum cc4xe6 lum 4xe6

0.6

0.4

0.2

0.0

0

10

20

30

40

50

60 70 80 radiant exposure [J cm –2]

Fig. 1. The photobleaching of the Fotolon 0.9% NaCl solution Normalized absorbance (rectangles) and emission (circles) intensity versus the radiant exposure delivered to the Fotolon solution (31 µg ml –1 – thin lines and 125 µg ml–1 – bold lines)

In the preliminary anti-bacterial tests (Fig. 2) in the three control groups (i) treated neither with PS nor light, (ii) exposed to light in the absence of PS, and (iii) bacteria treated with PS but not illuminated, no changes of the viable count were observed. The smallest survival fraction (a decrease by 5 orders of magnitude) was obtained for the chlorin e6 in concentration of 50 µg ml–1, 60 s of illumination, and 5 min of incubation. The Actinomyces naeslundii strain 52 was similarly sensitive to both the 25 µg ml–1 and the 50 µg ml–1 PS concentrations. In the case of Prevotella oralis isolate 9, the best antimicrobial effect was exhibited by the PDI with 5 min of incubation. Therefore these conditions (60s of activation preceded by 5 min of incubation with PS concentration 50 µg ml–1) were used for all further experiments on the clinical strains. The survival fractions of the tested clinical strains are shown in Fig. 3. There were no differences observed among the three control groups in spite of the relatively high light flux applied. The presented data are the number of colony forming units (cfu) normalised to the respective initial number of cfu to compare the different strains easily. The behaviour of the clinical strains could not be predicted from the Grampositive or Gram-negative character of the bacteria. Among the Gram-negative isolates, the Prevotella oralis strains were the most sensitive (4– 5 log of cfu decrease). Statistically significant susceptibility variations (p < .05) were observed for the P. oralis strains 9 and 32. The Prevotella denticola and Leptotrichia buccalis strains showed various susceptibilities to PDI with Fotolon, maintaining decreases in cfu of 2– 4 orders of

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Fig. 2. Effect of the incubation time and photosensitizer concentration on phototoxicity against (a) Prevotella oralis and (b) Actinomyces naeslundii Rectangles describe 30s of PS activation, circles describe 60s of PS activation and triangles 60s of activation preceded by 5min of incubation.

a) gram-negative strains 36 22 21 20 11 9 8 7 6 2 1

18 10 8 7 5 2 1

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magnitude. Among Prevotella denticola, isolate 44 was significantly (p < .05) more resistant to PDI than were strains 2, 5, 11, 17, and 22. The same dependency was observed between isolates 2 and 11. Statistically significant susceptibility variations (p < .05) were noticed for the 11 and 16 Leptotrichia buccalis strains. Most of the Veillonella sp. isolates demonstrated similar susceptibility of 3– 4 log cfu reduction, but only one isolate, 7, was significantly (p < .05) more resistant than the remaining isolates. We found that the clinical strains of Clostridium sp. and Actinomyces sp. behaved similarly to Gram-negative bacteria, with level decreases of 3– 4 and 3– 5 orders of magnitude, respectively. No significant susceptibility differences were observed between these two bacterial groups. The other Gram-positive isolates (Eubacterium sp., Propionibacterium sp., and Peptostreptococcus sp.) were more susceptible and showed 4– 6 log of cfu reduction. Statistically significant resistance (p < .05) was noticed for 18 compared with the 2, 5, 7 and 10 Eubacterium sp. strains. Discussion The spectroscopic investigation of the photostability of Fotolon exhibited a lack of reciprocity between light flux and treatment time. According to Wainwright (Wainwright, 1998), high power density over a short time period may give different anti-microbial effects from those of low power density over a longer time even though the light dose is the same in both cases. The smaller photobleaching rates for solutions with higher chlorin e6 concentrations can be explained by the self-shielding effect. When the dye concentration becomes higher, the distance travelled by the excitation light is reduced due to its loss in intensity down to e–1 of the initial value (defined by the absorption coefficient a). In such a case, superficial layers of the dye absorb the light very efficiently and thus block its penetration into deeper layers. As the photobleaching of superficial dye layers proceeds, these layers become transparent while deeper layers still strongly absorb the light. This is why radiant exposures for a highly concentrated photosensitizer solution may be underestimated, leading to reduced PDI efficiency. During the antibacterial experiments, 60-sec illumination was used, giving a 15 J cm–2 total light dose. This light dose does not completely photobleach chlorin e6 solution and, in fact, a higher light dose (e.g. ≥ 30 J cm–2) could be used for further enhancement of PDI. Several studies have demonstrated the efficacy of toluidine blue, methylene blue, and chlorin e6 derivatives in the photodynamic inactivation (PDI) of subgingival flora. Soucos (Soukos et al., 1998), Rovaldi (Rovaldi et al., 2000), and Pfitzner (Pfitzner et al., 2004) determined the antimicrobial activity of poly-Llysine chlorin e6 conjugates and new photosensitizers BLC 1010, BLC 1014 on anaerobic bacteria compared with pure chlorin e6. We have compared our results gained during preliminary tests with a concentration of 2.5 µg ml–1 (4.2 µM) chlorin e6 (see Fig. 2) with those of pure chlorin e6 obtained by the three other groups. As one could expect, the Gram-positive bacterial strains exhibited higher susceptibility to PDI with chlorin e6 than did the Gram-negative bacteria. This indicates that the crucial attack points of reactive species generated during photodynamic action are located on the cell cover , regardless of whether Grampositive or Gram-negative bacteria were treated. We have also performed experiments in which much higher concentrations of the PS were used than in experiments performed by Soukos (Soukos et al., 1998), Rovaldi (Rovaldi et al., 2000), and Pfitzner (Pfitzner et al., 2004). Since (i) Fotolon is not phototoxic up to 10 mg kg–1 of body weight, (ii) it does not accumulate in normal tissue up to 1 hour after administration, and (iii) the only “affinity” to bacteria comes from the shorter time needed for the PS compound to destroy crucial bacterial cell structures (mostly the cell membrane) compared with eukaryotic cells, there is no argument against using Fotolon concentrations as high as 80– 100 µM of chlorin e6. The highest concentration used by us was in fact the concentration of Fotolon infusion solution for standard cancer treatment. Both Gram-positive and Gram-negative bacteria were sensitive to PDI with Fotolon in higher concentration however, clinical isolates displayed various PDI susceptibilities. In our opinion, besides standard strains, larger clinical strain collections should be tested to assess the therapeutic effects of photosensitizers. The amount of cfu reduction after PDI was 3 to 6 logs (viable count < 0.01%) and 2 to 5 logs (viable count < 0.1%) for 20 Gram-positive and 30 Gramnegative wild strains, respectively. A reduction in cfu of over 99.9% is regarded as bactericidal and sufficient for bacteria eradication.

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Literature B e r t o l o n i G., F. R o s s i, G. V a l d u g a, G. J o r i and J. v a n L i e r. 1990 Photosensitising activity of water- and lipidsoluable phthalocyanines on Escherichia Coli. FEMS Microbiol. Lett. 59: 149–155. C h a n Y. and L. C h e r n - H s i u n g. 2003. Bactericidal effects of different laser wavelengths on periodontopathic germs in photodynamic therapy. Lasers Med. Sci. 18: 51–55. D o r t b u d a k O., R. H a a s, T. B e r n h a r t and G. M a i l a t h - P o k o r n y. 2001. Lethal photosensitization for decontamination of implant surfaces in the treatment of peri-implantitis. Clin. Oral. Implants Res. 12: 104–108. E m b l e t o n M.L., P.N. S e a n, B.D. C o o k s o n and M. W i l s o n. 2002. Selective lethal photosensitization of methicillin resistant Staphylococcus aureus using an IgG-tin (IV) chlorin e6 conjugate. J. Antimicrob. Chemother. 50: 857–864 H a m b l i n M.R., D.A. O’ D o n n e l l, N. M u r t h y, K. R a j a g o p a l a n, N. M i c h a u d, M.E. S h e r w o o d and T. H a s a n. 2002. Polycationic photosensitizers conjugates: effects of chain length and Gram classification on the photodynamic inactivation of bacteria. J. Antimicrob. Chemother. 49: 941–951. H a m b l i n M.R., T. Z a h r a, C.H. C o n t a g, A.T. M c M a n u s and T. H a s a n. 2003. Optical monitoring and treatment of potentially Lethal Wound Infections in vivo. J. Infect. Dis. 187: 1717–1725. J a c o b H.E. and M. H a m a n n. 1975. Photodynamic alterations of the cell envelope of Proteus mirabilis and their repair. Photochem. Photobiol. 22: 237–241. K l e i n f e l d e r J.W., R.F. M u l l e r and D.E. L a n g e. 1999. Antibiotic susceptibility of putative periodontal pathogens in advanced periodontitis patients. J. Clin. Periodontol. 26: 347–351. K ö m e r i k N., H. N a k a n i s h i, A.J. M a c R o b e r t, B. H e n d e r s o n, P. S p e i g h t and M. W i l s o n. 2003. In vivo killing of Porphyromonas gingivalis by toluidine blue-mediated photosensitization in an animal model. Antimicrob. Agents Chemother. 47: 932–940. M e n e z e s S., M.A.M. C a p e l l a and L.R. C a l d a s. 1990. Photodynamic action of methylene blue: repair and mutation in Escherichia coli. J. Photochem. Photobiol. B: Biol. 5: 505–517. M e r c h a t M., G. B e r t o l o n i, P. G i a c o m o n i, A. V i l l a n u e v a and G. J o r i. 1996. Meso-substituted cationic porphyrins as efficient photosensitizers of Gram-positive and Gram-negative bacteria. J. Photochem. Photobiol. 32: 153–157. P a r k h o t s M.V., V.N. K n y u k s h t o, G.A. I s a k o v, P.T. P e t r o v, S.V. L e p e s h k e v i c h, A. Y a. K h a i r u l l i n a and B.A. D z h a g a r o v. 2003. Spectral-luminescent studies of th “Photolon” photosensitizer in model media and in blood of oncological patients. J. App. Spectr. 70: 921–926. P f i t z n e r A., B.W. S i g u s c h, V. A l b r e c h t and E. G l o c k m a n n. 2004. Killing of periodontopathogenic bacteria by photodynamic therapy. J. Periodontol. 10: 1343–1349. R o v a l d i C.R., A. P i e v s k y, N.A. S o l e, P.M. F r i d e n, D.M. R o t h s t e i n and P. S p a c c i a p o l i. 2000. Photoactive porphyrin derivative with broad-spectrum activity against oral pathogen in vitro. Antimicrob. Agents Chemother. 44: 3364–3367. S a r k a r S. and M. W i l s o n. 1993. Lethal photosensitization of bacteria in subgingival plaque from patients with chronic periodontitis. J. Periodontal Res. 28: 204–210. S b o r d o n e L., I. R a m a g l i a, E. G u l l e t t a and V. I a c o n o. 1990. Recolonization of the subgingival microflora after scaling and root planning in human periodontitis. J. Periodontol. 61: 579–584. S l o t J. 1979. Subgingival microflora and periodontal disease. J. Clin. Periodontal. 6: 351–356. S o n c i n M., C. F a b r i s, A. B u s e t t i, D. D e i, D. N i s t r i, G. R o n c u c c i and G. J o r i. 2002. Approaches to selectivity in the Zn(II) phthalocyanine-photosensitized inactivation of wild-type and antibiotic-resistant Staphylococcus aureus. Photochem. Photobiol. Sci. 10: 815–919. S o u k o s N.S., L.A. X i m e n e z - F y v i e, M.R. H a m b l i n, S.S. S o c r a n s k y and T. H a s a n. 1998. Targeted antimicrobial photochemotherapy. Antimicrob. Agents Chemother. 42: 2595–2601. S o u k o s N.S., M. W i l s o n, T. B u r n s and P.M. S p e i g h t. 1996. Photodynamic effects of toluidine blue on human oral keratinocytes and fibroblasts and Streptococcus sanguis evaluated in vitro. Lasers Surg. Med. 18: 253–259. W a i n w r i g h t W. 1998. Photodynamic antimicrobial chemotherapy (PACT). J. Antimicrob. Chemother. 42: 12–28. W i l s o n M. 1994. Bactericidal effect of laser light and its potential use in the treatment of plaque-related diseases. Int. Dent. J. 44: 181–189. W i r k s t r ö m M., S. R e n v e r, T. J o h n s s o n and G. D a h l e n. 1993. Microbial association in periodontitis sites before and after treatment. Oral Microbial Immunol. 8: 213–218.

Polish Journal of Microbiology 2005, Vol. 54, No 4, 311– 316

Susceptibility Testing and Resistance Phenotypes Detection in Bacterial Pathogens Using the VITEK 2 System EL¯BIETA STEFANIUK*, AGNIESZKA MRÓWKA and WALERIA HRYNIEWICZ

Department of Epidemiology and Clinical Microbiology, National Institute of Public Health Che³mska str. 30/34, 00-725 Warsaw, Poland Received .23 August 2005, received in revised form 20 November 2005, accepted 21 November 2005 Abstract A set of well characterized strains, collected in Polish hospitals, including Gram-negative (n = 93) and Gram-positive (n = 90) isolates was used in the study. The VITEK 2 AST-cards were used in the analysis according to the manufacturer’s recommendations. Comparison of the susceptibility data obtained by the standard method and by VITEK 2 cards proved concordant in 99% of cases. Clinically important mechanisms were revealed by the VITEK 2 AES with > 95% agreement with reference data including methicillin resistance in staphylococci (98%), high-level aminoglycoside resistance in enterococci (100%), VanA and VanB phenotypes in enterococci (100%), and ESBLs in Enterobacteriaceae (93.8%). The VITEK 2 AES System appears a reliable tool for the detection and interpretive reading of clinically important mechanisms of resistance and can be recommended for routine work. K e y w o r d s: bacterial pathogens, antimicrobial agents, susceptibility testing, resistance mechanisms, VITEK 2

Introduction Increasing resistance in bacterial pathogens of high clinical relevance requires proper and rapid detection of the resistance phenotype. Dilution methods are the reference methods used in determining microorganisms susceptibility which allow the determination of the minimal inhibitory concentration of the drug (MIC) and thus the proper choice of antimicrobial agents for therapy. Following the emergence of many mechanisms of resistance special methods for their detection, allowing full expression of resistance mechanisms in vitro, are more often applied. Different phenotypic methods are used, supplemented by molecular biology techniques. However the main drawback in the majority phenotype methods used is the time required to obtain results. For many years the automation of microbiological diagnostics has been developing dynamically. New automated microbiology systems create opportunities for quickly obtaining complete microbiological results. Changes in the spectrum of etiological agents and their susceptibility profiles demand the continous improvement of such systems and their adjustment to new epidemiological situations. The VITEK 2 System (bioMérieux, USA) belongs to the new generation of automated microbiology systems designed to identify bacterial isolates to the species level and to determine their drug susceptibility. The system provides different types of card used for identification tests and for drug susceptibility determination. The aim of this study was to evaluate the VITEK 2 Automated System for antimicrobial susceptibility determination of the most important clinical pathogens and the Advanced Expert System (AES) for its interpretation of resistance mechanisms. The results of the analyses were compared with those obtained by reference microbiological methods. * Department of Epidemiology and Clinical Microbiology, National Institute of Public Health, Che³mska str. 30/34, 00-725 Warsaw, Poland; e-mail: [email protected]

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Experimental Materials and Methods Bacterial strains. A total of 183 clinically significant Gram-negative (n = 93) and Gram-positive (n = 90) bacterial pathogens were included in the study. Gram-negative organisms represented seven of the most commonly encountered species of the family Enterobacteriaceae (Escherichia coli n = 23, Klebsiella pneumoniae n = 18, Klebsiella oxytoca n = 3, Proteus mirabilis n = 2, Serratia marcescens n = 9, Enterobacter cloacae n = 13, Citrobacter freundii n = 5) and two species of nonfermenting rods (Acinetobacter baumannii n = 10, Pseudomonas aeruginosa n = 10). Gram-positive isolates belonged to four Staphylococcus species (S. aureus n = 42, S. epidermidis n = 4, S. haemolyticus n = 3, S. hominis n = 1), two species of Enterococcus (E. faecalis n = 10, E. faecium n = 10) and Streptococcus pneumoniae (n = 20). The isolates were recovered from specimens collected from patients hospitalised in different medical centers in Poland from 1996 to 2002. All strains were collected by the National Institute of Public Health. They were not epidemiologically related and represented one isolate per patient. A wide variety of clinically important antimicrobial resistance mechanisms were represented by the isolates, including extended-spectrum $-lactamases (ESBLs) in Enterobacteriaceae (48 isolates), methicillin resistance in staphylococci (37 isolates), decreased pneumococcal susceptibility to penicillin (8 isolates) and resistance to vancomycin and to high concentrations of aminoglycosides in enterococci (8 and 20 isolates, respectively). E. coli ATCC 25922, E. coli ATCC 35218, E. faecalis ATCC 29212, E. faecalis ATCC 51299, K. pneumoniae ATCC 700603 P. aeruginosa ATCC 27853, S. pneumoniae ATCC 49619, S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 43300 and S. aureus MR3 from the collection of the National Insitute of Public Health in Warsaw were used as reference strains. Identification methods. Gram-negative isolates were identified to the species level by API 20 E (Enterobacteriaceae) or API 20 NE (non-fermenters) biochemical tests (bioMérieux, Marcy l’Etoile, France). S. aureus was identified by coagulase production (bound and free), supplemented by DN-ase test, and coagulase-negative staphylococci were speciated by the API Staph test (bioMérieux, Charbonnieres-les-Bains, France). Identification of the enterococcal isolates was performed according to the method of Facklam and Collins (Facklam and Collins, 1989) and by the API 20 Strep test (bioMérieux, Charbonnieres-les-Bains, France), supplemented by potassium tellurite reduction, motility, and pigment production tests (Facklam and Collins, 1989). S. pneumoniae was identified using standard microbiology techniques including agar morphology, optochin test and bile solubility test (Ruoff et al., 1999). MIC evaluation. MICs of various antimicrobials were evaluated by agar or broth dilution methods in accordance with the Clinical and Laboratory Standards Institute (CLSI/ NCCLS – National Committee for Clinical Laboratory Standards) guidelines (NCCLS, 2003a.; CLSI, 2005). The same antimicrobial agents were tested as those that were present in the VITEK 2 cards used in the study, and specific sets of agents were selected for each of the species analysed. The following antimicrobials were included in the study: penicillin, cefotaxime, streptomycin and gentamicin (Polfa Tarchomin, Poland); amoxicillin, clavulanic acid and oxacillin and ceftazidime (Glaxo SmithKline, UK); piperacillin, and tazobactam (Wyeth, USA); cefepime and amikacin (Bristol-Myers Squibb, USA); meropenem (Zeneca, UK), ciprofloxacin (Bayer, Germany); vancomycin (Eli Lilly, USA); teicoplanin (Marion Merrell, UK). In $-lactam-inhibitor combinations, concentrations used were in accordance with the CLSI (CLSI, 2005). Detection of methicillin resistance. Methicillin resistance was detected in staphylococcal isolates by two methods recommended by the CLSI, using a 30mg cefoxitine disc and the agar screening method (CLSI, 2005; NCCLSb., 2003). The screening method was used only for S. aureus isolates. Results of the analysis were confirmed by the PCR amplification of the mecA gene in all methicillin-resistant isolates as described previously (Murakami et al., 1991). Detection of the high-level aminoglycoside resistance (HLAR) and vancomycin resistance in Enterococcus spp. Enterococcal isolates were tested for the presence of the HLAR phenotype using the agar screening method as described by CLSI (CLSI, 2005; NCCLSb., 2003). Vancomycin-resistant enterococci (VRE) were identified by the CLSI agar screening procedure (NCCLS, 2003a.; CLSI, 2005) and positive results of the test were confirmed by PCR detection of vanA or vanB genes as described previously (Dutka-Malen et al., 1995; Dahl et al., 1999). Detection of penicillin resistance in S. pneumoniae. Penicillin-resistant S. pneumoniae strains were identified by two methods recommended by CLSI: screening method with the 1mg oxacillin disc and the reference broth dilution method (NCCLS, 2003a.; CLSI, 2005). ESBL detection. ESBL activity was detected in Enterobacteriaceae isolates by the double disc synergy (DDS) test (Jarlier et al., 1988) with discs containing cefotaxime, ceftazidime and amoxicillin/clavulanate. The 48 DDS test-positive isolates included 15 Klebsiella pneumoniae, 2 Klebsiella oxytoca, 17 Escherichia coli, 5 Serratia marcescens, 7 Enterobacter cloacae and 2 Citrobacter freundii isolates. These were recovered in 7 different Polish hospitals in 1996–98, and were confirmed to be ESBL producers by biochemical and molecular methods (isoelectric focusing, bioassay, PCR and sequencing) published before (Pa³ucha et al., 1999; Fiett et al., 2000). VITEK 2 tests. Analysis was conducted with the use of antibiogram cards VITEK 2 (for Gram-positive cocci: AST-524, -526 and -GP56; for Gram-negative rods: AST-N019, -N020, -N021). The tests are in the form of small, waterproof cards with 64 wells containing antibacterial drugs (antibiogram tests) in different dilutions suitable for testing microorganisms according to the CLSI recommendations. Tests were performed according to the manufacturer’s instruction. Evaluated parameters. Characteristic parameters of the automatic method of drug susceptibility determination were defined: total concordance of resistance categories (CA), accuracy, sensitivity, specificity and type of errors (mE, ME, VME). Minimal error – mE was recognised when one of the methods – reference or evaluated (in this study – automatic method) identified the tested strain as “intermediate”, where the other method classified it as “resistant” or “sensitive”. Major error – ME – reference method – “sensitive” and automatic method – “resistant”. “Very major error” VME – reference method – “resistant”, whereas automatic method – “sensitive”. Additional parameters were also evaluated such as time taken to obtain results, functionality and level of difficulty in using the system.

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Results MIC values and interpretation. A high correlation was found between the results (n = 1882 susceptibility tests) obtained by the aid of VITEK 2 and by the dilution reference method. The total accordance of resistance interpretation was 99%. For the species of Enterobacteriaceae family CA was 98.8%. Single minor errors (mE) in MIC values were observed for gentamicin, amikacin and piperacillin. One major error (ME) appeared when testing E. cloacae susceptibility to piperacillin with tazobactam. In the group of nonfermenting rods CA obtained was 96.4% for A. baumannii (4 mE), and 99.2% for P. aeruginosa (1 mE). The interpretation of the carbapenems MIC value for nonfermenting rods was in 100% consistent with the interpretation of the MIC value defined with the reference methods. Susceptibility tests for Gram-positive cocci exhibited an accordance variation from 97.8% for pneumococci, 98% and 99% respectively for E. faecium and E. faecalis, to 99.5% for staphylococci. In this group of microorganisms one very major error VME (for S. aureus and oxacillin) was noted. Comparative analysis of the interpretation of glycopeptide MICs (vancomycin and teicoplanin) obtained for enterococci, and penicillin MIC values for pneumococci, showed one minor error in both cases. All types of errors encountered in the respective species are presented in Table I. Table I Discrepanciens between the VITEK 2 results and reference methods results Species [No of isolates with errors/no of all isolates] S. pneumoniae [n = 3/20] S. aureus [n = 1/50] E. faecalis [n = 1/10] E. faecium [n = 2/10]

E. coli [n = 3/23]

K. pneumoniae [n = 3/18] K. oxytoca [n = 1/3] E. cloacae [n = 2/13] S. marcescens [n = 2/9] A. baumannii [n = 3/10]

P. aeruginosa [n = 2/10] mE – minor error;

Antimicrobial agent / No and kind of errors

Reference method MIC [mg/l] / category of susceptibility

Penicillin / 1 × mE Ceftriaxon / 2 × mE Cefotaxime / 1 × mE Oxacillin /1×VME Ciprofloxacin /1 × mE Ciprofloxacin / 1 × mE Teicoplanin / 1 × mE Piperacillin / 1 × mE Gentamicin / 1 × mE Amikacin / 2 × mE Gentamicin / 1 × mE Amikacin / 2 × mE Gentamicin / 1 × mE Piperacillin/Tazobactam / 1 × ME Gentamicin / 1 × mE Gentamicin / 1 × mE Cefoxitin / 1 × mE Piperacillin/Tazobactam / 1 × mE Cefotaxim / 1 × mE Cefepime / 2 × mE Cefepime / 1 × mE

ME – major error;

VME – very major error;

1 2 0.5 64 2 4 32 256 32 64 16 8 32 16 8 2 4 8 16 128 32 16 16 16

S – sensitive;

I I S R I R R R R R S I I S I S S I I 16 R I I I

I – intermediate;

VITEK 2 MIC [mg/l] / category of susceptibility ≥2 ≥4 1 2 ≤ 0.5 2 16 64 8 32 32 ≥ 16 16 32 ≥ 16 ≥ 128 8 ≥ 16 32 64 ≥ 64 32 8 8

R R I S S I I I I I I R S I R R I R R I R R S S

R – resistant

Resistance phenotype detection and interpretation. ESBLs detection was not achieved by the VITEK 2 system in 3 isolates only. Two of these were E. cloacae and one C. freundii. The strains of these species produce cefalosporinase that is AmpC specific for those individual species, which when produced at a high level can mask the ESBL phenotype. In general good results were obtained, with 93.8% sensitivity, 95.9% accuracy and 100% specificity of detection of ESBL production by Enterobacteriaceae strains (for E. coli, K. pneumoniae and K. oxytoca – all parameters reached the level of 100%). In the case of staphylococci 97.3% sensitivity, 98% accuracy and 100% specificity in the detection of MRS strains (methicillin resistant Staphylococcus) was achieved. One isolate of S. aureus characterised

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by the presence of mecA gene showed an MIC value for oxacillin of 64 mg/l by the reference method and 2 mg/l by the VITEK 2 system. For enterococci, 100% accuracy, sensitivity and specificity was achieved for HLAR and VRE by VITEK 2 detection. The difference between teicoplanin MIC values described above had no influence on the susceptibility category. Detection of the resistance phenotype by the VITEK 2 system seldom resulted in a change of clinical interpretation of the sensitivity results for the respective drugs. High agreement between the results obtained with the automatic method and reference methods was achieved, as shown in Table II. Table II Characteristic features of VITEK 2 method in detection of selected resistance mechanisms VRE

ESBL ESBL (E. cloacae, (all species) K. pneumoniae, K. oxytoca)

MRSA

HLAR

Reference methods positive / VITEK 2 positive

36

20

8

45

35

Reference methods negative / VITEK 2 negative

13

0

12

25

9

Reference methods positive / VITEK 2 negative

1

0

0

3

0

Reference methods negative / VITEK 2 positive

0

0

0

0

0

Accuracy (%)

98

100

100

95.9

100

Sensitivity (%)

97.3

100

100

93.8

100

100

100

Specificity (%)

100

100

100

Times taken to obtain results by VITEK 2. The average time for results to be obtained using the VITEK 2 automatic system was 8 hours from the installation of the antibiogram cards. For Gram-positive cocci it required 9 hours and for Gram-negative rods – 7 hrs 45 minutes. The shortest time recorded was for Klebsiella pneumoniae (5.5 hours) and the longest – up to 17 hours for coagulase-negative staphylococci. Discussion The usefulness of microbiological diagnostics for patient care has been limited by the time taken for patient specimen processing and obtaining results. Thus a shortening and an improvement of this process is urgently needed in order to introduce etiologic agent specific therapy as early as possible. In this study VITEK 2, the new system offered by bioMérieux, was evaluated in regard to its usefulness for antibiotic susceptibility testing, one of the most important steps in routine diagnostic microbiology. The great majority of isolates tested in this study, representing the most clinically relevant bacterial species, showed a high concordance of the results with the reference methods. However, they were obtained in a much shorter time, on average in 8 hours. This compared with 16 to 24 hrs needed for the reference methods. Although some molecular techniques are able to detect resistance genes in less than 4 hrs they have several limitations. They can only detect mechanisms of resistance that are already known and need to use several primers since more and more bacterial pathogens show multiple drug resistance. Favourable results when testing the VITEK 2 system have been also obtained by several other workers. Aissa and Horstkotte reported the high sensitivity and specificity of the system in the detection of methicillin resistance in staphylococcal strains as well as in S. aureus and coagulase-negative staphylococci (Aissa et al., 2004; Horstkotte et al., 2002). All strains but one (MRSA) were properly identified in respect to methicillin resistance. Ligozzi also obtained good agreement between results obtained by the reference methods and the VITEK 2 system, ranging from 90– 100% for staphylococci, pneumococci and enterococci with an average of 96% (Ligozzi et al., 2002). In our study even higher concordance was obtained, 97.8% for pneumococci, 98– 99% for enterococci and 99.5% for staphylococci. The results published by Blondell-Hill demonstrated a high agreement of 96.2% between the VITEK 2 system and reference method in the interpretation of antimicrobial susceptibility for 300 isolates of the Enterobacteriacae family (Blondell-Hill et al., 2003). Similar results were obtained in this study, with an even higher agreement of 98.8% achieved between the two methods. It should be stressed that in the case of the isolates of the Enterobacteriacae family, not even a single VME was encountered, and only one ME was reported for piperacillin/tazobactam combinations (E. cloacae). Similar results for E. cloacae and

4

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315

piperacillin/tazobactam susceptibility were reported by Karlovsky on a very large number of isolates (Karlovsky et al., 2003). An important function of VITEK 2 is the interpretation of susceptibility data through the AES in reference to a major resistance mechanism. In our study, when ESBLs producing strains were tested, the mechanism was not identified in only 3 strains; all 3 strains belonged to AmpC producing species, with two representing E. cloacae and one representing C. freundii. The same problem was encountered in our previous study with BD Phoenix which was unable to detect ESBL in two strains of AmpC producing species (E. cloacae) (Stefaniuk et al, 2003). It seems that the presence of AmpC in those isolates makes detection of ESBL by AES impossible. Usually, ESBLs production is easily detectable in E. coli and Klebsiella spp. (Stefaniuk et al, 2003, Livermore, 1995, Leverstein-van Hall et al., 2002). However, Levenstein-van Hall comparing several methods of ESBL detection, such as E-test ESBL, BD Phoenix, Vitek 1 and Vitek 2, in 74 isolates of E. coli and K. pneumoniae, found an accuracy detection rate of 78% for the Vitek 2 (Leverstein-van Hall et al., 2002). This is in contrast to our study, which showed an excellent result of 100% ESBLs detection. The discrepancy between those two reports may result from the different bacterial populations under study, reflecting the complexity and diversity of these enzymes. The shorter time involved in getting susceptibility results, as compared to conventional methods, and their good correlation with those methods make the VITEK 2 automated microbiological system a useful device in clinical microbiological laboratory work. In addition, the VITEK 2 automated microbiological system is an aesthetically pleasing and userfriendly device. It should be operated by a qualified member the microbiological laboratory staff who has been trained by an experienced microbiologist. Accurate in vitro susceptibility testing methods are important for optional patient therapy, particularly for hospitalized patients, and for epidemiology study. As far as sensitivity, accuracy and specificity are concerned, antibiogram tests of the VITEK 2 system are comparable to other reference methods used to detect particular resistance mechanisms of clinically important bacterial strains. Acknowledgements. The authors wish to thank bioMérieux company for the system VITEK 2 and all materials used in the VITEK 2 testing they provided.

Literature A i s s a N., D. S t o l a r and P. L e g r a n d. 2004. Accuracy of four agar diffusion methods and Vitek 2 automated system for the detection of the methicillin resistance in coagulase negative staphylococci. Pathol. Biol. (Paris) 52: 26–32. B l o n d e l - H i l l E., C. H e t c h l e r, D. A n d r e w s and L. L a p o i n t e. 2003. Evaluation of Vitek 2 to analysis of Enterobacteriaceae using the Advanced Expert System (AES) versus interpretative susceptibility guidelines used at Dynacare Kasper Medical Laboratories, Edmonton, Alberta. Clin. Microbiol. Infect. 9: 1091–103. C l i n i c a l a n d L a b o r a t o r y S t a n d a r d s I n s t i t u t e. 2005. Performance standards for antimicrobial susceptibility testing; Fifteenth informational supplement: M100-S15. CLSI, Villanova, PA. D a h l K.H., G. S k o v S i m o n s e n, R. O l s v i k and A. S u n d s f j o r d. 1999. Heterogeneity in vanB gene cluster of genetically diverse clinical strains of vancomycin-resistant enterococci. Antimicrob. Agents Chemother. 43: 1105–1110. D u t k a - M a l e n S., S. E v e r s and P. C o u r v a l i n. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33: 24–27. F a c k l a m R. and M.D. C o l l i n s. 1989. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J Clin. Microbiol. 27: 731–734 F i e t t J., A. P a ³ u c h a, B. M i ¹ c z y ñ s k a, M. S t a n k i e w i c z, H. P r z o n d o - M o r d a r s k a, W. H r y n i e w i c z and M. G n i a d k o w s k i. 2000. A novel complex mutant beta-lactamase, TEM-68, identified in a Klebsiella pneumoniae isolate from an outbreak of extended-spectrum beta-lactamase-producing klebsiellae. Antimicrob. Agents Chemother. 44: 1499–1505. H o r s t k o t t e M.A., J.K.M. K n o b l o c h., H. R o h d e, S. D o b i n s k y and D. M a c k. 2002. Rapid detection of methicillin resistance in coagulase-negative staphylococci with the VITEK 2 system. J. Clin. Microbiol. 40: 3291–3295. J a r l i e r V., J.E. N i c o l a s, G. F o u r n i e r and A. P h i l i p p o n. 1988. Extended broad-spectrum $-lactamases conferring transferable resistance to newer $-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev. Infect. Dis. 10: 2141–2143. L i g o z z i M., C. B e r n i n i, M.G. B o n o r a, M. d e F a t i m a, J. Z u l i a n i and R. F o n t a n a. 2002. Evaluation of the Vitek 2 system for identification and antimicrobial susceptibility testing of medically relevant Gram-positive cocci. J. Clin. Microbiol. 40: 1681–1686. L i v e r m o r e D.M. 1995. $-Lactamases in laboratory and clinical resistance. Clin. Microbiol. Rev. 8: 557–58. L e v e r s t e i n - v a n H a l l M.A., A.C. F l u i t, A. P a a u w, A.T.A. B o x, S. B r i s s e and J. V e r h o e f. 2002. Evaluation of the Etest ESBL and the BD Phoenix, VITEK 1, and VITEK 2 automated instruments for detection of extended-spectrum $-lactamases in multiresistant Escherichia coli and Klebsiella spp. J. Clin. Microbiol. 40: 3703–3711.

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M u r a k a m i K., W. M i n i m i d e, K. W a d a, E. N a k a m u r a, H. T e r a o k a and S. W a t a n a b e. 1991. Identification of methicillin resistant strains of Staphylococcus aureus by polymerase chain reaction. J. Clin. Microbiol. 29: 2240–2244. N a t i o n a l C o m m i t t e e f o r C l i n i c a l L a b o r a t o r y S t a n d a r d s. 2003a. Methods for Dilution Susceptibility Tests for Bacteria That Grow Aerobically; Approved standard – Sixth Edition: M7-A6. NCCLS, Villanova, PA. N a t i o n a l C o m m i t t e e f o r C l i n i c a l L a b o r a t o r y S t a n d a r d s. 2003b. Performance Standards for Antimicrobial Disk Susceptibility Testing; Approved Standard – Eighth Edition M2-A8. NCCLS, Villanova, PA. P a ³ u c h a A., B. M i k i e w i c z, W. H r y n i e w i c z and M. G n i a d k o w s k i. 1999. Concurrent outbreaks of extendedspectrum beta-lactamase-producing organisms of the family Enterobacteriaceae in a Warsaw hospital. J. Antimicrob. Chemother. 44: 489–499. R u o f f K.L., R.A. W h i l e y and D. B e i g h t o n. 1999. Streptococcus, pp. 283–305. In: P.R. Murray, E.J. Baron, M.A. Pfaller, Tenover FC., Yolken RH. (eds): Manual of Clinical Microbiology, ed. 7. Washington, American Society for Microbiology. K a r l o v s k y J.A., M.K. W e a v e r, C. T h o r n s b e r r y, M.J. D o w z i c k y, M.E. J o n e s and D.F. S a h m. 2003. Comparison of four antimicrobial susceptibility testing methods to determine the in vitro activities of piperacillin and piperacillin-tazobactam against clinical isolates of Enterbacteriaceae and Pseudomonas aeruginosa. J. Clin. Microbiol. 41: 3339–3343. S t e f a n i u k E., A. B a r a n i a k, M. G n i a d k o w s k i and W. H r y n i e w i c z. 2003. The evaluation of the BD PHOENIXTM automated identification and susceptibility testing system in clinical microbiology laboratory practice. Eur. J. Clin. Microbiol. Infect. Dis. 22: 479–485.

Polish Journal of Microbiology 2005, Vol. 54, No 4, 317– 321

Bacteriological Urinalysis in Patients after Renal Transplantation BO¯ENA £AZIÑSKA1*, MICHA£ CISZEK2, ALICJA ROKOSZ1, ANNA SAWICKA-GRZELAK1, LESZEK P¥CZEK2 and MIROS£AW £UCZAK1 1 Chair

and Department of Medical Microbiology, Medical University of Warsaw T. Cha³ubiñskiego 5, 02-004 Warsaw, Poland 2 Transplantation Institute, Medical University of Warsaw, Nowogrodzka 59, 02-006 Warsaw, Poland Received 23 November 2004, received in revised form 3 October 2005, accepted 10 October 2005 Abstract The study consisted of microbiological urinalysis performed in 269 patients after renal transplantation who remained under medical care at the Outpatient Service of the Transplantation Institute in Warsaw. The patients enrolled into the study had undergone renal transplantation 6 to 72 months before urine samples were collected. 304 urinalysis were performed. In the group of 269 patients, 42 individuals had bacteria in their urine what was confirmed in 47 urine cultures. Cases of bacteriuria were divided into 5 groups: 5 cases of symptomatic urinary tract infection (5 individuals – 2% of all studied patients), 27 cases of asymptomatic bacteriuria in 22 individuals (8% of all studied patients), 5 cases of insignificant bacteriuria in 5 patients (2%), 10 cases of involuntary urine contamination in 10 cases (4%). Eventually, urinary tract infection (UTI) was established in 27 patients (5 cases of symptomatic UTI and 22 cases of asymptomatic UTI) what makes out for 10% of all studied patients. In cases where urinalysis showed significant bacteriuria, following pathogens were detected in urine cultures: Escherichia coli: 22 strains, Enterococcus faecalis – 4 strains, Enterobacter cloacae – 2 strains and 1 strains of Ralstonia picketii, Streptococcus uberis, Pseudomonas aeruginosa and Proteus mirabilis. Over 90% of Gram-negative bacteria were susceptible to ceftriaxone and ceftazidime, as well as to amikacin and aztreonam which are the drugs usually administered intravenously in hospitalized patients. The only drug of similar efficacy, which could be administered orally in outpatients was fosfomycin. K e y w o r d s: patients after renal transplantation, urinary tract infections, identification and drug susceptibility of uropathogens

Introduction The urinary tract in healthy individual is well protected against infections by both immunologic and nonimmunologic mechanisms. It has a capacity of self-sterilization due to mechanical washing out of bacteria with the urine stream, urine acidification and secretion of Tamm-Horsfall protein by the tubular cells. Renal allograft recipients represent a group of patients which is particularly susceptible to UTI. The renal transplantation as a surgical procedure, carries a higher risk of any infection, which is linked with hospitalization, the surgical techniques, anesthesia or specific procedures performed at the Intensive Care Unit. Anastomosis of transplant ureter to urinary bladder is challenging surgical procedure often leading to ureter structure or vesicoureteral reflux. Urinary stasis and the presence of vesicoureteral reflux predispose individuals to the multiplication of bacteria in the urinary tract which may be additionally facilitated by body temperature and chemical components of the urine. The 3-month period after renal transplantation makes transplant recipients even more susceptible to the development of UTI due to intensive immunosuppressive therapy. During that time patients often develop various viral, fungal, parasite as well as bacterial infections. Bacterial infections are most likely to occur in the first month after transplantation. In solid organ transplantation (kidney, liver, pancreas or lungs) infections are mainly detected in the respiratory tract, urinary tract, abdominal cavity and gastrointestinal tract. Thus, solid organ allograft recipients should be closely monitored especially during perioperative period. Microbiological monitoring should include * Correspondence to: Bo¿ena £aziñska, Chair and Department of Medical Microbiology, Medical University of Warsaw, T. Cha³ubiñskiego 5, 02-004 Warsaw, Poland, tel./fax + 48 (22)6282739, e-mail: [email protected]

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4

regular control measurements of sterility of drains, catheters (upon removal), analysis of sputum, urine, stool, blood and wound swabs (Dzier¿anowska and Jeljaszewicz, 1998). Urinary tract infection is the most common complication in patients after renal transplantation recipients and also is the most frequent cause of hospitalization in that group (Krieger et al., 1977). Urinary tract infections may have various clinical manifestations ranging from asymptomatic bacteriuria (positive urine culture without typical symptoms such as: fever, urgency, frequency or suprapubical tenderness) to symptomatic infection (urethritis, cystitis, acute and chronic pyelonephritis). Acute UTI could be complicated by sepsis, acute renal insufficiency, hydronephrosis, pyelonephrosis, renal or perirenal abscess. In many cases the primary renal disease may be a triggering factor for UTI like it is observed in reflux nephropathy, chronic pyelonephritis and polycystic kidney disease with episodes of infection localized in the upper urinary tract (Wetzel et al., 1993). Regardless of the primary cause of renal end-stage disease, patient’s own cirrhotic kidneys with regressive cysts may be a source of infection that descends down the urinary tract to reach renal allograft. That is why a renal transplant recipient should be closely monitored to establish as quickly as possible the occult infection. The frequency of UTI in early period after renal transplantation (3– 6 months after transplantation) is relatively high and according to various reports from the literature exceeds 50% (Renoult et al., 1994; Castelano et al., 1995; Maraha et al., 2001). There are, however, significant differences in assessment of infection risk in later periods after transplantation (Douglas et al., 1974; Kurijama et al., 1991; Goya et al., 1997). The aim of this study was the assessment of the rate of bacteriuria in patients after renal transplantation, identification of bacterial strains isolated from those patients and bacterial susceptibility to antibiotics. Experimental Material and Methods 269 patients (108 women, 161 men) after kidney transplantation with stable graft function and serum creatinine concentration below 2.0 mg/dl were part of the study. Patients, who were enrolled into the study, had been transplanted in the Department of General and Transplantation Surgery, Medical University of Warsaw and in the Department of General, Vascular and Transplantation Surgery, Medical University of Warsaw in the years of 1995–2001. The mean age of studied patients was 43.2 ± 9.6 years (median 47,2): 44.1 ± 11.1 in males (median 44,9) and 42.9 ± 12.3 in females (median 43,2). The mean time since the day of transplantation till the day when the urine sample was collected was 34.1 ± 18.9 months (median 32,6) in the whole studied group. Urine samples. Urine specimens for a colony count were obtained from patients on regular check-up visits at the Outpatient Service of the Transplantation Institute. Urine samples were collected 4 hours after previous urination. First morning urine samples could not be collected because most patients lived a long distance away from the Outpatient Service. Patients were provided with information forms where they could find instruction on how a urine specimen should be correctly collected. They were also asked to indicate the time of urine collecting and last micturition. All urine samples were obtained from a midstream into standardized, sterile containers and delivered to the laboratory at the Chair and Department of Medical Microbiology, Medical University of Warsaw within 2 hours after being collected. Microbiological examination of urine samples. In the first stage quantitative urine culture was performed where all urine samples were plated onto blood and MacConkey agar plates. Urine cultures that contained less or equal than 100 000 (≤ 105) CFU/ml of bacteria or less or equal than 10 000 (≤ 04) CFU/ml of fungi (one strain of pathogens in each case) were considered insignificant bacteriuria or insignificant funguria. Urine growth with two or more uropathogens was interpreted as contamination and was not further worked up. Patients in such cases were asked to provide another urine sample for correct assessment. In cases where the number of growing colonies of bacteria exceeded 105 CFU/ml (significant bacteriuria) or the number of growing fungal colonies exceeded 10 4 CFU/ml (significant funguria) the samples were further worked up. Identification of pathogenic strains. The biochemical identification of uropathogenic strains was performed in the automatic ATB Expression system (bioMerieux) with the use of specific test cards: ID 32 STAPH, API 20 STREP, ID 32 E, ID 32 GN, and ID 32 C. Antibiotic susceptibility testing of urine isolates. Antibiotic susceptibility of isolated Gram-negative bacilli was evaluated in the ATB expression system with the use of ATB UR test strips. Antibiotic susceptibility of isolated staphylococci, streptococci and enterococci was analyzed with the use of the disk diffusion method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Specific disk diffusion tests were used to detect Gram-negative bacilli producing the extended spectrum betalactamases (ESBL), methicillin-resistant staphylococci (MRS) and high-level aminoglycoside-resistant enterococci (HLAR). Reference strains such as: S. aureus ATCC 25923, S. aureus ATCC 29213, S. aureus MR3, E. faecalis ATCC 29212, E. coli ATCC 25922, K. pneumoniae ATCC 700603 and P. aeruginosa ATCC 27853 were used as controls in verification of antibiotic susceptibility.

Results Out of the group of 269 patients the presence of bacteria in urine was detected in 42 individuals (in 47 cultures). Cases of bacteriuria were classified into one of these groups: 5 cases of symptomatic urinary tract infection (UTI) in 5 patients (2% of patients), 27 cases of asymptomatic bacteriuria in 22 patients (8%),

4

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Bacteriological urinalysis in patients after renal transplantation

5 cases of non-significant bacteriuria in 5 patients (2%), 10 cases of urine contamination in 10 patients (10%). In total, urinary tract infection was detected in 27 (10%) patients (5 of them had symptomatic infection and 22 – asymptomatic). Gram-negative bacterial strains were isolated in most cases of urinary tract Table I Susceptibility to antibiotics of isolated Gram-positive bacterial strains Ampicillin

Amox\ Clav

Piperacillin

Nitrofurantoin

Vancomycin

Teicoplanin

1. Enterococcus faecalis

S

S

S

S

S

S

I

S

2. Enterococcus faecalis

I

I

I

S

S

S

R

R

3. Enterococcus faecalis

S

S

I

S

S

S

R

I

4. Enterococcus faecalis

S

S

S

S

S

S

I

R

5. Streptococcus uberis

S

S

S

S

S

S

S

S

Total (%)

80

80

60

100

100

100

20

40

Bacterial species

Ciprofloxacin

Tetracycline

Fosfomycin

Ciprofloxacin

Quinolones 2G

Quinolones 1G

Cotrimoxazole

Nitrofurantoin

Gentamicin

Tobramycin

Ceftazidime

Ceftriaxone

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

R

S

I

S

S

S

S

S

S

S

S

S

S

S

S

S

S

3. E. coli

R

S

I

S

S

S

S

S

S

S

S

S

S

S

S

S

S

4. E. coli

R

R

R

R

S

S

S

S

S

S

S

S

R

R

S

S

S

5. E. coli

R

I

S

R

S

I

S

S

S

S

S

S

R

R

S

S

S

6. E. coli

R

S

R

S

S

S

S

S

S

S

S

S

R

S

S

S

S

7. E. coli

R

R

S

R

S

S

S

S

S

S

S

R

S

R

S

S

R

8. E. coli

R

S

S

R

S

S

S

R

S

R

S

R

S

R

R

R

S

9. E. coli

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

10. E. coli

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

11. E. coli

I

S

I

I

S

I

I

I

S

S

S

S

I

I

S

S

S

12. E. coli

R

S

R

S

S

S

S

S

S

S

S

S

S

R

R

R

S

Netilmicin

S

2. E. coli

Amikacin

1. E. coli

Bacterial species

Aztreonam

Piperacillin

Cephalothin

Amox\Clav

Amoxycillin

Table II Susceptibility to antibiotics of isolated Gram-negative bacterial strains

13. E. coli

I

S

I

I

S

I

I

I

S

S

S

S

S

I

I

S

I

14. E. coli

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

15. E. coli

I

S

I

I

S

I

I

I

I

S

S

S

I

I

I

S

I

16. E. coli

I

I

I

I

I

I

I

I

S

S

S

S

I

I

S

I

I

17. E. coli

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

18. E. coli

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

S

19. E. coli

S

S

S

S

S

S

I

S

S

S

S

S

S

S

S

S

S

20. E. coli

R

I

R

I

S

S

I

S

S

S

S

S

R

R

S

S

S

21. E. coli

R

I

R

R

S

S

I

S

S

S

S

S

S

R

S

S

S

22. E. coli

R

S

I

I

S

S

I

S

S

S

S

S

S

S

S

S

S

23. E. cloacae

R

R

R

R

R

S

R

R

R

R

R

R

R

R

R

R

R

24. E. cloacae

R

R

I

R

I

I

I

I

I

R

R

R

R

I

S

S

I

25. Proteus mirabilis

S

S

S

S

S

S

S

S

S

S

S

R

S

S

S

S

S

26. P. aeruginosa

S

S

I

S

S

S

S

S

S

S

S

S

S

S

S

S

S

27. Ralstonia pickettii Total (%)

I

R

I

I

I

I

I

I

S

S

S

I

S

I

S

I

II

47

73

59

71

90

94

94

88

95

87

91

77

77

65

88

84

90

Quinolones 1G – nalidixic acid, Quinolones 2G – norfloxacin

320

£aziñska B. et al.

4

infection: Escherichia coli in 22 cases, Enterobacter cloacae in 2 cases, Proteus mirabilis, Pseudomonas aeruginosa and Ralstonia pickettii each in 1 cases. Gram-positive strains were isolated in 5 cases: Enterococcus faecalis in 4 and Steptococcus uberis in 1 case. Susceptibility of cultured bacterial strains is shown in Tables I and II. There was no HLAR strains detected among enterococci. In the group of Gram-negative bacilli there was no ESBL-positive strains. Multidrug resistant strains of Gram-negative rods can produce $-lactamases of AmpC type. These strains are resistant to all $-lactams, except carbapenems. In the group of patients with urinary tract infection 5 patients had diabetes mellitus (19% of the group): 2 patients were on insulin therapy and 3 on oral hypoglycemic agents. Diseases of native kidneys leading to end stage renal failure in the group of patients with urinary tract infection were: glomerulonephritis in 8 cases (30% of patients), chronic pyelonephritis in 7 (26%), reflux nephropathy in 4 (15%), diabetic nephropathy in 2 (7%), polycystic kidney disease in 3 (11%) and unknown in 3 (11%) cases. Discussion In the present study UTI was found in 10% of kidney transplant recipients during routine outpatient visits. The data of the frequency of UTI in kidney graft recipients differs among laboratories from 4.2 to 73.7%, depending on group of patients (hospitalized vs. ambulatory), time of observation (early period after transplantation vs. years of observation), chemoprophylaxis used and the definition of UTI (Hamshere et al., 1974; Belitsky et al., 1982; Cuvelier et al., 1985; Maddux et al., 1989). UTI after kidney transplantation is most common during hospitalization period, directly after transplantation procedure. The mean time from the procedure to clinical features of UTI lasts 4–7 days (Midtvedt et al., 1998), the mean period of hospitalization of patients with UTI after transplantation procedure lasts 36 days in comparison to 27 days in patients without UTI (Kentouni-Noly et al., 1994). In the early period, 1– 3 months after kidney transplantation, frequency of bacteriuria in patients is high – 39.5 to 73.7% which is confirmed by many laboratories (Renoult et al., 1994; Castelano et al., 1995; Maraha et al., 2001) and it can be even as high as 85% (Mroz et al., 1993). The lowest percentage of UTI was described in one of Japanese transplant center: 10% in perioperative period and 4.2% in ambulatory follow up (Goya et al., 1997). In this study the 5-day perioperative therapy with III generation cephalosporin intravenously and 4-month prophylaxis with trimethoprimsulphamethoxazole in relatively high dose (3 × 480 mg every second day) were administered to all patients. During later period after transplantation the frequency of UTI decreases but identification and management of this complication is a challenge in ambulatory medical care. Almost 80% of UTI cases in this group of patients are lower urinary tract infections, usually asymptomatic (Schmaldienst et al., 2002). In our study asymptomatic bacteriuria were diagnosed in relatively high rate (8% of study population) and symptomatic UTI in only 2% of patients. There is no evidence about the influence of asymptomatic bacteriuria on the function of transplanted kidney. In some reports, in which the aim was the assessment of the influence of symptomatic UTI on the function of transplanted kidney, it was showed that this influence exists, but it can be observed after long – over 3-year observation (Muller et al., 1998). Opinions about treatment of asymptomatic bacteriuria in patients after transplantation differ among authors. Some of them believe that this treatment is always necessary (Cormio et al., 2002; Raz, 2001), some – that the therapy is necessary only in early period after the procedure (Du³awa et al., 2001; Korzeniowski, 1991). The third group of authors suggests that the treatment is not required (Nicolle, 2000; Goya et al., 1991). In our study the incidence of diabetes mellitus in UTI group was 20% which is comparable to rate observed in whole population of kidney transplant patients. Chronic pyelonephritis and reflux nephropathy account for 30% diseases led to end stage renal failure in the group of patients with UTI. It is well known that patient’s own cirrhotic kidneys with regressive cysts may be a source of infection descending the urinary tract to the renal allograft. Our observation could support recommendation for more thorough monitoring for UTI in this groups of patients. The dominating bacteria in the cultures from tested urine specimens were Gram-negative microorganisms. Over 90% of isolated Gram-negative strains were sensitive to ceftriaxone, ceftazidime, amikacin and aztreonam – the drugs that can be administered intravenously in the hospitals. The only drug with similar effectiveness which can be administered orally in ambulatory medical care was fosfomycin. Ciprofloxacin and norfloxacin showed relatively high effectiveness (84% and 88% susceptible Gram-negative bacterial strains). These agents could be used in empiric treatment of UTI in patients after kidney transplantation. Although it was a small number of isolated strains, analysis of the data of susceptibility to antibiotics of Gram-positive bacteria showed that all of cultured enterococci were susceptible to nitrofurantoin. Urinary tract infection caused by enterococci is especially dangerous because it could lead to urosepsis, especially in patients with immunosuppres-

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321

sion-associated leukopenia (Caballero-Granado et al., 2001). Unfortunately, poor tissue penetration of nitrofurantoin and low urine concentration in patients with decreased glomerular filtration rate make this drug ineffective in most of kidney transplant recipients. In conclusion urine culture should be performed before starting UTI treatment in renal transplant recipients, especially caused by Gram-positive bacterial strains. The main aim of postoperative prophylaxis in graft recipients is eradication of Gram-negative bacilli of the family Enterobacteriaceae. Antibiotics that can be used for this purpose are orally administered fluoroquinolones and co-trimoxazole. In our study Gram-negative bacterial strains susceptibility to these agents was relatively high (88% and 77%, respectivelly) what makes them reasonable choice for postoperative prophylaxis. Overall, the antimicrobial therapy should be considered individually, the choice of the drug should be based on pharmacokinetics of the drug, specificity of the disease and interactions with immunosuppressive drugs (Dzier¿anowska and Jeljaszewicz, 1998). Literature B e l i t s k y P., S.G. L a n n o n, A.S. M a c D o n a l d, A.D. C o h e n, T.J. M a r r i e, P. H o u l i h a n and A. W h a l e n. 1982. Urinary tract infections (UTI) after kidney transplantation. Transplant Proc. 14: 696–699. C a b a l l e r o - G r a n a d o F.J., B. B e c e r r i l, J.M. C i s n e r o s, L. C u b e r o s, I. M o r e n o and J. P a c h o n. 2001. Casecontrol study of risk factors for the development of enterococcal bacteremia. Eur. J. Clin. Microbiol. Infect. Dis. 20: 83–90. C a s t e l a n o A.M., K. S o t o, J.M. G r i n y o, S. G i l v e r n e t, D. S e r o n, J. T o r r a s, L. R i e r a, J.M. C r u z a d o and J. A l s i n a. 1995. Prophylaxis of urinary tract infection in renal transplantation: comparison of three different protocols using ceftriaxone-cloxacillin, aztreonam-cloxacillin, or aztreonam-amoxycillin-clavulanic acid. Transplant Proc. 27: 2277–2279. C o r m i o L., B. B e r a r d i, A. C a l l e a, N. F i o r e n t i n o, D. S b l e n d o r i o, V. Z i z z i and A.T r a f i c a n t e. 2002. Antimicrobial prophylaxis for transrectal prostatic biopsy: a prospective study of ciprofloxacin vs piperacillin/tazobactam. BJU Int. 90: 700–702. C u v e l i e r R., Y. P i r s o n, G.P. A l e x a n d r e and C. v a n Y p e r s e l e d e S t r i h o u. 1985. Late urinary tract infection after transplantation: prevalence, predisposition and morbidity. Nephron 40: 76–78. D o u g l a s J.F., S. C l a r k e, J. K e n n e d y, J. M c E v o y and M.G. M c G e o w n. 1974. Urinary tract infection after renal transplantation. Lancet 2: 1015. D u ³ a w a J., M. M y œ l i w i e c and A. W i e l g o s z. 2001. Identification and treatment of urinary tract infections (in Polish). In: B. Rutkowski and S. Czekalski (eds), Standards in management and treatment of kidney diseases. MAKmed. D z i e r ¿ a n o w s k a D. and J. J e l j a s z e w i c z. 1998. Hospital infections (in Polish). "-medica press, Bielsko-Bia³a. G o y a N., K. T a n a b e, Y. I g u c h i, T. O s h i m a, T. Y a g i s a w a, H. T o m a, T. A g i s h i, K. O t a and K. T a k a h a s h i. 1997. Prevalence of urinary tract infection during outpatient follow-up after renal transplantation. Infection 25: 101–105. H a m s h e r e R.J., G.D. C h i s h o l m and R. S h a c k m a n. 1974. Late urinary-tract infection after renal transplantation. Lancet 2: 793–794. K e n t o u n i - N o l y J.C., P. C l o i x, X. M a r t i n, M. R a b o d o n i r i n a, N. L e f r a n c o i s, M. S e p e t j a n. 1994. Analysis of noscomial infections in renal transplant and pancreas transplant recipients. Transplant Proc. 26: 284. K o r z e n i o w s k i O.M. 1991. Urinary tract infection in impaired host. Med. Clin. North Am. 75: 391–401. K r i e g e r J.N., L. T a p i a, W.T. S t u b e n b o r d, K.H. S t e n z e l and A.L. R u b i n. 1977. Urinary infection in kidney transplantation. Urology 9: 130–136. K u r i j a m a M., T. N a g a i, H. U n o, Y. N i s h i d a, T. I s h i h a r a, K. K o b a y a s h i, Y. T a k a h a s h i, A. S a i t o and K. K a w a d a. 1991.Urinary tract infections after kidney transplantation. Acta Urol. Jpn. 37: 1173–1779. M a d d u x M.S., S.A. Ve r e m i s, W.D. B a u m a, R. P o l l a k and M.F. M o z e s. 1989. Effective prophylaxis of early posttransplant urinary tract infections (UTI) in the cyclosporine (CSA) era. Transplant Proc. 21: 2108–2109. M a r a h a B., H. B o n t e n, H. v a n H o o f f, H. F i o l e t, A.G. B u i t i n g and E.E. S t o b b e r i n g h. 2001. Infectious complications and antibiotic use in renal transplant recipients during a 1-year follow-up. Clin. Microbiol. Infect. 7: 619–625. M i d t v e d t K., A. H a r t m a n n, T. M i d t v e d t and I.B. B r e k k e. 1998. Routine perioperative antibiotic prophylactic in renal transplantation. Nephrol. Dial. Transplant. 13: 1637–1641. M u l l e r V., G. B e c k e r, M. D e l f s, K.H. A l b r e c h t, T. P h i l i p p and U. H e e m a n n. 1998. Do urinary tract infections trigger chronic kidney transplant rejection in man? J. Urol. 159: 1826–1829. M r o z E., J. U z a r, T. S z e p i e t o w s k i, J. S t a r e k and J. B a r t e l m u s. 1993. [Urinary tract infections in the first three months following kidney transplantation]. Pol. Tyg. Lek. 48: 448–451. N i c o l l e L.E. 2000. Asymtomatic bacteriuria-important or not? N. Engl. J. Med. 304: 1037–1039. R a z R. 2001. Asymptomatic bacteriria-clinical significance and management. Nephrol. Dial. Transplant. 16: 135–136. R e n o u l t E., F. A o u r a g h, D. M a y e u x, D. H e s t i n, A. L a t a s t e, J. H u b e r t, J. L’ H e r m i t e, M. W e b e r and M. K e s s l e r. 1994. Factors influencing early urinary tract infections in kidney transplant recipients. Transplant. Proc. 26: 2056–2058. S c h m a l d i e n s t S., E. D i t t r i c h and W.H. H ö r l. 2002. Urinary tract infections after renal transplantation. Curr. Opin. Urol. 12: 125–130. S t e i n G. and R. F u n f s t u c k. 1999. Asymptomatic bacteriuria-what to do. Nephrol. Dial. Transplant. 14: 1618–1621. W e t z e l O., M. H o r m i, L. L e N o r m a n d, G. K a r a m, J. G u e n e l, J. A u v i g n e and J.M. B u z e l i n. 1993. Autosomal dominant polycystic kidney disease: urologic complications and results of kidney transplantation: 217 patients. Prog. Urol. 3: 252–262.

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Polish Journal of Microbiology 2005, Vol. 54, No 4, 323– 330

The Action of Photosensitizers and Serum in a Bactericidal Process. II. The Effects of Dyes: Hypericin, Eosin Y and Saphranine O ANDRZEJ JANKOWSKI1,* STANIS£AW JANKOWSKI2, AGNIESZKA MIROÑCZYK1 and JOANNA NIEDBACH1 1 Institute

of Biotechnology and Environment Protection, University of Zielona Góra Monte Cassino 21 b, 65-561 Zielona Góra, Poland 2 Departament of Biology and Medical Parasitology, Medical University of Wroc³aw Mikulicza-Radeckiego 9, 50-367 Wroc³aw, Poland

Received 11 April 2005, received in revised form 12 July 2005, accepted 14 July 2005 Abstract The aim of the present work was to recognize the reasons for differences in the photodynamic action of dyes against various bacterial strains. It is expected that a better understanding of this problem may help in design of new photosensitizers. The sensitivity of 6 various bacterial strains to the photodynamic action of 5 photosensitizers was determined. The hydrophobicity of cell surface and susceptibility of bacteria to the natural defense mechanism of human serum, were estimated. The differences in the photodynamic efficiency of dyes could be contributed to various affinities of cell membrane to dyes, to known details of membrane architecture as well as to different mechanisms of photosensitization. K e y w o r d s: photosensitization, hypericin, saphranine O, eosin Y, bactericidal effect of serum

Introduction The photodynamic action of dyes against microorganisms consists in photosensitization of cells to the action of visible light. The effect is based on oxidative cleavage of cell membrane components initiated by light. Such dyes as porfirines (Doiron and Gomer, 1984; Jori and Preria, 1985; Gabor et al., 2001; Ashkenazi et al., 2003), porphycene (Lauro et al., 2002) phthalocyanines (Millson et al., 1996; Lacey et al., 2001), hypericin and hypocrellin (Chaloupka et al., 1999), saphranine O, eosin Y and others (Friedberg et al., 1991; Castael et al., 2003, Komerick and Wilson, 2002; Wilson, 2004) have been used as photosensitizers in photodynamic therapy of tumors and bacterial infections. The killing of bacteria by irradiation of photosensitizing dyes within absorption bands is due to production of oxygen containing radicals and/or other reactive oxygen species (ROS)1. Two mechanisms (I–II) are supposed to be responsible for this photodynamic action (Halliwell and Gutteridge, 1990). Superoxide anion radical generated in mechanism (I) in the presence of metal ion gives hydroxyl radical OH • that can readily oxidize some components of cell wall due to its extremely high reactivity (Bartosz, 1995). Another basic mechanism (II) of photosensitization consists in generation of singlet oxygen via triplet state of a photosensitizer. Hydroxyl radicals (OH•) and singlet oxygen (1O2) formed in mechanisms (I) and (II) respectively, as neutral species soluble in hydrophobic media, can permeate freely through the cell membranes, causing damage of some components of cell envelope. Since most photosensitizers are efficient only against selected bacterial strains it can be supposed that the photodynamic action of a given dye will depend not only on the dye spectral properties but also on the structure of bacterial cell wall. The reason for variation of sensitivity of bacteria is not completely clear at present. The question arises whether a photosensitizer, to be active, must be bound to the membrane, so that the affinity to the cell may decide of the efficiency. Though ROS species, generated by a dye anchored * E-mail: [email protected] 1 Abbreviations: DMF dimethylformamide, DMSO dimethylsulfoxide.

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on a cell, can reach the target place more rapidly than those present in bulk water solution, the binding site may be inaccessible to oxygen and the spectroscopic properties of the photosensitizer may be changed due to binding, leading to a decrease of the efficiency of ROS generation. The knowledge of the target site damaged by ROS in the membrane and its distance from the binding site is very important. It has been shown in our previous work that chlorophylls as photosensitizers (Jankowski et al., 2003) or even ozone (Doroszkiewicz et al., 1994) can enhance the bactericidal effect of serum. The bactericidal action of serum is a very important mechanism of resistance against infections of Gram-negative strains (Jankowski, 1995; Doroszkiewicz et al., 1994). A correlation between the photosensitization and the action of serum suggest that the place of action of photosensitizers may be close to the site of the membrane attack complex formed by the factors present in serum. The aim of our work is to recognize the interrelation between the structure of bacterial cell membranes and the properties of photosensitizers, their absorption ability on the cell surface and the kind of radicals produced during irradiation. The relation between the action of photosensitizers and of serum is also essential. We have found that some Shigella strains differ greatly with respect to the action of some dyes as well as to the bactericidal action of serum. Since the structure of antigen determinants of these strains is known (Simmons and Romanowska, 1987), it seems to be possible to correlate the properties of cell wall and the efficiency of photosensitization. Experimental Materials and Methods

PD effect

Strains and serum. Following strains obtained from Institute of Biotechnology and Environment Protection, University of Zielona Gora were used: Shigella flexneri 1a, S. flexneri 1b and S. flexneri 2a, Escherichia coli K12 No. 781 and Bacillus subtilis 003. The strain of E.coli K1 No. 959 was from Department of Biology and Parasitology, Medical Academy of Wroc³aw. Normal human serum (NHS) from 2 healthy donors not treated with any antimicrobial drugs was used. After separation from clot the serum was stored in 0.3 cm 3 portions at –70oC for no longer than 2 months. Photosensitizers. Solutions in water of following dyes were used, saphranine O (BDH, England), concentration c = 1.00 mM/dm 3, eosine Y c = 1.00 mM/dm 3 (BDH, England), chlorophyll (water soluble) was from C. Erba (Italy) c = 1.00 mM/dm3. Iron(II) sulfophthalocyanine (FePTS) was a kind gift from dr. £. Ostropolska (Chemistry Department, University of Wroc³aw) c = 5.00 mM/dm 3. Hypericin was obtained by extraction of Deprim (Pharmaceutical and Chemical Company, Ljubljana, Slovenija) by diethylether, evaporation of solvent in darkness and dissolving in DMF (c = 0.12 mM/dm 3). In the cases of hypericin and chlorophyll which are known (Chaloupka et al., 1999; Jankowski et al., 2003) to be sensitive to visible light, special attention was paid, to protect samples from light until photolysis. Each dye was used in the concentration range where saturation of photodynamic effect (PDE) was observed in a plot of PDE against dye concentration (Fig. 1). In the case of hypericin the results for concentrations of the photosensitizer higher than 0.2 mM/dm 3 could not be obtained because of toxic effect of the dye on most strains investigated.

100

80

60

40

20 -6

-5

-4

-3

-2

l

C

-1

log C

Fig. 1. Dependence of PDE of saphranine against S. flexneri 2a on the dye concentration

4

325

Action of photosensitizing dyes and serum in bactericidal process

Photolyses. The influence of photosensitizers on the survival of bacteria was tested by a modified procedure described by Wilson and Pratten (1994). To 0.1 cm3 of bacterial suspension (10 9 cells/cm3) placed in a well of pH plate 0.1 cm 3 dye solution and 0.3 cm3 physiological salt solution (PS) were added. The concentration of dyes in the photolysed samples was: saphranine, eosin and chlorophyll c = 2 × 10–4 M/dm3, FePTS 10–3M/dm3 and hypericin 2.4 × 10 –5M/dm3. Mean absorbance of the photolysed samples at 500 – 600 nm was similar for all dyes investigated. The samples were covered by glass plate to prevent evaporation of solvent and illuminated (by stirring) during 90 min by white non mutagenic light (5 W/cm2) in a special compartment enabling optimal illumination conditions and maintaining temperature near to 30°C (26 – 35°C). Another plate containing analogous samples was incubated in darkness (the pH plate was covered by black glass plate). The samples were incubated parallelly in light and in darkness in order to determine the PDE. The difference between the survival percent of bacteria in the illuminated and not illuminated sample was treated as the measure of photodynamic action. The compartment for photolyses, plates and other furniture were sterilized by UV bactericidal lamp overnight before the experiment. The survival of bacteria was determined by visible count method immediately after photolysis. For each sample at least 3 separate experiments and in each experiment 3 separate counts were performed. Bacterial strains showing survival percent higher than 50 were treated as resistant. The bactericidal effect of serum. To some samples of bacterial culture, before photolysis, 0.2 cm 3 of PS instead of 0.3 of PS were added. After the photolysis, 0.1 cm3 of serum diluted 1:1 by PS was added and the samples were incubated during 60 min (37oC) without illumination. Then the survival percent of cells was determined as described above. This protocol was aimed to avoid deleterious effect of photosensitizers and radicals on serum. Determination of OH• radicals. The amount of hydroxyl radicals, produced in the solutions of photosensitizers, was determined at the same conditions as in the experiments with bacteria, but instead bacterial suspension 0.1 cm3 of 4((9-acridinecarbonyl)amino)2,2,6,6 tetramethyl piperidin-1 oxyl, free radical (TEMPO, Molecular Probes Inc, USA, solution 1.7 mg/10 cm3 DMSO) was added to the samples. This nonfluorescent compound, by reaction with OH• is converted to strongly fluorescent 9-acridine derivative which can be detected by its fluorescence at 440 nm (excited at 300 nm, see Haugland, 1996). This system was standarized by measuring of acridine fluorescence (40 ng/dm3 –1 mg/dm3) and the fluorescence of TEMPO, at the same conditions in the presence of OH• generating system (Fe2+/ascorbate; see Yokoyama et al. 2002). We determined also lipid peroxidation products, as described previously (Jankowski et al., 2003). Cell surface hydrophobicity. It was determined by salting out test (Jankowski et al., 1997; Ljungh et al., 1985). This method is considered as a comparative test of cell surface affinity to hydrophobic compounds. The various amounts of (NH4)2SO4 (0–3.2 M/dm3) were added to bacterial suspension. Aggregation was observed 3 and 5 min after addition and the salt concentration needed to induce the aggregation was assumed to be inversely proportional to the hydrophobicity of cell surface.

Results Photodynamic effect of dyes. The influence of saphranine O and eosin Y on the survival of bacteria is shown in Tables I and II. The number of cells (N) in the sample with the dye, related to N in the control compartment (without dye), gives the survival percent (S%). S % in the presence of dyes but without illumination, is treated as the reference for determination of the photodynamic effect (PDE). Saphranine O is the most efficient photosensitizer tested at the given optimal experimental conditions. S. flexneri 1b, 2a, and E. coli K12 strains are sensitive to the photodynamic action of saphranine O but the effect is practically absent in the cases of S. flexneri 1a (Table I). The PDE of saphranine O on the strain S. flexneri 1b is lower than that of this photosensitizer on S. flexneri 2a or E.coli K12. B. subtilis 003 strain appears to be not liable to the PDE of investigated dyes though some weak susceptibility to saphranine O cannot be excluded. Table I The photodynamic effect (PDE) of saphranine O (1 mM/dm3) on the investigated strains Incubation type Strain

1)

Not illuminated sample Number of cells × 106

Illuminated sample Number of cells × 10 6

PDE

Control

dye

S%

Control

dye

S%

S. flexneri 1a

70.6

67.7

95.9

77.3

77.3

100.0

0

S. flexneri 1b

71.1

70.3

98.8

63.7

28.3

44.4

55.6 (p1 < 0.05)

S. flexneri 2a

60.3

57.5

95.3

75.6

9.6

12.7

87.3 (p1 < 0.05)

E. coli K12

73.0

65.1

89.1

57.3

14.1

24.6

75.4 (p1 < 0.05)

B. subtilis. 003

50.7

47.2

93.1

33.9

28.8

83.5

16.5

probability that the samples illuminated and not illuminated belong to the same population.

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Jankowski A. et al. Table II The photodynamic effect (PDE) of eosin Y (1 mM/dm3) on the investigated strains Incubation type Not illuminated sample Number of cells x10 6

Strain

1)

Illuminated sample Number of cells x10 6

PDE

Control

dye

S%

Control

dye

S%

S. flexneri 1a

39.5

37.1

93.9

27.7

20.5

74.1

25.9

S. flexneri 1b

43.9

39.9

90.9

38.7

15.1

39.1

60.9 (p1 < 0.05)

S. flexneri 2a

73.8

68.3

92.5

53.2

21.8

40.7

59.2 (p1 < 0.05)

E. coli K12

30.5

26.6

87.2

31.3

14.5

46.3

53.6 (p1 < 0.05)

B. subtilis. 003

53.2

52.2

98.7

40.7

39.1

96.1

3.9

see legend to Table I.

Eosin Y (Table II) exerts noticeable photodynamic action against S. flexneri 1b, S. flexneri 2a and E. coli K12 though the efficiency against these strains, seems to be lower than that of saphranine O except S. flexneri 1b appearing slightly more sensitive to eosin (the difference seems to be within experimental error). Fe(II) sulfophthalocyanine (anionic dye of strongly polar, hydrophilic character) and chlorophyll (water soluble preparation) are photodynamically inefficient against the strains investigated (results not shown). It is shown that the PDE greatly depends on the bacterial strain and for a given strain it varies with the type of photosensitizer. The observed differences in PDE of a given dye, between bacterial strains of the same species, must be connected with some special properties of cell membranes. The S. flexneri 1a strain undergoes aggregation by ammonium sulphate 0.2 M after 5 min while the strain 1b shows analogous behavior at 0.8 M and S. flexneri 2a strain at 3.2 M salt solution. These results indicate (Ljungh et al., 1985; Jankowski et al., 1997) that the cell envelopes of S. flexneri 1a strain are the most hydrophobic and S. flexneri 2a the most hydrophilic of the strains tested by us, while S. flexneri 1b strain shows intermediate hydrophobicity. This feature as well as the chemical nature of antigenic determinants (Simmons and Romanowska, 1987) rendering the cell surface of S. flexneri 1a strain more compact implies probably lower affinity of S. flexneri 1a cells to ionic dyes such as saphranine O and eosine Y than that of S. flexneri 2a and 1b. The difference between these serotypes resides in the structure of pentasaccharide repeating unit: S. flexneri 1a membrane has a sequence of 3 unsubstituted deoxymannose (rhamnose) residues and in 1b species rhamnose III is acetylated and in 2a serotype it is glycosylated. This structural elements may be responsible for the lower affinity to water soluble dyes and much lower PDE of hydrophilic dyes against S. flexneri 1a. The efficiency of water soluble dyes: saphranine O and eosine Y against S. flexneri 2a, S. flexneri 1b and S. flexneri 1a strains follows the order of affinity to water of the cells. This suggests that the efficiency of the photosensitizers against bacteria is proportional to the absorption ability of a dye to cell envelopes. The photodynamic effect of hypericin against the strains investigated is given in Table III. The most striking feature of the results presented is a great difference in the photodynamic action of hypericin on Table III The photodynamic effect of hypericin on the strains investigated Incubation type Strain

Not illuminated Number of cells x10 Control

6

Illuminated S%

1)

Hypericin

Number of cells x10 Control

6

Photodynamic effect S%

1)

PDE %

Hypericin

td2)

p3)

Hypericin

S. flexneri 1a

70.0

29.0

41.4

57.8

23.2

40.1

1.3

0.1

> 0.05

S. flexneri 1b

35.8

35.2

98.3

39.4

7.9

20.0

78.3

6.3

< 0.002

S. flexneri 2a

3.9

3.9

100

3.5

2.2

62.8

37.2

2.6

< 0.02

B. subtilis. 003

40.0

28.1

70.2

61.5

42.5

69.1

1.2

0.1

> 0.05

E. coli K12

58.8

39.6

67.3

93.2

18.4

19.7

47.6

3.4

< 0.01

E. coli K1

90.5

40.7

44.9

94.4

19.5

20.6

24.3

3.4

< 0.01

1)

S% is survival percent of cells surviving the treatment, with respect to control; 2) Student’s test; 3) see legend to Table I.

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327

Action of photosensitizing dyes and serum in bactericidal process

S. flexneri strains 1a, 1b and 2a. In the case of the strain 1b greater part of cells are killed in the presence of hypericin during illumination, while the strain 1a shows practically no photodynamic effect. Since hypericin is the most hydrophobic of the dyes tested by us, low PDE against this strain, showing high surface hydrophobicity, is unexpected. This apparent discrepancy between strength of PDE and affinity of a dye to cell surface of S. flexneri 1a is probably connected to the type of radicals produced and the explanation of this result will be presented below. S. flexneri 2a strain and both E. coli strains tested show lower sensitivity to the action of hypericin than the cells of S. flexneri 1b serotype in accord with the ranking of membrane surface hydrophobicity. B. subtilis 003 is resistant to the PD effect though some toxic action cannot be excluded. In most cases (Tables I, II and III) the number of cells (N) in the not illuminated samples, surviving the treatment, is close to the reference sample (without dye addition), so the survival percent (S%) in such samples is close to 100. These data indicate that the dyes are in most cases not toxic to the strains tested in our experimental conditions. However for chlorophyll (results not shown) and for hypericin (Table III), in the case of S. flexneri 1a and E. coli K1 and K12, the value of N in the not illuminated samples was essentially lower than in the control (without dye). This difference can be explained by some toxic effect of the dye on bacteria, independent on the presence of light. Hypericin, a substance insoluble in water, was used in our experiments in a form of a solution in dimethylformamide (DMF). It was shown in a separate experiment that DMF is not toxic for the strains investigated at our experimental conditions. Combined effect of serum and photosensitizers against bacterial strains. The impact of the photodynamic action of hypericin and the bactericidal effect of serum on survival of S. flexneri 1b and E. coli K12 seems to be comparable to the sum of the activities of these separate antibacterial factors (Table IV). It must be noticed however that an addition of serum leads to enhancement of antibacterial action against S. flexneri 1b and E. coli K12 that cannot be attained by increasing the concentration of the photosensitizer because of a saturation effect (Fig. 1). Table IV Combining of the bactericidal effect of hypericin and of serum against the investigated strainsa Strain Incubation conditions

Survival % relative to control

Control

Hypericin (H)

Serum (Se)

H + Se

illum.

dark

illum.

dark

PDE%

illum.

dark

illum.

dark

PDE%

25.6

28.9

18.4

94.1

75.7

31.2

38.1

14.4

35.6

23.7

S. flexneri 1a

22.0

23.0

45.9

47.4

1.5

60.4

52.2

24.1

33.5

9.4

E. coli K12

70.0

67.7

26.3

58.5

32.2

34.3

29.1

12.4

19.6

7.2

B. subtilis 003

50.0

59.7

81.0

81.0

0.0

88.6

93.8

81.2

83.1

1.9

S. flexneri 1b

a)

Number of cells [x10–6]

In all cases for the PDE in S. flexneri 1b and E. coli K12 p < 0.05 was found by means of Student’s test.

In the case of Shigella flexneri 1a which can be treated as resistant to the bactericidal action of complement (Survival > 50 %), photodynamic action of hypericin leads to a marked strengthening of the antibacterial effect, manifested by the fact that the number of cells in the illuminated sample with hypericin and after addition of serum (S = 24.1%) is much lower than in the sample with serum (S = 60.4%) or in the illuminated sample containing the photosensitizer (S = 45.9%). B. subtilis 003 strain seems to be resistant to the photosensitizer and serum and their combined action. The concentration of hydroxyl radicals. In order to get insight into the molecular mechanism of the effects observed it is necessary to asses the nature of radicals and reactive oxygen species generated during our experiments. The amount of hydroxyl radicals produced during 90 min illumination of photosensitizers solutions is given in Table V. A considerable amount of OH• radicals, in our experimental conditions is produced only in the cases of saphranine O and eosine Y. For the same dyes largest photodynamic effects against most strains tested, are observed (Tables I and II) so that the survival percent of bacteria is inversely proportional to the amount of hydroxyl radicals produced.

328

4

Jankowski A. et al. Table V Production of OHÿ radicals during photolysis of the dye solutions (90 min) Photosensitizer

)F1)

CA [nM/dm]2)

25 0 11 3 0

294.0 0 129.4 35.3 0

Safranine O Hypericin Eosine Y Chlorophyll (soluble) Fe(II) sulphophthalo-cyanine

Difference of the fluorescence intensity (at 440 nm) between illuminated and not illuminated sample; 2) The concentration of acridine derivative produced by reaction of TEMPO with OH • 1)

Our experiments suggest that OH• are efficient ROS species damaging cell membranes. The determination of the peroxidation products (results not shown) gives roughly the same result. Hypericin however appears to be inactive with respect to OH• production (Table V) though it gives strong antibacterial photodynamic effect against some strains (Table III). This apparent contradiction can be explained by another mechanism of the photosensitization: in the case of hypericin is operating probably the mechanism (II) mentioned in the introduction. This reaction sequence leads to production of singlet oxygen (1O2) rather than OH• (Mirossay et al., 1999; Weiner and Mazur, 1992). Another type of photosensitization might be resposnible for the fact that hypericin shows practically no PDE against S. flexneri 1a in spite of high affinity to cell surface while for other dyes tested usually there is a correlation between absorption on cell membrane and PDE. Discussion It is commonly known that the cells of various types may differ essentially in their sensitivity to the PD effect of dyes (Wilson, 2004; Weiner and Mazur, 1992; Halliwell and Gutteridge, 1990). In the present work high PD effect of hypericin against S. flexneri 1b and practically no photodynamic action of the same dye against S. flexneri 1a was found. Analogously in the case of 2 strains of E. coli (K12 and K1) various effect of chlorophyll (extract from leaves) has been reported (Jankowski et al., 2003). The Shigella strains 1a and 1b show also several sensitivity to the bactericidal action of serum (Table III) though the differences between strains is less marked than that of PD effect (Tables I and II). Combining of PDE and the bactericidal action of serum is relatively more efficient in the case of the S. flexneri 1a strain, less sensitive to both antibacterial actions while separate. The enhancement of the bactericidal efficiency of serum by addition of photosensitizers, observed in the present work, suggests that the membrane structure damaged by photosensitization is localized near to the site of complement (C) binding. The complex formed by C is probably bound to lipid A (Joiner, 1988; Jankowski, 1995) structure connected directly to the membrane outer layer containing the antigenic determinants in Gram-negative bacteria. The diversity of the PDE of hypericin within Shigella species can be explained taking into account high surface hydrophobicity of S. flexneri 1a strain shown in our test – a feature connected probably to the nature of repeating sugar sequences occurring in the membrane surface (Simmons and Romanowska, 1987) and responsible for antigenic properties. We suppose that hypericin, hydrophobic compound insoluble in water and soluble in DMF, is readily absorbed on the surface of 1a strain cell membranes, more hydrophobic than those of S. flexneri 1b and 2a species. Strong binding of hypericin to cell membrane of S. flexneri 1a cells leads to the toxic effect independent on light (see Table II). On the other hand such a strong binding on the cell surface hinders penetration of excited dye to the target site situated in an inner layer in the membrane which may be responsible for a lower PDE. In the case of 1b serotype, that probably has a lower affinity to hypericin than that of 1a strain, because of its lower hydrophobicity, the binding of the dye to cell membrane may be weaker but the excited dye can penetrate deeper into the membrane to the target site (perhaps lipid A) and therefore well marked PDE is observed (Table I). The fact that hypericin shows lower PDE against S. flexneri 2a strain that that in 1b serotype may be explained by more hydrophilic cell surface properties of species 2a.

4

Action of photosensitizing dyes and serum in bactericidal process

329

Such type of behavior – unexpected lack of PDE of hypericin against S. flexneri 1a that should have highest affinity of cell surface toward this dye, is noticeable in the case of hypericin, the dye giving probably singlet oxygen (1O2) in type II photosensitizing mechanism. On the other hand saphranine O and eosin, dyes of more hydrophilic character, and producing OH • radicals are more efficient against S. flexneri 2a and 1b than to S. flexneri 1a cells, in accord with expected order of affinities to cell membranes. If it is assumed that the strong binding of hypericin to S. flexneri 1a cells results in lack of PDE the question arises why strong binding does not prevent PDE in the case of hydrophilic dyes. It must be taken into account that the process of the dye binding to bacterial cell membrane proceeds by another way in the case of hypericin added in DMF solution than for photosensitizers soluble in water. It is possible that hypericin molecules aggregate in water suspension of microorganisms before binding disabling penetration into membrane. We suppose that hydrophilic dyes (eosin, saphranine) are bound closer to the target site (tentatively identified above as lipid A) while hypericin, to be effective photodynamically, must be transported to some distance through the membrane. Therefore strong binding on the outer membrane layer prevents PDE in the case of hypericin. 1O produced in the photodynamic action of hypericin is characterized by a lower oxidative potential, 2 higher lifetime in tissues and longer mean distance of penetration in the medium than that of OH • (Halliwell and Gutteridge, 1989; Weiner and Mazur, 1992). Its is also supposed that 1O2 formation in low polar media is inhibited Reddi et al., 1984). Specific properties of radicals generated can be the cause of the differences in effectivity between hepericin and other dyes tested. Conclusions: 1) The photodynamic efficiency of dyes depends essentially on the absorption site of photosensitizers on the target cell wall and on the kind of ROS produced. 2) Combined the action of photosensitizers and serum can essentially enhance the bactericidal effect especially in the case of less sensitive strains. Literature A s h k e n a z i H., Y. N i t z a n and D. G a l. 2003. Photodynamic effects of antioxidant substituted porphyrin photosensitizers on gram positive and negative bacteria. Photochem. Photobiol. 77: 186–191. B a r t o s z G. 1995. The Second Face of Oxygen (in Polish). PWN. Warszawa. C h a l o u p k a R., T. O b š i l, J. P l a š e k and F. S u r e a u. 1999. The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts. Biochim. Biophys. Acta 1418: 39–47. C a s t a e l M., A. G o l d, L. B a l l and M. S o b s e y. 2003. Photosensitization of hepatitis A virus (HAV) and other microbes by meso substituted porphyrins in water. Proceedings, Water Quality Technology Conference. p. 76–89. D o i r o n D. and Ch. G o m e r. 1984. Porphyrin Localization and Treatment of Tumors. A.R. Riss, N.York. D o r o s z k i e w i c z W., I. S i k o r s k a and S. J a n k o w s k i. 1994. Studies on the influence of ozone on complement mediated killing of bacteria. FEMS Immunol. Med. Microbiol. 9: 281–286. F r i e d b e r g J., R. T o m p k i n s, S. W a r r e n, A. F i s c h m a n and M. Y a r m u s h. 1991. Antibody targeted photolysis. Bactericidal effects of Sn(IV) chlorin e6 dextran monoclonal antibody conjugates. Annals N.Y. Acad. Sci. 618: 383–393. G a b o r F., K. S z o c s, P. M a i l l a r d, G. C s i k. 2001. Photobiological activity of cells. exogenous and endogenous porphyrin derivatives in Escherichia coli and Enterococcus hirae. Radiation and environmental biophysics. 40: 145–151. H a l l i w e l l B. and M. G u t t e r i d g e. 1989. Free Radicals in Biology and Medicine. 2 Ed. Oxford Univ. Press. Oxford . H a l l i w e l l B. and M. G u t t e r i d g e. 1990. Role of free radicals and catalytic metal ions in human diseases. An overview. Methods Enzym. 186: 1–85 H a u g l a n d R. 1996. Handbook of Fluorescent Probes and Research Chemicals (6 th ed). Molecular Probes. Eugene, USA. J a n k o w s k i A., S. J a n k o w s k i and A. M i r o ñ c z y k. 2003. Synergistic action of photosensitizers and of normal human serum in a bactericidal process. I. Effect of chlorophylls. Acta Microbiol. Polon. 52: 373–378. J a n k o w s k i S. 1995. Defense mechanisms protecting Gram negative bacteria against the bactericidal action of complement (in Polish). Post. Mikrobiol. 37: 23–44. J a n k o w s k i S., J. S a r o w s k a, H. ¯ a r c z y ñ s k a and A. C i s o w s k a. 1997. Hydrophobic properties of Pseudomonas strains (in Polish). Med. Dosw. Mikrobiol. 49: 187–190. J o i n e r K. 1988. Complement evasion by bacteria and parasites. Ann. Rev. Microbiol. 42: 201–209. J o r i G. and C. P r e r i a. 1985. Photodynamic Therapy of Tumors and other Diseases. Libreria Progetto, Padova. K o m e r i c k N. and M. W i l s o n. 2002. Factors influencing the susceptibility of Gram-negative bacteria to toluidine blue O-mediated lethal photosensitization. J. Appl. Microbiol. 92: 618–623. L a c e y J. and D. P h i l l i p s. 2001. The photosensitization of Escherichia coli using disulphonated aluminium phthalocyanine. J. Photochem. Photobiol. A. 142: 145–150. L a u r o F., P. P r e t t o, L. C o v o l o and G. J o r i. 2002. Photoinactivation of bacterial strains involved in periodontal diseases sensitized by porphycene-polylysine conjugates. Photochem. Photobiol. Sci. 1: 468–470. L j u n g h A., S. H j e r t e n and T. W a d s t r o m. 1985. High surface hydrophobicity of autoaggregating Staphylococcus aureus strains isolated from human infections studied with salt aggregation test. Infect. Immun. 47: 522–526.

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M i l l s o n C., M. W i l s o n, A. M a c r o b e r t, J. B e d w e l l and S. B o w n. 1996. The killing of Helicobacter pylori by low power laser light in the presence of photosensitizers. J. Med. Microbiol. 44: 245–252. M i r o s s a y L., A. M i r o s s a y, A. K o è i š o v a, I. R a d v a k o v a, P. M i š k o v s k y and J. M o j ž i š. 1999. Hypericin induced phototoxicity of human leukemic cell LineHL-60. Physiol. Res. 48: 135. R e d d i E., M. R o d g e r s, D. S p i k e s and G. J o r i. 1984. The effect of medium. polarity on the hematoporphyrin-sensitized photooxidation of L-tryptophan. Photochem. Photobiol. 40: 415–420. S i m m o n s D. and E. R o m a n o w s k a. 1987. Structure and biology of Shigella flexneri O antigens. J. Med. Microbiol. 23: 289–302 Y o k o y a m a S., T. T a k e d a, H. T a k a h a s h i, Y. O h t a, S. P a r k, T. N i s h i k a w a and M. A b e. 2002. Lipid peroxidation and the antioxidant effects. J. Oleo. Sci. 51: 63–75. W e i n e r L. and Y. M a z u r. 1992. EPR studies of hypericin. Photogeneration of free radicals and superoxide. J. Chem. Soc. Perkin Trans. 2: 1439–1447. W i l s o n M. and J. P r a t t e n. 1994. Lethal photosensitization of Staphylococcus aureus. Microbios 78: 163–168. W i l s o n M. 2004. Lethal photosensitization of oral bacteria and its potential application in the photodynamic therapy of oral infections. Photochem. Photobiol. Sci. 3: 412–418.

Polish Journal of Microbiology 2005, Vol. 54, No 4, 331– 334

Effect of Intensity of Feeding on the Intestinal Microflora of Pigs ANNA REKIEL1, JULITTA GAJEWSKA 2, KATARZYNA TOPOL2 and EWA SAWOSZ3 1 Department

of Animal Breeding and Husbandry and of Animal Nutrition and Feed Science, Faculty of Animal Sciences, Warsaw Agricultural University, Ciszewskiego 8, 02-786 Warsaw, 2 Department of Soil Environmental Science, Faculty of Agriculture and Biology, Nowoursynowska 159, 02-776, Warsaw, Poland 3 Department

Received 22 September 2005, accepted 15 October 2005 Abstract In individual, single-phase feeding animals were fed extensively (group E – 7 animals) or intensively (group I – 7 animals) in semi ad libitum system. The mixtures differed in composition as well as energy and nutritional value, with constant ratio of protein to energy of 13.12 : 1 in intensive feeding and 13.04 : 1 in extensive feeding. Fibre content per 1 kg mixture was 3.43% in group I and 12.3% in group E. For microbiological studies samples were taken from the duodenum, ileum, jejunum and large intestine and both quantitative and qualitative differences in the microflora of the differently fed groups was found. K e y w o r d s: fatteners, feed intensity, intestinal microflora

Introduction In order to obtain a high quality product both genetically and environmentally based approaches in the breeding of pigs are being undertaken. These include the selection of breeds and lines for crossing with indigenous breeds, the withdrawal of antibiotics used in feed and their replacement with biostimulators and the extensive husbandry and nutrition of animals (Libudzisz and Kowal, 2000; Fabijañska et al., 2001; Gajewska et al., 2001, 2002; Rekiel et al., 2004). Fibre can affect the length and capacity of select segments of the gastrointestinal tract in pigs (Turski and Batorska, 1991; Drochner and Coenen, 1986; Drochner, 1993; Schleicher, 1997). The type of fibre affects the development of the alimentary tract. A favourable increase in the volume of the large intestine of growing pigs was observed when the feed was supplemented with dry grass, whereas the use of wheat bran resulted in disadvantageous shortening and reduced volume of the small intestine (Leroch, 2003). The composition of the intestinal microflora is relatively constant in the individual stages of life but can change with the type of feed ingested (Stavric and Kornegay, 1995). It can thus be assumed that feed components, including the type and amount of fibre, can affect the quantitative and qualitative composition of the intestinal microflora. The aim of the current study was to determine the effect of varied feed intensity of growing pigs on the microflora of the small and large intestine. Experimental Materials and Methods The experiments embraced 14 young mixed-breed pigs, porkers and sows, divided into two groups. The mean body weight of the animals at the beginning of the experiment was 25 kg. The animals were fed intensively (group I) or extensively (group E). Fattening was single-phase and the feed was given individually in semi ad libitum system. The mixes used in the nutrition of the

332

4

Rekiel A. et al. Table I Composition of the mixes, their energetic and nutritional value Type of feeding intensive Feed components

extensive

participation of resources %

total protein* g

metabolic energy* MJ

participation of resources %

total protein* g

metabolic energy* MJ

Barley grits

64.0

75.65

7.507

64.0

75.65

7.507

Wheat grits

10.0

11.05

1.188

–

–

–

Soybean meal after extraction

13.5

62.57

1.667

–

–

–

Meat and bone meal

5.0

28.25

0.744

–

–

–

Lard

7.0

–

2.422

–

–

–

Premix

0.5

–

–

0.5

–

–

Dried plants of the Papilionaceae family Total

–

–

–

35.5

52.36

2.307

100.0

177.52

13.53

100.0

128.00

9.814

* – calculated according to Polish Norm of Pigs Nutrition (1993)

growing pigs differed in composition and energetic and nutritional value, with constant protein to energy ratio (Table I). In intensive feeding this ratio was 13.12 : 1 and in extensive feeding 13.04 : 1. The fibre content per 1 kg mixture was: in group I – 3.43%, in group E – 12.03%. Following fattening the pigs were slaughtered. For in vitro microbiological examinations approximately 1 g samples were taken under sterile conditions from the individual parts of the gastrointenstinal tract, that is the duodenum, jejunum, ileum and large intestine. After dilution, the removed material was used for plating, using poured plates and surface spread. Single colonies from the mixture of various microorganisms were obtained using the streak plate method. Morphological observations of the microorganisms embraced macroscopic examinations of the colonies, that is size, shape, surface, consistency, border type and colour, and microscopic observations after Gram staining of the cells. Examination and observations of microorganisms were made on King B medium (for growing bacteria belonging to the genus Pseudomonas), Sabouraud medium (for fungi), blood agar medium (for total bacterial count), on McConkey medium (for Enterobacteriaceae) and Eijkman medium (for acid-producing bacteria), (Kunicki-Goldfinger, 2001; Grabiñska-£oniewska, 1999; Duszkiewicz-Reinhard, 1999). After collecting samples from the intestines and spreading them on media, quantitative determinations were made. To allow for correct interpretation of the results determinations of dry weight of the intestinal mass were also carried out. For isolation and identification the following media were used: MPA agar (for plate streaking), McConkey, King and APT agar. Identification of bacteria to species was carried out using the following biochemical tests from bioMerieux: API 50CH for the identification of bacteria belonging to the genus Lactobacillus; API 20A for the identification of gram-positive and gram-negative anaerobic bacteria; API Staph for the identification of staphylococci and micrococci. Bacterial suspensions were prepared from single colonies. After confirming the purity of the cultures by streaking out, the strains were transferred to agar medium. After 24 hour incubation at 37 oC new suspensions were made. These were used to inoculate the API media API 20, API Staph, API 50CHL, API 50CHB (depending on the test) using the McFarland scale as recommended by the producer, which are then used to inoculate the test strips. The results were read using the APILAB computer program, which identifies the species or gives the percent probability of identification. The identity of the species was corroborated using Bergey’s Manual of Determinative Bacteriology (Buchanan and Gibbons,1974).

Results and Discussion Table II presents a quantitative compilation of the microorganisms from the individual sections of the digestive tract. In group E the number of bacteria belonging to the family Enterobacteriaceae was lower than in group I and moreover, the total number of microorganisms in the individual parts of the digestive tract was lower in this group. In the four studies segments of the intestines in pigs of the extensive groups the number of acid-producing bacteria, with potential probiotic activities, was more numerous than in the intensive group. These bacteria represented the genera Lactobacillus, Enterococcus and Leuconostoc (Rolfe 2000; Banach 2001). Several microorganisms representing auto- and allochtonous microflora were isolated. These were bacteria of the genera Lactobacillus, Escherichia, Enterococcus, Bacteroides, Sarcina, Pseudomonas, Bacillus and others (Table III). Some of them were found in both studied groups, some only in either group I or E. Bacteria belonging to the genera Escherichia or Pseudomonas grew on nutrient agar after 18– 24 hours, as opposed to bacteria of the genus Lactobacillus that grew much slower. Moreoever, the latter required specific growth media, such as APT agar.

4

333

Intensity of feeding and intestinal microflora of pigs

Table II Number of microbial cells in individual segments of the digestive tract in the intensively (I) and extensively (E) fed groups, per 1 g dry weight of intestine content Type of medium used Segment of digestive tract

King B

McConkey

Sabouraud group-feeding type:

I

E

8.7 × 107

5.7 × 107

8.5 × 107 2.1 × 106 5.6 × 103

2.3 × 103 7.2 × 106 3.0 × 108

Jejunum

9.2 × 107

1.3 × 108

1.2 × 108 3.5 × 106 9.0 × 103

5.3 × 103 7.4 × 106 3.0 × 108 1.7 × 1010 8.4 × 108

Ileum

1.7 × 10

5.9 × 10

5.0 × 10

3

5.6 × 103 8.0 × 106 6.1 × 108 1.8 × 1010 1.7 × 109

Large intestine

1.6 × 108

5.5 × 107 3.8 × 106 9.9 × 103

7.2 × 103 9.0 × 106 1.7 × 108 1.3 × 1010 7.9 × 108

I

8

6.2 × 108

7

I

2.1 × 10

7

5.2 × 10

E

Nutrient agar + blood

Duodenum

8

E

Eijkman I

E

I

E

7.1 × 109

3.3 × 108

Table III Bacteria isolated from animals fed intensively (I) or extensively (E) Group/feeding Intensive (I)

Identified bacteria Escherichia coli; Pseudomonas sp.; Sarcina; Micrococcus sedentarius (Kytococcus sedentarius); Staphylococcus cohnii subsp. cohnii; Bacillus subtilis;

Extensive (E) Lactobacillus acidophilus1; Leuconostoc mesenteroides subsp. mesenteroides Bacillus subtilis; Bacillus licheniformis; Bacteroides melaninogenicus subsp. intermedius (Prevotella intermedia); Escherichia coli; Pseudomonas sp.; Sarcina sp.;

From among the isolated bacteria several randomly selected strains, namely: Lactobacillus acidophilus 1, Leuconostoc mesenteroides subsp. mesenteroides, Bacillus subtilis, Bacillus licheniformis, Staphylococcus cohnii subsp. cohnii, Micrococcus sedentarius (Kytococcus sedentarius), Bacteroides melaninogenicus subsp. intermedius (Prevotella intermedia) were identified using Api tests and APILAB computer program. Moreoever, the bacteria: Escherichia coli, Pseudomonas sp. and Sarcina sp. were identified. The physiological, morphological and biochemical characteristics of the isolated strains ( e.g. Lactobacillus acidophilus 1, Leuconostoc mesenteroides subsp. mesenteroides, Bacillus subtilis, Bacillus licheniformis) points to their probiotic properties. Such organisms have a beneficial action on the organisms of animals (LonvaudFunel 1999; Reid 1999; Kailasapathy and Chin, 2000; Casula and Cutting, 2002; Heyman and Ménard, 2002; Fernández et al., 2003). These potentially probiotic strains were much more numerous in the case of the group E animals, which received feed containing greater fibre content. Moreover, the increase in number of probiotic species was accompanied by a decrease in the number of potentially pathogenic bacteria, such as E. coli and S. cohnii subsp. cohnii. Literature B a n a c h W., B. B u c h o l c and B. W ó j c i k. 2001. Characteristics of Lactobacillus strains in pharmaceutical preparations (in Polish). Med. Doœw. Mikrobiol. 53: 143–149. B u c h a n a n R.E. and N.E. G i b b o n s. 1974. Bergey’s Manual of Determinative Bacteriology. 8 th ed. The Williams and Wilkins Co., Baltimore. C a s u l a G. and S.M. C u t t i n g. 2002. Bacillus Probiotics: Spore germination in the gastrointestinal tract. Appl. Environ. Microbiol. 68: 2344–2352. D u s z k i e w i c z - R e i n h a r d W., R. G r z y b o w s k i and E. S o b c z a k. 1999. Theory and Exercises in General and Technical Microbiology (in Polish). SGGW, Warsaw. D r o c h n e r W. 1993. Digestion of carbohydrates in the pig. Arch. Anim. Nutr. 43: 95–97. D r o c h n e r W. and M. C o e n e n. 1986. The role of plant materials structure in swine nutrition. (Feeding aspects) (in German). Übers. Tierernährg. 14: 1–9. F a b i j a ñ s k a M., J. S i e d l e c k i, H. R e k o s z - B u r l a g a, E. G ó r s k a, W. J a n k o w s k i and J. G a j e w s k a. 2001. Results of young pigs breeding receiving promoting growth mixtures without antibiotic substituted with probiotics and synthetic zeolite (in Polish). Ann. Agri. Univ. Grodno 2: 231–237. F e r n á n d e z M.F., S. B o r i s and C. B a r b é s. 2003. Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J. Appl. Microbiol. 94: 449–455. G a j e w s k a J., M. F a b i j a ñ s k a, H. R e k o s z - B u r l a g a, J. S i e d l e c k i, W. J a n k o w s k i and E. G ó r s k a. 2001. Characterization of aerobic and anaerobic microflora of feeding pigs with fed mixtures containing probiotics and synthetic zeolite (in Polish). Ann. Warsaw. Univ. Spec. Nr. 230–235.

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G a j e w s k a J., M. F a b i j a ñ s k a and M. G a r b o l i ñ s k a. 2002. Microbiological studia of feed and faeces of fatteners fed mixtures containing naked oat and permutite. Acta Microbiol. Pol. 51: 71–78. G r a b i ñ s k a - £ o n i e w s k a A. 1999. Laboratory Exercises in General Microbiology (in Polish). Technical University, Warsaw. Heyman M. and S. Ménard. 2002. Probiotic microorganisms: how they affect intestinal pathophysiology. CMLS. Cell. Mol. Life Sci. 59: 1151–1165. K a i l a s a p a t h y K. and J. C h i n. 2000. Survival and therapeutic potential of probiotics organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp. Immunol. Cell Biol. 78: 80–88. K u n i c k i - G o l d f i n g e r W. 2001. Life of Bacteria (in Polish). PWN, Warsaw. L e r o c h S. 2003. Effect of various sources and level of crude fibres in mixes on the length and volume of the elinebtart tract of pigs slaughtered on 90 and 180 day of life (in Polish). Acta Scient. Polon. (Zoot). 2: 35–46. L i b u d z i s z Z. and K. K o w a l. 2000. Technical Microbiology (in Polish). Technical University, £ódŸ. L o n v a u d - F u n e l A. 1999. Leuconostoc. University Victor Segale, Academic Press, Bordeaux, France. P o l i s h N o r m o f P i g s N u t r i t i o n. 1993. (in Polish). Institute of Animal Physiology and Nutrition, PAN, Jab³onna, 1–87. R e i d G. 1999. The scientific basis for probiotics strains of Lactobacillus. Appl. Environ. Microbiol. 65: 3763–3766. R e k i e l A., J. G a j e w s k a and J. W i ê c e k. 2004. Microbiological state of pig intestine (in Polish). Trzoda chlewna. 46: 53–56. R o l f e R.D. 2000. The role of probiotics cultures in the control of gastrointestinal health. Symposium: Probiotic Bacteria: Implications for Human Health. Am. Society Nutr. Sci. 396–402. S c h l e i c h e r A. 1997. Nutritional and physiological evaluatuion of use of fodder with high participation of crude fibres in sows. Thesis (in Polish). Agricultural University. Wroc³aw. pp. 148 S t a v r i c S. and E.T. K o r n e g a y. 1995. Microbial probiotics for pigs and poultry. p. 206–231. In: Biotechnology in Animal Feeds and Animal Feeding. Wallace R.J. and A. Chesson (eds) VCH Verlagsgesellschaft mbH, Wienheim. RFN. T u r s k i W. and M. B a t o r s k a. 1991. Length and volume of the alimentary tract of pigs fed mixtures with high participation of rye. (in Polish). Zesz. Nauk. Przegl. Hod. 1: 179–185.

Polish Journal of Microbiology 2005, Vol. 54, No 4, 335– 338

Identification of Aeromonas culicicola by 16S rDNA RFLP ADAM KAZNOWSKI* and EDYTA KONECKA

Department of Microbiology, Institute of Experimental Biology, Fredry 10 A. Mickiewicz University, 61-701 Poznañ, Poland Received 16 May 2005, received in revised form 19 July 2005, accepted 22 July 2005 Abstract Studies were conducted on the improvement of A. culicicola identification. This species is phenotypically very similar to A. veronii biotype sobria, A. sobria, and A. allosaccharophila. The sequences of 16S rDNA of A. culicicola isolates show the highest similarity with A. jandaei, A. veronii, and A. caviae. Digestion of 16S rDNA PCR product with AluI and MboI restriction endonucleases allowed discriminating A. culicicola from all other Aeromonas species with the exception of A. jandaei. Additional digestion of 16S rDNA PCR product with BceAI showed a possibility of distinguishing A. jandaei from A. culicicola. K e y w o r d s: Aeromonas culicicola, 16S rDNA RFLP, identification

The genus Aeromonas comprises Gram-negative chemoorganoheterotrophic bacteria widely spread in the surface water, sewage (Schubert, 1991) and food (Hänninen and Sittonen, 1995; Palumbo, 1996). Some of the strains of the bacteria have been implicated as human pathogens causing gastroenteritis, soft-tissue and wound infections, pneumonia, and bacteraemia (Altwegg, 1999). Some members of Aeromonas sp. cause a broad range of infections in cold- and warm-blooded animals (Gosling, 1996). The taxonomy of the genus Aeromonas has undergone continual change due to addition of newly described species and reclassification of existing taxa. In Bergey’s Manual of Systematic Bacteriology, the genus has been divided into four species: A. hydrophila, A. caviae, A. sobria, and A. salmonicida (Popoff, 1984). DNA-DNA hybridizations have resulted in founding at least 19– 20 hybridization groups (HGs) within Aeromonas sp. Some of them have names: A. hydrophila (HG 1), A. bestiarum (HG 2), A. salmonicida (HG 3), A. caviae (HG 4), A. media (HG 5), A. eucrenophila (HG 6), A. sobria (HG 7), A. veronii (HG 8/10), A. jandaei (HG 9), A. schubertii (HG 12), A. trota (HG 14), A. allosaccharophila (HG 15), A. encheleia (HG 16), and A. popoffii (HG 17). Two genomic groups, HG 11 and HG 13 are unnamed (Altwegg, 1999; Martinez-Murcia, 1999). Recently, three new species have been described: A. culicicola (Pidyar et al., 2002), A. simiae (Harf-Monteil et al., 2004) and A. molluscorum (Miñana-Galbis et al., 2004). Phenotypic similarity of strains belonging to different Aeromonas sp. genomic groups creates many problems with their identification and requires confirmation by using the molecular methods (Kaznowski, 1997). Soler et al. (2003) have found that phenotypically only 14.5% or 20.3% of strains were correctly identified by MicroScan Walk/Away and BBL Crystal Enteric/Nonfermenter systems, respectively. Recently, Figueras et al. (2005) using 16S rDNA-RFLP obtained results that were completely different from those obtained by using API 20NE. Several molecular methods have been proposed to help distinguishing of Aeromonas spp.: rybotyping (Hänninen and Sittonen, 1995), multilocus enzyme electrophoresis (Altwegg et al., 1991), amplified fragment length polymorphism (AFLP) (Huys et al., 1996), restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer (£aganowska and Kaznowski, 2004), PCR-amplified length polymorphism in tRNA intergenic spacers (£aganowska and Kaznowski, 2005) and restriction fragment length polymorphisms of 16S rDNA (16S rDNA RFLP) (Borell et al., 1997; Figueras et al., 2000). * Corresponding author: tel. (48) 61 829 45 30; e-mail: [email protected]

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A. culicicola is a species proposed by Pidyar et al. (2002) upon the analysis of three strains. One of them was isolated from the midgut of mosquito Culex quinquefasciatus, whereas the other two from the midgut of Aedes aegyptii. Recently, Figueras et al. (2005) recovered 27 A. culicicola isolates from a drinking water supply in Spain. There are difficulties in the identification of this species because the isolates are phenotypically very similar to A. veronii biotype sobria, A. sobria, and A. allosaccharophila (Pidyar et al., 2002; Figueras et al., 2005). The presence of a cytolytic enterotoxin gene in A. culicicola strains, which is considered as a characteristic virulence factor in Aeromonas spp., indicate that this species may have significance for public health (Pidyar et al., 2003; Figueras et al., 2005). Characterization of the gene encoding 16S rRNA is now well established as a method for identification of species and genera of bacteria (Martinez-Murcia et al., 1992; Martinez-Murcia, 1999). Complete sequences of 16S rDNA of the members of Aeromonas sp. have been published (Martinez-Murcia, 1999; Pidyar et al., 2002) and are available in GenBank. Analysis of 16S rDNA sequences of all Aeromonas species showed differences from 1 to 33 substitutes. Pidyar et al. (2002) have found that sequences of their A. culicicola isolates show the highest similarity with A. jandaei (only one substitution), A. veronii, and A. caviae (5 substitutions). Recently, Figueras et al. (2005) revealed in 16S rDNA of six strains of A. culicicola five of the variation (positions 457 to 476) and other two at positions 1011 and 1018. The objective of our study was to improve A. culicicola identification by restriction fragment length polymorphism of 16S rDNA. Three strains of A. culicicola previously described by Pidyar et al. (2002) and 18 type or reference strains representing other 16 hybridization groups were used in this study (Table I). Bacterial DNA was extracted by using Nucleo-spin C + T kit (Macherey-Nagel, Germany). The primers 5’-AGA GTT TGA ATC ATG GCT CAG-3’ and 5’-GGT TAC CTT GTT ACG ACT T-3’ (Borrell et al. 1997) were synthesized by Genset Oligos (Paris, France). PCR amplifications were carried out in a final volume of 50 µl with 100 ng of template DNA, 5 µl of 10×PCR buffer with NH4(S04)2, 50 pmol of each primer, 200 µM of dNTP mix, 2.5 mM of MgCl2, and 2 U of Taq polymerase (Fermentas). The amplification involved initial denaturation step (93°C, 3 min), followed by 35 cycles of denaturation (94°C, 1 min), annealing (56°C, 1 min), and extension (72°C, 1 min). After the final cycle, extension at 72°C was allowed for 10 min. PCR products were precipitated by 96% cold (–20°C) ethanol, dried, and resuspended in 25 µl of sterile water (Borrell et al. 1997). Table I Bacterial strains used in the study Hybridization group

Hybridization group

Strain

1

A. hydrophila ATCC 7966T

9

2

A. bestiarum ATCC 51108 :

3

A. salmonicida subspecies salmonicida LMG 3780

4

A. caviae ATCC 15468

5

A. media ATCC 33907T

6

A. eucrenophila ATCC 23309

7

A. sobria CIP 7433T

T

T

T

T

Strain A. jandaei: ATCC 49568T, LMG 13065

11

Aeromonas sp ATCC 35941

12

A. schubertii ATCC 43700T

13

Aeromonas sp. LMG 17321

14

A. trota ATCC 49657T

15

A. allosaccharophila CECT 4199T

16

A. encheleia CECT 4342 T

8/10

A. veronii biotype sobria CDC 0437–84

17

A. popoffii LMG 17541T

10/8

A. veronii biotype veronii ATCC 35624

18

A. culicicola: MTCC 3249, SH 2/5, SLH 21/5

T

Abbreviations: ATCC – American Type Culture Collection, Manassas,VA, USA; CDC – Centers for Disease Control, Atlanta, USA; CECT – Collection Espanola de Cultivos Tipo, Universitad de Valencia, Spain; CIP – Collection bacterienne de l¢Institut Pasteur, Paris, France; LMG – Culture Collection, Laboratorium voor Microbiologie Universiteit Gent, Belgium; MTCC, SH, SLH – strains recived from dr Y. Shouche, Molecular Biology Laboratory, Pune University, Ganeshkhind, India.

Enzymatic digestions were performed by incubating 5 µl of the amplification product with 5 U of AluI and MboI (Fermentas) or BceAI (New England Labs) overnight at 37°C. Aliquots of 10 µl of each reaction mixture were mixed with 2 µl of loading buffer containing 0.09% bromophenol blue, 0.9% xylene cyanol FF, 60% glycerol and 60 mM EDTA, and the mixture was electrophoresed on 2.5% Micropore Nu agarose gel (Prona, Spain) in Tris-borate-EDTA buffer. Gels were stained with ethidium bromide (1 µg/ml) and documented with Bio-Print V.99 system (Vilbert Lourmat, France). Sizes of DNA fragments were cal-

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culated using GelCompar II software (Applied Maths, Belgium) with MassRuler DNA Ladder Mix (Fermentas) as a molecular size reference. We did not find a commercial restriction endonuclease that would allow one-step distinguishing of all Aeromonas species. A. culicicola can be identified in two steps. In the first step, the 16S rDNA amplicon is digested with AluI and MboI restriction endonucleases according to Borrell et al. (1997) and Figueras et al. (2000), who have elaborated a scheme for distinguishing Aeromonas species upon restriction analysis of 16S rDNA digested with several endonucleases. DNA fragments of 16S rDNA digested with AluI and MboI allowed distinguishing A. culicicola from all other Aeromonas species with the exception of A. jandaei. These two species gave identical DNA fragments of 207, 195, 188, 158 138, 78, and 69 bp. Sequences of 16S rDNA of six strains of A. culicicola have been described (Figueras et al. 2005). All the sequences contain thymine at position 254, whereas in A. jandaei there is cytosine at this position (Gen Bank). On-line analysis (http:// www.restrictionmapper.org) showed a possibility of distinguishing A. jandaei from A. culicicola by BceAI digestion (New England), which recognizes sequence 5’ACGGC (N) 123’; Fig. 1. 16S rDNA RFLP patterns of A. culicicola 3’TGCCG (N)145’. Electrophoresis of DNA fragments obtained and A. jandaei strains, obtained by using BceAI after digestion with BceAI empirically proved the capability of endonuclease this method. The sizes of the DNA fragments were 800, 322, Lanes: 1. A. culicicola MTCC 3249, 2 A. culicicola 285, and 110 bp for A. culicicola, and 520, 322, 285, and 110 SLH21/5, 3. A. culicicola SH2/5, 4. A. jandaei ATCC 13063, M. Molecular size bp for A. jandaei (Fig. 1). Identification of A. jandaei and 49568, 5. A. jandaei LMG reference. A. culicicola only upon digestion with BceAI, without preliminary differentiation from other Aeromonas species by AluI and MboI digestion, is not possible. PCR products of A. culicicola 16S rDNA digested with BceAI gave fragments similar in size to those for HGs 1 to 11, and 14– 17. A. schubertii (HG 12) and strain of unnamed HG 13 gave DNA fragments similar to A. jandaei. In conclusion, we propose BceAI treatment of PCR-amplified 16S rRNA gene for distinguishing A. culicicola and A. jandaei. This rapid method complements the identification scheme proposed by Borrell et al. (1997) and expanded by Figueras et al. (2000), which enables identification of Aeromonas sp. strains belonging to 17 hybridization groups. Literature A l t w e g g M., M.W. R e e v e s, R. A l t w e g g - B i s s i g and D. B r e n n e r. 1991. Multilocus enzyme analysis of genus Aeromonas and its use for species identification. Zbl. Bakt. 275: 28–45. A l t w e g g M. 1999. Aeromonas and Plesiomonas, p. 507–516. In: P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenower, R.H. Yolken. (eds), Manual of Clinical Microbiology. ASM Press, Washington. B o r r e l l N., S.G. A c i n a s, M.J. F i g u e r a s and A.J. M a r t i n e z - M u r c i a. 1997. Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. J. Clin. Microbiol. 35: 1671–1674. F i g u e r a s M.J., L. S o l e r, M.R. C h a c o n, J. G u e r r o and A.J. M a r t i n e z - M u r c i a. 2000. Extended method for discrimination of Aeromonas spp. by 16S rDNA RFLP analysis. Int. J. Syst. Bacteriol. 50: 2069–2073 F i g u e r a s M.J., A. S u a r e z - F r a n q u e t, M.R. C h a c o n, L. S o l e r, M. N a v a r r o, C. A l e j a n d re, B. G r a s a, A.J. M a r t i n e z - M u r c i a and J. G u a r r o. 2005. First record of the rare species Aeromonas culicicola from a drinking water supply. Appl. Environ. Microbiol. 71: 538–541 G o s l i n g P.J. 1996. Aeromonas species in disease of animals, p. 175–196. In: B. Austin, M. Altwegg, P.J. Gosling, S.W. Joseph (eds). The genus Aeromonas. J. Wiley and Sons, Chichester, New York, Brisbane, Toronto, Singapore. H a r f - M o n t e i l C., A. L e F l e c h e, P. R i e g e l, G. P r e v o s t, D. B e r m o n d, P.A.D. G r i m o n t and H. M o n t e i l. 2004. Aeromonas simiae sp. nov., isolated from monkey faeces. Int. J. Syst. Bacteriol. 54: 481–485. H ä n n i n e n M.L. and A. S i t t o n e n. 1995. Distribution of Aeromonas phenospecies and genospecies among strains isolated from water, foods or from humans clinical samples. Epidem. Infect. 115: 39–50. H u y s G., R. C o o p m a n, P. J a n s s e n and K. K e r s t e r s. 1996. High-resolution genotypic analysis of the genus Aeromonas by AFLP fingerprinting. Int. J. Syst. Bact. 46: 572–580.

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K a z n o w s k i A. 1997. Numerical taxonomy and DNA-DNA hybridizations of Aeromonas strains isolated from human diarrheal stool, fish and environment. System. Appl. Microbiol. 20: 458–467. £ a g a n o w s k a M. and A. K a z n o w s k i. 2004. Restriction fragment length polymorphisms of 16S-23S rDNA intergenic spacer of Aeromonas spp. System. Appl. Microbiol. 27: 549–557 £ a g a n o w s k a M. and A. K a z n o w s k i. 2005. Polymorphism of Aeromonas spp. tRNA intergenic spacer. System. Appl. Microbiol. 28: 222–229 M a r t i n e z - M u r c i a A.J., S. B e n l l o c h and M.D. C o l l i n s. 1992. Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations. Int. J. Syst. Bact. 42: 412–421. M a r t i n e z - M u r c i a A.J. 1999. Phylogenetic positions of Aeromonas encheleia, Aeromonas popoffii, Aeromonas DNA hybridization Group 11 and Aeromonas Group 501. Int. J. Syst. Bact. 49: 1403–1408. M i ñ a n a - G a l b i s D., M.M. F a r f á n, C. F u s t é and J. G a s p a r L o r é n. 2004. Aeromonas molluscorum sp. nov. isolated from bivalve molluscs. Int. J. Syst. Bact. 54: 2073–2078 P a l u m b o S.A. 1996. The Aeromonas hydrophila group in food, p. 287–310. In: B. Austin, M. Altwegg, P.J. Gosling, S.W. Joseph (eds). The genus Aeromonas. J. Wiley and Sons, New York, Chichester, Brisbane, Toronto, Singapore. P i d i y a r V., A. K a z n o w s k i, N. B a d r i N a r a y a n, M. P a t o l e and Y. S h o u c h e. 2002. Aeromonas culicicola sp. nov. from the midgut of Culex quinquefasiatus. Int. J. Syst. Evol. Microbiol. 52: 1723–1728. P i d i y a r V., K. J a n g i d, K.M. D a n y a n a n d a, A. K a z n o w s k i, J.M. G o n z a l e z, M.S. P a t o l e and Y.S. S h o u c h e. 2003. Phylogenetic affiliation of Aeromonas culicicola MTCC 3249T based on gyrB gene sequence and PCRamplicon sequence analysis of cytolytic enterotoixin gene. System. Appl. Microbiol. 26: 197–202. P o p o f f M. 1984. Genus III: Aeromonas, p. 545–547 In: N. R. Krieg, J. G. Holt (eds). Bergey’s Manual of Systematic Bacteriology, Wiliams and Wilkins, Baltimore, London. S c h u b e r t R.H.W. 1991. Aeromonads and their significance as potential pathogens in water. J. Appl. Bacteriol. Symp. Suppl. 70: 131S–135S. S o l e r L., F. M a r c o, J. V i l a, M.R. C h a c o n, J. G u e r r o and M.J. F i g u e r a s. 2003. Evaluation of two miniaturized systems. MicroScan W/A and BBL Crystal E/NF, for identification of clinical isolates of Aeromonas spp. J. Clin. Microbiol. 41: 5732–5734

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Analysis of the Murein of a Listeria monocytogenes EGD Mutant Lacking Functional Penicillin Binding Protein 5 (PBP5) DOROTA KORSAK1, MAGDALENA POPOWSKA and ZDZIS£AW MARKIEWICZ

Department of General Microbiology, Faculty of Biology, Warsaw University Miecznikowa 1, 02-096 Warsaw, Poland Received 21 September 2005, accepted 26 September 2005 after revision Abstract Cells of a mutant of Listeria monocytogenes lacking functional PBP5, an enzyme with DD-carboxypeptidase activity, make thicker cells walls. In this study we show that the muropeptide profile of the mutant, obtained after HPLC analysis of a muramidase digest of cell wall murein, differs from that for the wild type strain. The main differences embrace strongly reduced disaccharide-tripeptide content, strongly increased amounts of pentapeptide-containing muropeptides and a shift in profile from less cross-linked muropeptides (monomers, dimers) towards more highly cross-linked ones. K e y w o r d s: Listeria monocytogenes, penicillin binding protein (PBP), murein, HPLC

The murein of Listeria monocytogenes is of the A1g type, with direct cross-links between D-alanine and meso-diaminopimelic acid (Schleifer and Kandler, 1972), though cross-links between two meso-diaminopimelic acid residues in neighbouring peptide side chains, resembling the situation in Escherichia coli, have also been observed (Glauner, 1988; K³oszewska et al., this laboratory, unpublished). Like in other bacteria, the final stages of murein biosynthesis in L. monocytogenes are catalyzed by the so-called penicillin-binding proteins (PBPs), which are involved in transpeptidation and transglycosylation reactions (Ghuysen, 1991; van Heijenoort, 2001). During transpeptidation the terminal D-alanine is removed from the pentapeptide of the precursor (donor) with subsequent formation of a bond between D-alanine in position 4 of the precursor (or meso-diaminopimelic acid in position 3) and mesodiaminopimelic acid in position 3 of a second peptide chain (acceptor) (Höltje, 1996). The enzymes catalyzing murein polymerization reactions belong to the class A or B group of high molecular weight (hmw) PBPs. The low molecular weight (lmw) PBPs, on the other hand, as a rule are not directly involved in the biosynthesis of murein, but affect the final structure of the macromolecule, since they determine the length of the peptide side chains. Their activity thus decides how many peptides will serve as acceptors in transpeptidation reactions and consequently, the extent of murein cross-linking. Most of low molecular weight PBPs are DD-carboxypeptidases (Jamin et al., 1995; Ghuysen, 1997), though some of these proteins may have DD-endopeptidase activity, hydrolyzing peptide bonds in peptide bridges between adjacent sugar chains, formed as a result of earlier transpeptidation events (Goffin and Ghuysen, 1998). In L. monocytogenes 5 penicillin binding proteins have been identified, though analysis of the genome sequence of this bacterium shows that other putative PBPs may be synthesized. Four of these proteins, PBP1, 2, 3 and 4, belong to the hmw class of PBPs, whereas PBP5 is a lmw penicillin binding protein (Gutkind et al., 1989; Vicente et al., 1990; 1990a; Korsak et al., 2002). So far, the enzymatic properties of only hmw PBP4, which has transglycosylase and transpeptidase activities (Zawadzka-Skomia³ et al., submitted) and the lmw PBP5 (Korsak et al., 2005) have been determined. In the latter case, PBP5 has been found to be a DD-carboxypeptidase, and the preferred substrates for the enzyme in vivo are low molecular fragments of murein, that is monomeric muropeptides that are formed in the course of the metabolic turnover of the murein sacculus (that is in parallel processes of the degradation and synthesis de novo of the macromolecule) (Korsak et al., 2005). A mutant of L. monocytogenes lacking PBP5 was found to produce 1

Corresponding author, e-mail: [email protected]

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Korsak D. et al.

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a wall with greater thickness than the wild-type strain (Korsak et al., 2005). In view of this finding, the objective of the current study was to determine whether the absence of the DD-carboxypeptidase activity of PBP5 results in changes in the maturation of the cell wall of L. monocytogenes. L. monocytogenes EGD and the mutant strain DA01 lacking functional PBP5 were grown at 37°C in Tryptic Soy Broth (TSB) medium with shaking at 200 rpm. Cell cultures were cooled down and harvested by centrifugation (7 000 × g, 10 min, 4°C). The pellet was resuspended in 50 mM Tris/HCl buffer, pH 7.5. After adding 10 µg DNAse ml–1 and phenylmethanelsulfonyl fluoride (1 mM), the cells were broken in the ultrasonicator (Sonics and Materials, type VCX 600). Unbroken cells were removed by centrifugation (7 000× g, 10 min, 4°C). Hot 8% (v/v 1 : 1) sodium dodecyl sulfate (SDS) was added to broken material, which was then boiled for 30 min. The resulting insoluble wall preparation was then washed with warm distilled water (60°C) at least five times until free of SDS. Covalently attached protein was removed by treatment with 200 µg pronase ml–1 for 1.5 h at 60°C. The walls were then recovered by centrifugation (35 000 × g, 30 min, 4°C), washed once in distilled water, and suspended in trichloroacetic acid (final concentration 5%). The mixture was incubated at 4°C for 24 h. The insoluble material was collected by centrifugation (35 000 × g, 30 min, 4°C), and washed five times with cold distilled water. After final centrifugation the pellet was resuspended in 5 ml distilled water and kept frozen at –20°C. Samples containing muropeptides were digested with Cellosyl (Hoechst AG) as previously reported (Glauner, 1988). Soluble muropeptides were reduced by using sodium borohydride (Glauner et al., 1988). The reaction was stopped after 30 min by lowering the pH to 3.5 with phosphoric acid. The samples were analysed by HPLC according to the method of Glauner (1988) on a Hypersil ODS column (250 mm× 4 mm, particle size 3 mm diameter; Teknochroma). Elution buffers were 50 mM sodium phosphate containing 0.8 g sodium azide in 1 liter, pH 4.35 (A) and 15% methanol in 75 mM sodium phosphate, pH 4.95 (B). Elution conditions were 7 min isocratic elution in buffer A, 115 of linear gradient to 100% buffer B and 28 min of isocratic elution in buffer B. The flow rate was 0.5 ml/min and the column temperature was 55°C. Eluted compounds were detected by monitoring A205. The high performance liquid chromatography (HPLC) technique allows detailed analysis of the so-called muropeptide composition of murein, resulting in a specific pattern, depending on differences in number of sugar residues and number and type of amino acids In the individual muropeptides obtained after digestion of murein with a murolytic enzyme, such as the Cellosyl employed by us. This method allows the demonstrating of changes in the structure of murein, which frequently reflect altered biosynthetic processes, caused by the absence or overproduction of enzymes involved in these pathways, or by environmental factors, e.g. presence of certain amino acids or antibiotics in the growth medium (Glauner et al., 1988). We applied this technique for the analysis of the murein of the L. monocytogenes mutant lacking PBP5, compared to that of the wild type strain. In an earlier study of L. monocytogenes murein five major muropeptides, accounting for approximately 63% of the analyzed murein sample were identified (K³oszewska et al., this laboratory, unpublished). Muropeptides 1 and 2 are monomers – disaccharide-tripeptides, differing in the presence of an acetyl group on the glucosamine moiety of muropeptide 1 (Fig. 1A, peaks 1 and 2). Muropepties 3, 4 and 5 are dimers. Muropeptide 3 is a tri-tetrapeptide, in which the crosslink is between D-alanine and meso-diaminopimelic, with both glucosamine residues of the muropeptide being N-acetylated. The structure of muropeptide 4 is similar to that of muropeptide 3, the only difference being that one glucosamine residue is substituted with an acetyl group. In turn, muropeptide 5 is similar to muropeptides 3 and 4, but no glucosamine is acetylated (Fig. 1A, peaks 3, 4 and 5). No tetra monomers or tetra-tetra dimers, at least in significant amounts, were detected (K³oszewska, 2003). The muropeptide profile of mutant DA01 is strikingly different from that obtained in the case of the wild type L. monocytogenes strain (Fig. 1B). All the muropeptides identified in a murein digest of the wild type murein are absent but at a retention of approx. 25 min two new peaks appear that when compared with the characteristic and well identified muropeptides of a dacA mutant of Bacillus subtilis (lacking the gene coding for the major DD-carboxypeptidase synthesized by vegetative cells, which have a similar murein structure to that of L. monocytogenes); (Atrih et al., 1999) may correspond to monomers with disaccharidepentapeptide structure. The muropeptides that appear in the mutant profile with retention time between 55 and 70 minutes appear to be different forms of a dimer – a bis-disaccharide penta-tetra peptide, even though this has yet to be conclusively demonstrated. On the other hand, the muropeptides with retention time over 75 minutes correspond to trimers and higher oligomers. Even though the molecular identification of the muropeptides obtained on muramidase digestion of the murein of the mutant lacking functional PBP5 activity is lacking, certain conclusions can be drawn with

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absorbance 205 nm

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absorbance 205 nm

time (min)

time (min) Fig. 1. HPLC muropeptide elution patterns of murein from wild type Listeria monocytogenes EGD (A) and L. monocytogenes mutant DA01 lacking functional PBP5 (B) Purified Cellosyl-digested peptidoglycan samples were separated on a Hypersil octadecylsilane column, and the A205 of the eluate was monitored: 1, 2 disaccharide-tripeptide monomers; 3, 4, 5 bis-disaccharide tri-tetra peptide dimers.

absolute certainty. Analysis of the muropeptide profile of the mutant murein demonstrates the lack of muropeptides with peptide side chains composed of three amino acids. On the other hand, the appearance of muropeptides, both monomeric and dimeric, carrying pentapeptide side chains is observed. The muropeptide profile shows a dramatic shift towards higher muropeptides (dimers, trimers) which has to translate

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to higher overall extent of crosslinking in the murein of the murant. Similar changes have been observed in the murein of a mutant of B. subtilis lacking DD-carboxypeptidase activity (Atrih et al., 1999). The observed muropeptide profile of mutant DA01 can indicate that PBP5 of L. monocytogenes, similar to PBP5 of B. subtilis, is a DD-carboxypeptidase that plays a fundamental role in the maturation of the murein of the listerial cell wall. The course of murein turnover in mutant DA01 does not differ from that in the wild type strain, which indicates that PBP5 does not play a role in this process (data not presented). Of interest is the presence in the murein of wild type L. monocytogenes of monomers and dimers carrying tripeptides, which most probably reflects the presence of a protein with LD-carboxypeptidase activity. It thus seems highly probable that besides PBP5, which cleaves off the last D-alanine of the pentapeptide side chain, a penicillin-insensitive protein with LD-carboxypeptidase activity, also participates in determining the length (and role) of the peptide side chains in listerial murein. A protein with such enzymatic activity has been identified in Escherichia coli, also a bacterium with 1g type of murein (Ursinus et al., 1992). Further investigations on both the LD-carboxypeptidase of L. monocytogenes and the putative enzyme with LD-carboxypeptidase activity, as well as on the putative PBPs of the bacterium, not identified by treating the proteins of the cytoplasmic membrane with radiolabelled b-lactam antibiotic, are required. Literature A t r i h Z., G. B a c h e r, G. A l l m e i e r, M.P., W i l l i a m s o n and S.J. F o s t e r. 1999. Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation. J. Bacteriol. 181: 3956–3966. G h u y s e n J.-M. 1991. Serine b-lactamases and penicillin-binding proteins. Annu. Rev. Microbiol. 45:37–67. G h u y s e n J.-M. 1997. Penicillin-binding proteins. Wall peptidoglycan assembly and resistance to penicillin: facts, doubts and hopes. Int. J. Antimicrob. Agents 8: 45–60. G l a u n e r B., J.V. H o l t j e and U. S c h w a r z. 1988. The composition of the murein of Escherichia coli. J. Biol. Chem. 263: 10088–10095. G l a u n e r B. 1988. Separation and quantification of muropeptides with high-performance liquid chromatography. Anal. Biochem. 172: 451–464. G o f f i n C. and J.-M. G h u y s e n. 1998. Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol. Mol. Biol. Rev. 62: 1079–1093. G u t k i n d G.O., M.E. M o l l e r a c h and R.A. d e T o r r e s. 1989. Penicillin binding proteins in Listeria monocytogenes. APMIS. 97: 1013–1017. H ö l t j e J.-V. 1996. A hypothetical holoenzyme involved in the replication of the murein sacullus of Escherichia coli. Microbiology 142: 1911–1918 J a m i n M., J.M. W i l k i n and J.M. F r è r e. 1995. Bacterial DD-transpeptidases and penicillin. Essays Biochem. 29: 1–24. K ³ o s z e w s k a M. 2003. Doctoral thesis. Faculty of Biology. Warsaw University K o r s a k D., J.J. Z a w a d z k a, M.E. S i w i ñ s k a and Z. M a r k i e w i c z. 2005. Penicillin-binding proteins of Listeria monocytogenes – a re-evaluation. Acta Microbiol. Pol. 51: 5–12 K o r s a k D., W. Vo l l m e r and Z. M a r k i e w i c z. 2005. Listeria monocytogenes EGD lacking penicillin-binding protein 5 (PBP5) produces a thicker cell wall. FEMS Microbiol. Lett. S c h l e i f e r K.H. and O. K a n d l e r. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implication. Bacteriol. Rev. 36: 407–477. U r s i n u s A., H. S t e i n h a u s and J.V. H o l t j e. 1992. Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography. J. Bacteriol. 174: 441–446. v a n H e i j e n o o r t J. 2001. Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology 3:25R–36R. V i c e n t e M.F., J. B e r e n g u e r, M.A. d e P e d r o, J.C. P é r e z - D i a z and F. B a q u e r o. 1990. Penicillin binding proteins in Listeria monocytogenes. Acta Microbiol. Hung. 37: 227–231. V i c e n t e M.F., J.C. P é r e z - D i a z, F. B a q u e r o, M.A. d e P e d r o and J. B e r e n g u e r. 1990a. Penicillin-binding protein 3 of Listeria monocytogenes as the primary lethal target for $-lactams. Antimicrob. Agents Chemother. 34: 539–542.

Polish Journal of Microbiology formerly Acta Microbiologica Polonica

2005, Vol. 54, No 4

Instructions to authors I. General information Polish Journal of Microbiology publishes descriptions of all aspects of basic and applied research that focuses on topics of basic research of practical value in microbiology. Topics that are considered include microbiology of a genetic and molecular nature, foods, agriculture, industry, biotechnology, microbial ecology, public health and basic biological properties of bacteria, viruses, and simple eukaryotic microorganisms. Submit manuscripts directly to the Editorial Office, Polish Journal of Microbiology. The manuscript should be accompanied by a covering letter stating the address, fax number, e-mail of the corresponding author and “running head” of the manuscript (no longer than 47 characters). Submit two complete copies of each manuscript, including figures and tables. The manuscript should be either the original typescript from jet or laser printer (not dot matrix). Accepted papers are copy-edited as word-processor files, so authors are asked to provide their paper in this form on a disk when they submit the revised version. The text should be edited in Word 7 or higher or ASCII. Submission of figures in TIF or CorelDraw Format is appreciated. All manuscripts are subjected to peer review by the editors, by members of the editorial board and by qualified outside reviewers. When a manuscript is returned to the authors for modification, it should be returned to the editor within 2 months; otherwise it may be considered withdrawn. 15 reprints are sent free to the first author. II. Preparation of the manuscript The oryginal paper should be divided into the following sections written in sequence: Abstract, Introduction, Experimental: Materials and Methods and Results, Discussion, Acknowledgments, Literature. Type every portion of the manuscript double spaced with left hand margins, including figure legends, tables, table footnotes, and literature cited (type Literature sections on separate pages), and number all pages in sequence, including the abstract, tables and figure legends. The literature section must include all cited work. Arrange the citations in alphabetical order by first authors. Key words (no more than five) and the suggestion of runing head should be included. A paper in the form of a short communication must have an abstract of no more than 100 words. Do not use section headings in the body of the “Communication”; report introduction, methods, results, and discussion in a single section. The text should be concise, and the number of figures and tables should be kept to a minimum. Material and methods should be described in the text, not in figure legends or table footnotes. Present acknowledgments as in full-length papers. Minireviews are published in areas of particular interest and importance. They are usually invited, but authors wishing to submit a minireview should contact the scientific editor for further information. Before writing a manuscript authors are advised to consult a current issue of Polish Journal of Microbiology and carefully read the detailed “Instruction to authors” printed in number 1 of every volume in order to be familiar with the literature citations, preparation of figures and tables and the rules concerning chemical, biochemical, genetic etc. nomenclature recommended.

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