Polish Journal of Microbiology

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)

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Polish Journal of Microbiology formerly Acta Microbiologica Polonica

2005, Vol. 54, No 3

CONTENTS ORIGINAL PAPERS

Genetic diversity among Lactococcus sp. and Leuconostoc sp. strains using PCR-RFLP of insertion sequences ISS1-type, IS904, IS982

WALCZAK P., KONOPACKA M., OTLEWSKA A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

Lysostaphin as a potential therapeutic agent for staphylococcal biofilm eradication

WALENCKA E., SADOWSKA B., RÓ¯ALSKA S., HRYNIEWICZ W., RÓ¯ALSKA B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

gyrA mutations in ciprofloxacin-resistant clinical isolates of Pseudomonas aeruginosa in a Silesian hospital – in Poland WYDMUCH Z., SKOWRONEK – CIO£EK O., CHOLEWA K., MAZUREK U., PACHA J., KÊPA M., IDZIK B., WOJTYCZKA R.D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

The prognostic and diagnostic markers of invasive candidiasis in patients during chemotherapy

MAGRYŒ A., KOZIO£ – MONTEWKA M., STAROS£AWSKA E., GABCZYÑSKA B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

Seroepidemiological studies of Chlamydia pneumoniae infections in 1–36 months old children with respiratory track infections and other diseases in Poland

PODSIAD£Y E., FR¥CKA B., SZMIGIELSKA A., TYLEWSKA-WIERZBANOWSKA S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Occurence of serum class immunoglobulins interacting with specific antigens of Helicobacter pylori in patients with unstable coronary artery disease and in symptomless individuals

RECHCIÑSKI T., GRÊBOWSKA A., KURPESA M., RUDNICKA W., KRZEMIÑSKA – PAKU£A M., CHMIELA M. . . . . . . 221

Enzyme production and biotypes of vaginal Candida albicans

CYBULSKI Z., KRZEMIÑSKA – JAŒKOWIAK E., MAJEWSKI P., CHYLAK J., PAWLIK M. . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Purification and characterization of two extracellular lipases from Pseudomonas aeruginosa Ps-x

SAEED H.M., ZAGHLOUL T.I., KHALIL A.I., ABDELBAETH M.T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Extraction of milk-clotting enzyme produced by solid state fermentation of Aspergillus oryzae

SHATA H.M.A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

Cloning and preliminary characterization of a GATC-specific $2-class DNA:m 6A methyltransferase encoded by transposon Tn 1549 from Enterococcus spp.

RADLIÑSKA M., PIEKAROWICZ A., GALIMAND M., BUJNICKI J.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Co-occurence of urogenital mycoplasmas and group B streptococci with chlamydial cervicitis

FRIEDEK D., EKIEL A., ROMANIK M., CHELMICKI Z., WIECHU£A B., WILK I., JÓ¯WIAK J., MARTIROSIAN G. . . . . 253

INSTRUCTIONS TO AUTHORS

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Polish Journal of Microbiology 2005, Vol. 54, No 3, 183–189

Genetic Diversity among Lactococcus sp. and Leuconostoc sp. Strains Using PCR-RFLP of Insertion Sequences ISS1-type, IS904, and IS982 PIOTR WALCZAK*, MIROS£AWA KONOPACKA and ANNA OTLEWSKA

Institute of Fermentation Technology and Microbiology, Technical University of £ódŸ, £ódŸ, Poland Received 3 February 2005, received in revised form 23 June 2005, accepted 24 June 2005 Abstract PCR-RFLP analysis of commonly occurring insertion sequences ISS1-type, IS904 and IS982 in Lactococcus sp. and Leuconostoc sp. was used for the genetic differentiation of 17 strains of lactic acid bacteria. ISS1-type and IS982 were found in all analysed strains while IS904 was present exclusively in strains belonging to Lactococcus sp. Amplification of ISS1-type IS sequences resulted in formation of about 820 bp long amplicons, except of strains Lactococcus lactis ssp. lactis E and Leuconostoc lactis R where extra DNA bands about 370 bp long were observed. Similarly for strains of Leuconostoc lactis M and N, additional DNA bands about 280 bp long were present. TaqI digestion of ISS1-type amplicons revealed that all analysed sequences belonged to the restriction type (ii) or (iii) for which major restriction products were 543 and 147 bp long. Amplification of IS904 from all strains of Lactococcus sp. generated amplicons about 1260 bp long. In three strains of Leuconostoc sp. M, N and R, shorter amplicons about 880 bp were observed whereas strains O and P did not contained IS904. Amplification of IS982 resulted in formation of amplicons about 1000 bp long and no extra bands were observed for all tested strains. TaqI digestion of amplification products showed that for strains C, I and F, G, H, belonging to Lactococcus sp. smaller DNA bands were visible suggesting that they contain two different types of IS982. K e y w o r d s: PCR-RFLP, insertion sequences, Lactococcus sp., Leuconostoc sp.

Introduction Published data for genomes of Lactic Acid Bacteria (LAB) shows that insertion sequences are very common movable genetic elements present in the chromosome of bacteria as well as in plasmids. Common situation in LAB is that single strain carries from 3 to 8 different IS sequences, and each one can be present even in several copies (Klaenhammer et al., 2003). They contribute to genetic diversity of Lactic Acid Bacteria due to the conservative and replicative transpositions within the chromosome, causing negative or positive changes of bacterial phenotype. Bongers et al. (2003) observed that transposition of IS981, recovered activity of lactate production in a ldhB deficient strain of Lactococcus lactis by activation of transcription of that gene. Transposition of ISS1 into chromosome of Lactococcus lactis resulted in mutation manifested with increased sensitivity of mutants to UV light (Duwat et al., 1997). Presence of at least two identical IS sequences in bacterial chromosome can lead to large inversions of chromosomal DNA caused by homologous recombination between them (Daveran-Mingot et al., 1998). Activation or silencing of genes by spontaneous transposition of insertion sequences within bacterial chromosome may serve as a mechanism of strain adaptation to environmental changes (Duval-Valentin et al., 2001). The presence of IS sequences can also contribute to the genetic stability of particular strain since it may generate spontaneous mutations in growing bacterial population. Stability of biochemical profiles of LAB, especially lactose fermentation and protease activity, is very important in dairy industry where bacterial starters are used for the preparation of dairy products. IS mediated transposition leading to the induction of prophage excission * Corresponding author. Mailing address: Institute of Fermentation Technology and Microbiology, Technical University of £ódŸ, Wólczañska 171/173, 90-530 £ódŸ, Poland. E-mail address: [email protected]

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can be even more dangerous since it can stop fermentation process. ISS1-type insertion sequences belonging to IS6 family such as IS946, ISS1, iso-ISS1, IS1216, are usually associated with plasmids replicating via theta mechanism and therefore they are widely distributed among different bacterial genera, due to the plasmid exchange caused by conjugal transfer or plasmid uptake by competent cells (Romero and Klaenhammer, 1990; Schaefer et al., 1991). Insertion sequences of ISS1-type can be found in large plasmids of Bacillus thuringiensis (pBtoxis) (Berry et al., 2002), Listeria inocua (pLI100) (Glaser et al., 2001), as well as in Lactococcus lactis (pMRC01, pK214, pIL105) (Dougherty et al., 1998, Teuber et al., 1999, Anba et al., 1995). Plasmids containing ISS1-type insertion sequences can transpose to bacterial chromosome and form cointegrates. The resolution of cointegrate leads to the excision of plasmid containing ISS1 and the second copy of IS is left in the chromosome. This feature was used for the construction of vectors for insertional mutagenesis of pGh9:ISS1 type (Maguin et al., 1996). The second very common insertion sequence usually present in the chromosome or plasmid DNA of LAB is IS904 belonging to the IS3 family. Its iso- forms are IS1076 (L, R) (Huang et al., 1990), IS1069 (Rauch et al., 1994) iso-IS904 (Rauch, 1990). In the chromosome locations of Lactococcus lactis IL1403, this sequence accompanies another one IS1077 (Bolotin et al., 2001). IS904 can also be found in lactococcal plasmids such as pK214, pNZ4000 (Teuber et al., 1999; van Kranenburg and de Vos 1998; van Kranenburg et al., 1999; 2000) as well as in nisin sucrose conjugative lactococcal transposons Tn5276 and Tn5301 (Rauch and De Vos, 1992; Rauch et al., 1994; Horn et al., 1991). Transposons Tn5276 and Tn5301 belonging to the large conjugative transposons of LAB, which contains IS904 and genes responsible for nisin A or Z biosynthesis, sucrose metabolism, biosynthesis of N5-(L-1-carboxyethyl)-L-ornithine (gene ceo) as well as genes coding excision/integration system (Xis/Int) of the transposon (Dodd et al., 1982; 1990). Chromosome of the Lactococcus lactis IL1403 contains only one copy of IS982 which transposase is inactive due to the mutation prematurely terminating transcription of this gene (Bolotin et al., 2001). IS982 sequence belonging to the separate family often resides on plasmid DNA such as pNZ4000 and pCI658 and is associated with eps gene cluster responsible for production of extracellular polysaccharides (van Kranenburg et al., 1997; Forde and Fitzgerald, 2003). However pCI658 contains iso-IS982 which is about 50 bp shorter than the normal sequence and most likely its transposase is also inactive. Lactococcal citrate plasmid such as pKR223 from strain of Lactococcus lactis ssp.lactis var diacetylactis KR2 also contained IS982 sequence closely linked with restriction modification gene cluster (Twomey et al., 1998; 2000). Also citrate utilization genes found in other strain of Lactococcus lactis ssp. lactis var. diacetylactis CRL264 were located closely to the IS982 sequence (Lopez de Felipe et al., 1995). Knowledge about presence of particular insertion sequences and their stability in the chromosome of LAB is therefore of vital interest from the point of view of industrial application of certain strains. Distribution of IS sequences in strains of LAB may also contribute to better characterization of them and elimination of duplicates from strain collections. Experimental Materials and Methods Bacterial strains used in this study. Bacterial strains used in this study were originated from the collection of the Institute of Fermentation Technology and Microbiology, Technical University of Lodz or were isolated from kefir grains of polish origin (Table I). DNA preparation. Plasmid and chromosomal DNA was isolated according to the method described by Anderson and McKay (1983). PCR amplification of IS sequences. Primer sequences for amplification of ISS1-type, IS904 and IS982 were derived from data records published in NCBI Database (Table II). Underlined parts of oligonucleotides were identical to the beginning fragments of inverted repeats of corresponding IS sequences. In case where IRL and IRR sequences of IS were identical, only one primer was sufficient for the PCR procedure. Primer fragments located upstream of IR, marked in boldface, containing restriction sites for BamHI or EcoRI, were introduced additionally to facilitate possible cloning of PCR products. Amplification of ISS1 type IS sequences was performed in the following manner. About 20 ng of DNA template, 40 pmol of primer ISS1FR, 12.5 :l Red-Taq ReadyMix DNA polymerase (Sigma-Aldrich) were mixed together and supplemented with PCR grade water to a total volume of 25 :l. The amplification procedure consisted of one cycle of 2 min at 94°C, followed by 34 cycles for 1 min at 94°C, 1 min at 45°C and 3 min at 72°C with final extension cycle for 2 min at 72°C was performed using Uno II thermocycler, Biometra, with tube lid heating block set for 105°C. No overlay oil was added to the tubes. The reaction mix for amplification of IS904 was the same except of primers replaced by IS904F and IS904R in the concentration of 20 pmol each. Similarly for amplification of IS982, 40 pmol of primer IS982FR was used. The amplification procedure for IS904 and IS982 consisted of one cycle of 3 min at 94oC, followed by 29 cycles for 1 min at 94°C, 1 min at 40°C and 3min at 70°C with final extension cycle for 3 min at 70°C. Agarose gel analysis. PCR products of IS amplification were analysed on 1% [w/v] agarose gel in TBE buffer. Amplified PCR products of IS sequences in amount of 10 :l were digested by TaqI (MBI Fermentas) for 1 h at 65°C according to the product

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Table I Bacterial strains and their properties Strain symbol

Strain name

Description / genotype

A

Lactococcus lactis ssp. lactis var. diacetylactis

Industrial strain / lac+, cit+

B

Lactococcus lactis ssp. lactis var. diacetylactis

Industrial strain / lac+, cit+

C

Lactococcus lactis ssp. lactis

Industrial strain / lac+, cit–

D

Lactococcus lactis ssp. lactis

Industrial strain / lac+, cit–

E

Lactococcus lactis ssp. lactis

Industrial strain / lac+, cit–

F

Lactococcus lactis ssp. lactis

Derivative of ATCC 11454

G

Lactococcus lactis ssp. lactis

Kefir isolate / lac+, cit–

H

Lactococcus lactis ssp. lactis

Kefir isolate / lac+, cit–

I

Lactococcus lactis ssp. lactis

Kefir isolate / lac+, cit–

J

Lactococcus lactis ssp. lactis

Kefir isolate lac+, cit–

K

Lactococcus lactis ssp. lactis

Kefir isolate / lac+

L

Lactococcus lactis ssp.lactis

Kefir isolate / lac+

M

Leuconostoc lactis

Industrial strain / lac+, cit+

N

Leuconostoc lactis

Industrial strain / lac+, cit+

O

Leuconostoc mesenteroides

Kefir isolate / lac+

P

Leuconostoc mesenteroides

Kefir isolate / lac+

R

Leuconostoc lactis

Kefir isolate / lac+, cit+

Note. lac (+/–) – lactose fermentation, cit (+/–) citrate fermentation

Table II Primers used for PCR amplification of IS sequences IS sequence

Name

Primer

Sequence source

ISS1-type

ISS1FR

5’-GCGGATCCGGTTCTGTTGCAAAGTTT-3’

X62737, AF116286, AF036485

IS904

IS904F IS904R

5’-GCGGATCCTGGAAAGGTTATAATAAA-3’ 5’-CGAATTCTGGAAAGTCAACGAAAAA-3’

M27276, X52273, X78469, X92946

IS982

IS982FR

5’-GCGGATCCAWACCCGAATTGCTAGTT-3’

L34754, AF036485

instruction. Restriction fragments were analysed on 2% [w/v] agarose gel in TBE buffer. Gells placed on UV transiluminator were photographed with digital camera through yellow filter and obtained pictures were electronically inverted for better visualisation of separated DNA bands.

Results

Insertion sequences ISS1, iso-ISS1 and IS946 are very common in Lactic Acid Bacteria as well as in other gram positive organisms including Listeria, Staphylococcus, and Bacillus sp. Computer analysis of published sequences of ISS1 type, revealed their genetic diversity in respect to size (807–809 bp), nucleotide sequence of inverted repeats IR and transposase gene tnpA as well as presence of restriction sites for different enzymes. Insertion sequences ISS1 and IS946 contain 1, 3 or 4 restriction sites for TaqI and this enzyme generates fragments with the following length: (i) – 661 and 147 bp; (ii) – 543, 147, 72 and 48 bp; (iii) – 543, 147, 48, 42 and 30 bp. Restriction enzyme EcoRV may cut some of ISS1 type molecules in one site generating fragments of 377 and 431 bp whereas other types are not cut. Therefore, RFLP analysis of ISS1 type insertion sequences digested with one of these enzymes may be a simple method of their differentiation. Figure 1A shows results of agarose gel electrophoresis of PCR amplified ISS1-type insertion sequences from all tested strains of Lactococcus sp. and Leuconostoc sp. In one strain of Lactococcus lactis ssp. lactis (E) and in three strains of Leuconostoc sp. (M, N, R), normal size of ISS1-type amplicons as well as different shortened amplicons were observed. Size comparison of shortened amplicons revealed that strain Lactococcus lactis ssp. lactis (E) and Leuconostoc lactis (R) possessed extra amplicon of the same size (about 370 bp), whereas strains of Leuconostoc lactis (M) and (N) contained shorter fragments of the

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Fig. 1. A – Agarose gel electrophoresis of amplification products with primer ISS1FR specific for ISS1 type IS sequences. B – Agarose gel electrophoresis of TaqI digested amplification products of ISS1-type IS sequences. A, B, C, D, E, F, G, H, I, J, K, L – strains of Lactococcus sp., M, N, O, P, R – strains of Leuconostoc sp.

same length being about 280 bp. Size of short amplicons were calculated form the separate agarose gel electrophoresis experiment carried out in the presence of molecular size marker (BTL cat. No G004, pUC19 digested with RsaI, HinfI and PvuII). Presence of such shorter than normal ISS1-type sequence in a particular strain may be a unique marker differentiating it from other strains. However the nature and possible function of shorten amplicons is not known. They can represent truncated forms of ISS1 type sequences transpositionaly not functional due to the deletion of tnpA gene. This view strongly supports finding from the DNA sequence (Accession Number Z98171) published by Bourgoin (2002) for exopolysaccharide synthesis genes and insertion sequences of Streptococcus thermophilus. In the variable region of exopolysaccharide synthesis genes, three ISS1-type sequences (ISS1SA, ISS1SB and ISS1SC) were found of which ISS1SB was truncated form containing 62 first nucleotides and 270 last nucleotides of normal type ISS1 sequence. This truncated form being 332 bp long was named delta-ISS1SB. Figure 1B shows results of electrophoretic analysis of TaqI digested amplification products. RFLP analysis revealed presence of two major DNA bands with size of 550 and 150 bp which is very close to the expected values of 543 and 147 bp characteristic to the (ii) or (iii) restriction pattern. Fragments smaller than 100 bp were not visible. All analyzed IS sequences belonged to the restriction type (ii) or (iii) according to the proposed classification, for which the largest fragment had 543 bp. DNA bands with size between 543 and 147 bp visible in lanes B, C, E, I, J, L, M, N and R were most likely originated from digestion of “upper” amplicons visible above ISS1 bands or from shorten amplicons. It is also possible that those bands, are results of presence of two kinds of IS S1-type IS sequences in the same strain. Such situation was demonstrated by Bourgoin et al. (1996) for strain of Streptococcus thermophilus. Insertion sequence IS904 formerly named as IS1069 or IS1076 is very common in the chromosome of many lactococcal strains (7 copies in Lactococcus lactis IL1403), (Bolotin et al., 2002). Figure 2A shows results of agarose gel electrophoresis of PCR amplified IS904 insertion sequences from all tested strains. IS904 was present in all strains belonging to the genus Lactococcus and the size of obtained PCR amplicons were the same, being about 1260 bp. In two strains of Leuconostoc sp. (O, P) IS904 was not present and in the remaining ones (M, N, R), amplified sequence was shorter than that from strains of Lactococcus sp. Figure 2B shows results of electrophoretic analysis of TaqI digested amplification products. RFLP analysis of TaqI digestion products from strains of Lactococcus sp. showed their size of about 736 and 477 bp. Computer modeled restriction analysis of 5 published IS904 sequences or its iso- forms digested with TaqI,

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Fig. 2. A – Agarose gel electrophoresis of amplification products with primer IS904F and IS904R specific for IS904 sequences. B – Agarose gel electrophoresis of TaqI digested amplification products of IS904 sequences. A, B, C, D, E, F, G, H, I, J, K, L – strains of Lactococcus sp., M, N, O, P, R – strains of Leuconostoc sp.

revealed presence of two recognition sites for that enzyme located at positions 75 and 797 of 1245 bp long entire IS sequence. Taking into account extra nucleotides present in primers IS904F and IS904R, TaqI restriction fragments of PCR amplified sequences should have the following length 722, 456 and 82 bp. However the smallest fragment can be hardly visible when digestion products are analysed on 2% agarose gel. TaqI digestion of PCR products from strains of Leuconostoc sp. (M, N and R) were shorter than for original IS904 sequence (about 550 and 330 bp). This results indicate that analysed strains of Leuconostoc sp. possessed most probably a different type of an insertion sequence with front parts of inverted repeats having the same oligonucleotide sequence. The length of the new sequence was estimated to be about 300– 400 bp shorter than IS904. Question whether it is a new sequence or only deletion form of IS904 remains still opened. The lack of IS904 in strains of Leuconostoc sp. or the presence of its shorter form, may be diagnostic feature used for differentiation of Leuconostoc sp. and Lactococcus sp. associated with milk products. Insertion sequences IS982 has been found in both already sequenced chromosomes of Lactococcus lactis strains IL1403 and MG 1363 (Klaenhammer et al., 2002). Also Lactococcus lactis ssp. cremoris SK11 contains this sequence (Yu et al., 1995). Nothing is known about presence of IS982 sequence in strains of

Fig. 3. A – Agarose gel electrophoresis of amplification products with primer IS982FR specific for IS982 sequences. B – Agarose gel electrophoresis of TaqI digested amplification products of IS982 IS sequences. A, B, C, D, E, F, G, H, I, J, K, L – strains of Lactococcus sp., M, N, O, P, R – strains of Leuconostoc sp.

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Leuconostoc sp. The typical length of this sequence varies from about 950 to 1011bp. Published sequences contain a single restriction site for EcoRV and are not digested with TaqI enzyme. Figure 3A shows results of agarose gel electrophoresis of PCR amplified IS982 insertion sequences from all tested strains of Lactococcus sp. and Leuconostoc sp. This sequence was present in all strains and obtained amplicons were uniform with respect to their size. No extra bands were observed. Figure 3B shows results of electrophoretic analysis of TaqI digested amplification products. RFLP analysis showed in some strains of Lactococcus sp. (C, I) and (F, G, H,) that obtained amplicons were partially digested with TaqI enzyme, what may suggest the presence of two kinds of that sequence in one strain. Strains (C) and (I) had similar restriction pattern and strains (F), (G), (H) were characterized by the other type of restriction pattern. So far IS982 sequences published in the data bases do not contain TaqI recognition sites and therefore detected paralogs are still waiting for the recovery and sequencing. Discussion Detection of insertion sequences ISS1-type, IS904, and IS982 in the chromosome and plasmid DNA of LAB combined with RFLP analysis of TaqI digests of amplified sequences can be a valuable tool for analysis of genetic differences among closely related strains. Structural diversity of ISS1-type insertion sequences may be used for grouping strains according to their RFLP profile. The presence of certain type of ISS1 sequences as well as their degenerated shortened forms in particular strain is often associated with the specific plasmid which may function in strains of Lactococcus sp. (E) as well as in Leuconostoc sp. (R). Absence of IS904 or its presence in shorter form in strains of Leuconostoc sp. may be a method of strain differentiation and allows to distinguish them from strains of Lactococcus sp. RFLP analysis of TaqI digestion of IS982 amplification product revealed the existence in certain strains of Lactococcus lactis, the new type of such element which DNA is digested with this enzyme whereas, already known sequences, are not. It seems possible that such TaqI digested IS982 sequences are associated with the ability of strains to synthesize lantibiotic bacteriocin nisin. This view supports observation that strain of Lactococcus lactis ssp. lactis (H) having the same restriction pattern for TaqI digested IS982 as known nisin producer L. lactis ATCC 11454 (F) was also able to produce bacteriocin (data not published). However, the nature and structure of bacteriocin produced by strain Lactococcus lactis ssp. lactis (H) remains unknown. Also strain (G) represented the same TaqI restriction pattern of IS982 as (F) and (H) and therefore most probably it belongs to the same group. It seems possible that TaqI digested IS982 is therefore characteristic of nisin producing strains having nisin-sucrose transposons such as Tn5276 and Tn5301 carrying nisin biosynthesis operons. Similarity between strains (F), (G) and (H) and fact that last two of them were isolated from the kefir grains suggests that kefir microflora may produce nisin contributing to the probiotic properties of kefir. However these suggestions has to be confirmed experimentally.

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A transposon like element on the lactose plasmid of Lactococcus lactis subsp. lactis Z270. FEMS Microbiol. Lett. 61: 101–106. K l a e n h a m m e r T., E. A l t e r m a n, F. A r i g o n i, A. B o l o t i n, F. B r e i d t, J. B r o a d b e n t, R. C a n o, S. C h a i l l o u, J. D e u t s c h e r, M. G a s s o n, M. v a n d e G u c h t e, J. G u z z o, A. H a r t k e, T. H a w k i n s, P. H o l s, R. H u t k i n s, M. K l e e r e b e z e m, J. K o k, O. K u i p e r s, M. L u b b e r s, E. M a g u i n, L. M c K a y, D. M i l l s, A. N a u t a, R. O v e r b e e k, H. P e l, D. P r i d m o r e, M. S a i e r, W. d e Vo s, B. W e i m e r, M. Z a g o r e c and R. S i e z e n. 2002. Discovering lactic acid bacteria by genomics. Antonie van Leeuwenhoek 82: 29–58. L o p e z d e F e l i p e F., C. M a g n i, D. d e M e n d o z a and P. L o p e z. 1995. Citrate utilization gene cluster of the Lactococcus lactis biovar diacetylactis: organization and regulation of expression. Mol. Gen. 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Polish Journal of Microbiology 2005, Vol. 54, No 3, 191–200

Lysostaphin as a Potential Therapeutic Agent for Staphylococcal Biofilm Eradication EL¯BIETA WALENCKA1, BEATA SADOWSKA1, SYLWIA RÓ¯ALSKA2, WALERIA HRYNIEWICZ3 and BARBARA RÓ¯ALSKA 1* 1 Department of Immunology and Infectious Biology, of Industrial Microbiology and Biotechnology, Institute of Microbiology and Immunology, University of £ódŸ, Banacha 12/16, 90-237 £ódŸ, Poland, 3 National Institute of Public Health, Warsaw, Poland

2 Department

Received 24 February 2005, received in revised form 8 July 2005, accepted 11 July 2005 Abstract The aim was to study the activity of lysostaphin in monotherapy or in combination with oxacillin, towards biofilms built by clinical and reference S. aureus and S. epidermidis strains in the wells of microplate, in the chambers of a LabTekII chamber slide or on the polyethylene catheter. MICs of oxacillin and lysostaphin for planktonic bacteria were determined according to the standards of NCCLS. BIC (Biofilm Inhibitory Concentration) was estimated by the MTT assay. The integrity of biofilm treated with antimicrobials was also examined: by staining with FITC and laser scanning fluorescence confocal microscopy and visually by TTC reduction assay. Despite the fact that susceptibility of planktonic cultures of 25 staphylococcal strains to lysostaphin action was various, we have demonstrated the effectiveness of lysostaphin in the treatment of biofilm, built not only on the flat surface of the microplates but also on catheter’s surface. The synergistic effect of subBIC lysostaphin+oxacillin was observed for MSSA and MRSA biofilms but not for 1474/01 hVISA strain. Also BIC OXA for S. epidermidis RP12 and A4c strains, but not for 6756/99 MRSE biofilms was reduced when lysostaphin was simultaneously used. K e y w o r d s: staphylococcal biofilms, antibiotics, lysostaphin

Introduction Bacterial biofilms are described as polymer-dipped communities of cells which accumulate, in a precisely controlled manner, on the abiotic or biotic surfaces (Lewis, 2001; Fux et al., 2003; Boles et al., 2004). Some naturally existing biofilms have a protective role for the host tissue homeostasis (biofilms on urogenital epithelium, intestine epithelium, dental plaques), by preventing their colonization by exogenous pathogens (Prakash et al., 2003; Boles et al., 2004; Fux et al., 2003). However, it should be stressed that bacterial and fungal biofilms are also responsible for a number of diseases, such as native valve endocarditis, cystic fibrosis-associated pneumonia, middle ear infections, bone infections, bacterial prostatitis, periodontitis (Hall-Stoodley et al., 2004; Götz, 2002; Fux et al., 2003). Biofilms are also involved in the pathogenesis of various infections related to implanted medical devices (urinary and vascular catheters, prosthetic heart valves, prosthetic hip/knee, contact lenses etc.). Most of these infections have a chronic nature and, because of the intrinsic resistance of the biofilm cells to antibiotics and host defense sytems, such diseases are very difficult to treat effectively. Many hypotheses were considered to explain the high biofilm resistance to antimicrobial agents: restricted penetration, decrease in bacterial metabolism and growth rate, increase in antibiotic-degrading enzymes accumulation and enhancement of exchanging rate of genes encoding for resistance (Lewis, 2001; Hall-Stoodley et al., 2004; Fux et al., 2003). Currently, the most accepted view is that all the hypotheses are true, but none of them explains the resistance of various biofilms to killing by * Coressponding author: prof. Barbara Ró¿alska, Department of Immunology and Infectious Biology, Institute of Microbiology and Immunology, University of £ódŸ, Banacha 12/16, 90-237 £ódŸ, Poland, tel. 048-42-6354472, e-mail address: [email protected]

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different groups of antimicrobial agents. Nevertheless, additional and now dominating is molecular biology based explanation of antibiotic tolerance rather than resistance, induced within biofilm structure i.e. expression of stress response genes, phase variation, persister-state and biofilm-specific phenotype development (Hall-Stoodley et al., 2004). Many research groups investigated, alternative to antibiotics, potential strategies in preventing biofilm formation or its eradication. One possible approach is enzymatic removal of bacterial biofilms, but due to the heterogeneity of the extracellular matrix, in most cases a mixture of enzymes may be necessary for a sufficient degradation of biofilm structure. Nevertheless, this strategy is useful in the eradication of so called environmental biofilms, e.g. industrial pipelines (water, oil) or food processing equipment. Enzymatic biofilm disruption is a very attractive idea for the prevention or elimination of pathogenic biofilms causing various medical problems. Enzymatic activity can be directed to biofilm matrix, allowing better penetration of subsequent antimicrobials used or to be directed to pathogen’s cell wall components and causing their lysis (Johansen et al., 1997; Kaplan et al., 2004). Endopeptidases secreted by different bacterial species are examples of enzymes which can be used for this purpose. Two of them are now being thoroughly investigated, the LasA protease produced by Pseudomonas aeruginosa and lysostaphin secreted by Staphylococcus simulans, both specifically active towards staphylococcal strains (Barequet et al., 2004; Wu et al., 2003). Lysostaphin, a 27-kDa endopeptidase which degrades the pentaglycine bridges in peptidoglycan bone of the cell wall, was shown as a potent antistaphylococcal agent, however, it acts much more effectively against S. aureus than S. epidermidis strains due to differences in cell wall composition of these species. S. aureus and coagulase-negative staphylococci (CNS), mainly S. epidermidis, are known as the leading species in chronic polymer-associated infections of biofilm nature and resistant to antibiotic treatment. Therefore, we ask the question whether lysostaphin may be considered as an effective biofilm eradicating agent, since its strong activity against planktonic staphylococci is well documented (Climo et al., 1998, Kiri et al., 2002, von Eiff et al., 2003) and what advantages or limitations the use of lysostaphin alone or with antibiotics, creates. Experimental Materials and Methods Bacteria. The group of 25 staphylococcal strains chosen for investigation consisted of: clinical S. aureus (n = 10) and S. epidermidis (n = 9) isolates, S. aureus ATCC25923 and S. aureus ATCC29213 (the reference MSSA strains), S. aureus 1474/01 (clinical hVISA, NIPH), S. epidermidis ATCC12228 (the reference MSSE strain), S. epidermidis RP12 (slime producing clinical isolate, from the collection of A. Ljungh, Dept. Medical Microbiology, University of Lund, Sweden), S. epidermidis 6756/99 (clinical MRSE, NIPH). Most clinical strains were isolated from medical device-associated infections (Table I). The organisms were stored in TSB with 15% glycerol at –70°C, and in each experiment the cultures were established from the original stock. Antimicrobial agents and susceptibility testing against planktonic bacteria. The antibiotic oxacillin (disks 1 :g and tablets 0.1 mg) was purchased from Mast Diagnostics (United Kingdom). Recombinant lysostaphin (from S. simulans, No. L 0761) was obtained from Sigma, (St. Louis, USA). The susceptibility of staphylococcal strains to antimicrobial agents was determined by the standard NCCLS disk diffusion and microdilution methods (National Commitee for Clinical Laboratory Standards, M7-A5, 2000). The concentration range of oxacillin used in the study was 0.125– 128.0 :g mL–1 in CAMHB + 2% NaCl, whilst lysostaphin concentration range tested was 0.0625– 64.0 :g mL–1 in CAMH + 0.1% BSA (to prevent its nonspecific adherence to plastic surface). To specify the MICs, turbidometric (OD600) bacterial studies were carried out using the multifunction counter Victor2 (Wallac, Finland). MIC was estimated as the lowest concentration of antimicrobial agent which gave OD equal to the medium negative control (below 0.05). Biofilm formation. S. aureus or S. epidermidis from the stock cultures were grown for 24 h at 37°C on the agar plate. Next, a single colony of each strain was grown in 5 mL of TSB (Difco) supplemented or not with 0.25% D-(+)-glucose (TSBGlc). The overnight cultures were diluted 1:40 in TSBGlc. A final volume of 200 :L was added to each well of a 96-well tissue culture plate (Nunclon™ Surface, Nunc) or of 500 :L to each chamber of Lab Tek chamber slide II (Nalge-Nunc International, Napervile, Ill., USA). In order to allow bacteria to form biofilms the plates/chambers were incubated for 24 h at 37°C. Stains for biofilm visualization. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma), TTC (2,3,5-triphenyltetrazolium chloride, Sigma), FITC (Fluorescein isothiocyanate Isomer I, Sigma) were used. Biofilm MTT-staining. MTT assay was performed according to the method described by Kairo et al., (1999) with minor modifications. Briefly, after 24 h lasting biofilm formation the wells of microplate were emptied and filled with 150 :L of PBS per well, then 50 :L of MTT solution (0,3% in PBS) was added and plates were incubated for 2 h at 37°C. At the end of incubation period, MTT was replaced with 150 :L of DMSO and 25 :L of glycine buffer (0.1 M, pH 10.2). For complete dissolving of formed purple formazan crystals the plates were incubated for 15 min. at room temperature, with gentle agitation. The optical density of the wells containing biofilms was determined using a spectrophotometer (550 nm, Victor2 multifunction counter, Wallac, Finland). Biofilm FITC-staining. Biofilms formed on the slides surface of Lab Tek chamber slide II device were stained with FITC solution (0,1% in PBS) for 20 min. at room temperature. After staining chambers were emptied and very gently washed, once with

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Staphylococcal biofilm eradication Table I List of staphylococcal strains used in these studies Strain

Origin / characteristics

S. aureus

Strain

Origin / characteristics

S. epidermidis

ATCC25923

reference MSSA, ATCC*

ATCC12228

reference MSSE, ATCC

ATCC29213

reference MSSA, ATCC

RP12

slime positive, University of Lund, Sweden

1474/01

clinical hVISA, NIPH**

6756/99

clinical MRSE, NIPH

A3

drain

A4a

hip prosthesis

A7

hip prosthesis associated abscess

A4b

hip prosthesis

B1

orthopedic wound

A4c

hip prosthesis

C1

tracheostomic tube

C10

blood

D5

tracheostomic tube

C11

blood

D8

venous catheter’s tip

C12

blood

D13

drain

C13

blood

E1

tracheostomic tube

C15

blood

E4

tracheostomic tube

C27

blood

E7

tracheostomic tube

* – American Type Culture Collection, ** – National Institute of Public Health, Poland

PBS supplemented with 4% BSA and twice with PBS. After the washing, the chamber slides partitions were removed and the slides were covered with cover glasses. The effects of biofilm FITC-staining were observed using the laser scanning confocal microscope (LSCM, Zeiss). Biofilm bacterial cultures for antimicrobial test. Biofilms were prepared in 96-well microplates or in chamber slides device as described above. After 24 h of growth, the medium from the wells/chambers was removed by aspiration. The biofilms were treated either with various concentrations of oxacillin or lysostaphin, or with a combination of oxacillin and subMIC concentration of lysostaphin. Antimicrobial agents were diluted in CAMHB supplemented with 2% NaCl and 0,1% BSA. The incubation time was 24 h at 37°C, then the medium containing the antimicrobial agent was gently aspirated and the viability of the biofilm remaining on the surfaces of the wells or chamber slides was stained with MTT or FITC as described above. Confocal microscopy study. Examination of FITC-stained biofilms treated with antimicrobial agents was performed using a LSM5 (Pascal) Laser Scanning Confocal Microscope (LSCM), equipped with Axiovert 2 (Zeiss) microscope with objective Plan-Apochromat 100x (1.4 oil). Images were recorded at a 488 nm (argon laser) excitation and emission at 530 nm (long pass filter set). Digital image analysis of LSCM optical thin sections was performed with Pascal Zeiss software. “Catheter study”. Intravascular catheter’s (Venflon, PTFE, Becton Dickinson, USA) segments (1 cm length) were prepared using sterile instruments, placed into the eppendorf tubes (in triplicate) containing 1 mL of bacterial suspension (overnight culture of S. aureus A3- MRSA, diluted 1:40 in TSBGlc), and incubated for 72 h at 37°C. Then catheter’s segments were rinsed with PBS and transferred to the new tubes containing: i) medium, ii) oxacillin, iii) lysostaphin, or iiii) oxacillin and subMIC lysostaphin for 3 or 24 h incubation at 37°C. Next, biomaterial segments, gently rinsed with PBS were moved to the fresh TSBGlc medium with TTC (one drop of 1% TTC in PBS) and incubated for 24 h at 37°C. The presence of red stained bacterial biofilm (reduction by live bacteria of colourless TTC to the red insoluble formazan crystals) was estimated as described previously (Sadowska et al., 1998). Data analysis and presentation. Each assay was performed in duplicate or triplicate on at least two occasions. Data were expressed as the mean from 2–3 evaluations obtained from each experiment.

Results Most S. aureus and S. epidermidis strains used in this study were clinical isolates, mainly from patients suffering from medical-devices-associated infections, as presented in Table I. These strains, after their initial identification, were obtained from hospital microbiological laboratories. In our hands, strains were reidentified using selected typing methods (hemolysis, clumping factor and tube coagulase tests) and 32ID Staph (BioMerieux) system. Using the standard antimicrobial susceptibility tests, according to the 2000’ guidelines of NCCLS, the MICs of oxacillin and lysostaphin were determined. The obtained MICs values of each strain cell suspension tested on two occasions were similar (differences not exceeding one dilution), therefore single values are presented (Table II). For the suspension cultures of S. aureus and S. epidermidis strains, oxacillin MICs ranged from 0.250 to > 128 :g mL–1. However, most S. aureus clinical strains (7/10) were highly resistant, with MICOXA > 128 :g mL–1, whereas most S. epidermidis strains (10/12) had MICs range from 1.0 to

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Table II Susceptibility to oxacillin and lysostaphin of S. aureus and S. epidermidis strains. MIC for planktonic culture determined by disk-diffusion and broth microdilution methods Oxacillin Strain

Inhibition (∅mm)

MICOX :g mL –1

Lysostaphin

Oxacillin Strain

MICLYS :g mL –1

S. aureus

Inhibition (∅mm)

MICOX :g mL –1

Lysostaphin MICLYS :g mL –1

S. epidermidis

ATCC 25923 (MSSA)

16

0.250

0.125

ATCC 12228

22

0.125

ATCC 29213 (MSSA)

14

0.250

0.250

RP12

16

1.0

4.0 8.0

1474/01 (hVISA)

0

>128.0

0.0625

6756/99 (MRSE)

6

16.0

32.0

A3

0

>128.0

0.250

A4a

17

2.0

1.0

A7

0

>128.0

0.500

A4b

17

2.0

2.0

B1

14

2.0

0.250

A4c

17

1.0

1.0

C1

16

1.0

0.250

C10

0

16.0

8.0

D5

0

>128.0

0.250

C11

10

2.0

2.0

D8

15

1.0

0.250

C12

14

2.0

D13

13

2.0

0.250

C13

14

2.0

16.0

E1

0

>128.0

0.250

C15

0

128.0

2.0

E4

0

>128.0

C27

0

128.0

8.0

E7

6

>128.0

32.0

0.500

0.500

16 :g mL–1. These 25 staphylococcal strains underwent tests of susceptibility to lysostaphin. Within S. aureus group, lysostaphin MICs ranged from 0.0625 to 0.500 :g mL–1, with median MIC = 2.7 :g mL–1 for more than 92% of strains. One exception was S. aureus E4 strain whose MICLYS was 32 :g mL–1. The median MIC for 12 S. epidermidis strains was much higher – 7.0 :g mL–1, and for 91% of strains the range started from 0.5 and ended at 16 :g mL–1. Generally, for most staphylococcal strains there was no correlation between MIC OXA and MIC LYS values. However, S. aureus E4 which presented the highest MICLYS = 32.0 :g mL–1 was also highly resistant to oxacillin (MICOXA > 128 :g mL–1. A similar result was demonstrated for 6756/99 MRSE strain (MICLYS = 32.0 :g mL–1, MICOXA > 16.0 :g mL–1). On the other hand, 1474/01 hVISA strain, whose MICOXA exceeded 128 :g mL–1 had the lowest MICLYS among all 25 tested strains, which was 0.0625 :g mL–1. The preparation of S. aureus and S. epidermidis biofilms, on hydrophobic polystyrene (96-well microplate) or negatively charged glass (Lab Tek chamber slide II) surfaces, produced high bacterial yield. After MTT staining, optical density (OD550) readings ranged from 1.8 to 3.1 (Fig. 1), therefore the isolates were categorized as strong biofilm producers. The degree of biofilm formation was commonly higher when bacteria were initially and finally grown in TSBGlc than in TSB without glucose, however, for some strains (mainly among S. aureus group) the opposite effect was observed (data not shown). Cells growing as biofilms (in chamber slide devices) stained with FITC, were characterized without the disturbance of their structure using laser fluorescence scanning microscope. LSCM images showed multilayered clumps of bacteria, surrounded by less dense material, attached to the surface (Fig. 3-A, B). S aureus strains produced biofilms which were, on average, 2.5 times thinner than those formed by S. epidermidis. Biofilm of S. aureus ATCC29213, 24 h after set-up, reached 2.66 ± 0.56 :m of thickness, whereas that of S. epidermidis RP12 was 6.35 ± 0.86 :m. When cultures incubation time was prolonged, further growth of biofilms was observed and at the end (72 h) biofilm of S. aureus ATCC29213 and S. epidermidis RP12 reached the thickness of 4.09 ± 0.78 :m and 8.02 ± 0.61 :m, respectively. In order to test the hypothesis that lysostaphin and beta-lactam antibiotics act as synergistic agents toward staphylococci, the effect of the oxacillin or lysostaphin (alone or in combination) on cell viability in biofilms was examined. BICs (Biofilm Inhibitory Concentration) of antimicrobials were determined by MTT reduction assay measuring the active metabolism of bacteria that survived the antimicrobials action. Biofilms of all 13 S. aureus, as well as 12 S. epidermidis strains were highly resistant to oxacillin (BICs> 128– 256 :g mL–1). The absorbances of the oxacillin treated S. aureus biofilms were the same after 24 h as at the time of antibiotic application (OD550 2.9– 3.1). Similarly, the absorbances of S. epidermidis biofilms showed a minimal range of changes. One exception was biofilm of S. epidermidis RP12 strain treated with oxacillin at the

3

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Staphylococcal biofilm eradication

A

3.5 3 2.5

OD550

2 1.5 1 0.5

biofilm treated with LYS

E7

E4

E1

D13

D8

D5

C1

B1

A7

A3

1474/01

ATCC 29213

BIC ATCC 25923

0

biofilm non treated

3.5

B

3

OD550

2.5 2 1.5 1 0.5

biofilm treated with LYS

C27

C15

C13

C12

C11

C10

A4c

A4b

A4a

6756/99

RP12

BIC ATCC 12228

0

biofilm non treated

Fig. 1. Biofilm formation of S. aureus (A) and S. epidermidis (B) strains and their lysostaphin susceptibility, measured by the MTT reduction assay in the wells of 96-well microplate

concentration of 128 :g mL–1, where the absorbance dropped after 24 h from 2.2 to 0.49. For the evaluation of biofilm susceptibility to lysostaphin, two concentration ranges of the enzyme were used, chosen on the basis of different MICs values established for S. aureus and S. epidermidis strains growing in suspension. For generally more susceptible planktonic cultures of most S. aureus (MICsLYS 0.063– 0.5 :g mL–1), lysostaphin used against biofilms was at the concentrations of 2– 64 :g mL–1. For more resistant planktonic S. epidermidis (MICsLYS 0.5– 16 :g mL–1), lysostaphin concentration range used for biofilm eradication was 8–256 :g mL–1. S. aureus biofilms demonstrated various susceptibility to the lysostaphin; BICLYS range for 8/13 strains was from 4 to 32 :g mL–1, set when the absorbance dropped near the baseline established for negative control well (OD 550 = 0.05–0.09). On the other hand, for 5/13 S. aureus strains BICLYS exceeded the maximal concentration used – >64 :g mL–1. S. epidermidis biofilms were significantly more resistant to lysostaphin, BIC LYS determined for 10/12 strains was >256 :g mL–1, for one strain (RP12) it was 128 :g mL–1 (OD550 dropped from 2.237 to 0.092) and for the other one (A4c) – 16 :g mL–1 (OD550 dropped from 2.762 to 0.096) (Fig. 1). Further experiments concerned the possible synergistic effect of the antibiotic and subinhibitory concentration of enzyme, towards biofilm cultures. For this purpose, 3 S. aureus and 3 S. epidermidis strains,

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Walencka E. et al. Table III The influence of oxacillin or/and lysostaphin on biofilm viability, evaluated by MTT reduction assay in 96-well microplate Strain

BICOX (:g mL–1)

BICLYS BIC OX (+LYS) (:g mL–1) (:g mL–1)

Strain

BICOX (:g mL–1)

BICLYS BIC OX (+LYS) (:g mL–1) (:g mL–1)

S. epidermidis

S. aureus 4 (+4)

RP12

8

32 (+4)

A4c

>128

16

4 (+8)

4

>128 (+2)

6756/99 (MRSE)

>128

>128

>128 (+64)

ATCC 29213 (MSSA)

>128

8

A3 (MRSA)

>128

1474/01 (hVISA)

>128

>128

64

32 (+64)

whose biofilms were characterized as differently susceptible to lysostaphin, and, of course, highly resistant to oxacillin, were included in these studies. The applied “biofilm MTT viability” test, revealed non-significant differences between replicate wells, both within and between test dates, therefore single values are presented in Table III. When lysostaphin was used in subBIC (established earlier), together with different oxacillin concentrations, MTT reduction assay revealed that effective inhibition in biofilm growth could be achieved with a much lower antibiotic concentration. This was demonstrated for biofilms of S. aureus ATCC29213 (MSSA) and S. aureus A3 (MRSA), BICOXA dropped from > 128 to 4.0 and 32.0 :g mL–1, respectively. The synergistic action of oxacillin and lysostaphin was also demonstrated for S. epidermidis RP12 and clinical S. epidermidis A4c biofilms. Unfortunately, such a good effect was not observed for 1474/01 hVISA and 6756/99 MRSE strains included in this part of the study (Table III, Fig. 2). The activity of lysostaphin towards biofilm cultures was confirmed by laser scanning confocal microscopy. Microscopic examination of S. aureus and S epidermidis biofilms, prepared in chamber slides, which were treated with lysostaphin (16 :g mL–1) for 24 h, demonstrated a disruption of S. aureus biofilm and loosening of S. epidermidis biofilm structure (Fig. 3-A1, B1). Oxacillin alone had no effect on biofilm integrity, even when used at the highest concentration, whereas oxacillin used together with subBIC of lysostaphin caused partial biofilm disruption (data not shown). The incubation of polyethylene catheter’s segments, for 72 h at 37°C, with overnight culture of clinical MRSA strain (S. aureus A3) resulted in biofilm formation. Their presence on extra- and intraluminal surfaces of control segments was demonstrated as red stained bacterial deposit, which was a result of the reduction of colourless TTC to the red insoluble formazan crystals by live bacteria (Fig. 4a). When the colonized catheter’s segment was immersed in the medium with oxacillin (4 :g mL–1, according to NCCLS borderline concentration describing MRSA) and incubated at 37°C for 24 h, no visible effect of antibiotic action was seen (Fig. 4b). Lysostaphin alone used at a concentration of 8 :g mL–1 (BICLYS established earlier for S. aureus A3 strain) caused complete biofilm eradication as soon as after 3 h incubation (Fig. 4c). In order to demonstrate synergistic effect of antibiotic and lysostaphin, catheter’s samples colonized by S. aureus A3 were incubated at 37°C for 24 h with oxacillin (4 :g mL–1) together with lysostaphin in subBIC concentration (4 :g mL–1). Also in this case total biofilm eradication was achieved (lack of red stained bacterial deposit).

Fig. 2. Effect of lysostaphin (LYS), oxacillin (OXA) or both (LYS+OXA) on (A) S. aureus ATCC29213 – MSSA, (B) S. aureus A3 – MRSA, (C) S. aureus 1474/01 – hVISA biofilms, measured by the MTT reduction assay in the wells of 96-well microplate. 1 (A-C) – biofilm non-treated (positive control); 2 (A-C) – biofilm treated with OXA 128–2 :g mL–1; 3 – biofilm treated with LYS: (A, B) – 32–4 :g mL–1, (C) – 16–2 :g mL–1; 4, 5 – biofilm treated with OXA 128–2 :g mL–1 + LYS: (A, B) – 4 :g mL–1, (C) – 2 :g mL–1; 6 – medium + MTT (negative control)

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Fig. 3. Laser scanning confocal microscopy (LSCM) images of (A) – S. aureus A3 and (B) S. epidermidis A4c biofilms, formed in chamber slides and stained with FITC. A1, B1 – biofilms of S. aureus A3 and S. epidermidis A4c, respectively, treated for 24 h with LYS 16 :g mL–1. The square panel are a plain view and the side panels are vertical cross sections, respectively

Fig. 4. Eradication activity of oxacillin or lysostaphin alone and in combination, against S. aureus A3 biofilm formed on catheter’s extra- and intraluminal surfaces, measured by the TTC reduction assay. a – biofilm non-treated (positive control); b – biofilm treated for 24 h with OXA (4 :g mL–1); c – biofilm treated for 3 h with LYS (8 :g mL–1); d – biofilm treated for 24 h with OXA (4 :g mL–1) + LYS (4 :g mL–1)

Discussion It is now well accepted that bacteria form groups and respond as groups and that individual bacteria in biofilm community rapidly diversify, which increases the capability of the group and provides a form of “biological insurance” (Boles et al., 2004). Changeability of the bacteria within biofilm structure causes their extremely high resistance to antimicrobial agents and host immune system (Prakash et al., 2003; Fux et al., 2004; Hall-Stoodley et al., 2004). Biofilm infections are the major medical problems, with S. aureus

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and coagulase-negative staphylococci, mainly S. epidermidis, as the leading species responsible for chronic polymer-associated infections (Götz, 2002; Vuong and Otto, 2002; Costa et al., 2004; Lindsay and Holden, 2004). This was the main reason why in the present paper we wanted to test whether lysostaphin, alone or in combination with oxacillin, may be considered as an effective staphylococcal biofilm eradicating agent, which was indicated for planktonic cells in the reports of many authors (Climo et al., 1998, 2001; Kiri et al., 2002; von Eiff et al., 2003). Twenty five staphylococcal strains, 13 of S. aureus and 12 of S. epidermidis were included in this study (Table I and Table II). Within S. aureus group, lysostaphin MICs ranged from 0.0625 to 0.500 :g mL–1 and for S. epidermidis strains the range started from 0.5 and ended at 16 :g mL–1. Such lysostaphin MICs ranges, different for S. aureus and S. epidermidis strains were also demonstrated by Climo et al., (1998, 2001), Kiri et al., (2002), Wu et al., (2003). It is known that lysostaphin is capable of cleaving the crosslinking pentaglycine bridges in peptidoglycan of cell wall and that differences in the S. aureus and S. epidermidis susceptibility are mediated by increased incorporation to the muropeptide of serine and alanine, instead of glycine (Climo et al., 1998, 2001; Kiri et al., 2002; von Eiff et al., 2003). For all 25 staphylococcal strains their biofilm formation ability was evaluated using the MTT assay and laser scanning confocal microscopic (LSCM) observations. Optical density (OD 550) readings after MTT staining ranged from 2.1 to 3.1 (Fig. 1), therefore the isolates were categorized as strong biofilm producers, however, as revealed by LSCM, S. epidermidis formed more biomass than S. aureus (72 h after set-up 8.02 ± 0.61 :m and 4.09 ± 0.78 :m for S aureus ATCC29213 and S. epidermidis RP12, respectively). In many reports it is stressed that the main factors involved in S. epidermidis and S. aureus biofilm formation are not the same and they are not known to the same extent (Mack et al., 2004). The same methods (MTT, LSCM) were used for the evaluation of oxacillin or/and lysostaphin action on biofilm viabillity and integrity. A single standard method for the biofilm susceptibility testing is still lacking, so it is very difficult to compare the already published results obtained for biofilms assayed under different conditions. Since we wanted to know how many bacteria survive the incubation of biofilms with antimicrobial agent, our choice was to use MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) measuring active metabolism of cells. The reproducibility of our observations proved that this method was accurate and proper. It was demonstrated that biofilms of both species were highly resistant to oxacillin (BICs> 128–256 :g mL–1), which means that irrespective of different level of susceptibility (low or high MIC OXA of planktonic cells), none of the biofilms was killed by oxacillin used at such a concentration (Fig. 1). It should be pointed out that the first authors who demonstrated lysostaphin activity against staphylococcal biofilms were Wu et al., (2003). They also proposed the speculation on the possible explanation for the mechanisms of biofilm eradication by lysostaphin, such as rapid lysis of adherent cells, which may be sufficient to destabilize biofilm matrix and allow their detachment. Also, in our study lysostaphin was shown to be effective in biofilm eradication, however with different concentrations used. According to what was described earlier in many reports, the synergistic effect of lysostaphin with oxacillin combination works for planktonic cells (Kiri et al., 2002), and the same effect could be expected against biofilm, which was demonstrated in our study. We have shown that oxacillin at a concentrations of 4.0 and 32.0 :g mL–1, when applied for 24 h with subBIC of lysostaphin, was effective in the killing of biofilm formed by S. aureus ATCC29213 and A3 strains, respectively. A similar result (significant decrease in BICOXA values) was demonstrated for biofilms of two (RP12, A4c) S. epidermidis strains (Table III, Fig. 2). However, using lysostaphin in subBIC had also unexpected limitations, since applying it together with oxacillin did not result in the reduction of oxacillin concentration which could be effective in the killing of 1474/01 hVISA and 6756/99 MRSE biofilms. We suggest that unpredictable synergistic effect of lysostaphin low doses, combined with antibotics, could be the main limitation for such a therapeutic strategy. Similarly to Wu et al., (2003) we have examined biofilm microscopically. In our study laser scanning confocal microscopy of FITC-stained biofilm was used, which allowed us to observe not only differences in S. aureus and S. epidermidis biofilm thickness and architecture but also antibiotic and lysostaphin influence, more precisely than SEM used by these authors. Our results are in some discrepancy with theirs since lysostaphin used at a concentration of 16 :g mL–1 did not clear the biofilm from the surface, however it managed to disrupt it. Wu et al., (2003) demonstrated such an effect using a higher lysostaphin concentration. Nevertheless, we do hope that biofilm structure partially disrupted by the action of lytic enzyme probably could be enough for a more effective antibiotic activity and/or immune mechanisms of the host. The observed synergistic effect of lysostaphin and oxacillin, shown by MTT-reduction assay, was confirmed by the study on “catheter model”. It was another method successfully introduced to our study, based on the reduction by live bacteria of tetrazolium salt – TTC to unsoluble red formazan crystals. The presence

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of red-stained bacterial biofilm on extra- and intraluminal catheter’s surfaces and their disappearing after incubation with antimicrobials were easy to follow (Fig. 4 a-c). The findings of this in vitro study suggest that lysostaphin might be considered for treatment of implant or catheter-associated infections, caused by staphylococci, mainly S. aureus. However, it should be stressed that still we are far from being able to use lysostaphin in clinics, although many studies revealed that lysostaphin possesses a potent anti-staphylococcal activity, for example in in vivo models of rabbit aortic valve endocarditis and nasal colonization in a cotton rat model or in vitro against bacteria isolated from anterior nares and blood (Climo et al., 1998; Patron et al., 1999; Kokai-Kun et al., 2003; von Eiff et al., 2003). The promising results published by Wu et al., (2003) and the results of our study on the lysostaphin activity (alone or in combination with antibiotics) against the staphylococcal biofilms, should be considered with some caution due to: the unpredictable lysostaphin susceptibility of a given strain, which has to be established experimentally, the possibility of generation of lysostaphin-resistant strains when too low concentrations are used, or development of anti-lysostaphin antibodies when prolonged and repeated therapy with high lysostaphin doses is introduced (Boyle-Vavra et al., 2001; Climo et al., 2001; Dajcs et al., 2002; Kiri et al., 2002). Nevertheless, enzymatic detachment of medical biofilms seems to be a new way to increase or replace the ineffective in many cases antibiotic therapy (Barequet et al., 2004; Kaplan et al., 2004).

Literature B a r e q u e t I.S., G.J. B e n S i m o n, M. S a f r i n, D.E. O h m a n and E. K e s s l e r. 2004. Pseudomonas aeruginosa LasA protease in treatment of experimental staphylococcal keratitis. Antimicrob. Agents Chemother. 48: 1881–1687. B o l e s B.R., M. T h o e n d e l and P.K. S i n g h. 2004. Self-generated diversity produces “insurance effects” in biofilm communities. PNAS. 101: 16630–16635. B o y l e - V a v r a S., R.B. C a r e y and R.S. D a u m. 2001. Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate. J. Antimicrob. Chemother. 48: 617–625. C l i m o M.W., K. E h l e r t and G.L. A r c h e r. 2001. Mechanism and suppression of lysostaphin resistance in oxacillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 45: 1431–1437. C l i m o M.W., R.L. P a t r o n, B.P. G o l d s t e i n and G.L. A r c h e r. 1998. Lysostaphin treatment of experimental methicillinresistant Staphylococcus aureus aortic valve endocarditis. Antimicrob. Agents Chemother. 42: 1355–1360. C o s t a S.F., M.H. M i c e l i and E.J. A n a i s s i e. 2004. Mucosa or skin as source of coagulase-negative staphylococcal bacteraemia? Lancet Inf. Dis. 4: 278–286. D a j c s J.J., B.A. T h i b o d e a u x, D.O. G i r g i s, M.D. S h a f f e r, S.M. D e l v i s c o and R.J. O’ C a l l a g h a n. 2002. Immunity to lysostaphin and its therapeutic value for ocular MRSA infections in the rabbit. Invest. Ophthalmol. Visual Sci. 43: 3712–3716. F u x C.A., P. S t o o d l e y, L. H a l l - S t o o d l e y and J.W. C o s t e r t o n. 2003. Bacterial biofilms: a diagnostic and therapeutic challenge. Expert Rev. Anti-infect. Ther. 1: 667–683. G ö t z F. 2002. Staphylococcus and biofilms. Mol. Microbiol. 43: 1367–1378. H a l l - S t o o d l e y L., J.W. C o s t e r t o n and P. S t o o d l e y. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nature Rev. 2: 95–106. J o h a n s e n C., P. F a l h o l t and L. G r a m. 1997. Enzymatic removal and disinfection of bacterial biofilms. Appl. Environ. Microbiol. 63: 3724–3728. K a i r o S.K., J. B e d w e l l, P.C. T y l e r, A. C a r t e r and M.J. C o r b e l. 1999. Development of a tetrazolium salt assay for rapid determination of viability of BCG vaccines. Vaccine. 17: 2423–2428. K a p l a n J.B., C. R a g u n a t h, K. V e l l i y a g o u n d e r, D.H. F i n e and N. R a m a s u b b u. 2004. Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrob. Agents Chemother. 48: 2633–2636. K i r i N., G. A r c h e r and M.W. C l i m o. 2002. Combinations of lysostaphin with $-lactams are synergistic against oxacillinresistant Staphylococcus epidermidis. Antimicrob. Agents Chemother. 46: 2017–2020. K o k a i - K u n J.F., S.M. W a l s h, T. C h a n t u r i y a and J.J. M o n d. 2003. Lysostaphin cream erdicates Staphylococcus aureus nasal colonization in a cotton rat model. Antimicrob. Agents Chemother. 47: 1589–1597. L e w i s K. 2001. Riddle of biofilm resistance. Antimicrob. Agents Chemother. 45: 999–1007. L i n d s a y J.A. and M.T.G. H o l d e n. 2004. Staphylococcus aureus: superbug, supergenome? Trends Microbiol. 12: 378–385. M a c k D., P. B e c k e r, I. C h a t t e r j e e, S. D o b i n s k y, J.K.-M. K n o b l o c h, G. P e t e r s, H. R o h d e and M. H e r r m a n n. 2004. Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses. Int. J. Med. Microbiol. 294: 203–212. National Commitee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard fifth edition. M7-A5: 1–54. P a t r o n R.L., M.W. C l i m o, B.P. G o l d s t e i n and G.L. A r c h e r. 1999. Lysostaphin treatment of experimental aortic valve endocarditis caused by a Staphylococcus aureus isolate with reduced susceptibility to vancomycin. Antimicrob. Agents Chemother. 43: 1754–1755. P r a k a s h B., B.M. V e e r e g o w d a and G. K r i s h n a p p a. 2003. Biofilms: a survival strategy of bacteria. Curr. Sci. 85: 1299–1307.

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Polish Journal of Microbiology 2005, Vol. 54, No 3, 201– 206

gyrA Mutations in Ciprofloxacin-resistant Clinical Isolates of Pseudomonas aeruginosa in a Silesian Hospital in Poland ZENOBIA WYDMUCH1, OLGA SKOWRONEK-CIO£EK 1, KRZYSZTOF CHOLEWA2, URSZULA MAZUREK2, JERZY PACHA1, MA£GORZATA KÊPA1, DANUTA IDZIK1 and ROBERT D. WOJTYCZKA1 1 Department

of Microbiology and 2 Department of Molecular Biology and Medical Genetics, Medical University of Silesia, Sosnowiec, Poland

Received 24 March 2005, received in revised form 6 June 2005, accepted 7 June 2005

Abstract Among 73 clinical isolates of Pseudomonas aeruginosa 48 strains were ciprofloxacine (CIP) susceptible and 25 CIP resistant (Minimal inhibitory concentration – MIC > 32 :g/ml – 14 strains) or of intermediate susceptibility to CIP (MIC ≥ 1,5 – 32 :g/ml – 11 isolates). Mutations in the quinolone-resistance-determining region (QRDR) of gyrA gene were searched in groups of CIP resistant and of intermediate susceptibility to CIP isolates. Two methods: restriction fragment length polymorphism (RFLP) analysis and DNA sequencing analysis allowed to detect three different mutations. The nucleotide substitutions observed led to the following amino acid replacements: Thr-83 → Ile, Asp-87 → Asn, Asp-87 → Gly. One mutated strain among the group of mutants analyzed showed double mutation (Thr-83 → Ile, Asp-87 → Gly) and additional silent mutation (Val-103 → Val); whilst the rest of the isolates showed different single missense mutations. The most frequently detected mutation in the gyrA gene (16 out of 25 mutants) was the Thr-83 → Ile substitution. K e y w o r d s: Pseudomonas aeruginosa, ciprofloxacin, resistance, mutation, gyrA

Introduction Pseudomonas aeruginosa possesses intrinsic mechanisms of resistance to a wide variety of antibiotic. Fluoroquinolones (FQs) are broad-spectrum antibiotics, which are known to be effective in the treatment of a wide range of infections. Ciprofloxacin has emerged as one of the most effective quinolones against P. aeruginosa (Mouneimñe et al., 1999). However, the extensive use of fluoroquinolones has resulted in an increasing incidence of FQs resistance (Yonezawa et al., 1995). Opportunistic infections caused by Pseudomonas aeruginosa, especially quinolone-resistant isolates, pose a serious medical problem. The antibacterial effect of the FQs depends on the inhibition of bacterial topoisomerases: DNA gyrase and topoisomerase IV (Bearden and Danziger, 2001; Dricla and Zhao, 1997). DNA gyrase and topoisomerase IV are heterotetrameric proteins composed of two subunits designated A and B. The genes encoding the A and B subunits are referred to as gyrA and gyrB (DNA gyrase) or parC and parE (DNA topoisomerase IV). Both subunits constitute the active form of the enzyme: A 2B2 (Dricla and Zhao, 1997). DNA gyrase activity is strongly inhibited by quinolones. Alterations in DNA gyrase or topoisomerase IV caused by mutations in the QRDR appear to play a major role in fluoroquinolone resistance in clinical isolates of P. aeruginosa (Yoshida et al., 1990). Recently, several species of bacteria have been studied in order to determine the influence of gyrA mutation on quinolone resistance (Weigel et al., 1998). The authors have not found any Polish publications concerning mutations in gyrA gene in fluoroquinoloneresistant clinical isolates of P. aeruginosa. The aim of our study was to find mutations responsible for ciprofloxacin-resistance in gyrA gene of P. aeruginosa clinical strains, isolated in Silesia region.

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Experimental Materials and Methods Bacterial isolates. 73 clinical isolates of Pseudomonas aeruginosa were isolated at Microbiological Laboratory, Saint Barbara Hospital No 5 in Sosnowiec, Poland, in 2003 from various clinical materials, collected from infirmary and hospitalized patients, showing this species infections. Antimicrobial susceptibility testing. Initial MIC profiles were screened using the disc diffusion method in accordance with the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS, 2000). The MICs of ciprofloxacin were determined by the E-test (AB Biodisk, Solna, Sweden). The range of CIP concentration in susceptibility tests was between 0.002 and 32 :g/ml. The results were evaluated after 24 hours of incubation at 35°C. The Pseudomonas aeruginosa strain ATCC 27853 was included as a control. PCR analysis. Primers were designed to amplify the DNA fragment including the putative quinolone resistance-determining region (Yoshida et al., 1990). For the QRDR of gyrA (GenBank access number L29417), a pair of primers, PaGA 1 (5’-TGACGGCCTGAAGCCGGTGCAC-3’) and PaGA 4 (5’-TATCGCATGGCTGCGGCGTTG-3’) (Takenouchi et al., 1999), was used. These primers allowed amplification of the gyrA gene region, including codons 38 to 122, to encompass the region containing codons 67 to 106. The amplification procedure comprised of initial denaturation at 94°C for 3 min followed by 38 cycles of denaturation (30 s at 94°C), annealing (30 s at 55°C), and polymerization (60 s at 72°C), and then a final extension cycle: 10 min at 72°C. The reactions were conducted in a final volume of 50 :l with 2.5 U of Taq DNA polymerase (Fermentas). PCR-RFLP analysis. PCR products were treated with Cfr 42 I enzyme (an isoschizomer of SacII), (MBI Fermentas) at 37°C for 2 hours, and the restriction fragments were separated in 3% low-melting-point agarose gel and visualized by means of ethidium bromide staining (Takenouchi et al., 1999). The Cfr 42 I site (CCGC¯GG) is present in the wild-type gyrA gene between nucleotides 512 and 517, whereas it is absent in the mutant gyrA gene. Nucleotide sequencing. Prior to the sequencing reaction, the PCR products were purified by isopropanol precipitation with 2M NaClO4 and glycogen in low-TE buffer. Amplicons were analyzed by direct sequencing using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). DNA sequencing analysis was performed with the same primers as those used for PCR. Sequence reactions products were purified with the use of SigmaSpin columns (Sigma-Aldrich) and air-dried. Samples were heated at 96°C for 4 min in 5 :l of loading buffer (25 mM EDTA, pH 8,0 with 50 mg/ml blue dextran and deionized formamid with a ratio of 1:5) and chilled rapidly with ice before loading with 5% denaturing polyacrylamide gel in an Applied Biosystem 377 DNA Sequencer according to the manufacture’s instructions (Kureishi et al., 1994; Yonezava et al., 1995; Nakano et al., 1997; Takenouchi et al., 1999). The nucleotide sequences obtained both for sense and antisense primers were compared with the GeneBank Database (http://www.ncbi.nlm.nih.gov/blast) in order to check their homology.

Results and Discussion The first step of our study was to perform a drug susceptibility test. Using the E-test method, we evaluated the lowest ciprofloxacin concentration visibly preventing growth of P. aeruginosa, (MIC) (Fig. 1). The strains analyzed showed a various susceptibility to ciprofloxacin. Figure 1 presents ciprofloxacin MIC values for analyzed strains of P. aeruginosa. 48 isolates (65.7%) were CIP susceptible (MIC ≤1 :g/ml), whilst 14 strains (19.2%) were CIP resistant with MIC > 32 :g/ml, and 11 strains (15.1%) were of inter16

Numeber of strains

14 12 10 8 6 4 2 0 0.

06

4

0.

9 5 8 .5 64 5 5 94 4 32 09 0.12 0 . 1 0 . 2 0 . 0 . 3 0 0 . 0 . 7 0 . MIC (:g/ml)

1

1.

5

2

Fig. 1. Ciprofloxacin MIC profile for different strains of P. aeruginosa

3

32

3

203

gyr A mutations in CIP-resistant P. aeruginosa Table I Types of point mutations in gyrA and ciprofloxacin MIC range for the resistant or of intermediate susceptibility P. aeruginosa strains DNA sequencing method Amino acid replacement

Nucleotide replacement

Number and (%) of strains with mutation

–

–

–

2 (8)

Single mutation

Asp-87 → Asn

GAC→AAC

7 (28)

–

Cfr42 I site not detected Single mutation

Thr-83 → Ile

ACC→ATC

15 (60)

–

Double mutation

Thr-83 → Ile Asp-87 → Gly

ACC→ATC GAC→GGC

1 (4)

–

Silent mutation

Val-103 → Val

GTA→GTC 23 (92)

2 (8)

PCR-RFLP method Cfr42 I site present

Type of mutation Wild type

Total number of strains with and without mutation in the gyrA gene Total number of analyzed strains

Number and (%) of strains without mutation

25 (100)

mediate susceptibility to CIP with MIC ≥ 1.5– 32 :g/ml. Only the resistant strains and the isolates of intermediate susceptibility were subjects of our further studies (25 strains). The PCR reaction confirmed the presence of the gyrA gene in all of the strains. In order to detect mutations two methods were used for each examined analyzed strain: PCR-RFLP with Cfr 42 I (SacII) and DNA sequencing analysis. PCR-RFLP analysis with Cfr 42 I was performed as a screening method. The restrictase SacII recognizes the CCGC↓GG site, present only in a wild type gyrA gene between 512 and 517 nucleotides at codon 83, therefore this method allows mutation at the Cfr 42 I site to be detected, however it does not exclude the possibility of mutation elsewhere. In our study, using the PCR-RFLP method, we found that 9 out of 25 isolates (36%) have Cfr 42 I restriction site and 16 strains (64%) did not have the site recognized by this enzyme (Table I). Of the 16 isolates without Cfr 42 I restriction site, 14 showed resistance to CIP; furthermore, the MICs values were above the maximum E-test concentration (> 32 :g/ml) (Table II). In order to perform a more accurate analysis of the entire fragment of the gyrA gene, a DNA sequencing analysis was conducted for each examined strain of P. aeruginosa. Using this method we discovered gyrA mutation in 23 strains (92%). Gene alterations were located at codon 83 and 87. We found 15 strains with a single mutation Thr-83→ Ile (13 strains resistant to CIP, 2 of intermediate susceptibility to CIP); 7 strains with a single mutation Asp-87 → Asn (all of them of intermediate susceptibility to CIP), and 1 strain (#47) with a double mutation and additional silent mutation Thr-83 → Ile, Asp-87 → Gl, Val-103 → Val (resistant to CIP) (Table I, Fig. 2). Table II Correlation between MIC values and presence of mutations in the gyrA gene of P. aeruginosa Strain number

MIC :g/ml

Amino acid alteration in gyrA gene at codon 83 87 103

ATCC 27853

0.25

Thr (ACC) Asp (GAC) Val (GTA)

2

3

3

3

4

2

5

1.5

6

2

8

2

10

2

26

2

27

> 32

36

2

40

> 32

43

1,5

MIC :g/ml

Amino acid alteration in gyrA gene at codon 83 87 103

47

> 32

Ile (ATC)

Asn (AAC)

54

> 32

Ile (ATC)

Asn (AAC)

67

> 32

Ile (ATC)

Asn (AAC)

69

> 32

Ile (ATC)

70

> 32

Ile (ATC)

71

> 32

Ile (ATC)

76

> 32

Ile (ATC)

91

> 32

Ile (ATC)

93

> 32

Ile (ATC)

94

> 32

Ile (ATC)

95

> 32

Ile (ATC)

102

> 32

Ile (ATC)

119

2

Ile (ATC)

Asn (AAC) Ile (ATC) Asn (AAC) Ile (ATC) Asn (AAC) Ile (ATC) Asn (AAC)

Strain number

Gly (GGC) Val (GTC)

204 Wydmuch Z. et al.

Fig. 2. Nucleotide and amino acid sequence matching between isolate #47 with double mutation and silent mutation of gyrA gene and wild type of gyrA gene (GB ACC L29417, Kureishi et al., 1994)

3

3

gyr A mutations in CIP-resistant P. aeruginosa

205

Akasaka and co-workers (2001) collected clinical isolates of P. aeruginosa, and sequenced bacterial DNA to search for type II topoisomerase mutations. They found mutations in gyrA gene in 119 out of 150 isolates with reduced susceptibilities to levofloxacine (79.3%); the replacement(s) of amino acid(s) referred to: Thr-83 → Ile or Ala, Asp-87 → Asn, Gly or Tyr. The principal replacement observed by the researchers was Thr-83 → Ile (74.7%). In our study we found similar types of mutation in gyrA gene at codon 83 and 87. As a result of nucleotide substitutions we detected the following mutations: Thr-83 → Ile, Asp-87 → Asn, Asp-87→Gly. In addition to point mutations in the gyrA gene followed by a single amino acid change in 22 strains: 7 Asp-87 → Asn and 15 Thr-83 → Ile, we found one strain with one double mutation and additional silent mutation: Thr-83 → Ile, Asp-87 → Gly, Val-103 → Val. Moreover, the replacement Thr-83 →Ile was the most frequent (65.2%). The point mutation followed by Thr-83 → Ile (ACC?ATC) amino acid replacement was also reported to be the most frequent by Mouneimné et al. (1999), Yonezawa and co-authors (1995), and Nakano’s group (1997). Contrary to our study, mutation at codon 87: Asp-87→Asn has been reported only sporadically by other authors (Yonezawa et al., 1995; Nakano et al., 1997). The silent mutation Val-103→ Val observed in the present study was one of 7 different types of silent mutation in the gyrA gene reported by Takenouchi and co-authors (1999). Takenouchi’s group found two double mutations, three of which were previously unknown: Ala-67→ Ser, Asp-87 → Gly, Ala-84 → Pro and Gln-106 → Leu. One of them, Asp-87 → Gly, was also described by us in strain #47 carrying a double gyrA mutation, and showed resistance to CIP ≥32 :g/ml. The resistance of mutant Thr-83 → Ile to FQ, as mentioned in other articles, was also observed in our study (87.5%) (Mouneim né et al., 1999; Weigel et al., 1998; Akasaka et al., 2001; Nakano et al., 1997; Takenouchi et al., 1999). According to Ball (1994), suitable drug dosing and medical procedure supervision can minimize or even prevent bacterial resistance to quinolones. He emphasizes that bacteria causing intrahospital infections through steady contact with a wide range of chemotherapeutics easily develop resistance to drugs and, as a consequence, the drug resistance phenomenon occurs mostly among hospitalized patients. In our study, 10 of the 14 isolates (71.4%) recognized as CIP resistant (MIC ≥32 :g/ml) were obtained from hospitalized patients, which confirms Ball’s (1994) observation. Our results and the data of other authors show that the analyzed sequence of the gyrA gene plays a crucial role in counteracting ciprofloxacin and DNA gyrase, however, the participation of a mutation localized in different regions of P. aeruginosa genome cannot be excluded. A wide spectrum of genes has recently been analyzed with regard to FQ resistance, e.g. mutations in parC, mexR or nfxB genes have been the subject of intense discussion (Nakajima et al., 2002; Nakano et al., 1997). Further studies on the mechanism of P. aeruginosa resistance to FQs are necessary. Such studies may not only improve the efficiency of FQ therapy and help to prevent the new bacterial resistance phenomenon, but also can help to synthesize new drugs that enable initiation of the more effective therapy of P. aeruginosa infections. Literature A k a s a k a T., M. T a n a k a, A. Y a m a g u c h i and K. S a t o. 2001. Type II topoisomerase mutations in fluoroquinoloneresistant clinical strains Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrob. Agents Chemother. 45: 2263–2268. B a l l P. 1994. Bacterial resistance to fluoroquinolones: lessons to be learned. Infection 22: 140–147. B e a r d e n D.T. and L.H. D a n z i g e r. 2001. Mechanism of action and of resistance to quinolones. Pharmacotherapy. 21: 224S–232S. D r i l i c a K. and X. Z h a o. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61: 377–392. K u r e i s h i A., J.M. D i v e r, B. B e c k t h o l d, T. S c h o l l a a r d t, L.E. B r y a n. 1994. Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates. Antimicrob. Agents Chemother. 38: 1944–1952. M o u n e i m n é H., J. R o b e r t, V. J a r l i e r and E. C a m b a u. 1999. Type II topoisomerase mutations in ciprofloxacinresistant strains of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 43: 62–66. N a k a j i m a A., Y. S u g i m o t o o, H. Y o n e y a m a and T. N a k a e. 2002. High-level fluoroquinolone resistance in Pseudomonas aeruginosa due to interplay of the MexAB-OprM efflux pump and the DNA gyrase mutation. Microbiol. Immun. 46: 391–395. N a k a n o M., T. D e g u c h i, T. K a w a m u r a, M. Y a s u d a, M. K i m u r a, Y. O k a n o and Y. K a w a d a. 1997. Mutations in the gyrA and parC genes in fluoroquinolone-resistant clinical isolates of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 41: 2289–2291. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 5th edn. Approved standard. NCCLS document M7-A5. Wayne, Pennsylvania.

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T a k e n o u c h i T., E. S a k a g a w a, M. S u g a w a r a. 1999. Detection of gyrA mutations among 335 Pseudomonas aeruginosa strains isolated in Japan and their susceptibilities to fluoroquinolones. Antimicrob. Agents Chemother. 43: 406–409. W e i g e l L.M., Ch.D. S t e w a r d and F.C. T e n o v e r. 1998. gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae. Antimicrob. Agents Chemother. 42: 2661–2667. Y o n e z a w a M., M. T a k a h a t a, N. M a t s u b a r a, Y. W a t a n a b e and H. N a r i t a. 1995. DNA gyrase gyrA mutations in quinolone-resistant clinical isolates of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 39: 1970–1972. Y o s h i d a H., M. B o g a k i, M. N a k a m u r a and S. N a k a m u r a. 1990. Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli . Antimicrob. Agents Chemother. 34: 1271–1272.

Polish Journal of Microbiology 2005, Vol. 54, No 3, 207– 213

The Prognostic and Diagnostic Markers of Invasive Candidiasis in Patients During Chemotherapy AGNIESZKA MAGRYŒ1, MARIA KOZIO£-MONTEWKA 1, EL¯BIETA STAROS£AWSKA2 and BEATA GABCZYÑSKA2 1 Medical

University, Department of Clinical Microbiology, Lublin, Poland 2 Oncology Hospital, Lublin, Poland

Received 31 March 2005, received in revised form 25 May 2005, accepted 27 May 2005 Abstract The aim of the work was the early detection of Candida spp. in clinical samples of patients with carcinoma ovariorum undergoing chemotherapy by comparing three indicators of candidiasis: presence of mannan and yeast DNA in the bloodstream and colonization of mucosal membranes by Candida species as a prognostic marker of deep candidiasis. Thirty-one women with carcinoma ovariorum, during chemotherapy without symptoms of deep fungal infections, were examined twice over a six-day period. C. albicans was the dominant organism isolated from mucosal membranes. Two serum samples were positive for mannan on the first day of examination. All these patients were previously colonized by Candida spp. on mucous membranes. Four patients were positive on the last day of examination. Three of these patients were colonized by Candida spp. C. albicans infection was detected early in 4 out of 12 clinical samples by a combination of PCR and mannan-detecting methods. Colonization increases the risk of deep candidiasis. PCR and antigen detection are fast and reliable methods for early detection of Candida in bloodstream. For patients at risk, the clinical samples must be tested by at least two independent methods. K e y w o r d s: cancer, candidiasis, chemotherapy

Introduction Severe fungal infections, especially candidiasis, represent a serious medical problem in immunocompromised patients during anticancer chemotherapy. The mortality rates among patients of risk for Candida infection remain high, ranging from 50– 80%, despite the adequate treatment (Buchman et al., 1990; Flahaut et al., 1998; Pagno et al., 1999; Richardson and Kokki, 1999). Fungal infections appear mainly in people with dysfunctions of the immune system. In these patients the opportunistic and endogenous Candida strains that colonize superficial sites or non-sterile body fluids are the major etiologic agents of life-threatening infections. The factors predisposing to the systemic fungal infections are immunosuppresion, disturbance of residual bacterial flora by antibiotics, extensive surgical procedures, and AIDS. Systemic fungal infections are a great problem in patients receiving cancer therapy. In these patients profound neutropenia following chemotherapy is a serious risk factor for opportunistic fungal infections associated with high mortality risk (Holmes et al., 1994; Füsle, 1997; Rüchel, 1997; Pfaller, 1995; Murray et al., 1999). Disseminated candidiasis disturbs established schemes for chemotherapy and prolonging pause between courses reduces chances of successful treatment by about 20– 30%. Thus, in patients of risk, the empiric antifungal therapy is usually recommended. On the other hand, this therapy may be unnecessarily toxic and costly, and it may play a part in spreading resistance among Candida species (Buchman et al., 1990; Elie et al., 1998; Shin et al., 1997). High mortality rates in neutropenic patients treated for cancer may be reduced only by earliest possible diagnosis of systemic candidiasis with appropriate and effective treatment. Current methods for detection of candidiasis in clinical settings are poor at best. Corresponding author. Mailing address: Agnieszka Magryœ Department of Clinical Microbiology, Medical University of Lublin, ul. ChodŸki 1, 20-123 Lublin, Poland. Phone: (0-81) 740-58-33. Electronic mail address: [email protected]

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3

Too much time passes when employing microbiological cultures, while serological tests for antibody detection in immunocompromised patients are useless in clinical settings (Buchman et al., 1990; Flahaut et al., 1998; Richardson and Kokki, 1991; Shin et al., 1999). The standard laboratory diagnostic approach to systemic candidiasis is based on fungal growth in culture, and is often delayed due to the relatively slow growth of yeasts and inadequate sensitivity of the method in early recognition of infection. Thus, a lot of effort is put into developing rapid and more sensitive diagnostic methods of systemic fungal infections and identifying the virulence determinants of isolated strains. The invasive fungal infection occurs as a result of an increase in the biomass of the pathogenic fungi. The infection increases in aggressiveness as it spreads from an initial site of colonization, ultimately penetrating the host’s protective barriers and damaging tissues. In the processes within bloodstream, there is an increase in fungal specific products that can potentially be utilised as markers of systemic infection. In this regard, techniques have been developed to detect yeast’s proteins, metabolites, DNA and polysaccharides (Richardson and Kokki, 1999; Sendid et al., 1999). The tests for the detection of antigens in sera-glucan, a major polysaccharide of the cell wall and the highly immunogenic mannan, of the Candida are commercially available for few years. Early detection of systemic fungal infection by these methods, or by PCR assays, should improve the survival time in patients at risk. The crucial step in these patients is the timely start of therapy with antimycotic drugs before presentation of clinical symptoms. The aim of our work was the early detection of Candida spp. in clinical samples of patients with carcinoma ovariorum undergoing chemotherapy by comparing three indicators of candidiasis: 1) presence of mannan in the bloodstream; 2) presence of yeast DNA in the bloodstream; and 3) colonization of mucosal membranes by Candida species as a prognostic marker of deep candidiasis. Experimental Materials and Methods Patients and clinical data. Thirty-one patients aged 34 – 54 years (mean 42,6 years) with confirmed carcinoma ovariorum were included in our study. The patients were qualified for chemotherapy and treated with the aggressive anti-cancer drug Taxol, and cisplatin according to Polish standards for carcinoma ovariorum treatment. Presently, Taxol and cisplatin are considered the most effective treatment for carcinoma ovariorum, with haematological toxicity from level II to IV (WHO scale). The treatment, comprising six courses, involved standard doses of Taxol and cisplatin at intervals of 21 days. The study was performed after 3, 4 course of chemotherapy. Clinical samples and strains identification. In order to evaluate the colonization of mucosal membranes by Candida species, nasal, pharyngeal and vaginal swabs from 31 cancer patients were plated onto Sabouraud agar and CHROMagar Candida. The swabs were collected twice over a six-day period. The plated swabs were incubated at 35°C for 48 – 72 h and examined daily for evidence of growth. When a sample was found to be positive for Candida, the strain was identified. Isolates from CHROMagar Candida were identified according to the manufacturer’s instructions. Isolates from Sabouraud agar were first identified microscopically by Gram staining to assess the cell morphology and then the germ tube test was performed. Isolates were also identified on the basis of biochemical features using the ID 32C (bioMérieux) test. Detection of soluble antigen of Candida in serum. Sera for the serological tests comprised 62 samples from 31 cancer patients and 21 samples from 21 healthy donors. Two procedures were used to detect the soluble antigen of Candida in serum. The first method was the immunoenzymatic test (ELISA) for detection of mannan in serum (Platelia Candida; BioRad). Before starting the test all tested sera and standards were heat-treated in the presence of EDTA to dissociate the immune complexes and to precipitate the serum proteins that may interfere with the test. The supernatants were tested after a final, clarifying centrifugation. Microplate wells were coated with monoclonal antibodies directed against $ 1 – 5 oligomannosides of Candida. 50 µl of conjugate (peroxidase-labelled anti-mannan MAb) were simultaneously incubated with 50 µl of supernatant. After incubation, the wells were washed and the reaction was revealed by incubation in the dark with 200 µl chromogenic substrate. The enzymatic reaction was stopped by adding 1,5N H 2SO4. The optical density was determined at 450 nm. The limit of detection of the assay is 0,25 ng of mannan per ml of serum tested. All reactions were performed in duplicate. The second method was based on latex agglutination, using commercial test Pastorex Candida (BioRad). Briefly: 100 µl of treatment reagent was added to 300 µl of serum and placed on heat at 100°C for 3 minutes. After centrifugation at 10 000 g for 10 min., the supernatant was tested. 40 µl of supernatant fluid was deposited on the card, 10 µl of Candida latex was added and the card was shaked for 10 min. (160 rev./min.). A positive reaction was indicated by the agglutination of the latex particles. PCR detection of Candia albicans DNA in human whole blood samples. Isolation of genomic DNA: Clinical specimens (n = 62) examined for yeasts’ DNA presence were obtained from 31 cancer patients and included the whole blood samples. All samples were collected twice in a period of one week. In order to remove the erythrocytes from the blood specimens, 100 µl of the whole blood was added to 1 ml of sterile distilled water. Each sample was incubated on a shaker for 5 min. at RT. The lysate was centrifuged at 3,000 × g for 10 min. and the pellet was resuspended in 2 ml of sterile distilled water, vortexed and centrifuged again at 3,000 × g for 10 min. The erythrocyte-free pellet was washed in 2 ml of 20 mM Tris-HCl (pH 8.3), centrifuged for 10 min. at 3,000 × g, resuspended in 100 µl of RE lysis buffer (A&A Biotechnology) to which 10 µl of yeast lysis enzyme (ICN) was added.

3

Diagnosis of invasive candidiasis in cancer patients

209

The mixture was incubated for 1h at 37°C. After this step, 200 µl of universal lysis buffer LT (A&A Biotechnology) and 20 µl of protease K (1.7 mg/ml; Qiagen) were added and the mixture was incubated 20 min. at 37°C and then the sample was heated at 75°C for 5 min. The sample was centrifuged at 10,000 rpm for 3min. and supernatant was applied to QiAmp spin column (Qiagen), centrifuged at 10,000 rmp for 1 min. and washed twice with 500 µl of washing buffer A1 (A&A Biotechnology). DNA was eluted with 100 µl of eluting buffer RE (10 mM TRIS, pH 8.5; A&A Biotechnology) preheated to 75°C. The sample was incubated at RT for 5 min. and centrifuged at 10,000 rpm for 1 min. The purified DNA preparation was kept at –20°C until PCR. Primers and PCR amplification: Two oligonucleotides derived from Candida albicans rRNA fragments were used as the outer primers. The sequences of these oligonucleotides are the following: camt1 5’-CACCCGATCCGCCTCCTACCGAAG-3’ camt2 5’cGTCTGCCCGATCCGTACCTCCGT-3’. These primers amplify a 1039-bp fragment in the rRNA genes of C. albicans. The amplification was performed in a 50 µl reaction mixture containing: 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 100 µl (each) deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP; A&A Biotechnology) and 1.25 U of Delta2 DNA polymerase (A&A Biotechnology) and 10 pmol (each) primer. A 2.5 µl aliquot of the extracted DNA was added to the mixture. PCR was performed in a thermocycler (Peqlab, Biotechnologie GmbH) as follows. The reaction was initiated by incubation at 95°C for 5 min. Then, the first cycle included 30 sec. of denaturation at 94°C, 1 min. of annealing at 64°C and 1 min. of elongation at 72°C. This first step was followed by 30 cycles. The PCR product was then analysed on a 2% agarose gel stained with ethidium bromide. The positive samples were tested by nested-PCR. For nested-PCR, 1 µl of the PCR product from the first amplification and 10 pmol of Candida albicans-specific inner primers were mixed in a fresh reaction mixture. The sequences of these oligonucleotides are: camt3 5’-ATATATTAgTCTCCACCCGA-3’ camt4 5’-atagAGTATAACCACCCGAT-3’. These generated PCR product of 317-bp of Candida albicans. The second amplification was performed in similar conditions with the exception of annealing temperature (47°C for 1 min.) and elongation temperature (72°C for 30 sec.). The amplified product was analysed on a 2% agarose gel stained with ethidium bromide. Each reaction was carried out in duplicate. To avoid sample contamination, precautions suggested by Kwok and Higuchi (Kwok and Higuchi, 1989) were used. Cross-contamination by aerosols was reduced by physical separation of laboratory rooms used for reagent preparation, sample processing, and DNA amplification. Other precautions included UV irradiation for microcentrifuge tubes, racks, surfaces of laboratory benches, and instruments. Such laboratory procedures as autoclaving of buffers and distilled water, use of fresh lots of previously aliquoted reagents, combined use of positive-displacement pipetters and aerosol-resistant pipette tips, frequent changing of gloves, premixing reagents, addition of DNA as the last step, and testing negative controls, including omission of either the primer or the DNA template during PCR, were used. Appropriate negative controls were included in each test run, including controls omitting either the primer or the DNA template during PCR assay. In all experiments the negative controls always tested negative. Positive standard for PCR used 1ml of purified DNA for Candida albicans.

Results Routine culture of the swabs from mucosal membranes on Sabouraud agar demonstrated, that 4 patients (13%) had Candida spp. on at least two sites (Pfaller, 1995; Yeo and Wong, 2002). Candida strains colonized pharynx (4 cases) and vagina (3 cases) most frequently. Nose swabs were positive in 2 cases. Moreover, Candida spp. strains were present in 6 other patients at only one site. Of all Candida species isolated from patients, 10 were identified as C. albicans. These strains were isolated from 6 patients. 5 strains of C. glabrata were isolated from 4 patients. One strain of C. kruzei was isolated from 1 patient (Table I). Table I The positive culture results on the first day of examination No of occupied Patients sites

Site of Candida isolation vagina

nose

pharynx

4 1 3 6

C. albicans C. albicans C. glabrata –

C. albicans – – C. albicans

C. albicans C. albicans C. glabrata C. albicans

1 site

2 5 7 8 9 10 11

– – – C. albicans – C. glabrata –

C. glabrata C. albicans – – C. albicans – C. kruzei

– – C. glabrata – – – –

none

12–31

–

–

–

3or 2 sites

210

3

Magryœ A. et al. Table II Comparative identification of Candida spp. by routine phenotypic identification methods versus antigen detection and PCR/nested PCR method No Patients’ of occupied no sites 3 or 2 sites

First examination Second examination antigen conc. Routine antigen conc. latex PCR/nested latex PCR/nested by ELISA phenotypic by ELISA agglutin. PCR agglutin. PCR (ng/ml) identification (ng/ml)

4 1 3 6

C. albicans C. albicans C. glabrata C. albicans

< 0.25 1.5 >1 0

– + +/– –

+/+ +/+ – –

1 1.5 1 0

– + – –

+/+ +/+ – –

1 site

2 5 7 8 9 10 11

C. glabrata C. albicans C. glabrata C. albicans C. albicans C. glabrata C. kruzei

< 0.25 < 0.25 0 0 0 0 0

– – – – – – –

– – – – – – –

1 < 0.25 0 0 < 0.25 0 0

– – – – – – –

– – – – – – –

None

12–31

0

–

–

0

–

–

None

Thirty-one patients were classified into 3 groups according to the number of sites occupied by Candida spp. The first group (patients 4, 1, 3 and 6; 12.9%) comprised patients colonized by yeasts on two or three sites. The second group (patients 2, 5, 7, 8, 9, 10 and 11; 22.5%) corresponds to patients colonized at only one site by Candida spp. The third group (patients 12–31; 70.9%) comprised patients with no signs of Candida present on mucosal membranes (Table I). On the sixth day of examination we did not observe any other cases of yeast’s colonization on mucosal membranes. Each ELISA experiment for circulating mannan detection in sera was performed in duplicate. The repeatability of the optical density (OD) values on a single microtiter plate corresponded to a coefficient of variation of < 10%. The sensitivity of detection was 0.25 ng of mannan per ml. In this study we considered > 0.25 ng/ml to be the cutoff level since 100% of the healthy controls had mannanemia values that were equall or less than this value. The results obtained for each of the 31 individual patients are summarized in Table II. Table II compares the methods used to identify markers of candidiasis: routine culture indicating mucous membranes colonization, circulating mannan detection and detection of C. albicans DNA as markers of disseminated candidiasis. All patients have been classified into 3 groups according to the number of sites occupied by Candida spp. The patients’ classification was based on the mucosal membranes occupation by Candida spp., because colonization is regarded as one of the prognostic markers of deep candidiasis. In patients no 1 and 3 from the first group, a high mannan concentration was detected in the serum samples early (on the first day of examination). Mannnan concentration was above 1 ng/ml in both patients, while the latex agglutination method gave positive (no 1) and questionable (no 3) results. One of these patients (no 1) was colonized by C. albicans. For this patient PCR identification confirmed disseminated candidiasis caused by endogenous species. The phenotypic identification of the strain that colonized the mucosal membranes (C. albicans) matched the PCR reaction. Nucleic acid of C. albicans was also detected early by the PCR technique in the blood sample of patient no 4 six days before the appearance of mannan in the patient’s serum. It is worthy to note that in this case C. albicans colonized three anatomical sites. In the case of patient no 6, who was colonized by C. albicans on two sites, no other markers of candidiasis were detected on the first or the second day of examination. In the second group of patients it was not possible to detect antigenemia or C. albicans DNA in blood samples on the first day of examination. We observed one case of positive case of mannanemia six days later (patient no 2). In patients from the third group, we did not observe signs of Candida present on mucosal membranes or other markers of candidiasis in serum or blood samples. Disseminated C. albicans infection was detected early in 4 out of 12 (33%) clinical samples, by a combination of PCR and mannan-detecting ELISA methods. Moreover, the PCR method allowed us to detect 4 positive cases of deep C. albicans infections, whereas positive mannanemias were identified in 3 cases

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Diagnosis of invasive candidiasis in cancer patients Table III Results of mannan detection versus Candida albicans DNA detection in clinical samples No Candida spp./no of clinical of patients samples*

No of clinical samples positive for the following: Mannanemia of ≥ 1 ng/ml

PCR identification

I examin.

II examin.

I examin.

II examin.

12

1 (8%)

2 (16%)

2 (16%)

2 (16%)

C. glabrata/4

8

1 (12.5%)

2 (25%)

0 (0%)

0 (0%)

C. kruzei/1

2

0 (0%)

0 (0%)

0 (0%)

0 (0%)

0/ 20

40

0 (0%)

0 (0%)

0 (0%)

0 (0%)

C. albicans/6

* the clinical samples from each patient were collected twice

(Table III). All patients were at the higher risk of the candidiasis as the routine culture indicated fungal colonization of the mucosal membranes. In four patients with C. glabrata present on mucosal membranes, an increase in mannan concentration was observed in 2 clinical samples. PCR method did not identify DNA of C. albicans in the blood of these subjects. When microbiological and serological investigations were positive, antifungal treatment at standard dosages was performed. Discussion Infections due to Candida spp. remain the most frequent complications in cancer patients despite an increase in systemic fungal infections over the last few years. Candidiasis is the most prominent infection with a high mortality rate in patients at risk (Pagno et al., 1999). For many years Candida albicans was regarded as the most frequent etiologic agent involved in almost all cases of candidiasis, but recent reports indicate an increase in candidiasis sustained by non-albicans species: mainly Candida krusei, Candida tropicalis and Candida glabrata (Pagno et al., 1999; Shin et al., 1997; Yamamura et al., 1999; Abbas et al., 2000). Our study confirms this finding: Candida albicans was the predominant species isolated from mucosal membranes, but other non-albicans species were also present. Along with the appearance of life-threatening Candida non-albicans strains, the strains resistant to antifungal drugs appeared. It may be a direct result of the widespread use of antifungal agents for the prophylaxis and treatment of candidiasis (Pfaller, 1995; Yamamura et al., 1999; Morgenstern et al., 1999). In many oncological hospitals over the world, antimycotic prophylaxis is routinely prescribed and administrated at the start of chemotherapy, also in the absence of positive culture results. In order to reduce the mortality rate among the group of patients at risk there is a need for rapid, sensitive and specific tests for appropriate antifungal therapy before presentation of clinical symptoms. Nowadays patient evaluation, mycological cultures, diagnostic imaging, and biopsies are the standard clinical and laboratory approaches in diagnosis and therapeutic monitoring of systemic candidiasis. Unfortunately, these methods very often lack sensitivity in the early recognition of infection and are imprecise as markers of complete eradication (Richardson and Kokki, 1999). In the context of these disadvantages, new approaches to early diagnosis of disseminated candidiasis are being developed. In our work we compared traditional diagnostic methods with novel methods – serological and molecular. The routine culture allowed us to detect mucosal membranes colonization in 4 patients undergoing chemotherapy. Although the importance of colonization is unclear, in most instances precedes fungemia and is regarded as an independent risk factor for systemic fungal infection (Safdar et al., 2001). The probability of deep candidiasis was evaluated by serological methods detecting circulating mannan in patients’ blood. We tested 62 serum samples drawn from cancer patients, with detection of mannan in 6 cases. Subjects with mannanemia also showed signs of Candida sp. on mucosal membranes. Mannan antigenemia detection for the immunodiagnosis is now one of the most widely studied antigens in patients with candidiasis. Many investigators (Richardson and Kokki, 1999; Sendid et al., 1999; Yeo and Wong, 2002) suggest that positive mannan results may correlate with invasive candidiasis. Furthermore, studies have also shown a correlation between detectable mannanemia and tissue invasive by Candida spp. in

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patients’ with candidemia (Yeo and Wong, 2002). In our study, we used two tests to detect mannan in serum samples of examined persons. The latex agglutination test has been widely used as the first commercially available antigen detection test. The latex agglutination test has shown good specificity, but poor sensitivity, due to the rapid clearance of the antigen from patient’s sera and a low amount of circulating antigen (Richardson and Kokki, 1999; Füsle, 1997; Sendid et al., 1999). By using the ELISA method, the detection limit has been improved up to 0,25 ng of mannan per ml. This resulted in an increase in sensitivity, and allowed detection of mannanemia in 13% of patients at risk. However, these results are still disappointing because in 5% of patients with negative antigen detection, the symptoms of disseminated infection were clinically observed (data not shown). This illustrates one of the major limitations of the fungemia detection tests: the transient character of mannan circulation. Several mechanisms have been proposed to account for this observation, including the quick degradation of mannose oligomers by serum mannosidases, the binding of the mannose oligomers to soluble serum proteins (mannose binding protein C3) or membranous receptors of phagocytes (Sendid et al., 1999). As a consequence, sensitivity rises with the number of serum samples available from each patient. The performance of mannan detection in serum varies with the frequency of testing in a given patient. Regular antigen monitoring of high-risk patients is recommended as a means of increasing the sensitivity of the test and advancing the date of the first positive result (Yeo and Wong, 2002). The doubling time for yeasts is long – an hour or more, compared to 20 min. for bacteria. Furthermore, most clinical specimens are contaminated by bacteria that compete for nutrients and thereby can add to doubling times. For this reason, specimens that are believed to contain yeasts are generally plated out onto a selective medium that contains antibacterial agents. The specific identification of fungus requires 3 to 5 days (Buchman et al., 1990). With the PCR-based amplification procedure, contamination by the bacterial and human cells is avoided and because of the high sensitivity of the method it was possible to detect a small amount of yeast DNA in blood. In our work, 62 clinical samples from cancer patients undergoing chemotherapy were tested for the presence of C. albicans nucleic acid in blood. 33% (4 samples) were PCR positive and 25% (3 samples) were mannan-positive. All patients with disseminated Candida albicans fungemia were colonized by the same species. The PCR method appears to be more sensitive than mannan detecting immunoenzymatic method. In our work the difference in the sensitivity of both tests was not significant, but other authors (Buchman et al., 1990; Richardson and Kokki, 1999, Shin et al., 1999; Yeo and Wong, 2002) indicate the higher sensitivity of PCR based methods compared with other non-culture tests. In the presented work, we did not detect other than Candida albicans species by PCR. However, the high specificity of species-specific primers demonstrated by many investigators (Buchman et al., 1990; Holmes et al., 1994; Elie et al., 1998; Shin et al., 1997; Shin et al., 1999) confirm their value in early detection and differentiation of Candida species present in clinical materials. The conclusions from our work are: (i) colonization increases the risk of disseminated candidiasis occurrence and may be used as its prognostic marker; (ii) PCR and mannan detection are fast and reliable methods for early detection of Candida in bloodstream; (iii) in order to increase the rate of detection of systemic fungal infections, the clinical samples should be collected at least twice; (iv) for patients at risk, the clinical samples must be tested by at least two independent methods. Literature A b b a s J., G.P. B o d e y and H.A. H a n n a. 2000 Candida krusei fungemia: an escalating serious infection in immunocompromised patients. Arch. Intern. Med. 160: 2659–2664. B u c h m a n T.G., M. Rossier, W. M e r z and P. C h a r a c h e. 1990. Detection of surgical pathogens by in vitro DNA amplification. Part I. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene. Surgery 108: 338–347. E l i e C.H., T.J. L o t t, E. R e i s s and C.J. M o r r i s o n. 1998. Rapid identification of Candida species with species-specific DNA probes. J. Clin. Microbiol. 36: 3260–3265. F l a h a u t M., D. S a n g l a d, M. M o u o d, J. B i l l e and M. R o s s i e r. 1998. Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes. J. Clin. Microbiol. 36: 395–401. F ü s l e R. 1997. Diagnosis of fungal infections. Mycoses 40 (Suppl. 2): 13–15. H o l m e s A.R., R.D. C a n n o n, M.G. S h e p e r d and H.P. J e n k i n s o n. 1994. Detection of Candida albicans and other yeasts in blood by PCR. J. Clin. Microbol. 32: 228–231.

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K w o k S. and R. H i g u c h i. 1989. Avoiding false positives with PCR. Nature (London) 339: 237–238. M o r g e n s t e r n G.R., A.G. P r e n t i c e, H.G. P r e n t i c e, J.E. R o p n e r, S.A. S c h e y and D.W. W a r n o c k. 1999. A randomized controlled trial of itraconazole versus fluconazole for the prevention of fungal infections in patients with haematological malignances. Br. J. Haemathol. 105: 901–911. M u r r a y P.R., E.J. B a r o n, M.A. P f a l l e r, F.C. T e n o v e r and R.H. Y o l k e n. 1999. Manual of clinical microbiology. 7th Edition, ASM Press, Washington 1184–1241. P a g a n o L., A. A n t i o n o r i and A. A m m a s s i a r i. 1999. Retrospective study of candidemia in patients with haematological malignances. Clinical features, risk factors and outcome of 76 episodes. Eur. J. Haematol. 63: 77–85. P f a l l e r M.A. 1995. Epidemiology of candidiasis. J. Hosp. Inf. 30 (Suppl): 329–338. R i c h a r d s o n M.D. and M.H. K o k k i. 1999. New perspectives in the diagnosis of systemic fungal infections. Ann Med. 31: 327–335. R ü c h e l R. 1997. Clinical presentation of invasive Candida mycoses. Mycoses 40 (Suppl. 2): 17–20. S a f d a r A., V. C h a t u r v e d i, E.W. C r o s s, S. P a r k, E.M. B e r n a r d, D. A r m s t r o n g and D.S. P e r l i n. 2001. Prospective study of Candida species in patients at a comprehensive cancer center. Antimicrob. Agents Chemother. 45: 2129–213. S e n d i d B., M. T a b a u r e t, J.L. P o i r o t, D. M a t h i e n, J. F r u i t and D. P a u l a i n. 1999. New enzyme immunoassays for sensitive detection of circulating Candida albicans mannan and antimannan antibodies: useful combined test for diagnosis of systematic candidiasis. J. Clin. Microbiol. 37: 1510–1517. S h i n J.H., F.S. N o l t e and C.J. M o r r i s o n. 1997. Rapid identification of Candida species in blood cultures by a clinically useful PCR method. J. Clin. Microbiol. 36: 1454–1459. S h i n J.H., F.S. N o l t e, B.P. H o l l o w a y and C.J. M o r r i s o n. 1999. Rapid identification of up to three Candida species in a single reaction tube by a 5' exonuclease assay using fluorescent DNA probes. J. Clin. Microbiol. 37: 165–170. Y a m a m u r a D.L.R., C. R o t s t e i n, L.E. N i c o l l e and S. I o a n n o u. 1999. Candidemia at selected Canadian sites: results from the Fungal Disease Registry, 1992–1994. Can. Med. Assoc. J. 160: 493–499. Y e o S.F. and B. W o n g. 2002. Current status of nonculture methods for diagnosis of invasive fungal infections. Clin. Microbiol. Rev. 15: 465–484.

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Polish Journal of Microbiology 2005, Vol. 54, No 3, 215– 219

Seroepidemiological Studies of Chlamydia pneumoniae Infections in 1 – 36 Months Old Children with Respiratory Tract Infections and Other Diseases in Poland EDYTA PODSIAD£Y 1*, BEATA FR¥CKA2, AGNIESZKA SZMIGIELSKA2 and STANIS£AWA TYLEWSKA-WIERZBANOWSKA 1 1 National

2 Department

Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland, of Pediatrics and Pediatric Nephrology, Medical University of Warsaw, Marsza³kowska 24, 00-576 Warsaw, Poland

Received 20 April 2005, received in revised form 23 June 2005, accepted 11 July 2005 Abstract Presence of specific IgM, IgG and IgA antibodies against Chlamydia pneumoniae was evaluated in children aged 1 week to 36 months to investigate the role of C. pneumoniae in respiratory infections and other diseases. Serum samples were obtained from 150 hospitalized children, including 123 children presenting the clinical symptoms of various respiratory tract infections, two children with acute diarrhoea, two children with meningitis, 14 children with urinary tract infection, and 9 children with non-infectious diseases. Levels of specific C. pneumoniae IgM, IgG and IgA serum antibodies were measured by enzyme-linked immunoassay (ELISA). C. pneumoniae IgM antibodies were detected in 16 (13.0 %) of 123 children with respiratory tract infections. Specific IgG antibodies were found in sera of 11 children under 12 months old. Among 27 children without symptoms of a respiratory tract disease, specific C. pneumoniae IgM were found in two (7.4%) children, including one child with meningitis and another child with urinary tract infection. Specific IgA antibodies were not found in any tested child. All cases of C. pneumoniae infections were identified within two calendar years out of eight that were analyzed, i.e. in 1997 and 2000. The incidence of C. pneumoniae infections varied seasonally, with most children infected in autumn. C. pneumoniae IgM antibodies were detected more often in girls (17.9%) then in boys (7.2%). C. pneumoniae infections occur among small children in central Poland. The results of this study indicate that C. pneumoniae may play a role in the etiology of respiratory tract infections in infants and young children. K e y w o r d s: children, infants, respiratory tract infections, Chlamydia pneumoniae, antibodies

Introduction Chlamydia pneumoniae is a human pathogen with a wide distribution all over the world. This pathogen is responsible for upper and lower respiratory tract infections such as pharyngitis, sinusitis, bronchitis and pneumonia and probably myocarditis, erythema nodosum and reactive arthritis. The list of diseases associated with C. pneumoniae infection is growing. It is postulated that the microorganism is involved in the development of chronic diseases such as asthma, sarcoidosis, atherosclerosis, Guillain-Barre syndrome, Reiter’s syndrome, and Alzheimer disease (Dowel et al., 2001; Grayston, 2000; Kuo et al., 1995). It has been recognized worldwide that 50% of adults have significant levels of specific serum antibodies against C. pneumoniae (Kuo et al., 1995). Considerable data are available on C. pneumoniae respiratory tract infections in adults, but relatively little is known on the role of this pathogen in infants and young children. Thus the role of C. pneumoniae infections in pediatric patients is uncertain. Few reports on the association of C. pneumoniae with respiratory tract infections in children have been published so far. These studies have been performed in Gambia, Sudan, Thailand, the Philippines, Sweden, Finland, Germany, Switzerland, USA, and Chile (Forgie et al., 1991; Heiskanen-Kosma et al., 1999; Herrmann et al., 1994; Likitnukul et al., 2003; Lund-Olsen et al., 1994; Saikku et al., 1988; Tagle et al., 2000), with divergent and sometimes equivocal results. * Corresponding author: Edyta Podsiadly, National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland; tel. + 48 22 542 12 50; e-mail:[email protected]

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The aim of our study was to investigate the presence of specific C. pneumoniae IgM, IgG and IgA antibodies in Polish children with respiratory tract and other diseases, aged 1– 36 months. Experimental Materials and Methods Patients and specimens. A randomly selected group of 150 children aged from 1 week to 36 months admitted to the Department of Pediatrics and Pediatric Nephrology, Medical University of Warsaw in 1995 – 2003, with clinical symptoms of respiratory tract infections or other diseases was studied. The study group included 23 newborns (age 1 – 4 weeks), 75 infants (age 5 weeks12 months) and 52 children age 13 – 36 months. Sixty-seven girls and 83 boys were enrolled. Patients originated from the Mazowsze district – in central part of Poland. Diagnoses included recurrent respiratory tract infections in 37 patients, pneumonia in 28 patients, bronchitis in 28 patients, pharyngitis in 24 patients, laryngitis in two patients, otitis media in four patients, acute diarrhoea in two patients, purulent meningitis in two patients, and urinary tract infection in 14 patients. Nine children were diagnosed with a non-infectious disease, including dietary indiscretion, urinary tract malformation. Chest roentgenogram was performed in all children with respiratory infections. A diagnosis of pneumonia was based on radiological findings including hyperinflation, prominent bronchovascular makings or diffuse interstitial and patchy alveolar infiltrates. A diagnosis of bronchitis was based on clinical findings including wheezing, cough, dyspnea or tachypnea and normal chest radiograph. Children with more then six incidents of upper or lower respiratory tract infections within a year were categorized as having recurrent respiratory tract infections. Blood samples were taken from each patient prior to administration of antibiotic treatment. Laboratory tests included: leukocyte count and differential white blood cell count, CRP and serological tests. Serology. Anti-chlamydial IgM, IgG and IgA antibodies were determined by enzyme-linked immunoassay (ELISA), with a major outer membrane protein as an antigen. For IgM antibodies, Chlamydia pneumoniae IgM ELISA (Vircell, Granada, Spain) test was used. This test has 91% sensitivity and 98% specificity (Numazaki et al., 1996; Gutierrez et al., 2002). For IgG and IgA, ELEGANCE Chlamydia pneumoniae IgG & IgA ELISA (Bioclone, Sydney, Australia) tests were used. The ELEGANCE Chlamydia pneumoniae ELISA test has 71.7% sensitivity and 95.8% specificity for IgG and 73.9% sensitivity and 92.9% specificity for IgA (Kishimoto, 1990; Kishimoto, 1996;. Ekman, 1993). Presence of IgM antibodies was regarded as an indication of acute infection. The presence of C. pneumoniae IgG antibodies in children aged up to 12 months was assumed to be maternal origin.

Results Among 150 tested children, C. pneumoniae IgM and IgG antibodies were detected in sera of 18 (12%) and 12 (8%), respectively. In children with symptoms of a respiratory tract infection, specific serum C. pneumoniae IgM antibodies were detected in 16 (13.0%) of 123 patients. Specific IgG antibodies were found only in sera of 12 children younger then 12 months. Specific IgG antibodies were not detected in children with specific C. pneumoniae IgM. Specific IgA antibodies were not found in any of 150 tested children. Acute C. pneumoniae infection was detected in patients with various respiratory tract diseases (Table I). Most commonly, it was found in children with bronchitis – 32.1% (9/28), including 77.7% (7/9) of children with wheezing. C. pneumoniae infections were also detected in 10.8% (4/37) of children with recurrent respiratory tract infections, 8.3% (2/24) of children with pharyngitis, and in one child (out of two) with laryngitis. C. pneumoniae infection was detected neither in patients with otitis media (0/4) nor in patients with pneumonia (0/26). Among 27 children with a non-respiratory illness, specific serum C. pneumoniae IgM antibodies were found in two (7.4%) patients, including one child with meningitis and another one with urinary tract infection. Specific IgM and IgG antibodies were not detected in children with non-infections diseases and in children with acute diarrhoea (Table I). Serological evidence of C. pneumoniae infection was found in 6.3% of infants, 13.5% of children aged 1– 2 years and in 46.7% of those aged 2– 3 years. Specific C. pneumoniae antibodies were found more commonly in girls (12/67; 17.9%) then in boys (6/83; 7.2%). Only three children with C. pneumoniae infection presented fever, including a 10-month-old infant with pharyngitis, a 16-month-old child with bronchitis complicated by seizures and a 9-month-old infant with purulent streptococcal meningitis. In children with acute C. pneumoniae infection, average leukocyte count in peripheral blood was 12 100/mm3 with range 7 000– 27 000 (normal values 5 000– 17 500/mm 3 ). Leukocytosis of 27 000/mm3 was found in a 22-month-old girl with acute bronchitis. Differential white blood cell count showed predominant lymphocytes from 51 to 80% in 8 children in this group, CRP level was slightly elevated and averaged 0.6 with range 0.01–0.8 mg/dL (normal level < 0.01 mg/dL). Among the remaining children with symptoms of a respiratory tract infection and no specific C. pneumoniae IgM antibodies, 23 children presented fever, leukocyte count ranged from 5 000 to 24 000 and averaged 11 600/mm 3, again

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C. pneumoniae infections in children Table I Prevalence of C. pneumoniae serum antibodies in 1 – 36 months old children with various diseases No seropositives#

No (%) C. pneumoniae infections***

No tested

IgM

IgG$

Recurrent RTI *

37

4

0

4 (10.8)

Bronchitis

28

9

0

9 (32.1)

Pneumonia

28

0

6

0

Pharingitis

24

2

5

2 (8.3)

Otitis media

4

0

1

0

Laryngitis

2

1

0

1

2

1

0

1

14

1

0

1 (7.1)

Disease

Meningitis UTI ** Acuta diarrhoea

2

0

0

0

Non-infectious diseases

9

0

0

0

* RTI – respiratory tract infections, ** UTI – urinary tract infections, *** The presence of specific C. pneumoniae IgM antibodies was considerd an acute infection, # Specific C. pneumoniae IgA antibodies were not detected in any tested serum, $ Specific C. pneumoniae IgG antibodies were only detected in children under 12 months of age and were assumed to be maternal origin.

with differential white blood cell count showing predominant lymphocytes from 51 to 75% in 33 children, and CRP averaged 0.5 mg/dL. Serological testing revealed that all cases of C. pneumoniae infections occurred within only two out of eight analyzed years (Figure 1). In 1997, specific C. pneumoniae antibodies were detected in 4 of 16 tested children (25.0 %), and in 2000, 14 (25.0%) of 56 examined children were seropositive. In contrast, negative results were obtained in serum samples collected in other years (1995, 1996, 1998, 1999, 2001 and 2002) in all 78 tested children. In addition, it appears that the incidence of C. pneumoniae infections varies seasonally. Cases were detected mostly in November (1997 – 2/4, 2000 – 9/14), October (2000 – 2/14) and December 30

25

%

20

15

10

5

0 1995

1996

1997

1998

1999

2000

2001

2002

years

April

October

November

December

Years

Fig. 1. C. pneumoniae infections by years and months * Specific C. pneumoniae antibodies were not detected in any child in January, February, March, May, June, July , August and September

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(1997– 2/4, 2000 – 2/14). Among children tested in April, there was one child with C. pneumoniae infection. Specific C. pneumoniae antibodies were not found in serum samples collected in January, February, March, May, June, July, August and September. Discussion Among children younger than 3 years old with respiratory tract diseases, acute C. pneumoniae infections were detected in 13.0% of them. The youngest patient with C. pneumoniae infection detected serologically was one-month-old. The frequency of C. pneumoniae etiology in respiratory tract infections in pediatric populations varies from 0% up to more than 18% (Likitnukul et al., 2003; Lund-Olsen et al., 1994; Saikku et al., 1988; Tagle et al., 2000; Hammerschlag et al., 2003; Schmidt et al., 2002; Baer et al., 2003; Chirgwin et al., 1991; Block et al., 1995). These differences are probably related to the population studied and the presence or absence of local outbreaks in the respective communities during the investigation period as well as to applied specific diagnostic procedures (Hammerschlag et al., 2003). It is well known that C. pneumoniae infections spread as long-lasting epidemics that occur at irregular intervals of several years’ duration (Grayston, 2000; Kuo et al., 1995). There is a paucity of data on respiratory tract infections caused by C. pneumoniae in children in Poland. Similarly, there are no data on C. pneumoniae epidemics in Poland. Our results may suggest that such epidemics occurred in 1997 and 2000. Additionally, a seasonal trend for C. pneumoniae cases throughout a year was observed, with a peak of incidence in autumn. These results are in accordance with studies by T. Heiskanen-Kosma et al. who observed incidence peak of C. pneumoniae infections in October (Heiskanen-Kosma et al., 1998). Specific C. pneumoniae antibodies have been detected most frequently among children aged 2– 3 years. Serological evidence of C. pneumoniae infection was found in 46.7% children in this age group. Majority of cases were associated with recurrent respiratory tract infections. C. pneumoniae antibodies were also detected in children without symptoms of respiratory track diseases: a 9-month-old girl with meningitis and in a 3-month-old boy with a urinary tract infection. Detection of specific C. pneumoniae antibodies, in particular IgM class, in children without respiratory tract infections suggest the occurrence of asymptomatic chlamydial infections in early childhood. In Finland, it was shown that 4.0 to 6.0% of 2– 4 years old healthy children were seropositive. It confirms probability of the occurrence of asymptomatic C. pneumoniae infections in this age group (Tuuminen et al., 2000). The significance of asymptomatic C. pneumoniae infections and their sequelae requires further investigations. In adult populations, C. pneumoniae antibodies are present with significantly higher frequency in men then in women. This is, however, at variance with our results in a group of children aged 1– 36 months. C. pneumoniae antibodies were detected more often in girls then in boys. These results are in accordance with a study by Lin et al. who found higher prevalence of C. pneumoniae antibodies in females then in males among subjects aged 6 months to 20 years (Lin et al., 2004). Most of the seroepidemiological studies on C. pneumoniae infections in children were performed with the MIF method. According to the recent recommendations of Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada) (Dowell et al., 2001) this technique is considered a reference method, although there are studies showing low sensitivity of this method in children (Kutlin et al., 1998). Simultaneous application of MIF and culture in the examination of children shows that MIF is not a sensitive technique for detecting C. pneumoniae antibodies in children under 5 years old. Since positive culture results were achieved in seronegative children. According to some studies only 20% to 30% of children with culture-documented C. pneumoniae infection had antibodies detectable by MIF (Schmidt et al., 2002; Hyman et al., 1995; Emre et al., 1994). Kutlin et al. found that over 80% of culture-positive and MIF-negative children had antibodies to C. pneumoniae detected by immunoblotting (Kutlin et al., 1998). The lack of standardized methods, including serology and PCR, makes it difficult to perform a specific microbiologic diagnosis. Although there is currently no validated diagnostic marker for C. pneumoniae infections, and the value of the EIA method in diagnosis C. pneumoniae infections is an open issue, detection of C. pneumoniae IgM antibodies in several children below 3 years of age may suggest that C. pneumoniae infections occur in early childhood. Moreover, frequent detection of C. pneumoniae antibodies in children with respiratory tract infections suggests that the bacterium may be an etiological agent of respiratory diseases in infants and young children. Acknowledgement. The studies were supported by a research grant KBN 4PO5D 038 17.

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Literature B a e r G., G. E n g e l c k e, M. A b e l e - H o r n, U.B. S c h a a d and U. H e i n i n g e r. 2003. Role of Chlamydia pneumoniae and Mycoplasma pneumoniae as causative agents of community-acquired pneumonia in hospitalized children and adolescents. Eur. J. Clin. Microbiol. Infect. Dis. 22: 742–745. B l o c k S., J. H e d r i c k, M.R. H a m m e r s c h l a g, G.H. C a s s e l and J.C. C r a f t. 1995. Mycoplasma pneumoniae and Chlamydia pneumoniae in pediatric community-acquired pneumonia: comparative efficacy and safety of claritromycin vs. erythromycin ethylsuccinate. Pediatr. Infec. Dis. J. 14: 471–477. C h i r g w i n K., P.M. R o b l i n, M. G e l l i n g, M.R. H a m m e r s c h l a g and J. S c h a c h e r. 1991. Infection with Chlamydia pneumoniae in Brooklyn. J. Infect. Dis. 163: 757–761. D o w e l l S.F., R.W. P e e l i n g, J. B o m a n, G.M. C a r l o n e, B.S. F i e l d s, J. G u w n e r, M.R. H a m m e r s c h l a g, L.A. J a c k s o n, C.C. K u o, M. M a a s s, T.O. M e s s m e r, D.F. T a l k i n g t o n, M.L. T o n d e l l a and S.R. Z a k i. 2001. Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada). Clin. Infect. Dis. 33: 492–503. E k m a n M. 1993. Evaluation of serological methods in the diagnosis of Chlamydia pneumoniae during an epidemic in Finland. Eur. J. Clin. Microbiol. Infec. Dis. 12: 756–760 E m r e U., P.M. R o b l i n and M. G e l l i n g. 1994. The association of Chlamydia pneumoniae infection and reactive airway disease in children. Arch. Pediatr. Adolesc. Med. 148: 727–729.  F o r g i e I.M., K.P. O’ N e i l l, N. L l o y d - E v a n s, M. L e i n o n e n, H. C a m p b e l l and H.C. W h i t t l e. 1991. Etiology of acute lower respiratory tract infections in Gambian children: II. Acute lower respiratory tract infection in children ages one to nine years presenting at hospital. Pediatr. Infect. Dis. J. 10: 42–47. G r a y s t o n J.T. 2000. Background and current knowledge of Chlamydia pneumoniae and atherosclerosis. J. Infect. Dis. 181 (suppl 3): S402–411. G u t i e r r e z J., J. M e n d o z a, F. F e r n a n d e z, J. L i n a r e s - P a l o m i n o, M.J. S o t o and M.C. M a r o t o. 2002. ELISA test to detect Chlamydophila pneumoniae IgG. J. Basic Microbiol. 42: 13–18. H a m m e r s c h l a g M.R. 2003. Pneumonia due to Chlamydia pneumoniae in children: epidemiology, diagnosis, and treatment. Pediatr. Pulmonol. 36: 384–390. H e i s k a n e n - K o s m a T., M. K o r p p i, A. L a u r i l a, C. J o k i n e n, M. K l e e m o l a and P. S a i k k u. 1999. Chlamydia pneumoniae is an important cause of community-acquired pneumonia in school-aged children: serological results of a prospective, population-based study. Scan. J. Infect. Dis. 31: 255–259. H e i s k a n e n - K o s m a T., M. K o r p p i, C. J o k i n e n, S. K u r k i, L. H e i s k a n e n, H. J u v o n e n, S. K a l l i n e n, M. S t e n, A. T a r k i a i n e n, P.R. R o n n b e r g, M. K l e e m o l a, H. M a k e l a and M. L e i n o n e n. 1998. Etiology of childhood pneumonia: serologic results of a prospective, population-based study. Pediatr. Infec. Dis. J. 17: 986 –991. H e r r m a n n B., M.A. S a l i h, B.E. Y o u s i f, O. A b d e l w a h a b and P.A. M a r d h. 1994. Chlamydial etiology of acute lower respiratory tract infections in children in Sudan. Acta Paediatr. 83: 169–172. H y m a n C.L., P.M. R o b l i n, C.A. G a y d o s, T.C. Q u i n n, J. S c h a c h e r and M.R. H a m m e r s c h l a g. 1995. Prevalence of asymptomatic nasopharyngeal carriage of Chlamydia pneumoniae in subjectively healthy adults: assessment by polymerase chain reaction-enzyme immunoassay and culture. Clin. Infect. Dis. 20: 1174–1178. K i s h i m o t o H. 1990. Research into the comparison between healthy adults and those suffering from C. pneumoniae (TWAR) acute respiratory infection through serum based epidemiological examination. J. Infect. Dis. 64: 986–992. K i s h i m o t o H. 1996. The foundation and clinical serum based diagnosis of Chlamydia pneumoniae. Sphere Scien. Cure 12: 2250–2256. K u o C.C, L.A. J a c k s o n and L.A. C a m p b e l l. 1995. Chlamydia pneumoniae (TWAR). Clin. Microbiol. Rev. 8: 451–461. K u t l i n A., P.M. R o b l i n and M.R. H a m m e r s c h l a g. 1998. Antibody response to Chlamydia pneumoniae infection in children with respiratory illness. J. Infect. Dis. 177: 720–724. L i k i t n u k u l S., P. N u n t h a p i s u d and N. P r a p p h a l. 2003. Prevalence of Chlamydia pneumoniae infection in Thai children with community-acquired pneumonia. Pediatr. Infect. Dis. J. 22:749–750. L i n T.M., C.C. K u o, W.J. C h e n, F.J. L i n and H.L. E n g. 2004. 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K o g a n, P. R o j a s, L. R u b i l a r, R. V i d a l and E. P a y a. 2000. Diagnosis of Chlamydia pneumoniae in community-acquired pneumonia in children in Chile. Acta Paediatr. 89: 650–653. T u u m i n e n T., S. V a r j o, H. I n g m a n, T. W e b e r, J. O k s i and M. V i l j a n e n. 2000. Prevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae immunoglobulin G and A antibodies in a healthy Finnish population as analyzed by quantitative enzyme immunoassays. Clin. Diagn. Lab. Immunol. 7: 734–738.

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Polish Journal of Microbiology 2005, Vol. 54, No 3, 221– 225

Occurrence of Serum Class G Immunoglobulins Interacting with Specific Antigens of Helicobacter pylori in Patients with Unstable Coronary Artery Disease and in Symptomless Individuals TOMASZ RECHCIÑSKI1, ANETA GRÊBOWSKA2, MA£GORZATA KURPESA1, WIES£AWA RUDNICKA2, MARIA KRZEMIÑSKA-PAKU£A1 and MAGDALENA CHMIELA2 1 Second

Chair and Clinic of Cardiology, Medical University of £ódŸ, ul. Kniaziewicza 1/5, 91-347 £ódŸ of Infectious Biology, University of £ódŸ, ul. Banacha 12, 90-237 £ódŸ, Poland

2 Department

Received 7 January 2005, received in revised form 25 February 2005, accepted 28 June 2005 Abstract An impact of Helicobacter pylori on the process of atherogenesis may be related to the intensity of humoral response against selected specific antigens of this bacteria. We performed serological studies in which the recognition of 7 selected antigens was possible. The investigated group consisted of 56 patients hospitalized due to unstable angina pectoris. The control group consisted of 29 symptomless volunteers. The levels of class G serum immunoglobulins interacting with glycine extract (GE) of H. pylori antigens were assessed by ELISA test in both groups. The same sera were tested by the Milenia blot H. pylori IgG system. In this assessment the presence of IgG antibodies interacting with antigens of molecular weight of 120, 87, 64, 35, 30, 26, and 20 kDa was estimated separately for every listed antigen. The results revealed significant differences between investigated groups in the prevalence of anti-GE IgG (unstable angina – 100% vs. controls – 60%) and in the level of such antibodies expressed as total optical density units – OD450 (6.1 ± 3.0 vs. 3.4 ± 3.0 respectively, p < 0.05). However, anti-GE IgG detected in the sera of patients as well as controls reacted with similar frequency with selected H. pylori antigens: highly specific (120, 87, 64, 30 kDa) and specific (35, 26, and 20 kDa). We conclude, that although H. pylori infection is so common and mainly associated with gastroduodenal symptoms, it is also recognized by serological methods with high prevalence in patients with coronary artery disease, and less frequently in symptomless individuals. The humoral response against H. pylori in class G immunoglobulins in patients with unstable angina is characterized by higher levels of anti-H. pylori IgG but not by the higher prevalence of serum IgG interactions with the highly specific and specific H. pylori antigens. Such infection could be considered as a cofactor for atherogenesis by inducing strong humoral response against surface antigens of this bacteria. K e y w o r d s: immunity, H. pylori, atherosclerosis, unstable angina

Introduction Helicobacter pylori is one of the most common pathogens of humans causing a lifelong infection of gastric mucosa in > 50% of the global population. This infection has been proved to be an important pathogenic factor for some gastro-duodenal diseases like type B gastritis, ulcer disease, mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma (Rosenstock et al., 1997; Howden et al., 1996). Recent studies point to a possible role of H. pylori in extradigesive diseases, like Sjörgrene syndrome, thyroid diseases or Schönlein-Henoch purpura (Gasbarini et al., 1998). Also vascular diseases like Raynaud phenomenon or headaches are considered to be linked with this infection. Although human atherosclerosis is a multifactoral disease, some possible links between this chronic process, infections and inflammation have been emphasized (Watanabe et al.,1996). A relationship between H. pylori seropositivity and atherosclerosis in general and coronary heart disease in particular has been reported in mid 1990s (Mendall et al., 1994; Patel et al., 1995); however the role of this infection in atherogenesis is still controversial – in some large studies, there was no correlation shown between H.pylori, cardiovascular risk factors and ischaemic heart Corresponding author: T. Rechciñski, MD, 2 nd Chair and Clinic of Cardiology, Medical University of £ódŸ, Biegañski Hospital, Kniaziewicza 1/5, 91-347 £ódŸ, Poland, e-mail: [email protected]

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disease (Murray et al., 1995; Ridker et al., 2001). Since then, the methods of serological detection of H. pylori have become more sophisticated and accurate. This is a problem of great importance, as this bacteria shares a number of antigens with other microorganisms, giving false-positive cross-reactions in serological studies (Johansen et al., 1995; Paziak-Domañska et al., 2000). In the recent studies it was found that patients with unstable angina demonstrated enhanced humoral response to a glycine acid extract of H. pylori (Rechciñski et al., 2002). Glycine extract is a mixture of H. pylori surface antigens whose molecular weight ranges from 14 to 120 kDa. It is to be emphasized that in previous studies, the composition of circulating IgGs in respect to different specific and highly specific H. pylori antigens in patients with angina pectoris was not estimated or compared with symptomless individuals. In our present study, we intended to find out whether the enhancement of humoral response was generalized or rather limited to the immunoglobulins of a strictly limited idiotype (antigenic specificity). The aim of this study was also to find out how prevalent the patients with unstable angina are when infected by more virulent strain of H. pylori, characterized by the presence of cytotoxin-associated protein A (CagA), highly specific for this bacteria. Experimental Material and Methods Fifty-six males (age 30–65 years) hospitalized due to chest pains in the Cardiology Department were included in the study after an unstable angina pectoris according to Braunwald definition was diagnosed. Coronary angiography confirmed the atheromatic background of symptoms (Braunwald, 1989). In this group, the prevalence of previous myocardial infarction was 52%, arterial hypertension – 72%, diabetes mellitus – 18%, hyperlipidemia – 62%, nicotinism – 18%, rate of the revascularisation (percutaneous transluminal coronary angioplasty or coronary artery by-pass graft) – 58%. The control group consisted of 29 symptomless age-matched males. All controls had negative history of ischaemic heart disease or gastric symptoms. The comparison of prevalence of risk factors of atherosclerosis in investigated group and controls is presented in Table I. Table II shows the pharmacological treatment obtained by patients and controls prior to blood sampling. ELISA. Blood from antecubital vein was obtained from all study participants for serological tests. The sera were stored at a temperature of –70°C. The level of anti-H.pylori IgG was estimated using ELISA method with GE of H. pylori CCUG 17874 as the coating antigen and with rabbit anti-human IgG antibodies labeled with horseradish peroxidase HRP (Dako, Glostrup, Denmark). The serum samples for anti-GE IgG ELISA were diluted from 1:500 to 1:128000. The results were expressed as total optical density

Table II Comparison of pharmacological treatment in patients with unstable angina and controls

Table I The comparison of risk factors of atherosclerosis in investigated group and controls Risk factor

Unstable angina pectoris

Controls

Compound

Unstable angina pectoris

Controls

Diabetes mellitus

18%

3%

Aspirin

100%

6%

Arterial hypertension

72%

13%

Statins

60%

0

Hypercholesterolaemia

62%

12%

Beta-blockers

90%

0

Nicotinism

18%

33%

ACE* – inhibitors

74%

0

*ACE – angiotensin converting enzyme

values measured in 450 nm wave length. The details of this assay are described elsewhere (Rechciñski et al., 1997). The sera diluted 1:500 were also tested for optical density (OD450) of IgG reacting with the recombinant cytotoxin-associated protein A CagA using ELISA (A. Covacci, IRS Siena, Italy). Another method used in this study to assess the presence of IgG antibodies against four highly specific antigens and three specific antigens of H. pylori was Milenia blot H. pylori IgG test (DPC Biermann GmbH, Bad Nauheim, Germany). The antigens investigated in this kit were: cytotoxin-associated protein A (CagA) of molecular mass 120 kDa, vacuolising cytotoxin A (VacA) – 87 kDa, urease subunit A (UreA) – 30 kDa, urease subunit B (UreB) – 64 kDa, as well as antigens of molecular weight of 35, 26, and 20 kDa. The combination of positive results for separate antigens made it possible to diagnose H. pylori infection, as recommended by the manufacturer. Statistical methods. Chi2 test was used to assess the significance of differences in results observed between the studied groups.

Results The IgG antibodies reacting with GE of H. pylori antigens were detected by ELISA in all patients with unstable angina and in 60% of symptomless individuals (p< 0.05). The level of anti-GE IgG expressed as total optical density for the wave length of 450 nm (OD total) ranged in the group of patients with unstable

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H. pylori IgG system and atherogenesis

angina from 0.585 to 13.922 (mean 6.1 ± 3.0) and in the controls – from 0.422 to 8.308 (mean 3.4 ± 3.0) – p< 0.05 (Table III). Using Milenia blot, 75% of cardiac patients, and 48% of controls were H. pylori positive – p< 0.05. The prevalence of anti-CagA IgG assessed by ELISA was observed in 66% of cardiac patients and in 70% of controls – with the difference being nonsignifficant – p> 0.05. The total optical density in ELISA test for anti-CagA ranged in the group of patients with unstable angina from 0.09 to 2.489 (mean 0.8 ± 0.70), in controls – from 0.067 to 2.485 (mean 0.6 ± 0.7) and did not vary significantly between two groups (Table IV). Despite the significant differences between both studied groups in the prevalence of H. pylori infection detected by ELISA or Milenia blot, no statistical difference was found in the occurrence of IgG antibodies against selected highly specific antigens when they were analyzed separately (Table V). Antibodies interacting with CagA were detected by Milenia blot in 59% patients with unstable angina and in 48% controls (p> 0.05), the prevalence of IgG interacting with VacA was 57% and 38%, respectively (p>0.05). Also IgGs interacting with two subunits of urease were detected with a similar rate in both groups – 71% vs. 48% for UreA (p > 0.05) and 50% vs. 38% for UreB (p> 0.05), respectively. Also, the rate of detection of IgG antibodies against specific 35 kDa protein did not vary significantly between the compared groups: 21% vs. 14%, respectively (p > 0.05). Antibodies of IgG class against protein of molecular weight of 26 kDa were Table III The prevalence of anti-H. pylori GE IgG and anti-CagA IgG assessed by ELISA or Milenia blot Percent of positive results ELISA

Group

Milenia blot

GE 14-120kDa IgG

CagA IgG

20-120kDa IgG

CagA IgG

100*

66

75

+

59

60*

70

48+

48

Angina pectoris Controls

* and + – significance p < 0.05

Table IV The levels of anti-H. pylori GE IgG and anti-CagA IgG expressed as optical density ELISA total OD 450 1:500–1:128000

Group

IgG anti-GE range

OD 450 1:500

IgG anti-GE mean IgG anti-CagA range IgG anti-CagA mean

Angina pectoris

0.585 – 13.922

6.1 ± 3.0*

0.09 – 2.489

0.8 ± 0.7

Controls

0.422 – 8.308

3.4 ± 3.0*

0.06 –2.485

0.6 ± 0.7

* – significance p < 0.05

Table V The prevalence of serum IgG interactions with selected H. pylori antigens detected by Milenia blot H. pylori IgG system. Analysis performed for all subjects in compared groups and restricted only to H. pylori-seropositive individuals in both groups Antigens

Percent of positive serum interactions with selected H. pylori antigens detected by Milenia blot Angina pectoris all subjects – (seropositive ones)

Controls all subjects – (seropositive ones)

CagA 120kDa

59 – (80)

48 – (100)

VacA 87kDa

57 – (76)

38 – (79)

ure A 30kDa

71 – (95)

48 – (100)

ure B 64kDa

50 – (67)

38 – (79)

35kDa

21 – (29)

14 – (29)

26kDa

71* – (95)

45* – (93)

20kDa

73* – (98)

41* – (96)

* – significance p < 0.05

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detected in 71% of patients with unstable angina and in 45% of controls (p< 0.05), and the antibodies against 20 kDa protein were found in 73% vs. 41% (p< 0.05), respectively. However, when the analysis of the occurrence of IgG antibodies specific for 26 and 20 kDa proteins was only limited to H. pylori-positive individuals in both groups (Milenia blot), the prevalence of such antibodies ranged from 86 to 98%, and there were no statistical differences between these subgroups. Discussion The differentiation of various antigens interacting with the sera of the investigated humans made the serodiagnosis of H. pylori infection more reliable (Faulde et al., 1993; Nilsson et al., 1997), but it was not used in clinical practice due to the fact that this method seemed to be expensive and laborious. The results of our studies concerning the similar prevalence of anti-CagA IgG in patients with coronary artery disease in comparison with controls, remain in accordance with previous reports (Koenig et al., 1999; Whincup et al., 2000). Such high prevalence of this type of antibodies even in the group of healthy individuals, may be justified by the asymptomatic H. pylori infections. In our previous study, the frequency of asymptomatic infections in healthy donors ranged from 50 to 60% as detected by 13C-urea breath test (Wiœniewska et al., 2002). Another possible explanation of this phenomenon is antigenic mimicry with host proteins detectable in both normal and atherosclerotic arteries tissues (Franceschi et al., 2002). In previous studies it was already suggested that generalized elevated levels of IgG, IgA, IgE (but not IgM), help to predict first myocardial infarction or sudden cardiac death in population of healthy men with abnormalities in the lipid profile (Kovanen et al., 1998), but the specificity of these immunoglobulins was not investigated. In our studies it was confirmed that patients with the documented coronary atherosclerosis in comparison with symptomless individuals had higher levels of IgG reacting with H. pylori antigens. Previously, we showed that the highest titers of anti-GE IgG were even more prevalent in the patients with atherosclerosis than in H. pyloriinfected patients with gastroduodenal symptoms (Rechciñski et al., 2002; Chmiela et al., 2003). The tendency of the patients with coronary artery disease to intensively produce IgG to various antigens was considered. However, we could not see any significant difference in the production of IgG to mycobacterial Hsp65 among such patients, patients with active tuberculosis, dyspeptic patients and healthy controls (Chmiela et al., 2003). In the present study we did not find any significant difference in the prevalence of serum IgG interaction with H. pylori highly specific and specific antigens between the group of coronary atherosclerosis and controls. Obviously, we are not free to assume the absence of atheromatic plaques in coronary vessels of symptomless individuals in the control group, since recently it has been proved that coronary atherosclerosis is a process which begins already in asymptomatic teenagers (Tuzcu et al., 2001). Nevertheless, according to the clinical interview, the plaques in coronary arteries of these individuals, if present, were stable. The role of immunoglobulins in the process of initiation or destabilization of atheromatous lesions may be elucidated by recent studies of Sims et al., 2001. These authors found in their immunohistological studies, that gamma globulin leakage from the lumen into arterial wall is observed when arteries exhibit a breakdown of sub-endothelial lamina, which results in subsequent lipids and inflammatory cells entry. Class G immunoglobulins seem to have the best properties to participate in this process, as they have the smallest sizes and the longest clearance in comparison with IgA and IgM. One cannot rule out the fact that also the size of the reacting antigen may be important for this phenomenon. Interestingly enough, it was previously found, that the smallest antigens of investigated bacteria are recognized by serum antibodies predominantly in the organisms of infected children, in contrast with the infected adults, whose organisms used to recognize mainly the larger bacterial proteins (Chmiela et al., 1998). Although we did not observe such a phenomenon in the patients with atherosclerosis or in symptomless subjects, it is possible that gastrotoxic activity of aspirin used for treating the patients with coronary artery disease may lead to the failure of mucosal barrier and an easier penetration of some bacterial antigens to the circulatory system and the induction of stronger humoral response. We conclude, that class G immunoglobulins interacting with H. pylori surface antigens, and circulating in blood vessels of the patients with unstable angina pectoris, can be characterized by higher concentration than in symptomless individuals, and by a similar specificity. The strong humoral response against H. pylori antigens may play a role in the maintenance of coronary heart disease. Acknowledgements. We thank Dr. Antonello Covacci (IRIS, Siena, Italy) for providing us with flCagA protein and Mrs. Aleksandra Siwicka for her linguistic assistance. This study was supported by grant from KBN 3P05E04524

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225

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Polish Journal of Microbiology 2005, Vol. 54, No 3, 227– 231

Enzyme Production and Biotypes of Vaginal Candida albicans ZEFIRYN CYBULSKI1, EL¯BIETA KRZEMIÑSKA-JAŒKOWIAK, PRZEMYS£AW MAJEWSKI 2, JERZY CHYLAK3 and MAREK PAWLIK4 1 Wielkopolska

3 Department

Cancer Center, 2 Department of Clinical Pathomorphology, of Medical Microbiology, University of Medical Sciences, Poznañ, 4 Central Hospital, Lutycka str., Poznañ, Poland

Received 28 December 2004, received in revised form 4 March 2005, accepted 17 June 2005 Abstract Candidial vulvovaginitis is one of the most common forms of vaginal infection. However, the origin of the infecting organism is sometimes doubtful. Therefore, epidemiological investigation can help to recognize routes of infection spreading. The aim of the present study was to determine the ability to produce esterases by clinical isolates of C. albicans and to find the relationship between their serotypes. Also, it was intended to determine the ability of these strains to produce proteases and lipases as well as the ability of the strains to assimilate carbohydrates. 46 strains of C. albicans isolates from the vagina of women suffering from vulvovaginitis were examined. Three main kinds of esterases were distinquished by their spectra of hydrolytic activity toward "-naphthyl acetate, $-naphthyl propionate and indoxyl acetate. The strains were grouped into four categories: three categories in which esterase patterns were observed and one category in which esterase bands were not observed. On the basis of the 20 carbon sources assimilated, the C. albicans strains were categorized into 11 biotypes with the major biotype accounting for 21 (45.7%) strains. The examination of proteolytic activity using casein and albumin enabled to divide the strains into four groups. All of the examined strains belonged to serotype A and all of them expressed lipolytic activity. Esterase electrophoretic patterns and biotypes based on proteolytic activities were compared with the ability to assimilate carbon from various sources. K e y w o r d s: C. albicans, vulvovaginitis, biotypes, esterases

Introduction The use of epidemiological markers to type C. albicans in communicable diseases is recognized as being very important for the following purposes: detection of sources and routes of infections; distinction between recurrent and chronic infection; identification of endemic strains; selection of specific immunoprophylaxis and immunotherapy (al Rawi and Kavanagh, 1999; Kubota, 1998; Magliani et al., 2002; Mendling et al., 2000; Moraes et al., 2000). In the case of Candida vulvovaginitis, the origin of the infecting organism is unclear. It has been suggested that there are many women who carry C. albicans in the vagina without subjective symptomatology, often with low Candida concentration. These observations are in agreement with the view that C. albicans may be either human commensal or a pathogen (Kubota, 1998; Marai, 2001). C. albicans is a common opportunistic pathogen in HIV-infected patients. Diabetes mellitus increases the rate of vaginal colonization and infection with C. albicans spp. (Goswami et al., 2000; HaberlandCarrodeguas et al., 2002; Taylor et al., 2000). However, C. albicans is a potent allergen in some situations and it has been suggested that local hypersensitivity to these fungi is a factor which prolongs recurrent vaginal candidiasis (Moraes et al., 2000). Various methods have been used to type Candida isolates, with the objective of developing epidemiological tools. The most widely used methods include biotyping, serological typing, comparison of the susceptibility or resistance response to antifungal chemicals, and the ability of the strains to produce hydrolytic enzymes (Kantarcioglu 2002; Kurnatowska, 1998; Mendling et al., 2000; Mercure et al., 1996; Quindos et al., 1996). Hydrolytic enzymes of C. albicans have been implicated as virulence factors, and particular emphasis has been given to the extracellular protease, esterase and lipase (De Bernardis et al., 1999; Kantarcioglu 2002; Kurnatowska, 1998). The goals of the present study were: firstly, to determine the patterns of esterase

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electrophoretic types (zymotypes) among clinical isolates of vaginal C. albicans, secondly, to find out whether the zymotypes could be used to define strains of C. albicans for epidemiological purposes, thirdly, to determine the serotype of the strains. Moreover, the aim was to compare C. albicans strains with respect to their esterase patterns, their capability of secreting protease and lipase and their ability to assimilate carbohydrates. Experimental Materials and Methods 46 strains of Candida albicans were isolated from vagina of women suffering from vulvovaginitis. The age of patients ranged from 17 to 67 years. Samples were taken from the wall or fluid (pool) of the vaginal canal with a sterile cotton swab and immediately streaked on Sabouraud glucose plates agar. Isolates were found to be C. albicans if they showed germ tube production in rabbit serum and if they were able to produce chlamydospores on Nickerson-Mankowski medium. Apart from that, strains of Candida albicans were typed using API 20C AUX (bioMerieux). Proteolytic activities of the strains examined were determined using human albumin and bovine casein as substrates according to the procedure described by Staib (1965). Lipolytic activities were determined using the medium containing Tween 80 according to Werner (1966). API 20 C AUX test was performed according to the manufacturer’s instruction. For each of the isolates the serotype was determined by slide agglutination test according to Hannula et al. (2001). The determination of the esterase patterns was performed using the techniques described by Goullet and Picard (1991) and Branger et al. (1990) which were adapted in order to examine strains of C. albicans. The single colony of each examined strain of C. albicans growing on Sabouraud agar was inoculated into Sabouraud broth then used to determine the esterase pattern. C. albicans was grown overnight at 37°C in 40 ml of Sabouraud broth in Erlenmayer flasks being vigorously shaken. After centrifugation the yeasts were washed in 0.075 M Tris-0.06 M glycine buffer (pH 8,7), resuspended in 1.5 ml of the same buffer and then disrupted 10 times by refrigeration and thawing. Debris was removed at 10.000 × g for 15 min. at 4°C. The supernatants containing at least 6 mg of protein per 1 ml were stored at – 20°C until used for electrophoresis. Each strain was cultured and extracted at least twice. Horizontal slab polyacrylamide-agarose gel electrophoresis and an estimation of electrophoretic mobility were performed as described by Goullet and Picard, 1991 and Uriel 1966. The detection of hydrolytic activity to a naphthyl acetate, b naphthyl propionate and indoxyl acetate was carried out by staining with the dye Fast Blue.

Results All of the examined strains belonged to serotype A and all of them showed lipolytic activity. The examination of esterase electrophoretic motility allowed to differentiate the examined strains into four groups of which one group was constituted by strains not producing esterases detected in the present study. All of the three substrates used: "-naphthyl acetate, $-naphthyl propionate and indoxyl acetate, were hydrolysed by fungal esterases. 15 of the C. albicans strains exhibited an esterase pattern consisting of two distinctly stained bands. 7 strains showed a simple esterase pattern consisting of a single band and the pattern of 5 strains consisted of three bands (zymotypes A, B, C, respectively). The mobility of these enzymes and esterase patterns of the examined strains are shown in Fig. 1. 19 strains of C. albicans did not exhibit esterase activity detected in this examination. Basing on proteolytic activities, the strains were divided into four groups. 23 of 46 examined strains exhibited proteolytic activity to human albumin and bovine casein, 10 strains were active with respect to only bovine casein and 11 strains were active only with respect to human albumin. 2 strains did not exibit proteolytic activity with respect to both substrates used (Table I). On the basis of 20 carbon sources assimilated, the C. albicans isolates were categorized into 11 biotypes Fig. 1. Esterase patterns of C. albicans with the major biotype accounting for 21 (45.7%) strains Electrophoretic mobility of Candida albicans esterases in horizontal slab polyacrylamide – agarose gel electrophoresis. Three of numerical profile 2 576 174. 13 of them hydrolyzed esterase patterns of C. albicans are presented. All patterns show both substrates: human albumin and bovine casein. 8 of esterases which hydrolysed a-naphthyl acetate, b-naphthyl the strains from these 21 strains belonged to pattern A propionate and indoxyl acetate. 27 of 46 examined C. albicans of esterase production. The relationship between esterase strains exhibited esterase activity detectable in used method.

3 Table I Biotypes according to proteolytic activity of C. albicans Biotype I I III IV

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C. albicans biotypes

Proteolytic activity H.A.a B.C.b + + – + + – – –

Number of strains 23 10 11 2

H.A.a – human albumin, B.C.b – bovine casein (+) – presence of proteolysis (–) – absence of proteolysis

Table II The comparison of 21 strains of C. albicans exhibiting the same biochemical properties Proteolytic activity Numerical profile Lack of with respect to (2576174) of proteolysis a b 21 strains examined HA BC HA&BC

A

B

C

0

No of strains

8

2

2

9

3

4

13

1

Esterase pattern

HA a human albumin, BCb bovine casein A, B, C – zymogram characterized by two, one and three esterase bands, respectively 0 – lack of esterase pattern.

patterns and proteolytic activity of the strains exhibiting the most common biotype is shown in Table II. The remaining 25 strains belonging to ten different biotypes showed a variety of esterase paterns and they differed in their proteolytic activities to the substrates used, making any comparison impossible. Discusion Candida albicans is an important cause of a wide spectrum of diseases including vulvovaginitis (Farina et al., 2000; Linhares et al., 2001; Marai, 2001; Mendling et al., 2000). The presence of Candida vulvovaginitis cannot be definitively identified by clinical criteria (Linhares et al., 2001; Novikova et al., 2002). Among methods used in yeast infections diagnosis, the most important are cultures of these microorganisms (Linhares et al., 2001). The identification of C. albicans is based on a number of phenotypic characteristics, of which carbohydrate assimilation is of primary importance. For these purposes we used the commercial API 20 C AUX system, which is commonly used for diagnosis of Candida spp. (Ellabib et al., 2001; Mercure et al., 1996; Smith et al., 1999; Wadlin et al., 1999). When microbial strain typing methods are compared, the most important characteristics are typeability, reproducibility and discriminatory power. The results of the present study confirm those of other authors, and namely that the API system shows good reproducibility, sensitivity and specificity (Wadlin et al., 1999). However, the discriminatory power of API system is not high, and, if epidemiological investigation has to be performed, results of examination with the use of Api system should be supplemented with other methods. For example electrophoretic typing of esterases and biotyping based on proteolytic activity can be used as additional methods for differentiating strains. Other authors have already indicated the value of using a combined typing system (Quindos et al., 1996). We tentatively grouped the C. albicans strains into four categories, three with esterase patterns, and one without an esterase band. Because the C. albicans isolates show serological differences according to their geographical origin (Hannula et al., 2001; Mercure et al., 1996), serological examination of C. albicans seems to be very important from the epidemiological point of view. In our investigation, all examined strains belonged to the A serotype. It must be pointed out that the application of esterase zymogram in bacterial taxonomy and epidemiology is often used (Branger et al., 2003; Chetoui et al., 1998; Gilot and Andre, 1995 and 1996; Giver et al., 1998). The examination of properties of C. albicans enzymes is widely applied (De Bernardis et al., 1990; Pichova et al., 2001; Tsuboi et al., 1996; Vazquez-Reyna et. al., 1999) and may be useful for epidemiological purposes. Tsuboi et al 1996 showed that the induction level of C. albicans extracellular esterase was found to be correlated with fungal growth. Protease production is one of the most relevant factors of Candida spp. virulence in mucosal diseases, including vaginitis (De Bernardis et al., 1990 and 1999; Kantarcioglu and Yucel, 2002; Pichova et al., 2001; Rodrigues et al., 1999; Smolenski et al., 1997). C. albicans is significantly more proteolytic than the non-albicans yeasts (Wu and Samaranayake, 1999). Vaginal candidiasis may be observed as an acute disease which is characterized by a presence of inflamation symptoms. The cause of development of vaginal candidiasis may be reinfection from a gastrointestinal reservoir, sexual transmission, or impaired host defence mechanisms and enhanced Candida virulence (Kubota, 1998). There is clear evidence that proteolytic C. albicans strains were more virulent than the non proteolytic ones (De Bernardis et al., 1999). In our study, 95.7% of the investigated strains exhibited proteolysis, when albumin, albumin and casein or casein were used as substrates (Table I). Similar results of

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M o r a e s P.S., S. d e L i m a G o i a b a and E.A. T a k e t o m i. 2000. Candida albicans allergen immunotherapy in recurrent vaginal candidiasis. J. Investig. Allergol. Clin. Immunol. i10: 305–9. N o v i k o v a N., E. Y a s s i e v i c h and P.A. M a r d h. 2002. Microscopy of stained smears of vaginal secretion in the diagnosis of recurrent vulvovaginal candidosis. Int. J. STD AIDS. 13: 318–22. O d d s F.C., A.B. A b b o t t, R.L. S t i l l e r, H.J. S c h o l e r, A. P o l a k and D.A. S t e v e n s. 1983. Analysis of Candida albicans phenotypes from different geographical and anatomical sources. J. Clin. Microbiol. 18: 849–57. P i c h o v a I., L. P a v l i c k o v a, J. D o s t a l, E. D o l e j s i, O. H r u s k o v a - H e i d i n g s f e l d o v a, J. W e b e r, T. R u m l and M. S o u c e k. 2001. Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae. Inhibition with peptidomimetic inhibitors. Eur. J. Biochem. 268: 2669–77. Q u i n d o s G., V. L i p p e r h e i d e, B. B a r t u r e n, R. A l o n s o, J. B i k a n d i, R. S a n - M i l l a n, M. T e l l a e t x e, L. R i b a c o b a and J. P o n t o n. 1996. A new method of antibiotyping yeasts for subspecies discrimination and distribution in human clinical specimens. Eur. J. Epidemiol. 12: 55–62. R o d r i g u e s A.G., P.A. M a r d h, C. P i n a - Va z, J. M a r t i n e z d e O l i v e i r a and A.F. d a F o n s e c a. 1999. Is the lack of concurrence of bacterial vaginosis and vaginal candidosis explained by the presence of bacterial amines? Am. J. Obstet. Gynecol. 181: 367–70. S m i t h M.B., D. D u n k l e e, H. V u and G.L. W o o d s. 1999. Comparative performance of the RapID Yeast Plus System and the API 20C AUX Clinical Yeast System. J. Clin. Microbiol. 37: 2697–8. S m o l e n s k i G., P.A. S u l l i v a n, S.M. C u t f i e l d and J.F. C u t f i e l d. 1997. Analysis of secreted aspartic proteinases from Candida albicans: purification and characterization of individual Sap1, Sap2 and Sap3 isoenzymes. Microbiology 143: 349–56. S t a i b F. 1965. Serum-proteins as nitrogen source for yeastlike fungi. Sabouraudia 4: 187–193. T a y l o r B.N., C. F i c h t e n b a u m, M. S a a v e d r a, J. S l a v i n s k y I I I, R. S w o b o d a, K. W o z n i a k, A. A r r i b a s, W. P o w d e r l y and P.L. F i d e l J r. 2000. In vivo virulence of Candida albicans isolates causing mucosal infections in people infected with the human immunodeficiency virus. J. Infect. Dis. 182: 955–9. T s u b o i R., H. K o m a t s u z a k i and H. O g a w a. 1996. Induction of an extracellular esterase from Candida albicans and some of its properties. Infect. Immun. 64: 2936–40. U r i e l J. 1966. Method of electrophoresis in acrylamide-agarosa gels (in French). 1966. Bull. Soc. Chim. Biol. (Paris). 48: 969–82. Va z q u e z - R e y n a A.B., P. P o n c e - N o y o l a, C. C a l v o - M e n d e z, E. L o p e z - R o m e r o and A. F l o r e s - C a r r e o n. 1999. Purification and biochemical characterization of two soluble alpha-mannosidases from Candida albicans. Glycobiology 9: 533–7. W a d l i n J.K., G. H a n k o, R. S t e w a r t, J. P a p e and I. N a c h a m k i n. 1999. Comparison of three commercial systems for identification of yeasts commonly isolated in the clinical microbiology laboratory. J. Clin. Microbiol. 37: 1967–70. W e r n e r H. 1966. Studies on the lipase activity in yeasts and yeast-like fungi (in German). Zbl. Bakt. I Orig. 200: 113–124. W u T. and L.P. S a m a r a n a y a k e. 1999. The expression of secreted aspartyl proteinases of Candida species in human whole saliva. J. Med. Microbiol. 48: 711–20.

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Polish Journal of Microbiology 2005, Vol. 54, No 3, 233– 240

Purification and Characterization of Two Extracellular Lipases from Pseudomonas aeruginosa Ps-x HESHAM M. SAEED 1, TAHA I. ZAGHLOUL1, AHMED I. KHALIL2 and MOHAMED T. ABDELBAETH1 1 Institute

for Graduate Studies and Research, Department of Bioscience and Technology University of Alexandria, Chatby 21526, Alexandria, Egypt, 2 Institute of Graduate Studies and Research, Department of Environmental Studies, University of Alexandria, Chatby 21526, Alexandria, Egypt Received 2 March 2005, received in revised form 25 May 2005, accepted 27 May 2005 Abstract Two different extracellular lipases were isolated and purified from Pseudomonas aeruginosa Ps-x to apparent homogeneity using ammonium sulfate precipitation followed by ion exchange chromatography on Q- and S-Sepharose column. Both of the purified lipases are monomeric protein with molecular weight of 15.5 and 54.97 KDa respectively. The optimal activities of the enzymes were at 45 and 50°C and pHs 10.0 and 9.0. Calcium ions increase thermostability of both purified lipases I and II. The purified lipase I showed no metal ion dependence for its activity since EDTA up to 10 mM has no effect on the enzyme activity. However purified lipase II showed slight inhibition by EDTA at the same concentration. Moreover, a serine protease inhibitor, PMSF showed an inhibitory effect on both purified enzymes. K e y w o r d s: Lipases, Pseudomonas aeruginosa Ps-x, 16S rRNA

Introduction Lipases (triacyl glycerol acylhydrolases, EC 3.1.1.3) constitute a diverse and ubiquitous family of enzymes that in biological system initiate the catabolism of fats and oils by hydrolyzing the fatty acylester bonds of acylglycerols (Carriere et al., 1994 and Jose et al., 2004). Most of lipases remain active in a variety of organic solvents, where they can catalyze various transformations other than hydrolytic reaction by which they are defined (Margolin and Klibanov, 1987). Lipases are widely distributed in nature and have been found in many species of animals, plants, bacteria, yeast and fungi. Although their wide distribution, the enzymes from microorganisms are most interesting because of their potential application in various industries ranging from the use in laundry detergent to stereospecific biocatalysts (Maliszewska and Przemyslaw, 1992). Most of the microbial lipases are secreted into the culture medium and they differ from one another in their physical and biochemical properties. Since each industrial application requires specific properties of lipases, there is still an interest in additional lipases that could be used in new applications (Jaeger et al., 1994; Lambit and Goswami, 2002 and Kyu et al., 2005). The synthesis and secretion of extracellular lipases by various microorganisms appear to be controlled in a variety of ways which are only now beginning to be elucidated and the limited data on this subject have been reviewed by Jaeger et al., 1994. One of the best studied case, P. aeruginosa, lipase only appears in the culture medium at the end of logarithmic growth when a number of other hydrolytic exoenzymes are also released. However, lipases are produced during logarithmic growth in a minimal media when growth is dependent for carbon and energy upon hydrolysis of either a triglyceride or a detergent such as a Tween (Jaeger et al., 1994 and Stuer et al., 1986). This study described the production, purification and characterization of two extracellular lipases from P. aeruginosa Ps-x. Abbreviations: SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis, Q-Sepharose; Quaternary amino methyl Sepharose, EDTA; ethylene diamine tetraacetic acid. 1 Corresponding author: tel: 002034297005, fax: 002034285792, e-mail: [email protected]

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Experimental Materials and Methods Bacterial isolate identification. The bacterial isolate used in this study was a gift from Dr. Yossry Gohar, Department of Microbiology, Faculty of Science, Alexandria University, Egypt. This isolate was tested for lipase production using nutrient agar medium, pH 7.5 containing per liter: peptone, 10.0 g; NaCl, 5.0 g; CaCl2, 0.1 g; and agar, 20.0 g. The medium was sterile by autoclaving and let to cool to 45°C after that 1% of sterile Tween-20 or Tween-80 was added to the medium and mixed well then poured (20 – 25 ml) into 100 mm Petri dishes. The identification of the bacterial isolate was based on cell morphology, colony morphology, growth on nutrient broth and nutrient agar as well as several biochemical tests. The identification process was performed at the Fermentation and Biotechnology Center, El-Azhar University, Cairo. To confirm the biochemical tests results for isolate identification, 16S rRNA (rDNA) technique was carried out. DNA was isolated and purified according to Sambrook et al., 1989. Amplification of the 16S rDNA gene from the genome was carried out by polymerase chain reaction (PCR) using primers designed to amplify 16S rRNA gene. The forward primer was 5’-AGAGTTTGATCMTGGCTCAG-3’ and the reverse primer was 5’- TACGGYTACCTTGTTACGACTT-3’. The polymerase chain reaction analysis was performed with 100 ng of genomic DNA in a final volume of 50 :l, including a reaction buffer 1x, 30 pmole of each primer, and 2 units of Taq polymerase. Thermocycling consisted of an initial denaturation of 5 minutes at 94°C and of 30 cycles of 1 minute at 94°C (denaturation), 1 minute at 55°C (primers annealing), and 1.5 minutes at 72°C (extension). Polymerase chain reaction products were analyzed for purity check on 1% agarose gel by electrophoresis, stained with ethidium bromide (0.5 :g/ml), and visualized using ultraviolet transillumination. DNA sequencing. DNA was sequenced by the dideoxy chain termination method according to Sanger et al., 1977 using ABI Prism Ready Reaction Dye Terminator Sequencing Kit and analyzed on an ABI 377 automated sequencer. The nucleotide sequence obtained about 326 base pairs were then analyzed using nucleotide Blast search data base and have been deposited in the GenBank sequence data base and have the accession number AF419219. Lipase assay. Lipase activity was determined colorimetrically according to Kordel et al., 1991, where two solutions were prepared for the assay. Solution 1 contained 90 mg of pNPP (p-nitrophenyl palmitate), dissolved in 30 ml propane-2-ol. Solution 2 contained 2 g Triton X-100 and 0.5 g gum Arabic dissolved in 450 ml (Tris-HCl 50 mM) buffer at pH 8.0. The assay solution was prepared by adding 1 ml of solution 1 to 9 ml of solution 2 drop wise to get an emulsion that remained stable for 2 hrs. The assay mixture contained 900 :l of the emulsion and 100 :l of the appropriately diluted enzyme solution. The liberated p-nitrophenol was measured at 410 nm using Novospek, Pharmacia spectrophotometer. One unit of enzyme was defined as the amount of enzyme that releases 1 :mol of p-nitrophenol from the substrate. Protein analysis. Total protein concentrations of cell free supernatant and purified samples were assayed by the method of Bradford (Bradford, 1976) using a calibration curve established with bovine serum albumin as a standard. Proteins in these preparations were analyzed by SDS-PAGE on 10% gels according to the method of Laemmli (Laemmli, 1970). Gel analysis and molecular weight determination was done using Alpha Imager 1200 Tm gel documentation system. Monitoring bacterial growth and extracellular lipase production. The growth and activity of the extracellular lipase was monitored throughout the growth of the P. aeruginosa Ps-x strain on modified Williams Basal medium II (Williams et al., 1990) containing per liter: NH4Cl, 5g; NaCl, 5g; K2HPO4, 3.0 g; KH2PO4, 4.0 g; MgCl2 × 6H2O, 1.0 g and yeast extract 1.0 g. Deionized water was added to approximately 1 L, then the pH was adjusted to 7.5 with 10 N NaOH and autoclaved. Cells were activated by growing them overnight at 37°C on nutrient agar plates. Several recently growing colonies were transferred to a 50 ml of production medium and incubated at 37°C overnight with agitation at 150 rpm. Then, 1 ml was taken to inoculate 100 ml of the production medium and allowed to grow at 37°C with shaking at 150 rpm. Growth was monitored by measuring the absorbance at 420 nm. At the indicated time, 1.0 ml of the growing cultures was taken and centrifuged in a microcentrifuge at 8,000 rpm for 2 minutes and the supernatants were assayed for extracellular lipase using p-nitrophenylpalmitate as a substrate as described earlier. Enzyme production and purification. The extracellular lipase produced by P. aeruginosa Ps-x was first purified by salting out precipitation using ammonium sulfate at 80% saturation. P. aeruginosa cells were cultured aerobically in 500 ml production medium containing per liter: 5 g NaCl, 0.05 g CaCl2 × 2H2O, 10 g yeast extract and 10 ml Tween 20 for 18 hours at 37°C with agitation at 150 rpm. The culture was then centrifuged at 8,000 rpm for 20 minutes at 4°C using the small SS-34 small rotor. Solid ammonium sulfate was then added slowly to the culture filtrate at 70% saturation with gentle stirring on ice bath. After that, the mixture was allowed to stand at 4°C for overnight. The mixture was then centrifuged at 12,000 rpm for 30 minutes using the above rotor. Pellet was dissolved in 5 ml 0.02 M Tris-HCl pH 8.0 and dialyzed overnight against 2 liters of the same buffer. The protein content and the lipase activity were determined as described earlier. The concentrated dialyzed cell free supernatant was then applied to a column (2.5 × 7 cm) containing Quaternary amino methyl Sepharose fast flow (Q-Sepharose), which previously was equilibrated with 20 mM Tris-HCl, pH 8.5. The column was washed with 3 bed volumes of the same buffer at a flow rate of 60 ml/hour and the bound proteins were eluted with a linear gradient of NaCl (0 – 1.0 M) in the same buffer. Active fractions that contain lipase enzyme were then polled and concentrated using ammonium sulphate as mentioned before. The concentrated and dialyzed enzyme was further purified using S-Sepharose fast flow column (2.5 × 5 cm) prequilibrated with 20 mM Tris-HCl, pH 8.5 at a flow rate of 45 ml/hour. The column was washed with three bed volumes of the same buffer and the bound proteins were eluted using linear gradient of NaCl (0–0.5 M) in the same buffer. Lipase containing fractions were polled and concentrated as described before. pH optima, temperature and thermostability studies. Extracellular lipase optimum pH was determined over a pH values range from 6.0 to 10.0. Sodium phosphate buffer 0.1 M was used for pH 6.0 and 7.0, Tris-HCl 0.1 M for pH 8.0 and 9.0 and carbonate buffer 0.1 M for pH 10.0 and 11.0. The temperature optimum of the purified P. aeruginosa lipases was determined over a temperature range of 25 – 90°C in 50 mM Tris-HCl buffer pH 8.0. Thermostability of the purified enzymes was examined at a temperature range 40 – 70°C for different time intervals (10 – 60 minutes) in 50 mM Tris-HCl buffer pH 8.0 in absence and in the presence of 5.0 mM CaCl2. The residual activity was determined by taking 25 ml of the enzyme solution after specified time (10 – 60 minutes) and the assay was carried out as described before. Effect of some compounds on the activity of the purified enzymes. To examine the effect of EDTA (ethylenediamine tetraacetic acid), SDS (sodium dodecylsulphate), PMSF (phenylmethane sulfonyl fluoride) and DMSO (dimethyl sulfoxide) on

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the activity of the purified lipases, different concentrations of these compounds were prepared and the enzymes solution were pre-incubated with these compounds for 30 minute on ice after which the residual activity was determined as described before using p-nitrophenyl palmitate substrate. Effect of some metal ions on the activity of the purified enzymes. For determining the effect of metal ions on lipase activity, the purified enzymes were pre-incubated with 1–10 mM of the following metal ions MgCl2, ZnCl2, CaCl2 and NaCl for 30 minute on ice and then the residual activity was determined.

Results and Discussion Identification of bacterial isolate. Based on cell morphology and colony morphology, growth on nutrient broth and nutrient agar as well as several biochemical tests, the bacterial isolate was identified as Pseudomonas aeruginosa. To confirm the biochemical tests results for bacterial isolate identification, 16S rRNA methodology was carried out. The sequencing data obtained utilizing this strategy indicated that the isolate under study was 98% P. aeruginosa and the nucleotide sequence was deposited in the GenBank database, and given the accession number AF419219. Monitoring the bacterial growth and extracellular lipases enzymes production. The growth and extracellular lipase production level of P. aeruginosa Ps-x was monitored on modified William’s Basal medium II supplemented with 2% Tween-20. Figure 1 illustrates the growth behavior with incubation time and lipase production. The level of extracellular lipase enzyme production of P. aeruginosa increased gradually at the end of log phase (6 hours after inoculation) and reached its maximum level (74.66 U/ml) after 48 hours of inoculation. It was reported that the extracellular lipase production normally appears in the fermentation medium when the bacterial cell growth reach to the end of the logarithmic. Moreover, the observation of similar growth rates for Pseudomonas strains during the logarithmic growth phase is in accordance with the concept that the production of these enzymes is advantageous to the microorganism only when nutrients become limiting i.e., during the late log phase or early stationary phase (Lee and Rhee, 1993). 10

80 Ab .420 nm .

U /ml

70

Ab 420 nm

50 40 30

0 .1

Lipase activity

60 1

20 10 0 .01

0 0

2

5 7 Time after inoculation

9 (hrs )

12

48

Fig. 1. Monitoring of growth and extracellular lipase production by P. auroginosa strain Ps-x on modified basal medium

Purification and characterization of P. aeruginosa extracellular lipases. P. aeruginosa lipases enzymes were purified using (NH4)2SO4 selective precipitation (70%) followed by ion exchange chromatography on Q and S-Sepharose Fast Flow columns. It was found that the unbound proteins were eluted first using 20 mM Tris-HCl, pH 8.5 buffer giving only one protein peak that overlapped with the lipolytic activity. Upon using NaCl gradient (0.0– 1.0 M) in the same buffer, one major and one minor protein peaks appeared as shown in Figure 2. Surprisingly, the minor protein peak showed a lipolytic activity as well. This result indicated that, at least P. aeruginosa Ps-x strain under study produced two extracellular lipases of different biochemical characters. Thus, upon using Q-Sepharose Fast Flow anion exchanger, lipase enzyme contained in the first peak (LipI) was purified to 6.36 fold with specific activity of 165.5 (Units/mg protein/ml). While lipase enzyme contained in the second minor peak that eluted with NaCl gradient (LipII)

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Ab.280 nm.

5

U/ml

NaCl gradient (M)

70

0.8

60

4

50 3

40

2

30

0.6

Activity

Ab 280 nm

1.0

80

0.4

20

1

NaCl

6

0.2

10

0

0.0

0 1

11

21

31

41

51

61

71

81

Fraction Number Fig. 2. Fractionation pattern of the extracellular lipase enzyme produced by P. aeruginosa upon using Q-Sepharose Fast Flow anion exchange column

0.1

80 Ab.280 nm.

Units/ml

NaCl gradient (M)

0.8

60 40

0.04 20

0.02 0

0.2

0.0

0 1

11

21

31

41

51

0.4

NaCl

0.6

0.06

Activity

Ab 280 nm

0.08

1.0

61

Fraction Number

Fig. 3. Fractionation pattern of extracellular lipase II (LipII) from P. aeruginosa on S-Sepharose Fast Flow column

was found to be purified to almost 3.77 fold and with specific activity of 98.2 (Units/mg protein/ml) as indicated in Table 1. Interestingly, the eluted lipase I (LipI) was found to be almost pure enzyme as judged by SDS-PAGE as shown in Figure 4 just by selective precipitation with ammonium sulphate followed by anion exchanger chromatography on Q-Sepharose column. Figure 3 show the fractionation pattern of lipase II on S-Sepharose cation exchanger. It was found that; one distinctive protein peak was appeared in the elution buffer 20 mM Tris-HCl pH 7.4 that overlapped with the lipase activity. By applying NaCl gradient Table I Purification of extracellular lipases produced by P. aeruginosa Ps-x Purification steps (NH4)2SO4 concentrated and dialyzed enzyme

a

Lipase Volume activity (ml) Units/ml a

Specific Fold Recovery activity purification (%) U/mg protein

Total Units

Protein content mg/ml

7448

19

26

18

496.5

Q-Sepharose Column First Peak (Lipase I)

8

503.2

4025.6

3.04

165.5

6.36

54

Second Peak (Lipase II)

20

88.4

1768.6

0.9

98.2

3.77

23.7

S-Sepharose Column Second Peak (Lipase II)

18

38.4

691.2

0.1

384

14.76

9

One unit of enzyme is the amount of enzyme that liberate one micromole of D-nitrophenol per minute at 37°C

1

100

3

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Lipases from P. aeruginosa KDa

1

2

3

4

1

5

97.4

2

97.4

66.2 66.2 43.0 43.0 30.0 30.0 20.1

14.2 Fig. 4. Sodium dodecyl sulfate polyacrylamide gel (10%) electrophoresis of P. aeruginosa cell free supernatant (Lane 2), ammonium sulfate concentrated and dialyzed sample (Lane 3), Q-Sepharose purified lipase I (Lane 4), and Q-Sepharose unbound eluted proteins (Lane 5). Lane 1 represents molecular weight markers protein

20.1

Fig. 5. Sodium dodecyl sulfate gel (10%) electrophoresis of purified lipase II from P. aeruginosa (lane 2). Lane 1 represents molecular weight markers protein

(0–1.0 M) in 20 mM Tris-HCl pH 7.4, two major protein peaks appeared that retained no lipolytic activity. Ion-exchange chromatography on S-Sepharose Fast Flow resulted in increase in both the specific activity and fold purification, 384 and 14.74 versus 98.2 and 3.77 after Q-Sepharose Fast Flow column (Table I). SDS-PAGE showed that both of the purified lipases were composed of a single type of subunit with a molecular weight of 15.5 (lipase I) and 54.97 KDa (lipase II) respectively as shown in Figure 4 and 5. pH and temperature optima of the purified lipase. The pH range of purified P. aeruginosa lipase I and II was detectable over a wide range between 6–10 with an optimum pH value at 9.0 and 10 respectively. It has been reported that most of lipases produced by Pseudomonas sp. have pH optimum around pH 7.0– 9.5 (Lee and Rhee, 1993; Dong et al., 1999). It was found that, P. aeruginosa purified lipase I showed lipolytic activity over a wide range of temperature from 40 to 70°C with an optimum temperature of 50°C which is in agreement with that of P. aeruginosa EF2 and significantly higher than that of Pseudomonas PACIR and psychrotrophic strain of P. fluorescens (Gibert et al., 1991; Yong and Rhee, 1993 and Lee et al., 1993). On the other hand, purified lipase II showed an optimum temperature of 45°C. Temperature and pH stability of the purified enzymes. Temperature stability of the purified P. aeruginosa lipase I and lipase II was investigated for a period of one hour at temperature range from 40– 70°C in absence and in the presence of calcium ions. It was noticed that, the residual activity for both lipase I and lipase II decreased as the exposure time and temperature increased in absence of calcium chloride. It was found that, after 60 minutes of exposure to 70°C, purified lipase I retained about 47.74% of its original activity. On the other hand purified lipase II retained about 36.7% of its original activity after exposure to 70°C for 60 minutes. Thermostability in the presence of calcium ions indicated that, at a temperature of 40 and 50°C, CaCl2 not only stabilize lipase I against thermal inactivation but also enhanced the lipolytic activity of the purified enzyme (146.19 and 116.4% respectively after exposure time of 60 minutes). On the other hand, CaCl 2 at the same concentration and under identical assay condition had no effect on thermostability of purified lipase II at all studied temperatures (from 40–70°C). These results indicated that P. aeruginosa lipase I that have a molecular weight of 15.5 KDa, can be stabilized against thermal inactivation by the addition calcium chloride ion while calcium chloride ion has no effect on the thermostability of the purified lipase II (molecular weight 54.97 KDa). The effect of metal ions on thermostability and activity of some enzyme has been investigated before and it has been reported that metal ions such as Ca2+, Mn2+ and Co2+ can stabilize and activate some enzymes such as xylose isomerases which bind two metal ions, one cation is directly involved in catalysis and the second is mainly structural (Whitlow et al., 1991 and Kasumi et al., 1982). The role of calcium ions in relation to thermostability was also investigated

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and extensively studied in Thermoactinomyces vulgaris subtilisin-type serine-protease thermitase and it was found that, thermitase contains three Ca2+-binding sites; one of them is not present in its mesophilic homologues (Teplyakov et al., 1990). A thermophilic homologue of thermitase, the Bacillus AK1 protease, contains one more Ca2+ than thermitase does, and it is significantly more kinetically stable than thermitase in the presence of Ca2+. It has been reported that, in certain P. aeruginosa lipase, the Ca2+ ion might be involved in the correct positioning of the histidine residue of the catalytic triad, since three of the Ca 2+ ion ligand are contained in a loop together with the histidine residue (Mohamed et al., 2003 and Umesh et al., 2003). Thus, it was clear from these results that, both P. aeruginosa purified lipase I and II can be affected by the exposure to temperature in presence and in absence of CaCl2 metal to different degree. Difference in thermostability of both enzymes may be explained by the presence of metal binding site that can bind for example calcium ions and hence increase thermostability and enhance the lipolytic activity of purified lipase I but not lipase II that lack this binding site. One of the most important characteristic features of the enzymes involved in detergent industries is the pH stability of these enzymes. Stability of P. aeruginosa lipases against pH was examined at pH’s range from 6– 10 for 20 days (480 hours) at room temperature and it was found that, both enzymes are stable at pHs from 6– 9 for 20 days. Interestingly, both enzymes showed some sort of activation after 36 hours of storage at pHs from 6– 8 and this activation phenomenon increased gradually with the incubation time at indicated pHs. On the other hand both purified lipase I and II showed pH instability at pH 10 and were gradually inactivated as the incubation time increases at pH 10 and the residual activities were found to be 28.69 and 50.0 % for lipase I and II respectively after 20 days. One possible explanation for this observation is that different pH values affected the ionization properties of some amino acid residues in the enzyme, which may be resulted in the variation of the activity and stability of the enzymes (Dong et al., 1990). Effect of some metal ions and some compounds on lipase activity. The effect of some metal ions on the activities of P. aeruginosa purified lipases was also investigated. It was found that, NaCl, CaCl2 and MgCl2 enhance the lipolytic activity of purified lipase I and II and the residual activity was higher in the case of CaCl2 than that of NaCl and MgCl2 (Table II). This observation is also exhibited by other Pseudomonas lipases (Fox and Stepaniak, 1983 and Yamamoto and Fujiwara, 1988). The observation that lipase activity was significantly enhanced in the presence of these metal ions probably reflects the ability of these salts to react with free fatty acids adhering to the oil droplets to diminish interfacial charge effects and/or to increase droplet surface area. Interestingly, ZnCl 2 at a concentration of 10 mM inhibits the lipolytic activity for both lipase I and II and the inhibition was higher in the case of lipase I than lipase II as shown in Table II. This inhibitory effect of Zn+2 is also exhibited by the other Pseudomonas lipases (Yamamoto and Fujiwara, 1988) and could probably be due to the direct interaction of metal with the catalytic site, although the alteration of the properties of the interface must be considered and it may be attributed to their binding of the thiol group of the enzyme or sulfhydryl groups which may be present in the active center of some microbial lipases (Schrag et al., 1991). Table II Effect of some compounds on the activity of P. aeruginosa purified lipase I and II Compound None

Lipase residual activity* (%) Lipase I 100

Lipase II

Compound

Lipase residual activity* (%) Lipase I

100

EDTA

20.03

Lipase II 100

NaCl

111.04

101.01

DMSO

173.5

173.5

CaCl2

122.04

270.32

SDS

94.54

101.21

108.89

PMSF

15

MgCl2 ZnCl2

97 106.72

41.84

30.64

* Lipase residual activity (%) reflects the percentage of enzyme activity at a given compound concentration when compared to the enzymatic activity at zero concentration and under identical assay conditions.

The effect of EDTA, DMSO, SDS and PMSF was also studied. Data of Table II indicated that the divalent metal-chelating agent, EDTA at a concentration of 10 mM showed slight inhibitory effect on the activity of lipase I and no effect on the activity of lipase II. It was reported that lipase enzyme produced by P. aeruginosa EF2 is not affected by metal chelating agent, EDTA which indicate that this kind of lipolytic activity is independent of metal ions (Gibert et al., 1991) in contrast to some other metal ions dependent

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Pseudomonas lipases (Fox and Stepaniak, 1983 and Yamamoto and Fujiwara, 1988). Dimethyl sulfoxide (DMSO) enhanced the lipolytic activity of both purified lipase I and II and this observation was agreed with the lipase of P. aeruginosa YS-7. It was found that sodium dodecyl sulphate (SDS) at a concentration of 10 mM showed little inhibition of purified lipase II while the same concentration had no effect on the activity of purified lipase I (94.541% and 101.219% respectively). Since most of the microbial true lipases contain a catalytic triad Ser-Asp-His, inhibition study using PMSF was carried out. PMSF at a concentration of 10 mM showed a drastic inhibitory effect on both lipases as shown in Table II and the residual activity was found to be 15% and 41.84% for lipase I and II respectively. This inhibitory effect of PMSF also exhibited for some Pseudomonas sp. (Schrag et al., 1991; van Oort et al., 1989 and Svendsen et al., 1995). Conclusion. In the present work, two lipases from Pseudomonas aeruginosa Ps-x were isolated, purified and biochemically characterized. When compared with lipases from other Pseudomonas sp. (Stuer et al., 1986; Brady et al., 1990; Sugiura, 1977 and Mencher and Alford, 1967), these lipases differ in the following properties: molecular weight, wider stable pH and temperature ranges and purified lipase II being of higher molecular weight compared to other Pseudomonas sp. lipases. The satisfactory pH and thermal stability will make these two lipases very attractive for future synthetic applications. Genomic library was constructed from purified P. aeruginosa DNA and screened for extracellular lipases. A number of potentials positives lipases producing clones were obtained. Future research will focus on the sequencing, subcloning and overexpression of these genes in E. coli. Moreover, studies of the substrate specificities, stereospecificities and some aspects of the kinetics of their secretion into the fermentation medium will be undertaken. Literature B r a d y L., A.M. B r z o z o w s k i, Z.S. D e r e w e n d a, E. D o d s o n, G. D o d s o n, S. T o l l e y, J.P. T u r k e n b e r g, L. C h r i s t i a n s e n, B. H u g e j e n s e n, L. N o r s k o v, L. T h i m and U. M e n g e. 1990. A serine protease triad forms the catalytic center of a triacylglycerol lipase. Nature 343: 767–770. B r a d f o r d M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254. C a r r i e r e F., Y. G a r g u r i, H. M o r e a u, S. R a n s a c, E. R o g a l a s k a and R. Ve r g e r. 1994. Gastric lipases: cellular, biochemical and kinetic aspects. pp. 181–205. In: P. Woolley, S.B. Petersen (eds). Lipases-their structure, biochemistry and application. Cambridge, UK.Cambridge University Press. D o n g H., S. G a o, S. H a n and S. C a o. 1990. Purification and characterization of a Pseudomonas sp. Lipase and its properties in non-aqueous media. Biotechnol. Appl. Biochem. 30: 251–256. F o x P.F. and L. S t e p a n i a k. 1983. Isolation and some properties of extracellular heat stable lipases from Pseudomonas fluorescens strain AFT 36. J. of Diary Research. 50: 77–89. G i b e r t E.J., A. C o r n i s h and C.W. J o n e s. 1991. Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2. J. General Microbiol. 137: 2223–2229. J a e g e r K.E.H., S. R a n s a c, B.W. D i j k s t r, C. C o l s o n, M. v a n H e u v e l and O. M i s s e t. 1994. Bacterial lipases. FEMS Microbiol. Rev. 15: 29–63. J o s e M.P., C. O r t i z, M. F u e n t e s, G. F e r n a n d e z, J.M. G u i s a n and R.F. L a f u e n t e. 2004. Use of immobilized lipases for lipase purification via specific lipase-lipase interaction. J. Chrom. A. 1038: 267–273. K o r d e l M., B. H o f m a n n, D. S c h o m b u r g and R.D. S c h m i d. 1991. Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data. J. Bacteriol. 173: 4836–4841. K a s u m i T., K. H a y a s h a i and N. T s u m u r a. 1982. Roles of magnesium and cobalt in the reaction of glucose isomerase from Streptomyces griseofuscus S-41. Agric. Biol. Chem. 9: 21–30. K y u R.K., D.Y. K w o n, S.H. Y o o n, W.Y. K i m and K.H. K i m. 2005. Purification, refolding and characterization of recombinant Pseudomonas fluorescens lipase. Protein Expression and Purification. 39: 124–129. L a m b i t K. and P. G o s w a m i. 2002. Isolation of a Pseudomonas lipase produced in pure hydrocarbon substrate and its application in the synthesis of isoamyl acetate using membrane immobilized lipase. Enz. Microb. Tech. 31: 727–735. 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. L e e Y.P., G.H. C h u n g and J.S. R h e e. 1993. Purification and characterization of Pseudomonas fluorescens SIK W1 lipase expressed in E. coli. Biochem. Biophys. Acta 1169: 156–164. L e e S.Y. and J.S. R h e e. 1993. Production and partial purification of lipase from Pseudomonas putida 3SK. Enz. Microb. Tech. 15: 617–623. M a r g o l i n A.L. and A.M. K l i b a n o v. 1987. Peptide synthesis catalyzed by lipases in anhydrous organic solvents. J. Am. Chem. Soc. 109: 3802–4. M a l i s z e w s k a I. and M. P r z e m y s l a w. 1992. Production and some properties of lipase from Penicillium citrinum. Enz. Micrb. Tech.. 14: 190–193. M o h a m e d K., P. Va n G e l d e r, W. B i t t e r and J. T o m m a s s e n. 2003. Role of the calcium ion and the disulfide bond in the Burkholderia glumae lipase. J. Mol. Cat. B: Enzymatic 22: 329–338. M e n c h e r J.R. and J.A. A l f o r d. 1967. Purification and characterization of the lipase of Pseudomonas fragi. J. Gen. Microbiol. 48: 317–328.

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S a m b r o o k J., E.F. F r i t s c h and T. M a n i a t i s. Molecular cloning. A laboratory Manual. Cold Spring Harbor Laboratory, NY. 1989. S a n g e r F., S. N i c k l e n and A.R. C o u l s o n. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74: 5463–5467. S t u e r W., K.E. J a e g e r and U.K. W i n k l e r. 1986. Purification of extracellular lipase from Pseudomonas aeruginosa. J. Bacteriol. 168: 1070–1074. S c h r a g J.D., L. Y u n g e, W. S h a n g and C. M i r o s l a w. 1991. Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum. Nature 351: 761–764. S v e n d s e n A., K. B o r c h, M. B a r f o e d, T.B. N i e l s e n, E. G o r m s e n and S.A. P a t k a r. 1995. Biochemical properties of cloned lipases form Pseudomonas family. Biochem. Biophys. Acta 1259: 9–17. S u g i u r a M., T. O i k a w a, K. H i r a n o and T. I n u k a i. 1977. Purification, crystallization, and properties of triacylglycerol lipase from Pseudomonas fluorescens. Biochim. Biophys. Acta 488: 353–358. T e p l y a k o v A.V., I.P. K u r a n o v a, E.H. H a r u t y u n y a n, B.K. V a i n s h t e i n, C. F r o m m e l, W.E. H o h n e and K.S. W i l s o n. 1990. Crystal structure of thermitase at 1.4 A resolution. J. Mol. Biol. 214: 261–279. U m e s h K., A.U. J i n w a l, R. R o y, C. A b h i j i t, A. C h o w d h u r y, A.P. B h a d u r i c and P.K. R o y. 2003. Purification and characterization of an alkaline lipase from a newly isolated Pseudomonas mendocina PK-12CS and chemoselective hydrolysis of fatty acid esters. Bioorg. Med. Chem. 11: 1041–1046. v a n O o r t M.G., A.M. D e v e e r, R. D i j k m a n, M.L. T j e e n k, H.M. V e r h e i j, G.H. d e H a a s, E. W e n z i g and F. G ö t z. 1989. Purification and substrate specificity of Staphylococcus hyicus lipase. Biochemistry 28: 9278–9285. W h i t l o w M., A.J. H o w a r d, B.C. F i n z e l, T.L. P o u l o s, E. W i n b o r n e and G.L. G i l l i l a n d. 1991. A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose. Proteins 9: 153–173. W i l l i a m s C.M., C.S. R i c h t e r, J.M. M a c k e n z i e and J.C.H. S h i h. 1990. Isolation, identification, and characterization of a feather-degrading bacterium. J. App. Environ. Microbiol. 56: 1509–1515. Y a m a m o t o K. and N. F u j i w a r a. 1988. Purification and some properties of a castor-oil hydrolyzing lipase from Pseudomonas sp. Agri. Biological. Chem. 52: 3015–3021. Y o n g S.L. and J.S. R h e e. 1993. Production and partial purification of a lipase from Pseudomonas putida 3SK. Enz. Microb. Technol. 15: 617– 623.

Polish Journal of Microbiology 2005, Vol. 54, No 3, 241– 247

Extraction of Milk-clotting Enzyme Produced by Solid State Fermentation of Aspergillus oryzae HODA M.A. SHATA

Microbial Chemistry Department, National Research Center, El Behoos Street, Dokki, Cairo, Egypt Received 9 March 2005, received in revised form 7 June 2005, accepted 10 June 2005 Abstract Studies on the extraction of milk-clotting enzyme after solid-state fermentation (SSF) of wheat bran by a local strain of Aspergillus oryzae LS1 were done. The extraction of the enzyme was found to be depended on different parameters like nature of extractant, soaking time, temperature etc. From different inorganic and organic extractants, calcium chloride (0.05%) and glycerol (40%) were found to be the best solvents for leaching out milk-clotting enzyme. The optimum volume of calcium chloride was 5 ml/g biomass. An extraction time of 180 min. at 30°C and 100 rpm was sufficient to extract out nearly 28% of the enzyme (2666.7 U/g biomass). Most of the enzyme (about 98.71%) was recovered in four repeated extractions. K e y w o r d s: milk-clotting enzyme, extraction, solid-state fermentation, Aspergillus oryzae

Introduction Solid-state fermentation is the one in which microorganisms secret the necessary enzyme for degradation of the available substrate molecules in order to meet their nutritional requirement. In this system the fermented mass consists of non-utilized solid substrate containing microbial cells, spores and product for which a number of co-metabolites are formed during the course of fermentation. Solid-state fermentation is the fermentation in the absence of free liquid and recovery of fermentation products requires its extraction from the solid fermented medium with a suitable solvents or solutions (Tunga et al., 1998). From the bulky solid mass, getting the product out of the system has many problems (Lonsane and Krishnaiah, 1992). Attempt has been made by researchers to isolate the desired product from the fermented mass by various techniques due to its implication on process economics (Bjurstrom, 1985 and Calton et al., 1986). With the initial moisture levels used for fermentation, squeezing of the solid medium itself hardly yields any extract and even if there is any, the volume might not be sufficient for complete extraction. The medium must therefore, be soaked for some time in an adequate amount of a suitable extractant for total recovery of the product. A common extractant is distilled or deionized water (Silman, 1980; Wang et al., 1984; Yano et al., 1991 and Ghidyal et al., 1993). Bhumibhamon (1986) used distilled water for extraction of glucoamylase enzyme from solid-state on rice bran. Malathi and Chakraborty (1991) also used distilled water for extraction of alkaline protease from solid wheat bran fermentation. Tunga et al., 1998 also reported that fermented mass was soaked with water for two hours at room temperature to extract proteolytic enzyme. Other extractants have also been used for extract other enzymes. Yang and Chiu (1987) used sodium chloride solution for protease extraction. Shata (1999) extracted glucoamylase by sodium chloride solution. RiveraMunoz et al. (1991) used 0.002 M succinate buffer for lipase and protease while Castilho et al. (2000) extracted pectinases from wheat bran by acetate buffer at pH 4.4. This paper presents, studies on milk-clotting enzyme extraction from wheat bran, a cheap agro-residue, fermented by a local strain of Aspergillus oryzae LS1.The study includes the effect of some factors which influence the efficiency of leaching out of the enzyme and its efficacy in the leaching technique.

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Experimental Materials and Methods Microorganisms. Aspergillus oryzae LS1 a local strain obtained from the Microbial Resource Center at Cairo (MIRCEN), Ain Shams University was used through out this work. Inoculum preparation. Fungal spores were prepared on Czapek’s Dox agar medium in Petri dish incubated for 7 days at 30°C to assure good sporulation. The amount of inocula determined as colony-forming units (CFU) was prepared by scraping agar discs of 1.5 cm diameter aseptically from agar plate. Fermentation. Wheat bran (10 gram) in 500 ml conical flask sterilized at 121°C for 20 min. were mixed with 15 ml of a sterile salt solution containing (g/l): magnesium chloride 15 × 10–3, and fructose, 8 previously sterilized at 110°C for 10 min. Each flask was inoculated with 186 × 10 6 colony-forming units. Incubation was carried out at 30oC for three days. Extraction. The extraction of the enzyme from the fermented biomass was carried out with distilled water, tap water and different salt solutions of, potassium chloride, magnesium chloride, calcium chloride and sodium chloride. The investigation was also, carried out to see the effect of different organic solvents like glycerol, methanol, ethanol and acetone in concentration of 5%. Unless otherwise stated, the extraction was conducted in 250 ml conical flask containing one gram of fermented biomass and 5 ml of solvent solution. Each type of solvent was added separately to the fermented biomass and kept for 180 min. on a rotary shaker at 100 rpm at 30°C. Then the extract was collected by filtration through Whatman No 1 filter paper and the clear solution containing milk-clotting enzyme was assayed. To optimize the extraction process, different experiments were carried out by varying the volume and solvent concentration in addition to time, temperature and physical state (agitation or stationary) keeping all other conditions at optimum levels. Milk-clotting enzyme assay. The assay of milk-clotting enzyme activity was carried out according to the standard procedure described by Carlson et al. (1985) with some modifications using 12% (w/v) dried skim milk in 0.01 M of calcium chloride solution as substrate. The reaction mixture contained 0.5 ml of enzyme and 1 ml of substrate and incubated at 70°C. The enzyme activity was calculated according to Otani et al. (1991) as follows: Milk-clotting activity units = 2400/TXS/E where T is the time necessary for the crude fragment formation, S is the volume of milk, and E is the volume of enzyme. Protein estimation. Protein content was estimated by the method of Ohanistti and Bar (1978).

Results Influence of solvent type. Effect of different solvents on milk-clotting enzyme extraction from solidstate fermented wheat bran is presented in Figs. 1 and 2. Inorganic and organic solvents beside tap and distilled water were used.

MgCl2 NaCl KCl CaCl2 ###

Milk-clotting activity (U/g biomass)

2500

2000

1500

1000

500

0.00

0.02

0.04

0.06

0.08

0.10

Salt conc. g/100 ml

Fig. 1. Effect of different concentrations of magnesium chloride, sodium choride, potassium choride and calcium choride on extraction of milk-clotting enzyme from SSF

3

243

Milk-clotting activity U/g biomass

Milk-clotting enzyme extraction from SSF by A.oryzae

1500

1000

500

0

Acetne

Ethanole

Methanol Glycerol Solvents

Tap water

Dist. water

Fig. 2. Effect of different organic solvents and water extraction of milk-clotting enzyme from SSF

Among four inorganic salt solutions tested (sodium chloride, potassium chloride, calcium chloride and magnesium chloride), calcium chloride in concentration of 0.05% (w/v) gave the best extraction of milkclotting enzyme from the fermented solids. As the concentration of calcium chloride increased, the amount of extracted enzyme increased reaching its maximum value at 0.05% (w/v). Addition of different concentrations of potassium chloride, sodium chloride or magnesium chloride, gave no significant results compared to calcium chloride solution. Among four organic solvents (methanol, glycerol, ethanol, and acetone) investigated in concentration of 5% (v/v), glycerol gave the maximum leaching of milk-clotting enzyme. To optimize the concentration of glycerol in the solution used as solvent, experiment was carried out by varying the glycerol concentration from 5% to 60% keeping other parameters constant. As the concentration of glycerol increased milk-clotting enzyme recovery increased (Fig. 3). The increasing trend was observed up to 40%, beyond which there was a slight decrease. Therefore, calcium chloride of 0.05% (w/v) was used through the next experiments. Optimization of solvent volume. Fig. 4 shows the effect of different volumes of 0.05% calcium chloride solution used as extractant on recovery of milk-clotting enzyme from the fermented wheat bran in SSF. 2200

Milk-clotting activity U/g biomass

2100 2000 1900 1800 1700 1600 0

10

20

30

40

50

60

Glycerol % (v/v)

Fig. 3. Effect of different concentrations of glycerol on extraction of milk-cloting enzyme from SSF

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Shata H.M.A.

2200

6

1800

4

C

1600

Protein mg/ml

2000

1400

B

Milk-clotting activity (U/g biomass)

Milk-clotting activity U/g biomass

2400

2

1200 1000

0

5

10

15

20

25

Calcium chloride volume (ml)

Fig. 4. Effect of calcium chloride volume on extraction of milk-cloting enzyme from SSF

Milk-clotting activity (U/g biomass)

3000 2500 2000 1500 1000 500 0 Aggitation

Static

Fig. 5. Effect of phisical state on extraction of milk-cloting enzyme from SSF

The range investigated was 2.5–30 ml/g biomass with 3 h soaking time at 30°C and 100 rpm. The total activity of the extract increased up to 5 ml/g biomass, above which it remained more or less constant. However, the amount of extracted protein increased with the increase of volume of calcium chloride up to 20 ml. Effect of physical state. Fig. 5 shows the effect of physical state on extraction of milk-clotting enzyme. It was found that agitation was quite satisfactory for maximum recovery of the enzyme. So it was selected as the best condition for enzyme recovery. Effect of extraction time. Soaking of the fermented solid substrate with calcium chloride solution at the optimum ratio 5 ml/g biomass was done at 30°C and at 100 rpm for different periods varying from 15 min to 48 hours. As shown from Fig. 6 maximum amount of milk-clotting enzyme was extracted after 180 min. Longer extraction time did not result in significant gain of recovery. Effect of temperature on extraction process. To evaluate the effect of temperature on leaching process, a series of experiments were carried out at 10, 30, 40, 50, 60 and 70°C, keeping the other experimental conditions at optimum. The results presented in Table I show that 30°C was the optimum temperature for extraction of the enzyme. At higher temperature above 30°C the activity decreased.

3

245

Milk-clotting enzyme extraction from SSF by A.oryzae

Milk-clotting activity (U/g biomass)

2800 2600 2400 2200 2000 1800 1600 1400 1200

0

50

100

150

200

250

Time (min.)

Fig. 6. Effect of soaking time on extraction of milk-cloting enzyme produced in SSF

Repeated extraction. Repeated extractions were carried out for recovery of the most milk-clotting enzyme from the fermented solid mass. From Table II, it was observed that most of milk-clotting enzyme was recovered during four repeated extractions. About 28% of the total activity was found in each of the first three extractions. Table I Effect of temperature on extraction of milk-clotting enzyme MCA

Temperature °C

U/ml

U/g

10

16

80

Remaining activity %

Loss of activity %

3

97

20

26

133

5

95

30

505

2526

100

0

45

231

1154

46

54

50

53

267

11

89

60

20

100

4

69

70

0

0

0

0

MCA = Milk-clotting activity

Table II Milk-clotting activity recovery in five repeated extractions with fresh calcium chloride solution Number of recovery stage

MCA U/ml

U/g

Cumulative MCA U/ml

MCA U/ml %

Cumulative MCA U/ml %

Recovery in the 1st washing

533

2667

533.3

28

28

Recovery in the 2nd washing

527

2637

1060.8

29

57

Recovery in the 3rd washing

527

2637

1588.3

28

86

Recovery in the 4th washing

264

1319

1852.0

13

99

Recovery in the 5th washing

5

27

1857.3

1

100

MCA = Milk-clotting activity

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Discussion Solid-state fermentation is fermentation in the absence of free liquid, and recovery of the fermentation product requires its extraction from the solid fermented medium. Among the salt solutions tested, calcium chloride (0.05% w/v) gave the best extraction of milk-clotting enzyme from the fermented solids. However, it’s interesting to notice that the extraction of milk-clotting enzyme increased with all salt solutions, as compared with tap or distilled water. The above observation was recorded for the protease enzyme produced by Rhizopus oryzae in solid-state fermentation of wheat bran, (Aikat and Bhattacharyya, 2000). They mentioned some weak ionic binding of the enzyme with either cell or substrate. On the other hand Wang (1967) reported from the studies on Mucor hiemalis that most of the proteolytic activity is cell surface bound and could be released by elution with sodium chloride suggesting that the enzyme was probably bound to the cell by weak ionic bonds. Castilho et al. (1999 and 2000) reported that acetate buffer gave better results for extraction of pectinases and from the fermented solids by Asp. niger than glycerol or distilled water. The poor performance of glycerol can be explained by its higher viscosity, which has a negative effect on mass transfer. Corpe and Winters (1972) found most of the protease activity to be cell bound, largely being associated with the cell envelop. They mentioned also that about 50% of the specific protease activity of the cell envelope could be removed with three washes with sea water, indicating a fairly weak binding. The effect of calcium chloride concentration on enzyme extraction was a remarkable observation. Up to 0.05% (w/v), extraction increased probably due to the salting-in effect of electrostatic effect of the salt (Aikat and Bhattacharyya, 2000). Beyond the above mentioned concentration a slight salting-out effect due to hydrophobic effect began to show up resulting in a slight decrease in enzyme activity. In the present investigation the presence of organic solvent in water was more effective in milk clotting enzyme extraction than tap or distilled water. Tunga et al. (1999) reported that in SSF protease was present in solid fermented mass due to some binding force. Water has the highest dielectric constant compared with other organic solvents. They mentioned that as the dielectric constant decrease, the force of interaction between the enzyme and solvent may increase. Therefore, leaching out of the enzyme by inorganic solvent was more effective. However, it was noticed that with different concentrations of glycerol, a small increase in enzyme activity was recorded at higher concentration as shown in Fig. 3. This was due to the protective effect of glycerol on enzyme activity. Scopes (1982) mentioned that glycerol forms strong hydrogen bonds with water, reducing the water activity. On the other hand, Tunga et al. (1999) reported that protease extraction from SSF was maximally achieved using ethanol-glycerol mixture. They suggested that the hydroxyl group of ethanol and glycerol may form hydrogen bonding with protein molecules and this gives the stability of the enzyme molecule. As the literature report (Stryer, 1975, and Bailey and Ollis 1986) the stability of enzymes can be improved by using sorbitol as solvent. A contact time of 180 min. at 30°C provided the best enzyme activities for most of the conditions tested. Periods of 15, 30 or 60 min. seem not to be enough for total solubilization of milk-clotting enzyme present in wheat bran medium. Increasing the time of extraction up to 24 hours has no promising value on extraction of the enzyme. This loss could have been due to the prolonged mechanical or to a greater extraction of denaturant agents (Ghildyal et al., 1991). Castilho et al. (2000) investigated the effect of incubation time on protease extraction in SSF and they found that 30 min. provided the best time for enzyme extraction. Also, Ikasari and Mitchell (1996) studied the effect of incubation time on extraction of protease in SSF. They concluded that maximum enzyme recovery was achieved at 22°C with 60 min. contact time. According to Aikat and Bhattacharyya (2000) the amount of solute increases with the increase of solvent volume. Our results showed that the level of milk-clotting activity reaches the maximum at 5 ml/g biomass and then decreased. These results can be explained from the calculated specific activity since more solvent cause the release of non specific protein. Excessively large volume of extractant used for greater extraction would also yield enzyme solutions to be too dilute to be profitably utilized. Agitation of the fermented biomass with the extractant at 30°C and 100 rpm gave appreciable amount of milk-clotting enzyme compared with stationary condition. Agitation helps to reduce enzyme adhesion to cell biomass and also disperses the fermented mass uniformly in the continuous phase of the solvent (Tunga et al., 1999). Maximum yield of enzyme recovery was obtained at 30°C but at higher temperature it was less. Higher temperatures may have inhibitory effect on the enzyme activity and make it less stable. This fact could be explained by the two opposite effects on enzyme extraction. On one hand, greater temperatures increase

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the solute solubility and diffusivity, therefore, a higher activity is attained in the extract. On the other hand, enzyme is susceptible to deactivation, which will increase as temperature and contact time increase. In order to maximize enzyme recovery from solid-state culture, repeated extractions were investigated. It was verified that about 98.7% of milk-clotting activity were recovered in four repeated extractions using calcium chloride solution. As the extract obtained from the fifth washing contained about 1.3%, the recovery of the enzyme was very small. Therefore, four repeated extractions are sufficient, although dilute extracts were obtained. Literature A i k a t K. and B.C. B h a t t a c h a r y y a. 2000. Protease extraction in solid-state fermentation of wheat bran by a local strain of Rhizopus oryzae and growth studies by soft gel technique. Process Biochemistry. 35: 907–914. B a i l e y J.E. and D.E. O l l i s. 1986. Biochemical engineering fundamentals. McGraw Hill, New York. B h u m i b h a m o n O. 1986. Glucoamylase of fungus isolated from rotting cassava tuber by Aspergillus niger. Mircen-J. App. Microbiol. Biotech. 2: 443–482. B j u r s t r o m E.E. 1985. Biotechnology. Fermentation and down stream processing. Chem. Eng. 92: 120–158. C a l t o n G.J., G.S. C o b b s and J.P. H a m m a n. 1986. Manual of industrial. microbiology and biotechnology; p. 436–445. In: A.I. Demain and M.A. Solomn (eds), American Society of Microbiology, Washington, USA. C a r l s o n A., C.G. H i l l and N.F. O l s o n. 1985. Improved assay procedure for determination of milk-clotting enzyme. J. Dairy Sci. 68: 290–299. C a s t i l h o L.R., T.L.M. A l v e s and R.A. M e d r o n h o. 1999. Recovery of pectolytic enzymes produced by solid-state culture of A. niger. Process Biochemistry. 34:181–186. C a s t i l h o L.R., R.A. M e d o n h o and T.I. A l v e s. 2000. Production of pectinases obtained by solid-state fermentation of agro-industrial residues with Aspergillus niger. Bioresource Technology 71: 45–50. C o r p e W.A. and H. W i n t e r s. 1972. Hydrolytic enzymes of some periphytic marine bacteria. Can. J. Microbiol. 18: 1483–1490. G h i l d y a l N.P., M. Ramakrishna, B.K. I o n s a n e, N.G. K a r a n t h and M.M. K r i s h n a i a h. 1993. Temperature variations and amyloglucosidase levels at different bed depths in a solid-state fermentation system. Chem. Eng. J. 51: 17–23. G h i l d y a l N.P., M. R a m a k r i s h n a, B.K. L o n s a n e and N.G. K a r a n t h. 1991. Efficient and simple extraction of mouldy bran in a pulsed column extractor for recovery of amyloglucosidase in concentrated form. Process Biochemistry 26: 235–241. I k a s a r i L. and D.A. M i t c h e l l. 1996. Leaching and characterization of Rhizopus oligosporus acid protease from solid-state fermentation. Enzyme and Microbial Technology 19: 171–175. L o n s a n e B.K. and M.M. K r i s h n a i a h. 1992. Leaching of the product and further down stream processing. In: solidsubstrate cultivation, Doelle, H.S.; Mitchell, D.A. and C.E. Rolz. (eds), Elsevier Science Publishers, Essex, U.K. M a l a t h i S. and R. C h a k r a b o r t y. 1991. Production of alkaline protease by a new Aspergillus flavus isolate under solidsubstrate fermentation conditions for use as a depilation agent. Applied and Environmental Microbiology. 57: 712–716. O h a n i s t t i S.T. and J.K. B a r. 1978. A simplified method of quantitating protein. The buriet and phenol reagents. Anal. Biochem. 86: 193–200. O t a n i M., M. I w a g a k i and A. M o n s o n o. 1991. The screening trees having milk-clotting activity. Animal. Sci. Technol. (Jap). 62: 417– 423. R i v e r a - M u n o z G., J.R. T i n o c o - V a b n i c, S. S a n c h e z and A. F a r r e s. 1991. Production of microbial lipase in a solid-state fermentation system. Biotech. Lett. 13: 274–280. S c o p c s R.K. 1982. Protein purification: Principles and Practice. Springer. New York. S h a t a M.A.H. 1999. Ph.D. Thesis Microbiology Cairo University, Egypt. S i l m a n R.W. 1980. Enzyme formation during solid-substrate fermentation in rotating vessels. Biotech. Bioeng. 22: 411–420. S t r y e r L. 1975. Biochemistry; W. H. Freeman and company; New York; 2 nd Edition. T u n g a R., R. B a n e r j e e and C.B. B h a t t a c h a r y y a. 1999. Some studies on optimization of extraction process for protease production in SSF. Bioprocess Engineering 20: 485–489. T u n g a R., R. B a n e r j e e and B.C. B h a t t a c h a r y y a. 1998. Optimizing some factors affecting protease production under solid-state fermentation. Bioprocess Engineering 19: 187– 190. W a n g H.L. 1967. Release of proteinase from mycelium of Mucor hiemalis. J. Bact. 93: 1794–1799. W a n g H.L., E.W. S w a i n and C.W. H e s s e l t i n e. 1984. Isolation, purification and characterization Glucoamylase of Amylomyces rouxil J. Food. Sci. 49: 1210–1211. Y a n g S.S. and W.F. C h i u. 1987. Protease production with starchy agricultural wastes by solid-state fermentation. Microbe. 283. 86 Int. Cong.. Microbiol, 14 Meet. Y a n o T., S. A s h i d a, T. T a c h i k i, H. K u m a g a i and T. T o c h i k u r a. 1991. Development of a soft gel cultivation method. Agric. Biol. Chem. 55: 379–385.

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Cloning and Preliminary Characterization of a GATC-specific $2-class DNA:m6A methyltransferase Encoded by Transposon Tn1549 from Enterococcus spp. MONIKA RADLIÑSKA1,*, ANDRZEJ PIEKAROWICZ1, MARC GALIMAND2 and JANUSZ M. BUJNICKI3 1 Institute

of Microbiology, University of Warsaw, ul. Miecznikowa 1, 02-096 Warszawa, Poland des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15, France 3 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland 2 Unité

Received 13 April 2005, received in revised form 8 June 2005, accepted 10 June 2005 Abstract A recent study revealed a subfamily of N6-adenine (m 6A) methyltransferases that comprises a few functionally studied eukaryotic members acting on mRNA and prokaryotic members acting on DNA as well as numerous uncharacterized open reading frames. Here, we report cloning and functional characterization of a prokaryotic member of this family encoded by transposon Tn1549 from Enterococcus spp. K e y w o r d s: DNA methyltransferase, sequence specificity

DNA of prokaryotic and eukaryotic organisms and their viruses is often modified by methylation, carried out by S-adenosyl-L-methionine (AdoMet)-dependent DNA methyltransferases (MTases). In Eukaryota DNA methylation plays a role in crucial regulatory processes, such as regulation of gene expression, embryonic development, genomic imprinting, and carcinogenesis (reviewed by Scarano et al., 2005). Most DNA MTases in prokaryota belong to the restriction-modification (RM) systems, where they serve to protect the host genome against the cleavage by a cognate restriction endonuclease (REase). However, some prokaryotic MTases (here termed “solitary”) are not associated with REases and are involved in processes distinct from restriction, such as DNA mismatch repair, regulation of gene expression, and control of timing of DNA replication (reviews: Dryden, 1999; Noyer-Weidner and Trautner, 1993). The best studied solitary MTase is Dam of Escherichia coli, which specifically methylates adenine residues within the palindromic sequence GATC, to yield N6-methyladenine (m6A) (Herman and Modrich, 1982). It is noteworthy that GATC methylation serves as a regulator of gene expression including virulence factors. Accordingly, the Dam activity was found to be necessary for both in vitro and in vivo virulence of numerous pathogenic bacteria (Chen et al., 2003; Heithoff et al., 1999; Watson et al., 2004). All known DNA MTases are homologous (i.e. they evolved from one common ancestor by accumulating divergent mutations) but their sequences are strongly divergent. One of the characteristic features of DNA MTases is the variability of the linear arrangements of nine common motifs (I–VIII and X). According to the possible linear arrangements of conserved and variable regions, DNA MTases were subdivided into 6 classes: ", $, (, *, g and . (Malone et al., 1995). The majority of DNA MTases fall into the classes ", $, and ( (Bujnicki, 2002; Malone et al., 1995). Recently, a subfamily of $-class enzymes was identified that include structurally unusual DNA:m 6A MTases M.MunI and M.AvaI as well as m6A MTases acting on mRNA (homologs of the MT-A70 protein) (Bujnicki et al., 2002; Matveyev et al., 2001). These enzymes Abbreviations: RM, restriction-modification; MTase, methyltransferase; REase, restriction endonuclease; TRD, target recognition domain; m6A, N6-methyladenine; m4C, N4-methylcytosine; m 5C, C5-methylcytosine; AdoMet, S-adenosyl-L-methionine * to whom correspondence should be addressed, e-mail: [email protected]

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lack the large variable region involved in the target sequence recognition, which is present between motifs VIII and X in the “orthodox” $-class MTases (Bujnicki et al., 2002). For clarity, this subclass of MTases will be referred to hereafter as $2, as proposed earlier (Matveyev et al., 2001). Phylogenetic analyses and structure prediction suggested that M.MunI and M.AvaV are related to mRNA MTases more closely than to any other known DNA MTases. Interestingly, these two bacterial MTases are functionally dissimilar: M.MunI is a part of a restriction-modification system (Siksnys et al., 1994) while M.AvaV is a solitary MTase (Matveyev et al., 2001). Their specificity is also different: M.MunI methylates the second adenine in the hexanucleotide CAATTG while M.AvaV methylates the GATC tetranucleotide (i.e. exhibits the Dam specificity). Thus, a comprehensive characterization of this intriguing subfamily of DNA MTases requires functional characterization of other prokaryotic members.

Fig. 1. Sequence alignment of the M.MunI/M.AvaV/M.EfaBMDam family of prokaryotic $2-class MTases, including many uncharacterized members Protein names follow the REBASE format, and in the first panel include the Gene Identification numbers (NCBI). N-terminal extensions of M.MunI (15 aa) and MspMCORF3385P (210 aa, including a predicted ParB-like nuclease domain) have been omitted for clarity. Dashes represent insertions or deletions. Identical and conservatively substituted residues are shown on dark background (black and grey, respectively). Conserved motifs (Malone et al., 1995) are indicated; it is noteworthy that motifs III and IX are missing.

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Among the proteins closely related to M.MunI and M.AvaI we found A B C M D E F an uncharacterized open reading frame (ORF18; dubbed M.EfaBMORFAP) in REBASE (Roberts et al., 2005) encoded by transposon Tn1549, which confers VanB-type resistance in Enterococcus spp. (Garnier et al., 2000); GenBank Acc.no. AF192329. Neither phylogenetic studies reported earlier (Bujnicki et al., 2002) nor the detailed comparison of sequence similarities between the ORF18 protein and M.MunI or M.AvaI (Figure 1) could reveal whether ORF18 may share sequence specificity with any of these MTases. Therefore, we cloned the corresponding gene and tested its activity in vivo. DNA manipulations, general techniques, and standard reactions were done according to protocols described for E. coli (Sambrook et al., 2002) or following recommendation of the enzyme’s suppliers. The plasmid expressing the putative M.EfaBMORFAP MTase was constructed as follows: the 444 bp region encompassing open reading frame 18 was amplified by PCR using Tn1549 DNA from Enterococcus faecalis 268–10 and the following primers: Primer Ofr18Nde: 5'-GAAGGAGATATACATATGTTGTTTATTTCAACGTACAACATC-3' corresponds to the 5' terminal part of the orf18 gene and possesses an optimized Shine-Dalgarno sequence (underlined), the NdeI site (italic) with translation codon ATG (bold). Primer Orf18Mun: 5'-CAATTGCTACGTCAGGATAAGGTCACATTCCAC-3' is complementary to the 3' terminal part of the orf18 gene and possesses a stop codon (bold) and the MunI restriction site (italic). The amplified bluntended PCR fragment was inserted in the SmaI and Ecl136II cleaved Fig. 2. Cleavage of pEfaORF18KS and pBluescript KSII(+) [Stratagene] generating plasmid pEfaORF18KS car- pBluescript DNA isolated from E. coli rying the orf18 gene cloned in-frame with the $-galactosidase promoter GM2163 cells. of the pBluescript vector. The nucleotide sequence of orf18 was verified. Aliquotes of 0.4 :g DNA were digested in Plasmids encoding an active DNA MTase are methylated in vivo and 20 :l reaction volumes with 10 u (25-fold hence resistant to digestion by the corresponding restriction endo- excess) of enzymes in buffers recommended by the manufacturers for 8 hrs at nuclease, while the DNA from cells lacking the MTase activity are 37oC. Lane A: non-digested DNA of completely cleaved. The degree of resistance of the plasmid DNA to pBluescript; lane B: pBluescript digested digestion by the restriction enzyme can be used as an indicator of the with MboI; lane C: pBluescript digested with lane D: pEfaORF18KS digested relative in vivo activity of the MTase clones. For this assay, pBluescript Bsp143I; with Bsp143I; lane E: pEfaORF18KS diplasmid carrying orf18 gene and the empty vector pBluescript were gested with MboI; lane F: non-digested grown in E. coli GM2163 (dam dcm) cells. Plasmid DNA from over- DNA of pEfaORF18KS; lane M DNA Ladder Mix (GeneRuler™ – Fermentas). night cultures was prepared using DNA minipreparation kits [Sigma] according to the instruction of the supplier. The purified DNA was tested for sensitivity to cleavage by several restriction enzymes (including MunI and MboI) known to be inhibited by activity of m6A MTases (data not shown). pEfaORF18KS DNA was completely digested with all enzymes used except MboI. The digestion reaction for pEfaORF18KS was repeated using isoschizomeric restriction enzymes MboI and Bsp143I, which differ in their sensitivity to Dam methylation. The cleavage by Bsp143I is not affected by adenine methylation in the 5’GATC3’ sequence, whereas MboI depends on by this modification (Hermann and Jeltsch, 2003; McClelland et al., 1994). The results presented in Figure 2 show that plasmid pEfaORF18KS DNA was totally resistant to cleavage by MboI but sensitive to Bsp143I. On the other hand, the pBluescript vector DNA was cleaved by MboI and Bsp143I, indicating that the cloned insert of pEfaORF18KS exerts the GATC-specific DNA:m6A MTase activity. In this study, we have cloned and characterized the in vivo activity of M.EfaBMORFAP a M.MunI/M.AvaV homolog from Enterococcus spp. transposon Tn1549. Preliminary studies suggest that the product of orf18 encodes a DNA:m6A MTase with a Dam-like sequence specificity (GATC) – i.e. like M.AvaV and unlike M.MunI. Therefore, we suggest to rename M.EfaBMORFAP as M.EfaBMDam. It will be interesting to determine the function and sequence specificity of other members of the M.MunI/M.AvaV/M.EfaBMDam subfamily. The identification of a Dam-like MTase on mobile genetic element Tn1549, which confers antibiotic resistance in clinical isolates of Enterococcus spp., suggests that this important determinant of bacterial virulence may be transmitted by horizontal gene transfer. It remains to be determined if M.EfaBMDam may

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be essential for the virulence of Enterococcus spp. In the light of the fact that Dam MTases are considered as potential drug targets it is important to note that MTases from different classes, such as the “orthodox” Dam from E. coli and T-even phages – " class (Herman and Modrich, 1982), Dam MTases from phages such as HP1, VT-2 or T1 (Bujnicki et al., 2001; Piekarowicz and Bujnicki, 1999; Radliñska and Bujnicki, 2001) – ( class and M.EfaBMDam and M.AvaV – $2 class (Matveyev et al., 2001) are structurally very divergent, and therefore may require the development of different specific inhibitors. Acknowledgments. This work was supported by the Polish State Committee for Scientific Research (grant 2P04B00827)

Literature B u j n i c k i J.M. 2002. Sequence permutations in the molecular evolution of DNA methyltransferases. BMC Evol. Biol. 2: 3. B u j n i c k i J.M., M. F e d e r, M. R a d l i ñ s k a and R.M. B l u m e n t h a l. 2002. Structure prediction and phylogenetic analysis of a functionally diverse family of proteins homologous to the MT-A70 subunit of the human mRNA: m6A methyltransferase. J. Mol. Evol. 55: 431–44. B u j n i c k i J.M., M. R a d l i ñ s k a, P. Z a l e s k i and A. P i e k a r o w i c z. 2001. Cloning of the Haemophilus influenzae Dam methyltransferase and analysis of its relationship to the Dam methyltransferase encoded by the HP1 phage. Acta. Biochim. Pol. 48: 969–83. C h e n L., D.B. P a u l s e n, D.W. S c r u g g s, M.M. B a n e s, B.Y. R e e k s and M.L. L a w r e n c e. 2003. Alteration of DNA adenine methylase (Dam) activity in Pasteurella multocida causes increased spontaneous mutation frequency and attenuation in mice. Microbiology 149: 2283–90. D r y d e n D.T. 1999. Bacterial DNA methyltransferases. p. 283–340. In: Cheng X, Blumenthal RM (eds) S-Adenosylmethioninedependent methyltransferases: structures and functions. World Scientific Publishing, NJ. G a r n i e r F., S. T a o u r i t, P. G l a s e r, P. C o u r v a l i n and M. G a l i m a n d. 2000. Characterization of transposon Tn1549, conferring VanB-type resistance in Enterococcus spp. Microbiology 146 1481–9. H e i t h o f f D.M., R.L. S i n s h e i m e r, D.A. L o w and M.J. M a h a n. 1999. An essential role for DNA adenine methylation in bacterial virulence. Science 284: 967–70. H e r m a n G.E. and P. M o d r i c h. 1982. Escherichia coli dam methylase. Physical and catalytic properties of the homogeneous enzyme. J. Biol. Chem. 257: 2605–2612. H e r m a n n A. and A. J e l t s c h. 2003. Methylation sensitivity of restriction enzymes interacting with GATC sites. Biotechniques 34: 924–6, 928, 930. M a l o n e T., R.M. B l u m e n t h a l and X. C h e n g. 1995. Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyltransferases, and suggests a catalytic mechanism for these enzymes. J. Mol. Biol. 253: 618–632. M a t v e y e v A.V., K.T. Y o u n g, A. M e n g and J. E l h a i. 2001. DNA methyltransferases of the cyanobacterium Anabaena PCC 7120. Nucleic. Acids. Res. 29: 1491–506. M c C l e l l a n d M., M. N e l s o n and E. R a s c h k e. 1994. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic. Acids. Res. 22: 3640–3659. N o y e r - W e i d n e r M. and T.A. T r a u t n e r. 1993. Methylation of DNA in prokaryotes. EXS 64: 39–108. P i e k a r o w i c z A. and J.M. B u j n i c k i. 1999. Cloning of the Dam methyltransferase gene from Haemophilus influenzae bacteriophage HP1. Acta Microbiol. Pol. 48: 123–9. R a d l i ñ s k a M. and J.M. B u j n i c k i. 2001. Cloning of enterohemorrhagic Escherichia coli phage VT-2 dam methyltransferase. Acta Microbiol. Pol. 50: 161–7. R o b e r t s R.J., T. V i n c z e, J. P o s f a i and D. M a c e l i s. 2005. REBASE-restriction enzymes and DNA methyltransferases. Nucleic Acids Res. 33 Database Issue: D230–2. S a m b r o o k J., D.W. R u s s e l l and J. S a m b r o o k. 2002. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. S c a r a n o M.I., M. S t r a z z u l l o, M.R. M a t a r a z z o and M. D’ E s p o s i t o. 2005. DNA methylation 40 years later: Its role in human health and disease. J. Cell. Physiol. S i k s n y s V., N. Z a r e c k a j a, R. V a i s v i l a, A. T i m i n s k a s, P. S t a k e n a s, V. B u t k u s and A. J a n u l a i t i s. 1994. CAATTG-specific restriction-modification munI genes from Mycoplasma: sequence similarities between R.MunI and R.EcoRI. Gene 142: 1–8. W a t s o n M.E., Jr., J. J a r i s c h and A.L. S m i t h. 2004. Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence. Mol. Microbiol. 53: 651–64.

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Co-occurrence of Urogenital Mycoplasmas and Group B Streptococci with Chlamydial Cervicitis DANIELA FRIEDEK1, ALICJA EKIEL1, MA£GORZATA ROMANIK1, ZBIGNIEW CHELMICKI2, BARBARA WIECHULA1, IWONA WILK1, JAROS£AW JÓZWIAK3 and GAYANE MARTIROSIAN 1,3* 1 Department

1 Department

of Medical Microbiology, Medical University of Silesia, Katowice, Poland and Clinic of Gynecology and Endocrinology, Medical University of Silesia, Katowice, Poland 3 Department of Histology and Embryology Center of Biostructure Research, Warsaw Medical University, Warsaw, Poland

Received 8 March 2005, received in revised form 21 June 2005, accepted 23 June 2005 Abstract The aim of our study was to evaluate whether in women with chlamydial cervicitis urogenital mycoplasmas and group B streptococci (GBS) colonization is found more often than among women with non-chlamydial cervicitis. This study included 351 (mean age 31.7 ± 6.82) not pregnant, menstruating, sexually active women. We confirmed a high frequency (49.3%) of C. trachomatis infection among women with cervicitis. Cervical ectopia was confirmed in 26.5% of examined women, in half of them ectopia was associated with chlamydial infection. We did not notice differences in frequency of colonization by urogenital mycoplasmas and GBS among women with chlamydial and non-chlamydial cervicitis. K e y w o r d s: Chlamydia trachomatis, GBS, urogenital mycoplasmas, cervicitis, ectopia

Urogenital mycoplasmas and group B streptococci (GBS) are microorganisms colonizing female urogenital tract and playing an important role in the pathology of fetus and newborn. Urogenital mycoplasmas are often isolated, even in 54% of tested sexually active women of childbearing age (Schlicht et al., 2004). Sexual transmission of GBS is questioned (Honig et al., 2002), while it is recognized in urogenital mycoplasmal infection (Keane et al., 2000, Nunez-Troconis, 1999). Today Chlamydia trachomatis is on the first place among sexually-transmitted bacteria (Millman et al., 2004). Cervicitis, often with co-occurring ectopia, is the dominating clinical finding during C. trachomatis infection (Critchlow et al., 1995, Giedrys-Kalemba et al., 1994). Thus, the aim of our work was to evaluate, whether in women with chlamydial cervicitis urogenital mycoplasmas and GBS colonization is found more often than among women with non-chlamydial cervicitis. This study included 351 (mean age 31.7 ± 6.82) not pregnant, menstruating, sexually active women who attended the Department and Clinic of Gynecology and Endocrinology, Medical University of Silesia in Katowice between 2001 and 2004. Cytological examination of cervix was performed in each case. All studied women had symptoms of cervicitis: mucopurulent endocervical discharge and/or greater or equal to 30 neutrophils per × 1000 field on the cervical Gram stain, and/or bleeding contact. Patients with gonococcal infection and those receiving antibiotic therapy within the month before consultation were excluded from the study. Sterile swabs were used to obtain material for testing/culturing of expected microorganisms (Friedek et al., 2004). First swab (no 1) from vaginal fornix for GBS culturing was inoculated on Columbia sheep blood agar plate and incubated aerobically for 24– 48 hours at 37°C. Identification of GBS was based on latex Slidex Streptokit (bioMerieux, France). Susceptibility of isolated GBS to antibiotics (ampicillin, * Corresponding author: Gayane Martirosian1,3, Department of Medical Microbiology, Medical University of Silesia, 18 Medyków str. 40-752 Katowice, Poland, phone/fax: +48 32 262 5075, e-mail: [email protected], [email protected]

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erythromycin and clindamycin) was examined by disk-diffusion method. Second swab (no 2) from endocervical canal for isolation of genital mycoplasmas was inoculated in urea-arginine-broth transport medium (bioMerieux, France). Culturing of genital mycoplasmas was performed by using Mycoplasma IST (bioMerieux, France), according to manufacturer’s instruction. Mycoplasma IST shows good sensitivity and specificity for U. urealyticum (100% and 90%, respectively), and for M. hominis (100% and 85% respectively) (Rastawicki et al., 2004). Third swab (no 3) from endocervical canal for detection of C. trachomatis was fixed by acetone on a slide. Chlamydia Direct IF – DIF (bioMérieux, France) was used for C. trachomatis antigen detection, according to manufacturers instruction. Slides were examined in Nikon Model HB – 10101AF fluorescent microscope (x40 objective). In studied group chlamydial etiology of cervicitis was confirmed in 49.3% (173/351). Genital mycoplasmas were isolated in 25.9% of women with cervicitis. There were statistically insignificant differences between occurrence of urogenital mycoplasmas in women with and without chlamydial cervical infection. U. urealyticum was a more frequently isolated species than M. hominis (Table I). Table I Prevalence of cervical ectopia, urogenital mycoplasmas and GBS among women with chlamydial and non-chlamydial cervicitis C. trachomatis – positive C. trachomatis – negative women (n = 173) women (n = 178) No

%

No

%

ectopia

42

24.3

51

28.7

Ureaplasma urealyticum

36

20.8

31

17.4

Mycoplasma hominis

4

2.3

3

1.7

Ureaplasma urealyticum and Mycoplasma hominis

7

4.0

10

5.6

23

13.3

26

14.6

GBS

The frequency of GBS isolation was 13.3% in chlamydia-positive and 14.6% in chlamydia-negative women. All isolated GBS strains were sensitive to ampicillin, only 9.6% of strains were resistant to erythromycin and 7.7% – to clindamycin. Cervical ectopia was confirmed in 26.5% (93/351) of examined women. In 42 out of them ectopia was associated with cervical chlamydial infection. In regions, where early screening program for detection of C. trachomatis infection was established, percent of infection is very low: in the USA – 4.7%, in Sweden – 5.4%, in Norway – 2.4% (Bakken et al., 2004, Egger et al., 1998, Miller et al., 2004). In Poland, frequency of C. trachomatis infection in studied groups of symptomatic and asymptomatic women is around 20– 40% (Choroszy-Król et al., 1994, GiedrysKalemba et al., 1994, Zbroch et al., 2004). It is a well-known fact that cervicitis may be a predisposing factor for cervical ectopia (Critchlow et al., 1995, Giedrys-Kalemba et al., 1994). In our study in 45.2% of women with cervical ectopia we showed co-existence of C. trachomatis infection. It is in concordance with the data of other authors: 46.9% reported by Giedrys-Kalemba et al. (1994) and 39.7% by Barnes et al. (1990). However, when analyzing cervical ectopia rate in women with and without C. trachomatis infection, we obtained similar results (24.3% and 28.7%, respectively). Urogenital mycoplasmas are frequently isolated from clinical samples. We did not notice differences in frequency of colonization by urogenital mycoplasmas among women with chlamydial and non-chlamydial cervicitis. The ratio was 27.2% and 24.7%, respectively (Table I). Maeda et al. (2004) did not observe statistically significant differences in the frequency of isolation of mycoplasmas among NGU patients withand without chlamydial infection. U. urealyticum was isolated much more often than M. hominis, which agrees well with the results of other authors (Keane et al., 2000, Schlicht et al., 2004). Schlicht et al. (2004) showed high prevalence of genital mycoplasmas among sexually active young women with cervicitis (54% for ureaplasmas and 26% for M. hominis). They also observed a high level (16%) colonization of healthy female volunteers by mycoplasmas. High level of mycoplasmal colonization in asymptomatic women was also reported by Keane et al. (2000): appropriatly 29% for U. urealyticum and 12% for M. hominis. In our study we demonstrated 14% of GBS-positive swabs obtained from vaginal fornix. We did not observe any significant correlation between occurrence of C. trachomatis and GBS or urogenital mycoplas-

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mas and GBS. Honig et al. (2002) did not demonstrate any correlation of vaginal colonization with GBS and chlamydial infection or other STIs. Frequency of isolation for these streptococci from the urogenital tract of healthy women was estimated to be 7% to 34% (Bayo et al., 2002, Manning et al., 2001). In spite of long-time using of penicillins in the treatment of streptococcal infections GBS are still sensitive to this group of antibiotics. The sensitivity of GBS to penicillins and percentage of resistance to erythromycin (9.6%) and clindamycin (7.7%) in our study was similar to that reported by others (Stiller et al., 2003, Weisner et al., 2004). Our study confirms high frequency of C. trachomatis infection among women with cervicitis in the region of Upper Silesia. However we demonstrated that C. trachomatis infection does not influence urogenital colonization by mycoplasmas and GBS. Literature B a k k e n I.J., F.E. S k j e l d e s t a d, T. O v r e n e s s, S.A. 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Polish Journal of Microbiology formerly Acta Microbiologica Polonica

2005, Vol. 54, No 3

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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