Polish Journal of Microbiology

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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 [email protected] biol.uw.edu.pl

Archives of Acta Microbiologica Polonica, from 2004 Polish Journal of Microbiology online www.microbiology.pl\pjm\ at PTM Journals online www.microbiology.pl Visit the home page to browse contents, gallery, links page and instructions to authors in HTML and PDF formats

Editorial correspondence should be addressed to Editors of Polish Journal of Microbiology 02-096 Warsaw, Miecznikowa 1, Poland Correspondence regarding subscription and spedition of Polish Journal of Microbiology should be addressed to National Institute of Public Health, Division of Clinical Microbiology and Prevention of Infections 00-725 Warsaw, Che³mska 30/34 tel. 48 (22) 841 33 67, fax 48 (22) 841 29 49, e-mail: cls.edu.pl

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

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

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

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

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

Polish Journal of Microbiology formerly Acta Microbiologica Polonica

2005, Vol. 54, No 2

CONTENTS IN MEMORIAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ORIGINAL PAPERS

Numerical analysis of electrophoretic periplasmic protein patterns of Aeromonas sp. strains SZCZUKA E., KAZNOWSKI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Receptors for endogenous and heterogenous hydroxamate siderophores in Staphylococcus aureus B47 WYSOCKI P., LISIECKI P., MIKUCKI J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

91 97

Extended-spectrum $-lactamase-producing Klebsiella pneumoniae in a neonatal unit: control of an outbreak using a new ADSRRS technique

KRAWCZYK B., SAMET A., CZARNIAK E., SZCZAPA J., KUR J. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Detection of Clostridium difficile and its toxin A (TcdA) in stool specimens from hospitalized patients

WRÓBLEWSKA M.M., SWOBODA-KOPEÆ E., ROKOSZ A., NURZYÑSKA G., BEDNARSKA A., £UCZAK M. . . . . . . . . 111

Slime production and cell surface hydrophobicity of nasopharyngeal and skin staphylococci isolated from healthy people

MALM A., BIERNASIUK A., £OŒ R., KOSIKOWSKA U., JUDA M., KORONA-G£OWNIAK I., GÓRNIEWSKI G. . . . . . . . 117

Resistance patterns of Streptococcus pneumoniae strains isolated in the west Pomerania province in 2001–2003

NOWOSIAD M.M., GIEDRYS-KALEMBA S.T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Whole cell-derived fatty acid profiles of Pseudomonas sp. JS150 during naphthalene degradation

MROZIK A., £ABU¯EK S., PIOTROWSKA-SEGET Z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

A potent chitinolytic activity of Alternaria alternata isolated from Egyptian black sand

SHARAF E.F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Purification and characterization of a glutathione S-transferase from Mucor mucedo

HAMED R.R., ABU-SHADY M.R., EL-BEIH F.M., ABDALLA A-M .A., AFIFI O.M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Surface active properties of bacterial strains isolated from petroleum hydrocarbon-bioremediated soil

P£AZA G.A., ULFIG K., BRIGMON R.L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Biotransformation of phosphogypsum by bacteria isolated from petroleum-refining wastewaters

WOLICKA D., KOWALSKI W., BOSZCZYK-MALESZAK H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

BOOK REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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Polish Journal of Microbiology 2005, Vol. 54, No 2, 89–90

IN MEMORIAM Edmund Strzelczyk 1930 – 2005 Edmund Strzelczyk, Full Professor in the Institute of General Biology and Microbiology of Nicolaus Copernicus University in Toruñ, Poland, passed away on March 8, 2005; he was the founder and for many years the head of the Department of Microbiology. Professor Strzelczyk was born on May 3, 1930, in Micha³kowice (Siemianowice) near Katowice. In 1950 he graduated from the Adam Mickiewicz Gymnasium in Katowice and subsequently became a student of the Faculty of Mathematics and Natural Sciences at the Copernicus University in Toruñ. In 1953 he completed there his bacheloriate, and decided to continue his study of microbiology at Maria Curie-Sk³odowska University in Lublin. He graduated in 1955 with a Master’s Degree in microbiology. As a student of the fourth year he was hired as a younger assistant in the Division of Microbiology of the University in Lublin. After graduation he was a member of the faculty of the Division of Agricultural Microbiology at the Agricultural School in Lublin until 1965. He was the favorite student of Professor Jadwiga MarszewskaZiemiêcka, an outstanding microbiologist who was very well known in Poland and abroad. She spent her scientific practice in the Pasteur Institute in Paris as a result of a private invitation of Sergiusz Winogradski, a collaborator of Pasteur. In Lublin, Professor Strzelczyk met for the first time Professor W³adys³aw Kunicki-Goldfinger, intellectualist and renown authority in the field of microbiology, a philosopher of nature, and a scientist with very broad and astonishing knowledge. While a young assistant in Lublin, Professor Strzelczyk, as a fellow of the Rockefeller Foundation, left for two years to work in the Microbiology Institute in Ottawa, Canada. During his stay there he continued his research on nitrogen fixing bacteria. After returning from Canada, still in Lublin, he completed his Ph.D. in 1961, and within the next four years he finalized his so-called habilitation (equivalent to tenure). In 1965, as a young docent he moved to Toruñ as a head of the Microbiology Laboratory, which, very soon became the Department of Microbiology. In 1967–68 he again left for Canada to work in the Institute of Cell Biology in Ottawa, this time as a scholar funded by the National Research Council of Canada. In 1972, as one of the youngest scientists of his field, he was promoted to ordinary Professor; he received the title of the Full Professor in 1978. Professor Strzelczyk created at Nicolaus Copernicus University a unique scientific center of research devoted to the microbiology of soils and forest trees. He was intrigued by the forest from early years; in fact just after graduating from the gymnasium he initially planned to study at the Forestry Faculty of the Agricultural Academy in Kraków. This love for the forest and the passion for research resulted in his professional successes and personal satisfaction. Research work of Professor Strzelczyk was dealt with mutual interactions  between plant roots and soil microorganisms. He was also interested in the importance of soil microbes in sustainable functioning  of forest ecosystems. In order to analyze this complex ecological phenomenon, it was necessary to first learn

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the physiological and biochemical properties of microorganisms, both of bacteria and fungi. Therefore, he investigated the microbial production of plant hormones, vitamins, enzymes, amino acids, organic acids, sugars and siderophores. He also studied the influence of detrimental factors (heavy metals, pesticides) on the metabolic activity of soil microorganisms. One of his pioneering findings was to point out that bacteria, the so-called mycorrhization helper bacteria, are active participants in the sustainable functioning of the mutual interaction between the tree roots and mycorrhizal fungi. The Department of Microbiology became the only department in Poland to work on this kind of microorganisms. Professor Strzelczyk published in 19 international and 16 domestic scientific journals. He is the author, or co-author, of 200 publications and two text books. He was distinguished two times by receiving a diploma of appreciation from the U.S. Department of Agriculture; three times he was awarded by the Ministry of High Education of Poland. He also received the award of the Secretary of Polish Academy of Sciences, and more than a dozen awards from the Chancellor of the Nicolaus Copernicus University in Toruñ. For many years Professor Strzelczyk was a member of the Committee of Microbiology of the Polish Academy of Sciences, the Committee of Soil Science and Agricultural Chemistry of Polish Academy of Sciences, and of the Scientific Council of the Institute of Microbiology Warsaw University. He served also on the editorial board of Acta Microbiologica Polonica and the Polish Journal of Soil Science. He collaborated with many important scientific institutions such as the U. S. Department of Agriculture, Forestry Sciences Laboratory (Oregon, U.S.A.), and the pharmaceutical company Hoffman – La Roche & Co (Switzerland). He taught and lectured in Canada, U.S.A., Switzerland, Italy and Germany. He also collaborated with Polish scientific institutions for example with the Institute of Dendrology of Polish Academy of Sciences in Kórnik, Forest Research Institute in Warsaw/Sêkocin, and the Faculty of Forestry of Agricultural Academies in Kraków and Poznañ. He never turned down any request for professional help in the identification of microbial contaminations, determination of bactericidal agents, and protection of the production processes against microbial corrosion. For many years he was the head of the Laboratory of Intravenous Injection Solutions of the District Hospital in Toruñ. He was the thesis advisor of more than 200 Master’s Degree students, and 13 graduate students who completed the Ph.D. program; two of his students received the title of professor. He was rather a demanding teacher and head of the Department; to get his approval and appreciation was always very rewarding. Professor Les³aw Badura from the Silesian University in Katowice standing at the coffin during the last farewell of Professor Strzelczyk, with deep grief said: “... I would like to point out another, maybe the most important feature of the characteristic of your nature – you were always very humble and well wishing to all people. In addition, you were characterized by the honesty, rightness and extraordinarily touching demeanor. In spite of the fact that in the scientific matters you were demanding and you did not accept the compromises, always you were able to find time to explain complex scientific problems, and also those taken from life” We believe that the life achievements of Professor Strzelczyk will be remembered throughout the scientific world. Did he realize all of his professional desires? Perhaps not. He planned to complete a book on “Microbiology of the forest”... Desires are the last to die. Hanna Dahm

Polish Journal of Microbiology 2005, Vol. 54, No 2, 91–95

Numerical Analysis of Electrophoretic Periplasmic Protein Patterns of Aeromonas sp. Strains EWA SZCZUKA and ADAM KAZNOWSKI*

Department of Microbiology, Institute of Experimental Biology, Adam Mickiewicz University, ul. Fredry 10, 61-701 Poznañ, Poland Received 1 December 2004, received in revised form 1 March 2005, accepted 4 March 2005 Abstract A total of 103 strains of Aeromonas spp. isolated from clinical and from environmental samples was compared by using SDS-PAGE of periplasmic proteins patterns. Strains isolated from Polish children suffering from gastroenteritis did not appear similar to strains isolated from human living in Hong-Kong. Aeromonas sp. strains did not show a tendency to cluster according to their origin. Our results have demonstrated no species-specific periplasmic protein profiles. A significant protein electrophoretic heterogeneity was observed within the species A. hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, and A. veronii biotype sobria. K e y w o r d s: Aeromonas spp., electrophoretic periplasmic protein patterns

Introduction Members of the genus Aeromonas occur widely in the aquatic environment including freshwater, estuaries and marine (Altwegg, 1999). Aeromonas spp. are also isolated from diseased cold- and warm-blooded animals and from humans (Altwegg, 1999). The most common infection in humans is gastroenteritis, with frequent isolation of Aeromonas spp. from diarrhoeal stool (Altwegg, 1999). On the basis of DNA-DNA hybridization, 18 hybridization groups (HG) of Aeromonas sp. have been identified (Kaznowski, 1998; Altwegg, 1999; Pidyar et al., 2002). A variety of methods have been used to type Aeromonas sp. for epidemiological and ecological purposes (Altwegg, 1996). Genotyping methods such as RAPD, ERIC-PCR and PFGE of total chromosomal DNA after restriction with rare-cutting endonucleases have been used with success in differentiation of isolates (Talon et al., 1996; Hänninen and Hirvelä-Koski, 1997; Davin-Regli, et al., 1998; Szczuka and Kaznowski, 2004). Gargallo-Viola and Lopez (1990) have proposed electrophoretic periplasmic protein patterns for epidemiological study. The objective of our investigation was to evaluate the potential of this method for identification and differentiation of strains within Aeromonas spp. isolated of various origins. We also wanted to compare clinical strains isolated in Europe and Asia. We wanted to found out if strains showed a tendency to cluster according to their origin. Experimental Materials and Methods Bacterial strains. The study was performed on 103 Aeromonas spp. strains, including type and reference strains (Tables I and II). Preparation of periplasmic protein samples, electrophoresis and staining. The methods used for cultivation of bacteria, preparation of periplasmic protein samples, gel preparation and staining have been described previously (Ames et al., 1984; Costas, 1992). The gels were visualized on a UV light transilluminator and documented with V.99 Bio-Print system (Vilber Lourmat, France). Computer analyses was determined using GelCompar II version 3.0 software (Applied Maths, Belgium). Cluster analysis was performed using the unweighted pair group method with average linkages (UPGMA). * Corresponding author: phone: (61) 8294529; fax: (61) 8295550; e-mail: [email protected]

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Szczuka E., Kaznowski A. Table I Strains of Aeromonas sp. isolated from human Genospecies

HG

Strain no

Source of isolation

A. hydrophila

1

RK 70363, RK 226254, RK 217215 ATCC 49140

human stool, Hong Kong human

A. caviae

4

AK 375, AK 376, AK 377, AK 378, AK 379, AK 380, AK 383, AK 384, AK 385, AK 386, AK 388, AK 390, AK 393 RK 25447, RK 27611, RK 65541, RK 66942, RK 77620, RK 217455, RK 220132

human stool, Poland human stool, Hong Kong

A. veronii biotype sobria

8/10 AK 382, AK 387, AK 389, AK 391, AK 392 RK 43939, RK 66113, RK 77343

human stool, Poland human stool, Hong Kong

A. veronii biotype veronii

10/8 ATCC 35624T

sputum

A. jandaei

9

ATCC 49568

Aeromonas sp.

11

ATCC 35941

A. schubertii

12

ATCC 43700T

A. trota

14

ATCC 49657

Aeromonas sp.

*

RK 61871

human stool

T

abscess abscess human stool

T

human stool, Hong Kong

AK, Culture Collection of Department of Microbiology A. Mickiewicz University, Poznañ, Poland; RK, strains obtained from Dr R. Kong, Hong Kong University; ATCC, American Type Culture Collection, Manassas, Va.; USA; * isolate not included in any of Aeromonas sp. HG.

Table II Strains of Aeromonas sp. isolated from environmental sources Genospecies

HG

Strain no

Source of isolation

A. hydrophila

1

AK 44 ATCC 7966T

lake water canned milk

A. bestiarum

2

AK 1, AK 41 AK 115 ATCC 23213 ATCC 23211 ATCC 13444 ATCC 51108T

lake water drinking water, Konin river water drinking water surface water diseased fish

A. salmonicida

3

AK 46, AK 50, AK 76, AK 130, AK 131 AK 106, AK 117, AK 125 AK 400, AK 401, AK 402 AK 409, AK 410 CDC 0434-84

lake water rolling-mill emulsion sea water drinking water, Poznañ fresh water

A. caviae

4

AK 48, AK 335, AK 338, AK 339 AK 276, AK 296 AK 104, AK 126 AK 220, AK 232 AK 404, AK 405 AK 406, AK 407, AK 408 ATCC 15468T

lake water sewage drinking water, Konin rolling-mill emulsion river water sea water guinea pig

A. media

5A

AK 42 AK 403 CDC 0862-83

lake water sea water fish

5B

ATCC 33907T

fresh water

A. eucrenophila

6

AK 65 ATCC 23309T

lake water fresh water

A. sobria

7

CIP 7433T

fish

AK 12, AK 59 AK 100, AK 102, AK 120 AK 156, AK 160, AK 164, AK 180 AK 165, AK 167, AK 176 AK 411, AK 412, AK 413 CDC 0437-84

lake water drinking water, Konin dead fish healthy fish drinking water, Poznañ fish

A. veronii biotype sobria

8/10

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Aeromonas sp. periplasmic protein patterns Table II continued Genospecies

HG

Strain no

Source of isolation

A. allosaccharophila

15

CECT 4199

A. encheleia

16

CECT 4342T

fish

A. popoffii

17

LMG 17541

drinking water, Belgium

T

T

diseased eel

AK, Culture Collection of Department of Microbiology A. Mickiewicz University, Poznañ, Poland; ATCC, American Type Culture Collection, Manassas, Va., USA; LMG, Culture Collection, Laboratorium voor Microbiologie Universiteit Gent, Belgium; CECT, Coleccion Espanola de Cultivos Tipo, Universitad de Valencia, Spain; CIP, Collection bacterienne de l¢Institut Pasteur, Paris, France; CDC, Centers for Disease Control, Atlanta, Ga., USA

Results and Discussion One dimensional SDS-PAGE of periplasmic protein extracts of 103 cultures of Aeromonas sp. strains produced patterns containing 10–26 discrete bands corresponding to molecular size of 20 to 100 kDa (Fig. 1). The reproducibility limits of protein patterns from different gels were r = > 0.92. We identified seven clusters at the 90% S level (Fig. 1). Two clusters (2 and 7) contained strains isolated from Polish children suffering from gastroenteritis. Our previous genetic study using RAPD and ERIC-PCR methods demonstrated that strains included in cluster 2 and 7 are genetically different, (Szczuka and Kaznowski, 2004) which is in disagreement with the results obtained by SDS-PAGE method. Protein patterns of the remaining 13 strains isolated from Polish children suffering from gastroenteritis were clearly different. Strains isolated from stool of people living in Hong-Kong also showed distinct patterns. It is interesting that none of clinical strains isolated in Poland showed high degree of similarity to clinical strains originated from Hong-Kong. This is in accordance with our previously genetic analysis (Szczuka and Kaznowski, 2004). Our results demonstrated that the majority of protein patterns of strains isolated from Polish children suffering from gastroenteritis did not match with those of the strains isolated from water supply. The present study revealed the existence of strains of A. veronii biotype sobria AK 411, AK 412, and AK 413 with very similar protein patterns in drinking water collected from city distribution system in Poznañ (cluster 3). Domination of these strains in the water distribution system could be a result of being a component of biological membranes (Gavriel et al., 1998). Our results also suggested colonisation of local industrial water distribution system in Konin by widespread strains of A. veronii biotype sobria AK 102, AK 100, and AK 120 (cluster 4). We found that seven strains of A. veronii biotype sobria isolated from healthy and dead fish Rutilus collected from the same lake did not form a separate group. Only two strains, AK 176 and AK 167 (cluster 5), had very similar protein patterns. However they are not clonally related (Szczuka and Kaznowski, 2004). Patterns of the rest of strains isolates from fish were strain-specific. We observed considerable heterogeneity in protein profiles among strains isolated from the lake. Seven strains originated from seawater generated highly distinct profiles. No specific profile was obtained for strains isolated from sewage. However, we identified two clusters, cluster 1 and cluster 6, containing strains isolated from rolling emulsion. Strains of A. salmonicida AK 106, AK 125 and AK 117 (cluster 1) are clonally related as previously determined by RAPD and ERIC-PCR methods (Szczuka and Kaznowski, 2004). Strains of A. caviae AK 232 and AK 220 belonging to cluster 6 are not clonally related. This indicated that genetically different strains produce very similar proteins when they are isolated from very specific sources. This study demonstrated also that protein patterns reflect genome information of the bacteria because strains belonging to different species isolated from rolling emulsion did not group together. We observed considerable heterogeneity in periplasmic protein profiles of isolates within A. hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, and A. veronii biotype sobria. Previous analysis also revealed genetic heterogeneity within these species (Szczuka and Kaznowski, 2004). We did not obtain protein bands specific for the species and therefore the analysis of SDS-PAGE of periplasmic proteins can not be used for distinguishing Aeromonas species. By using SDS-PAGE of periplasmic protein patterns we were able to recognise related strains (cluster number 1, 3, 4). However, some genetically different strains isolated from the same source also showed very similar patterns. Ruimy et al. (1994) suggested that bacteria contain the products of many regulated

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Fig. 1. Dendrogram showing periplasmic protein similarity of 103 strains of Aeromonas sp. determined by the SDS-PAGE protein pattern analysis using Dice similarity coefficient and UPGMA cluster method

2

Aeromonas sp. periplasmic protein patterns

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genes that are expressed according to the environment in which they grew. Environmentally-linked phenotypes could be reflections of the ecosystems from which the bacteria were obtained and they could occur for more than several cell cycles. Literature A l t w e g g M. 1996. Subtyping methods for Aeromonas species. p. 109–125. In: B. Austin, M. Altwegg, P.J. Gosling and S.W. Joseph (eds), The Genus Aeromonas. Chichester, New York, Brisbane, Toronto, Singapore: John Wiley and Sons A l t w e g g M. 1999. Aeromonas and Plesiomonas. p. 507–516. In: P. Murray, E. Baron, M. Pfallen, F. Tenover and R. Yolken (eds), Manual of Clinical Microbiology. Washington, D.C: ASM Press A m e s G.F.-L., C. P r o d y and S. K u s t u. 1984. Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J. Bacteriol. 160: 1181–1183. C o s t a s M. 1992. Classification, identification and typing of bacteria by the analysis of their one-dimensional polyacrylamide gel electrophoretic protein patterns. p. 351–408. In: A. Chrambach, M.J. Dunn and B.J. Radda (eds) Advances in Electrophoresis. Weinheim, New York, Basel, Cambrige: VCH D a v i n - R e g l i A., C. B o l l e t, E. C h a m o r e y, V. C o l o n n a D’ I s t r i a and A. C r e m i e u x. 1998. A cluster of cases of infections due to Aeromonas hydrophila revealed by combined RAPD and ERIC-PCR. J. Med. Microbiol. 47: 499–504. G a r g a l l o - V i o l a D. and D. L o p e z. 1990. Numerical analysis of electrophoretic periplasmic protein patterns, a possible marker system for epidemiological studies. J. Clin. Microbiol. 28: 136–139. H ä n n i n e n M.L. and V. H i r v e l ä - K o s k i. 1997. Pulsed-field gel electrophoresis in the study of mesophilic and psychrophilic Aeromonas spp. J. Appl. Microbiol. 83: 493–498. G a v r i e l A.A., J.P.B. L a n d r e and A.J. L a m b. 1998. Incidence of mesophilic Aeromonas within a public drinking water supply in north-east Scotland. J. Appl. Microbiol. 84: 383–392. K a z n o w s k i A. 1998. Identification of Aeromonas strains of different origin to the genomic species level. J. Appl. Microbiol. 84: 423–430. P i d i y a r V., A. K a z n o w s k i, N.B. N a r a y a n, M. P a t o l e and Y.S. S h o u c h e. 2002. Aeromonas culicicola sp. nov., from the midgut of Culex quinquefasciatus. Int. J. Syst. Evol. Microbiol. 52: 1723–1728. R u i m y R., V. B r e i t t m a y e r, P. E l b a z e, B. L a f a y, O. B o u s s e m a r t, M. G a u t h i e r, R. C h r i s t e n. 1994. Phylogenetic analysis and assessment of the genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas deduced from smallsubunit rRNA sequences. Int. J. Syst. Bact. 44: 416–426. S z c z u k a E. and A. K a z n o w s k i. 2004. Typing of clinical and environmental Aeromonas sp. strains by random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus sequence PCR. J. Clin. Microbiol 42: 220–228. T a l o n D., M.J. D u p o n t, J. L e s n e, M. T h o u v e r e z and Y. M i c h e l - B r i a n d. 1996. Pulsed-field gel electrophoresis as an epidemiological tool for clonal identification of Aeromonas hydrophila. J. Appl. Bacteriol. 80: 277–282.

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Polish Journal of Microbiology 2005, Vol. 54, No 2, 97–103

Receptors for Endogenous and Heterogenous Hydroxamate Siderophores in Staphylococcus aureus B 471 PIOTR WYSOCKI2, PAWE£ LISIECKI and JERZY MIKUCKI

Chair of Biology and Biotechnology, Department of Pharmaceutical Microbiology, Medical University of £ódŸ, Pomorska 137, 90-235 £ódŸ, Poland Received 27 October 2004, received in revised form 9 February 2005, accepted 12 February 2005

Abstract In Staphylococcus aureus B47 grown in iron-restricted medium, six new, iron-regulated proteins occurred in cytoplasmic membrane. Protein of 14kDa has bound two complexes of iron: Fe(III)-staphylobactin and Fe(III)-acinetoferrin. Complexes of Fe(III)-ferrichrome and Fe(III)-rhodotorulic acid were not bound to any of new membrane protein. Iron of Fe(III)-staphylobactin and Fe(III)-acinetoferrin complexes was transported into the cells. K e y w o r d s: Staphylococcus sp., siderophores, iron regulated proteins.

Introduction Microorganisms produce siderophores, high affinity iron chelating molecules, that solubilize Fe(III) and present it to the bacterial surface, where the complex may by transported across the bacterial envelope so that the iron can be used for biological processes. Staphylococcus aureus may synthesize siderophores belonging to different chemical classes. Staphylobactin is a hydroxamate class chelator (Lisiecki and Mikucki, 1996; Lisiecki et al., 1994; Lisiecki et al., 2001), aureochelin belongs to the catecholate class (Courcol et al., 1997) and staphyloferrin A and B have been classified as aminohydroxypolycarboxylic acids (Drechsel et al., 1993; Konetschny-Rapp et al., 1990). Heterogenous siderophores-bacterial and fungal, utilized by staphylococci as a source of iron, may be hydroxamate chelators-acinetoferrin, aerobactin, schizokinen, rhodotorulic acid, ferrioxamine B, ferrichrome and catecholates ones – N-(2,3-dihydroxybenzoyl)-glycine, N-(2,3-dihydroxybenzoyl)-L-serine, 2,3-dihydroxybenzoic acid and enterobactin (Lisiecki and Mikucki, 1996; Sebulsky et al., 2000). The transport system of Fe(III)-siderophore complex in bacterial cell is composed of a receptor, binding protein dependent transport (BPT) and permease complex (Clarke et al., 2001). In S.aureus several proteins involved in Fe(III)-hydroxamate complexes uptake have been detected. FhuD1 and FhuD2 proteins form binding proteins transport system. Two proteins – Fhu B and Fhu G are a complex of permease and the third one Fhu C – ATP binding protein with traffic ATP-ase activity (Cabrera et al., 2001; Sebulsky and Heinrichs, 2001; Sebulsky et al., 2000). The aim of this study was to detect and localize siderophore receptors in the cell and estimate their specificity for Fe(III)-hydroxamate siderophore complexes. Abbreviations: AB – p-azidobenzoyl analog of siderophore, AC – acinetoferrin, FC – ferrichrome, RA – rhodotorulic acid, SB – staphylobactin. 1 Presented at Congress of the Polish Society of Microbiologist, Bydgoszcz 2004 2 Corresponding author: Piotr Wysocki. Phone, fax +426779302. E-mail [email protected]

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Experimental Materials and Methods Bacterial strain. Staphylococcus aureus B47 isolated from the human nasopharynx was used. The strain was identified with API-STAPH (bioMerieaux) and stored at –70°C in 50% glycerol solution in 3.7% Brain Heart Infusion Broth (Difco). Siderophores. Acinetoferrin AC (gift from Professor Shigeo Yamamoto, University of Yokohama), ferrichrome FC (Sigma), rhodotorulic acid RA (Sigma) and staphylobactin SB, extracted and purified in our laboratory were used. Medium and growth conditions. The chemically defined medium CDM of pH 7.2, sterilized with filtration through a membrane filter of 0.22 mm pore width (Milipore) was used (Lisiecki et al., 1994). The iron content was reduced using polyaminocarboxyl resin Chelex 100 (200– 400 mesh) (BioRad). The bacteria were grown in iron-deficient CDM medium (CDM-Chelex) and with excess of iron content (1× 10–4M, CDM-Chelex +Fe) in the form of FeSO 4 ×7H2O. A portion of an appropriate medium was inoculated with 18 hour, iron starved culture in CDM-Chelex medium constituting 5% of total volume. The culture was incubated for 24 hours in temperature of 37 oC with constant shaking (120 strokes/min). The culture was centrifuged (1600×g, 15min, 4oC), supernatant was assayed for siderophore content and cells were subjected to the process of protoplast formation and membrane preparation. Suspensions optical density. The optical density of suspension and cultures was measured in UV/Vis Cary 1 (Varian) spectrophotometer at 540 nm. Viable count. Viable count was estimated by using serial dilutions in buffered 0.155 M NaCl, pH 7.2 (Biomed) and standard plate method on 4% Tripticase Soy Agar (Difco). Protoplast formation and cytoplasmic membrane preparation. Protoplasts were prepared using lysostaphin (Sigma) and lysozyme (Serva) according to the method of Theodore et al. (1971) as modified by Lindberg (1981). The protoplast lysate in 0.01 M Tris – HCl (pH 7.2) buffer containing membranes and cytoplasm was pre-centrifuged (3000×g, 15 min, 4°C), the pellet discarded and the supernatant was centrifuged 100 000×g, 60 min, 9°C (Beckman, L8-70M). The collected membranes were washed three times with deionised water and lyophilized. Siderophores determination. Total activity of siderophores was assayed by the Schwyn and Neilands (1987) method with Chrome Azurol S. Hydroxamate siderophore-staphylobactin was assayed according to Csaky (1948) and Emery and Neilands methods (1962). The results were expressed as :g of the desferrioxamine mesylate (Desferal, Ciba-Geigy) and calculated for millilitre of culture supernatant. Staphylobactin isolation and purification. Siderophore was isolated and purified according to the method of Okuyo et al. (1994) modified by Lisiecki et al. (1994). The bioactivity of the siderophore during purification was assayed according to the method of Reissbrodt and Rabsch (1988). The culture supernatant of S.aureus B47 strain was adjusted to pH 6 with 60% citric acid. An 80 g of resin Amberlite XAD 7 (Aldrich) was added to 4000 ml of supernatant and suspension was shaken slowly for 1 hour. The suspension was filtered and the pellet was washed three times with 1000 ml of distilled water. The resin was eluted with 200 ml of methanol and incubated at room temperature for 30 min, filtered and then washed with 100 ml of methanol. This procedure was repeated twice more. The eluate was evaporated at 30°C and the residue was dissolved in 100 ml of water. The solution adjusted to pH 2.0 with solid citric acid was extracted three times with 200 ml of ethyl acetate. After washing three times with 0.1 M sodium citrate, pH 5.5, the organic layer was evaporated to give a crude siderophore fraction. It was dissolved in 5 ml of methanol and the insoluble materials were removed by centrifugation. The resulting solution was subjected to preparative paper chromatography on Whatman No 3 paper in mixture of solvents n-butanol-acetic acid-water (63:25:12). A strip of paper was taken and sprayed with Schwyn and Neilands (1987) reagent containing Chrom Azurol S to locate the siderophore. The colored spot of Rf = 0.2 exhibited siderophore activity. Corresponding regions of the remaining papers were excised and extracted with methanol-water (1:1) solution. After evaporation of the solvent, the residue was deferrated 1.0 M KOH, centrifuged (9000×g, 20°C, 10 min) and lyophilized. This material was positive in the plate bioassay with S. aureus B47 strain and the Csaky (1948), Emery and Neilands (1962) and Schwyn and Neilands (1987) tests. Photoaffinity labels of siderophores. Photoaffinity labelling of siderophores was performed according to Nelson et al. (1992) methods. Siderophores were referrated with 59FeCl3: 500 :g of siderophore was first dissolved in 50 :L of methanol to which 50 :Ci 59FeCl3 (NEN) and 100 :L of dimethylformamide (Sigma) were added and, to AC solutions only, 100 :L of triethylamine (Ubichem). The reaction mixtures were gently shaken to aid dissolution. The N-hydroxysuccinimidyl-4-azidobenzoate (Sigma) was then dissolved in these solutions and the reaction was allowed to proceed for 24 hours at 25°C. All preparations involving photoreactive compounds were done under very low light conditions. The particular solutions contained photoreactive p-azidobenzoyl analogues of the siderophores labeled with 59Fe(III). Photolabelling of the membrane proteins. Lyophilized membranes were diluted with HEPES buffer of pH 7.2 and 20 :L of each sample was mixed with 5 :M labelled siderophore solution. The mixture was chilled in ice-water bath for 10 min and photolysed for 2 min using UV lamp. The membranes were pelleted and washed three times with HEPES buffer by centrifugation (100 000× g, 60 min, 9°C). Electrophoresis. Membrane proteins were separated electrophoretically according to Laemmli (1970) method in Multiphor 2 (Pharmacia Biotech) apparatus using 12.5% Excel Gel SDS (Pharmacia Biotech) and buffered strips (Pharmacia Biotech). The set of standard proteins Low Molecular Weight Calibration Kit (Pharmacia) 14.4 to 94 kDa was used. The investigated samples contained about 15 :g of protein in 10 :L. All the samples and the standard proteins were boiling for 2 min in a 100 oC water bath. Non-radioactive gels were stained with Coomassie Blue (Merck). Gels of the labelled siderophores were subjected to autoradiography using Kodak X-Omar film for 24 hours. Following autoradiography these gels were stained with Coomassie blue and photographed. The analysis of electrophoretic separation was performed with computer programme Image Master (Pharmacia Biotech). Time-dependent uptake of radioactive iron sources. S. aureus B47 strain was grown in CDM-Chelex medium. The cells were harvested after 24 hours of incubation and washed three times with buffered 0.155 M NaCl, pH 7.2 (Biomed) by centrifuga-

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tion (1600×g, 20 min., 4°C). The pellet was suspended in CDM-Chelex medium to give a final cell concentration of about 3×108 cfu ml–1. Suspension was then divided into 200 :L portions. 10 :L of radioactive iron sources containing 10 :M 59Fe ferrated siderophore was added to series of 7 tubes. At various intervals, after 5, 10, 15, 20, 25, 30 and 40 minutes one tube was withdrawn. The suspension was filtered through a membrane filter of 0.22 mm pore (Milipore), washed three times with buffered saline (Biomed) pH 7.2. The radioactivity of the cells was measured in a Wallac 1470 Wizard gamma counter. Blocking of receptors and uptake of radioactive iron sources. S. aureus B47 strain was grown in CDM-Chelex medium. The cells were harvested after 24 hours of incubation, washed three times with buffered 0.155 M NaCl, pH 7.2 (Biomed) by centrifugation (1600× g, 20 min, 4°C) and resuspended in appropriate volume of fresh CDM-Chelex medium. The density of suspension corresponded to 3× 108 cfu ml–1. To 1 ml of suspension, 10 :L solution containing 10 :M photoreactive p-azidobenzoyl analogues of siderophores saturated with Fe(III) was added. The suspension was poured to Petri dishes and immediately, using UV lamp, photolysed for 2 minutes. After photolysis the suspension was centrifuged (3000× g, 5 min, 4°C), washed three times with HEPES buffer, pH 7.2, suspended in the initial volume of CDM-Chelex medium and viable count (cfu ml–1) was estimated. 10 :L of solution containing 10 :M labeled 59Fe (III) siderophore was added per milliliter of suspension. After 25 minutes of incubation the suspension was filtered through 0.22 :m membrane filter (Milipore) and washed three times with buffered 0.155 M NaCl, pH 7.2 (Biomed). The radioactivity of cells was measured in a Wallac 1470 Wizard gamma counter. Analytical methods. Protein was assayed with Lowry et al. (1951) method. Iron content was determined with Gadia and Mehra method (1977). Statistical analysis was performed with the Statistica PL computer programme (StatSoft).

Results The iron content in CDM-Chelex medium varied in range of 9.5×10–7 to 1.2×10–6 M. The detectable siderophores activity in CDM-Chelex medium was found in the 5 th – 6th hour of incubation: in the middle of the exponential growth phase. The highest activity has occurred in the 15th – 22th hour, in the stationary growth phase. The siderophores were not produced in CDM-Chelex + Fe medium. The investigated strain harvested from CDM-Chelex and CDM-Chelex + Fe media, after 25 hours of incubation was subjected to protoplast formation. In a typical experiment, after 60 minutes the optical density of the suspension was maintained at the level equal to 32% of the initial value and over 90% of gram-negative cells were found. The cytoplasmic membrane protein profiles of S. aureus B47 strain grown in CDM-Chelex and CDMChelex + Fe media were compared with a computer programme Image Master. This technique revealed six new band-iron regulated proteins that were expressed only in cytoplasm membranes from cells grown under iron limitation in CDM-Chelex medium. They corresponded to proteins with apparent molecular masses: 14 kDa with Rf = 0.92 which might be a complex of two peptides and at the region of 96–43 kDa with molecular masses of 96, 88, 80, 69, 43 kDa and Rf values of 0.25, 0.29, 0.34, 0,38 and 0.54, respectively (Fig. 1; lane 1A). Searching for proteins which might be siderophore receptors, autoradiograms of gels containing cytoplasmic membrane proteins of cells grown under iron deficiency (CDM-Chelex) and labelled by p-azidobenzoyl complex [59Fe(III)]AB were analysed and referred to electrophoretic separations of cytoplasmic membrane proteins. Then they were compared with electrophoregrams of the membranes of cells

Uptake of 59Fe [nM×mg–1]

20

15

10

5

0 0

10

20

30

40

Time [min]

Fig. 2. Iron uptake by S. aureus B47 from [59Fe(III)]-staphylobactin -¡-, [59Fe(III)]-acinetoferrin -■-, [59Fe(III)]-ferrichrome-x-, [59Fe(III)]-rhodotorulic acid -◆- complexes.

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Fig.1. SDS-PAGE profiles of cytoplasmic membrane proteins of S. aureus grown in CDM-Chelex medium (lane1A) and CDM-Chelex+Fe medium (lane 2A). Autoradiograms of membrane proteins (lane1A), labelled with [59Fe(III)]-staphylobactin (lane 4B), [59Fe(III)]-acinetoferrin (lane 5B), [59Fe(III)]-rhodotorulic acid (lane 6B) complexes

grown in the presence of iron excess (CDM-Chelex+Fe) (Fig.1; lanes B). Acinetoferrin complex ([59Fe(III)]ABAC) was bound to the cytoplasmic membrane protein with the molecular mass of 14 kDa and Rf of 0.92, constituting 4.3% of total membrane proteins. Staphylobactin complex ([59Fe(III)]ABSB) was also bound to the protein with the molecular mass of 14 kDa. Rhodotorulic acid complex ([59Fe(III)]ABRA) has not bound any of new iron-regulated proteins, because a signal of Rf=1 in autoradiograms did not correspond to any on electrophoregrams. The Rf value of this signal has shown that it occurs in the region of low-molecular membrane proteins. Thus, the movement in electric field was determined by binding to low-molecular protein that, however, could have a non-specific character. Ferrichrome complex ([59Fe(III)]ABFC) did not occur on autoradiograms, thus this siderophore was not bound to any of membrane proteins. The control electrophoresis of protein-unbound 59Fe(III)AB-siderophore complexes revealed that they do not move in the electric field, remaining on the autoradiograms at the start line. The control of viable count of cells after the photolysis showed that in the suspension containing about 3.6×108 cfu ml–1 an average 70% of cells remained. To prove participation of the studied siderophores and their receptors in iron Fe(III) transport into the cell, kinetics of iron [59Fe(III)]-siderophore complexes uptake by resting cells was investigated (Fig. 2). Complexes of [59Fe(III)]AC and [59Fe(III)]SB after binding by the receptor were transported into the cell. Following the initial phase of fast uptake of [59Fe(III)]AC complex, until 25 min of incubation, the linear phase of slow iron uptake occurred. After 25 min the cells contained 9% initial dose of isotope. After 30 and 40 min the isotope content in cells was 8.1% and 10.2% of isotope added in the to time of the experiment, respectively. When staphylobactin was ligand of 59Fe(III), after 25 min of incubation the amount of assimilated isotope was 10.3% of the initial dose. After 30 min it increased slightly and at the end of the observation it reached 11.5% of the initial isotope dose. The examination of 59 Fe(III) iron uptake from ferrichrome ([ 59 Fe(III)]FC) and rhodotorulic acid 59 ([ Fe(III)]RA) was a control because these fungal chelators were not heterogenous siderophores for S. aureus B47 strain. Flat uptake curves without maximum reflected very small amounts of the isotope, which were quite different in comparison with the iron uptake from the acinetoferrin and staphylobactin complexes bound to receptors. When 14 kDa receptor was blocked by p-azidobenzoyl analogue of Fe(III)-staphylobactin (Fe(III)ABSB), after 25 min of incubation cells of S. aureus B47 strain contained only 0.9% and 0.8% of the initial isotope doses of [59Fe(III)]SB and [59Fe(III)]AC respectively per mg of dry bacteria mass. After blocking this receptor by p-azidobenzoyl analogue of Fe(III)-acinetoferrin complex (Fe(III)ABAC) the cells contained 0.8% and 0.7% of the initial doses of [59Fe(III)]AC and [59Fe(III)]SB per mg, respectively (Table I).

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Table I Iron uptake from [59Fe(III)]-staphylobactin and [59Fe(III)]-acinetoferrin complexes by S. aureus B47 cells with blocked and unblocked 14 kDa receptor Unblocked receptor

Blocked receptor S. aureus B47 Fe(III)-AB**)staphylobactin 59

[ Fe(III)]staphylobactin

[ Fe(III)]acinetoferrin

59

cpm*) nM mg–1***) mg–1

59

Fe(III)-AB**)acinetoferrin

Fe(III) initial dose

[ Fe(III)]staphylobactin

[59Fe(III)]acinetoferrin

59

[59Fe(III)]acinetoferrin

[59Fe(III)]staphylobactin

% ini% ini% ini% ini% ini% inicpm*) nM cpm*) nM cpm*) nM cpm*) nM cpm*) nM tial tial tial tial tial tial mg–1***) mg–1 mg–1***) mg–1 mg–1***) mg–1 mg–1***) mg–1 mg–1***) mg–1 dose dose dose dose dose dose

12192 178.3 100 ±1207

13624 178.3 100 ±1720

1251 ±91

1223 ±273

13106 178.3 ±1700

100

13106 178.3 ±1700

100

13106 178.3 ±1700

100

13106 178.3 ±1700

100

Iron uptake 18.29 10.3

*) cpm, counts per minute;

16.01

9.0

114 ±16

1.55

0.9

**) AB, p-azydobenzoyl analogue;

106 ±22

1.44

0.8

101 ±24

1.39

0.8

91 ±15

1.24

0.7

***) mg dry weight of bacteria

The statistical analysis has proved that differences between mean values of iron uptake in time from Fe(III)AC, Fe(III)SB complexes and Fe(III)FC, Fe(III)RA complexes were significant (p< 0.05). The differences between mean values of iron uptake from Fe(III)AC, Fe(III)SB complexes by cells with blocked and unblocked receptors were also significant (p< 0.05). All these results were confirmed with the LSD test (least significant differences) (p < 0.05). Discussion Staphylobactin belonging to the citrate-based hydroxamate class chelators has been found to be the endogenous siderophore of S. aureus B47 strain (Lisiecki et al., 1994; Lisiecki and Mikucki, 1996; Lisiecki et al., 2001). Heterogenous siderophores utilised by this strain belonged to the catecholate class chelators – N-(2,3-dihydroxybenzoyl)-glycine and N-(2,3-dihydroxybenzoyl)-L-serine as well as to chelators of the hydroxamate class of citric acid derivatives-aerobactin, schizokinen and acinetoferrin (Lisiecki et al., 2001). Most of bacterial siderophore systems are subjected to derepression at iron Fe(III) concentrations of 10–7 M but the concentration of 10–6 optimally regulates their expression (Hider, 1984). Iron concentrations in CDM-Chelex medium were within the range between 9.5× 10–7 M and 1.2× 10–6 M fulfilling condition of derepression. Under iron-limitation conditions S. aureus B47 strain has demonstrated simultaneous expression of siderophores and receptor proteins. There is no reports about iron-regulated proteins in S. aureus, which could be receptors for Fe(III)siderophore complexes. In cell lysates of blood-isolated S. aureus, at least one protein was detected in the region of 36–39 kDa, occurrence of which correlated with siderophores release (Lindsay and Riley, 1994). Proteins with higher molecular mass were also found (Courcol et al., 1997; Trivier et al., 1995). The presence of such proteins in the region of 81–17 kDa was observed in cell lysates from two S. aureus strains, DES and DAU, isolated from cases of mucoviscidosis. One of them, 81 kDa, was repressed in the presence of iron excess. Two other proteins – 23 and 17 kDa occuring together with siderophores did not undergo repression (Trivier et al., 1995). The expression of three proteins with masses of 120, 88 and 57 kDa occurred exclusively under iron-deficient conditions (Courcol et al., 1997). In the isolated cytoplasmic membrane of S. aureus B47 strain grown in iron-deficient CDM-Chelex medium there was always the same profile of new, iron regulated proteins with masses of 96, 88, 80, 69, 43 and 14 kDa. The 14 kDa protein was formed with two closely localized bands in the electrophoregram. These iron-regulated proteins were probably anchored in the cytoplasmic membrane as cell fractionation by lysostaphin did not remove them, similarly as in S. epidermidis ABC transporter lipoprotein (Cockayne et al., 1998). The protein with the mass of 43 kDa was identified as the ferrioxamine B receptor (Wysocki et al., 2003). The second receptor protein – 14 kDa bound two chelators i.e. staphylobactin and acinetoferrin.

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There were no receptors for ferrichrome and rhodotorulic acid. Subsequent blocking 14 kDa receptor by Fe(III)-AC and Fe(III)-SB complexes gave the evidence that they were bound to only one of these two proteins forming in the electrophoregrams the band of 14 kDa mass. It also demonstrated that this receptor protein participated in Fe(III)-siderophore complex transport into the cell. So, this protein met the criteria for receptors function. It not only recognised and bound Fe(III)-siderophore complex but also took part in its transport to the cells presenting it to the subsequent component of the siderophore system. In E. coli outer membrane protein Fhu E is the receptor for two siderophores also. It has bound coprogen and rhodotorulic acid, structurally related to coprogen, both linear fungal chelators (Hantke, 1983). Another protein – Cir was capable of transporting iron complexes very similar structurally siderophores: dihydrobenzoic acid-enterobactin precursor and dihydrobenzoylserine-enterobactin breakdown product (Hantke, 1990). In order to use hydroxamate siderophores by S. aureus an operon fhuCBG, another two genes – fhuD1 and fhuD2 are required (Cabrera et al., 2001; Sebulsky and Heinrichs, 2001; Sebulsky et al., 2000). Genes fhuD1 and fhuD2 encode proteins belonging to binding protein dependent transport system (BPT) (Sebulsky and Heinrichs, 2001). Two homologous proteins of this system FhuD1 and FhuD2, have overlaping but different specificity, lower than receptor proteins (Sebulsky and Heinrichs, 2001). The FhuD1 co-operates with linear hydroxamate siderophores ferrichrome and ferrioxamine B while FhuD2 co-operates with ferrichrome, ferrioxamine B and coprogen, also linear siderophores and aerobactin, symmetric derivative of citric acid. Affinity of various Fe(III)-hydroxamate siderophore complexes to FhuD1 and FhuD2 proteins is diverse and depends on the environmental conditions (Sebulsky and Heinrichs, 2001). The FhuD2 protein transports aerobactin being a symmetric derivative of citric acid (Sebulsky and Heinrichs, 2001). Staphylobactin, and acinetoferrin also have such a structure. Purified staphylobactin contained citric acid and amino acids-glycine, alanine and leucine. The initially proposed its structure was citric acid as central backbone linked to two lateral chains of amino acids (Lisiecki and Mikucki, 1996). The backbone of acinetoferrin is citric acid also, which is linked to two lateral chains of 1,3-diaminopropane (Okuyo et al., 1994). In bioassays these two siderophores, like aerobactin, have promoted growth of two indicators strain – E. coli LG 1522 and A. flavescens JG-9 requiring for growth different hydroxamate chelators, proving that they have belonged to hydroxamate siderophores group-symmetric derivatives of citric acid (Reissbrodt and Rabsch, 1988; Lisiecki and Mikucki, 1996). Thus, both siderophores are hydroxamate chelatorscontaining hydroxamic groups and additionally iron-binding ligands in form alpha-hydroxycarboxylate residues which coordinate iron Fe(III) (Winkelman and Drechsel, 1997). These siderophores can be thus transported into the cell by FhuD2 protein. The transport of these siderophores by the same BPT protein, and, especially, binding by the same receptor confirms structural similarity of staphylobactin, acinetoferrin and aerobactin. The FhuD2 protein transports the adequate Fe(III)-siderophore complex to the subsequent transport link – Fhu CBG proteins encoded by the fhuCBG operone (Cabrera et al., 2001; Sebulsky et al., 2000). FhuC is ATP – binding protein with traffic ATP-ase activity, while FhuB and FhuG form the complex of transmembrane permease with a wide range of specificity, passing Fe(III)-siderophopre complexes across the cytoplasmic membrane and are necessary for hydroxamate siderophores uptake (Cabrera et al., 2001; Sebulsky et al., 2000). There is alack of one important link in this system iron assimilation – i.e. receptor. The recognition of the complex and binding to the membrane receptor is an essential prerequisite for transport of Fe(III)-siderophore complex into the cell. Acinetoferrin and staphylobactin were bound to 14 kDa protein receptor and transported with permease as [59Fe(III)]AC and [59Fe(III)]SB complexes were detected in S. aureus B47 strain cells. Ferrichrome and rhodotorulic acid for which S. aureus B47 strain did not have membrane receptors, were not transported into the cell. In the previous investigations it has been found that the growth of S. aureus B47 strain was promoted by its endogenous siderophore-staphylobactin and heterogenous one-acinetoferrin and was not promoted by heterogenous ones-ferrichrome and rhodotorulic acid (Lisiecki et al., 2001). The presented study allowed to prove that S. aureus B47 strain growth was the result of binding these siderophores to the receptor and stimulating growth transport Fe(III) into the cell. Acknowledgements. This research was supported by grant from Medical University N° 502-13-705.

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Polish Journal of Microbiology 2005, Vol. 54, No 2, 105–110

Extended-spectrum $-lactamase-producing Klebsiella pneumoniae in a Neonatal Unit: Control of an Outbreak Using a New ADSRRS Technique BEATA KRAWCZYK1, ALFRED SAMET2, EL¯BIETA CZARNIAK3, JERZY SZCZAPA4 and JÓZEF KUR1* 1 Department

of Microbiology, Gdañsk University of Technology, ul. G. Narutowicza 11/12, 80-952 Gdañsk, Poland; 2 Department of Clinical Microbiology, Clinical Hospital No. 1, Medical University of Gdañsk, ul. Dêbinki 7, 80-952 Gdañsk, Poland; 3 Department of Epidemiology, Clinical Hospital No. 1, Medical University of Gdañsk, ul. Dêbinki 7, 80-952 Gdañsk, Poland; 4 Department of Neonatology, Clinical Hospital No. 1, Medical University of Gdañsk, ul. Kliniczna 1a, 80-402 Gdañsk, Poland Received 11 January 2005, received in revised form 25 February 2005, accepted 1 March 2005 Abstract Extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae (EPKP) strains are frequently implicated in outbreaks in neonatal units. From April 2002 to January 2003, 149 neonates were colonized/infected with EPKP in the Neonatal Clinic of the Teaching Hospital at the Medical University of Gdañsk, Poland. A novel assay based on suppression of PCR, ADSRRS-fingerprinting, was successfully evaluated for typing EPKP isolates. The results showed that the genotypes of all outbreak-related strains were identical, which suggested that the outbreak originated from a single clone. This conclusion was confirmed by using different methods – RAPD and PFGE. The outbreak was stopped by adopting improved hygiene and instituting outbreak control measures. K e y w o r d s: Enterobacteriaceae, ESBL, PCR-fingerprinting, RAPD, ADSRRS-fingerprinting

Introduction Klebsiella pneumoniae has been recognized as an important cause of infections in hospitalized neonates (Hart, 1993). Various environmental reservoirs have occasionally been identified in outbreaks caused by K. pneumoniae in such settings (Gaillot et al., 1998; Lalitha et al., 1999). Irrespective of the primary source, it seems that the most significant reservoir of the microorganism is the digestive tract of colonized patients, and that transmission occurs mostly via the hands of nursing staff (Coovadia et al., 1992; Hart, 1993). During the past decade, K. pneumoniae strains exhibiting resistance to newer cephalosporins due to the production of extended-spectrum $-lactamases (ESBLs) have been frequently implicated in outbreaks in pediatric hospitals and neonatal intensive care units (Bingen et al., 1993; Royle et al., 1999; Szabo et al., 1999; Venezia et al., 1995). These strains usually exhibit cross-resistance to other antibiotics, such as aminoglycosides. A report of imipenem-resistant Klebsiella pneumoniae is also very alarming (Ahmad et al., 1999). Therapeutic options are, therefore, limited. This problem is still emerging and occurring throughout the world, even at the beginning of the twenty-first century. This wide geographic spread of ESBLs was occurred due to transmission of strains between hospitals, horizontal transfer of resistance plasmids, or clonal expansion of epidemic strains. Rigorous compliance to the infection control program is one of the most important conditions in the control of outbreaks, and personnel education is a cardinal element. Moreover, education should be modified accordingly to the situation and infection control procedures should be improved. The success of the control of an outbreak depends also on the possibility of using quick, sensitive and discriminative tests as accurate epidemiological markers that permit identification of epidemic strains or spread of resistance plasmids. * Corresponding author: Józef Kur, Gdañsk University of Technology, Department of Microbiology, ul. G. Narutowicza 11/12, 80-952 Gdañsk, Poland, e-mail: [email protected]; tel/fax: +48 58 3471822

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In recent years, genotyping methods have gained popularity and are frequently used to support and/or initiate epidemiological investigation. Macrorestriction analysis of genomic DNA, followed by pulsed-field gel electrophoresis (PFGE) has become the “gold standard” for molecular typing. PFGE, however, is limited in its resolving power (Gerner-Smidt et al., 1998), and this contributes to difficulties with gel-to-gel and interlaboratory reproducibility (Van Belkum et al., 1998). Consequently, several novel methods for DNA fingerprinting of medically important bacteria have received considerable attention for their suitability in epidemiological studies. Recently, we presented the performance and convenience of a novel assay based on the fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) for its potential usefulness in epidemiological investigation (Krawczyk et al., 2003a; Krawczyk et al., 2003b). This method is rapid, offers good discriminatory power and also demonstrates excellent reproducibility. The aim of this study was to present an epidemiological investigation of the population of K. pneumoniae in the neonatal unit over a ten-month period. The isolates were analyzed in the context of clinical data and antimicrobial susceptibility. The performance and convenience of an ADSRRS-fingerprinting method for its potential usefulness in epidemiological investigation of K. pneumoniae outbreak is shown. Experimental Materials and Methods Hospital setting. The outbreak occurred in the Neonatal Clinic of the Teaching Hospital of the Medical University of Gdañsk, Poland. The clinic consists of four neonatal wards: a Neonatal Intensive Care Unit, Pathology Unit, Septic Unit and Rooming-in Unit. This clinic serves an average of about 160 patients per month. Up to the year 2001, extended-spectrum $-lactamase (ESBL)-producing Klebsiella pneumoniae (EPKP) strains were rarely isolated in the clinic. However, during 2002, a relatively high rate of isolation of EPKP strains in the clinic was observed. The infection surveillance reported here covers a period of ten months: from April 2002 to January 2003. During this period, 1582 neonatal patients were hospitalized in the clinic. Patients and bacterial strains. After searching the computer microbiology database, we found 149 records of patients testing positive in cultures with ESBL-producing Klebsiella pneumoniae, and they were included in the study. The clinical data of patients were retrospectively collected from the medical records. The following sociodemographic variables and potential risk factors were assessed: gender, gestational age, mode of delivery, Apgar score, birth weight, mechanical ventilation, parenteral nutrition, length of hospital stay, type and length of antimicrobial treatment. Attempts to isolate EPKP strains from the environment were also made. Environmental screening was performed by using swabs moinstened with sterile saline and included work surfaces, sinks, incubators, solutions, and equipment used in intubation. Hand impressions were also taken in order to examine carriage of EPKP by the medical and nursing staff of the clinic. Of all the 101 isolates of the micro-organism included in the analysis, 99 isolates were from neonates and 2 isolates were recovered from environmental examinations. Eighty-eight clinical isolates were non-repetitive (replicate) (first from each patient) and from 11 patients duplicate isolates were included in the study (from different specimens and different dates, the same specimen and different dates or different specimens and the same dates). Of all the 99 clinical isolates, 39 (39.4%) were obtained from throat swab specimens, 35 (35.4%) from rectal swab specimens, 9 (9.1%) from blood, 10 (10.1%) from urine, 5 from the respiratory tract and 1 from cerebrospinal fluid. Infection control measures. After the reorganization of The Infection Control and Surveillance System in January 2003, the Epidemiology Department was set up. An infection control and surveillance system was modified and new priorities were established. The control of an outbreak due to ESBL-producing Klebsiella pneumoniae in the Neonatology Clinic was one of them. An outbreak was confirmed by epidemiological studies using molecular methods. New surveillance procedures were introduced and old procedures were improved. Extensive environmental, medical staff and patient microbiological screening were performed. Routine microbiological examinations from throat and rectal sites were performed in each newly admitted neonate at every 3 days of hospitalization from all patients without colonization/infection with the emerging strain, and at every 7 days from patients with established colonization/infections. A rigorous contact precaution was introduced, and separate nursing staff for colonized/infected neonates were designated. Five new nurses were employed in the clinic. Routine preventive use of disposable gloves for each new patient contact was implemented. Deficient invasive equipment of the Intensive Care Unit was purchased. Training sessions about surveillance methods for health care personnel (appropriate in content and vocabulary to the educational level) were organized, underlining the importance and the proper use of gloves, gown and hand hygiene. Microbiological examinations from all patients were reviewed by the Hospital Epidemiologist and referred to every day to the medical personnel of the clinic. Identification and antimicrobial susceptibility tests. Species identification was performed with the use of a Vitek system (bioMérieux Vitek, Inc, Hazelwood, USA) and additional test (indol) recommended by the manufacturer. Susceptibility to common antibiotics was determined by disc diffusion methods with Mueller-Hinton agar according to the NCCLS. The following antimicrobials were tested: cefotaxime (30 :g), ceftazidime (30 :g), pipreracillin-tazobactam (100 + 10 :g), amoxicillin-clavulanate (20 + 10 :g), imipenem (10 :g), meropenem (10 :g), trimethoprim-sulfamethoxazol (1.25+ 23.75 :g), netilmicin (30 :g), amikacin (30 :g), aztreonam (30 :g) and ciprofloxacin (5 :g). Screening and phenotypic confirmatory tests for ESBL. Routine susceptibility testing by the disc diffusion method with cefotaxime (30 :g) and ceftazidime (30 :g) was used for screening for ESBL (zone diameter: for ceftazidime = 22 mm and for cefotaxime = 27 mm).

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The double disk synergy (DDS) test (Jarlier et al., 1988), carried out with disks containing amoxicillin-clavulanate (20 + 10 :g), cefotaxime (30 :g) and ceftazidime (30 :g), was used as a phenotypic confirmatory test for ESBL production (increased size of inhibition zone or a new inhibition zone). DNA isolation and typing methods. Total DNA isolation and ADSRRS-fingerprinting were performed as described previously (Krawczyk et al., 2003a). The randomly amplified polymorphic DNA (RAPD) with primer RAPD-4 from the RAPD Analysis Primer Set (Pharmacia Biotech, St Albans, UK) and pulsed-field gel electrophoresis (PFGE) were performed according to the procedure described previously (Krawczyk et al., 2003b).

Results Outbreak description. One hundred and forty nine neonates hospitalized in the Neonatal Clinic of the large Teaching Hospital from April 2002 to January 2003 were colonized/infected by ESBL-producing Klebsiella pneumoniae. In the reported period, 86 (5.4%) neonates were colonized and 63 (4%) were found to be infected. The average percent of colonized/infected patients during each month was from 6.2% to 22.4% (Table I), and the increase in colonized infants seen in April and December 2002 was due to active surveillance. Mean gestational age of the study group was 32 weeks, most of them were born by cesarean section (69.1%) with mean birth weight about 1921 g and Apgar score 6.4 (see Table II). In about 86% of Table I Percent of colonized/infected patients in ten months of study period and four months follow up Month and year

No of hospitalized No of colonized/ patients infected patients

April 2002

173

Percent of colonized/ infected patients

25

14.4

May 2002

166

11

6.6

June 2002

164

22

13.4

July 2002

163

19

11.7

August 2002

168

17

10.1

September 2002

135

16

11.8

October 2002

161

26

16.1

November 2002

150

21

14.0

December 2002

156

35

22.4

January 2003

146

9

6.2

February 2003

169

6

3.5

March 2003

175

12

6.9

April 2003

174

1

0.6

May 2003

175

none

0.0

Table II Clinical data of the neonates including to the study Genotyping (+)

Genotyping (–)

All study group

No. of neonates

88

61

149

Gender: male

49

38

87

39

23

62

female Gestational age, mean, wk Mode of delivery: cesarean section, n (%) Apgar score at 1 min, mean Birth weight, mean, g

33.1

33.5

32

62 (70.4%)

41 (67.2%)

103 (69.1%)

6.3

6.6

6.4

1883.9

1975.5

1921.4

Mechanical ventilation, n (%)

29 (32.9%)

21 (34.4%)

50 (33.5%)

Parenteral nutrition, n (%)

76 (86.4%)

52 (85.2%)

128 (85.9%)

41,3

36.3

393

78 (88.6%)

52 (85.2%)

130 (87.2%)

Length of hospital stay, mean Antimicrobial treatment, n (%)

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them parenteral nutrition was performed in the first few days, and 87.2% of them had undergone antimicrobial treatment. More than one-third (33.5%) of the neonates needed mechanical ventilation and their mean length of hospital stay was 39.3 days (min. 4 days, max. 130 days) (Table II). ESBL-producing Klebsiella pneumoniae was isolated in 6.7% of patients from blood, 20.8% had urinary tract infections, and pneumonia was recognized in 12.8%. From 149 neonates included in the study, 63 (57.7%) were found to be only colonized by Klebsiella pneumoniae in the throat and/or alimentary canal. Only two strains of EPKP were isolated during the environmental screening carried out several times (165 samples) in the reported time period. Identification and antimicrobial susceptibility. All of the isolates were identified as Klebsiella pneumoniae. As determined by disc-diffusion antibiotic susceptibility testing, the isolates, including those two from environmental sites, exhibited almost the same pattern of resistance. The percentage susceptibility rates to cefotaxime, ceftazidime and azteronam were 0%, 99% and 0%, respectively. All isolates were susceptible to meropenem, imipenem and ciprofloxacin. However, susceptibility to pipreracillin-tazobactam varied, and 82% of isolates were susceptible. Only 8% were susceptible to amoxicillin-clavulanate, 5% to amikacin and netilmicin and 4% to trimethoprim-sulfamethoxazol. Genotyping of isolates using ADSRRS-fingerprinting, RAPD and PFGE techniques. We investigated the clonally relatedness among outbreak ESBL-producing Klebsiella pneumoniae strains. One hundred and one isolates were included for typing by ADSRRS-fingerprinting, RAPD and PFGE methods. Four additional, epidemiologically unrelated EPKP strains were included for control purposes. The obtained ADSRRSfingerprinting patterns for representative isolates are presented in Fig. 1. Each pattern consisted of approximately 12 to 15 fragments in a size range of 200 to 1000 bp. The ADSRRS-fingerprinting patterns of the 99 K. pneumoniae isolates recovered from patients found only one unique profile, and this confirmed that a clonal spread of the emerging pathogen was established. Two isolates recovered from environmental examinations (sink) also belonged to the same cluster of genotypes. The RAPD and PFGE results were exactly in accordance with ADSRRS-fingerprinting patterns and revealed also one predominant pattern (results not shown). The isolates not-related to the outbreak and reference strains showed completely different patterns by using ADSRRS-fingerprinting (Fig. 1), RAPD and PFGE (results not shown) methods.

Fig. 1. ADSRRS fingerprints of the K. pneumoniae isolates; representative results The lane designated M1 contained molecular mass marker (501, 489, 404, 331, 242, 190, 147 and 110 bp); lane K, negative control; lane 1– 8, representative isolates from patients; lane 9– 10, environmental isolates; lane 11, reference strain, lanes 12– 13, related K. pneumoniae strains isolated from other clinic. The DNA fragments were electrophoresed in 8% polyacrylamide gel by using 1× Tris-borate EDTA running buffer at a field strength of 8 V cm–1

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Control measures. In the epidemiological inquest, we found that hygiene insufficiency (no adherence to the hand hygiene procedure, medical equipment sterilization, contact precautions of colonized patients) led to an outbreak, and extensive training of health care personnel was begun. In the next five months we reduced the transmission of endemic strain progressively, to succeed in May 2003 when none of the neonates were colonized/infected with ESBL-producing Klebsiella pneumoniae. Discussion Klebsiella pneumoniae often leads to nosocomial outbreaks due to its ability to spread among patients, mainly because of lack of adherence by medical personnel to infection control guidelines (Asensio et al., 2000; Podschun and Ullmann, 1998; Szabo et al., 1999). In general, the use of a third-generation cephalosporins was reported as a risk factor for acquisition of ESBLs (Asensio et al., 2000; Jain et al., 2003; Naumovski et al., 1992). In our clinic, third-generation cephalosporins were used only occasionally. According to the antibiotic policy, amoxicillin with clavulanic acid, with or without aminoglicosides (mostly amikacin) was used as the first-line therapy. According to some investigators, antimicrobial combinations may also be a risk factor for colonization by multi-resistant pathogens (Pessoa-Silva et al., 2003; Podschun and Ullmann, 1998). In our study 78.5% of neonates received amoxicillin with clavulanic acid and 45.6% of neonates amoxicillin/clavulanic acid with aminoglicoside (amikacin in 80.9%). However, we controlled an outbreak without changing the antibiotic policy, and, therefore, we concluded that a lack of adherence to infection control guidelines led to the spread of ESBL-producing Klebsiella pneumoniae, and that antibiotic treatment was only conducive in colonization of the digestive tract. During the study period ESBL-producing Klebsiella pneumoniae were found in the hospital environment (sink), but we thought that it was simply another site of contamination rather than a source of pathogen. The gastrointestinal tract of neonates was the principal reservoir of the pathogen, and there was no adherence to hygiene procedures which were crucial. In spite of extensive screening of hands because we suspected transmission of the emerging strain by health care personnel, we didn’t find this pathogen. However, we know it can be found for a short period of time on the skin surface (Podschun and Ullmann, 1998). During the hospital epidemiologist control study, some non-adherence to the guidelines was found: lack of contact precautions, improper use of gloves and gowns, inappropriate hand hygiene, no sterilization of nipples, wrong storage of laryngoscopes, and insufficient intensive care equipment. All of the infractions were eliminated, and the outbreak was controlled. We would like to emphasize that supplementation of deficient invasive equipment of the Intensive Care Unit and employing five new nurses was very important. Good communication between microbiologists, epidemiologists and neonatologists was crucial too, which has been emphasized previously by others (Asensio et al., 2000). Molecular epidemiology is irreplaceable in the identification of an outbreak. The clonal spread of an emerging pathogen was established, and it was very convincing to the medical personnel, which led to rigorous compliance with infection control guidelines. The origin of the resident strain and its mode of spread in the clinic were not elucidated. Differentiation of bacterial isolates by DNA fingerprinting facilitates epidemiologic studies and disease control. In the examined K. pneumoniae outbreak, ADSRRS-fingerprinting, RAPD and PFGE methods showed that the genotypes of all outbreak-related strains were identical, which suggested that the outbreak originated from a single clone. Although a particular typing method may have high discriminatory power and good reproducibility, the complexity of the method and interpretation of results, as well as the costs involved in setting up and using the method, may be beyond the capabilities of the laboratory. The choice of the molecular typing method, therefore, will depend upon the needs, skill level, and resources of the laboratory. The ADSRRS-fingerprinting is generally a simple technique with high discriminatory power and is low cost and may be most suitable for epidemiological studies (Masny and P³ucienniczak, 2001). This method may be equally attractive in comparison to PFGE or AFLP for storage of genetic profiles and for the creation of reference databases of organisms to which new outbreak strains can be compared across laboratories in order to monitor changes in microbial populations. Here we show the evaluation of a novel fingerprinting method (ADSRRS-fingerprinting) for epidemiological studies of K. pneumoniae outbreak. Using three genotyping methods (ADSRRS-fingerprinting, RAPD and PFGE), we showed that in an outbreak one genotype was responsible for the infections/colonisations, and although the ADSRRS-fingerprinting method may appear to be more complex than the RAPD technique,

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we found it fast and reproducible, as we showed previously for epidemiological studies of E. faecium (Krawczyk et al., 2003a) and S. marcescens (Krawczyk et al., 2003b). Acknowledgement. This study was supported by the grant of Gdañsk University of Technology to J.K.

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Fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites. BioTechniques 31: 930–936. N a u m o v s k i L., J.P. Q u i n n, D. M i y a s h i r o, M. P a t e l, K. B u s h, S.B. S i n g e r, D. G r a v e s, T. P a l z k i l l and A.M. A r v i n. 1992. Outbreak of ceftazidime resistance due to a novel extended-spectrum beta-lactamase in isolates from cancer patients. Antimicrob. Agents Chemother. 36: 1991–1996. P e s s o a - S i l v a C.L., B. M e u r e r M o r e i r a, V. C a m a r a A l m e i d a, B. F l a n n e r y, M.C. A l m e i d a L i n s, J.L. M e l l o S a m p a i o, L. M a r t i n s T e i x e i r a, L.E. V a z M i r a n d a, L.W. R i l e y and J.L. G e r b e r d i n g. 2003. Extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit: risk factors for infection and colonization. J. Hosp. Infect. 53: 198–206. P o d s c h u n R. and U. U l l m a n n. 1998. 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Polish Journal of Microbiology 2005, Vol. 54, No 2, 111–115

Detection of Clostridium difficile and Its Toxin A (TcdA) in Stool Specimens from Hospitalised Patients MARTA M. WRÓBLEWSKA1*, EWA SWOBODA-KOPEÆ1,2, ALICJA ROKOSZ1, GRA¯YNA NURZYÑSKA 2, AGNIESZKA BEDNARSKA2 and MIROS£AW £UCZAK1,2 1 Chair

and Department of Medical Microbiology, Medical University of Warsaw, 5 Chalubinskiego Street, 02-004 Warsaw, Poland 2 Microbiology Laboratory, Central Clinical Hospital of the Medical University of Warsaw, 1 Banacha Street, 02-097 Warsaw, Poland Received 1 December 2004, received in revised form 21 January 2005, accepted 25 January 2005 Abstract The study has been carried out to determine the frequency of C. difficile recovery in stool cultures and the rate of C. difficile toxin A detection in faecal specimens of patients with nosocomial diarrhoea. Clinical specimens comprised 4414 stool samples collected from 1998 to 2002 from adult patients hospitalised in different wards of a universityaffiliated hospital (1200 beds) and suspected of C. difficile-associated disease (CDAD). There have been 1308 (29.6%) specimens positive for C. difficile culture (15.1% in 1998, 29.5% in 1999, 33.8% in 2000, 31.2% in 2001 and 32.0% in 2002). The highest number of C. difficile strains was cultured from stool samples of patients hospitalised in the haematology/oncology ward (51.1% of all isolates), neurology (8.3%), nephrology (8.0%), gastrointestinal surgery (7.0%) and neurosurgery (6.2%) wards. The testing for C. difficile toxin A yielded 847 (19.2%) positive samples and 3567 (80.8%) toxin A-negative results. The percentage of C. difficile toxin A-positive samples was 29.4% in 1998, 17.5% in 1999, 23.2% in 2000, 17.1% in 2001 and 15.0% in 2002. In the analysed period we observed an increase in the number of stool specimens tested for C. difficile and an increase in the number of C. difficile culture-positive samples. A decrease in the number of C. difficile toxin A-positive samples was noted in the last 2 years of the study. This phenomenon may be due to an improved antibiotic policy of the hospital. K e y w o r d s: Clostridium difficile, C. difficile toxin A, antibiotic-associated diarrhoea, pseudomembranous colitis

Introduction In the recent years Clostridium difficile strains have been isolated with increasing frequency from the clinical specimens obtained from hospitalised patients (Wilcox and Smyth, 1998). It is therefore regarded as an emerging pathogen of the hospital-acquired infections. The bacterium spreads easily between patients due to transmission by the hospital environment or healthcare personnel. Furthermore, intensive therapy with broad-spectrum antibiotics and chemotherapeutic agents favour colonisation of the patients and subsequently development of disease. In healthy adults asymptomatic carriage rate is 2–3%, but upon hospitalisation increases to over 20%, especially if antibiotic therapy has been administered to the patient (Kyne et al., 1998). In adults with nosocomial diarrhoea C. difficile is the most commonly detected agent (Decre et al., 2000). Therapy with clindamycin or third generation cephalosporins has been predominantly reported as a predisposing factor to C. difficile-associated disease (Mylonakis et al., 2001). Furthermore, the infection causes prolonged hospitalisation and significantly increases its costs, even by over 50% (Wilcox et al., 1998; Wilcox and Dave, 2000; Kyne et al., 2002). C. difficile is an etiological agent of C. difficileassociated disease (CDAD). This entity comprises antibiotic-associated diarrhoea (AAD), antibiotic-associated colitis (AAC) and the most severe clinical presentation – pseudomembranous colitis (PMC), which * Corresponding author: Marta M. Wróblewska, Department of Medical Microbiology, Medical University of Warsaw, 5 Cha³ubiñskiego Street, 02-004 Warsaw, Poland. Tel./fax: (+48-22) 628-27-39. E-mail: [email protected]

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Table I Current methods for detection of C. difficile toxins and genes encoding C. difficile toxins Toxin/toxin encoding genes

Detection methods

Toxin A (TcdA)

immunoenzymatic assay (EIA), latex test

Toxin B (TcdB)

tissue culture cytotoxicity assay (cell lines: Vero, McCoy’s, etc.) cytotoxin neutralization assay (tissue culture + antitoxin)

Toxin A/toxin B (TcdA/TcdB) immunoenzymatic assay (EIA) Binary toxin (CDT)

not available

Toxin A gene (tcdA)

polymerase chain reaction (PCR)

Toxin B gene (tcdB)

polymerase chain reaction (PCR)

Binary toxin gene (cdt)

polymerase chain reaction (PCR)

can be fatal particularly in immunocompromised patients (Kato et al., 1991). Toxin A-producing strains of C. difficile have been mainly incriminated in these conditions. Majority of toxigenic C. difficile strains produce two types of toxins – A (TcdA) and B (TcdB). Some strains (7–8%) may produce a third toxin called binary toxin (CDT). The most sensitive and specific test available for diagnosis of C. difficile infection, which remains the “gold standard”, is a tissue culture assay for cytotoxicity of toxin B (Decre et al., 2000; Mylonakis et al., 2001). However it is not used in most of routine laboratories, since it requires tissue culture facilities. Also detection of binary toxin is not done routinely so far. Recently an immunoassay has been developed to determine simultaneously the presence of both toxin A and toxin B in a clinical sample, however without their discrimination from one another (Decre et al., 2000; Aldeen et al., 2000). In hospitalised patients with severe diarrhoea immunoassays for detection of toxin A (enterotoxin) appear at present to be important laboratory tests helping the clinicians with the diagnosis of infections caused by C. difficile (Jacobs et al., 1996). In patients with hospital-acquired diarrhoea it is therefore necessary to test stool specimens for the presence of C. difficile toxin A (Gerding et al., 1995; Poutanen and Simor, 2004). Diagnostic methods used for detection of C. difficile toxins and toxin encoding genes are listed in Table I. The aim of the study was to evaluate the frequency of recovery of C. difficile in culture and to determine the frequency of C. difficile toxin A detection in the stool specimens of patients hospitalised in a tertiary care hospital in view of an increasing number of cases of nosocomial postantibiotic gastrointestinal disorders. Experimental Materials and Methods The study comprised retrospective analysis of faecal specimens from adult patients suspected on clinical grounds of CDAD. The patients were hospitalised in the Central Clinical Hospital in Warsaw (1200 beds) over a period of five years (1998–2002). Duplicate specimens were excluded. The samples were collected into sterile containers. Inoculation of culture media and testing for C. difficile toxin A was done within 5 hours of specimen collection. Whenever possible, both the culture of C. difficile and TcdA detection were done on clinical specimens comprised in the study. Isolation of C. difficile strains. The stool samples were cultured for C. difficile by inoculation of Columbia blood agar containing cefoxitin, cycloserine and amphotericin B (CCCA medium). The plates were incubated at 37°C for 48 h in an anaerobic chamber “Heraeus” (85% N 2, 5% H2 and 10% CO2) and isolates identified by standard methods for these anaerobic bacteria (colony morphology, characteristic smell of the colonies, microscopic appearance of bacteria and their fluorescence in the UV lamp). The identification of C. difficile was confirmed with a latex agglutination assay for C. difficile antigen “Culturette Brand CD” test (Becton Dickinson). C. difficile toxin A detection in stool samples. The stool specimens were examined for the presence of C. difficile toxin A using a commercial immunoassay “Clostridium difficile toxin A test” (Oxoid, England).

Results In total, 4414 samples have been cultured in the studied period (1998–2002). Out of them, 1308 specimens (29.6%) have yielded growth of C. difficile. This comprised 1053 (80.5%) samples from patients hospitalised in internal medicine wards and 255 (19.5%) specimens from surgical wards (Table II). Among

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Table II Number of C. difficile culture-positive stool specimens in patients hospitalised in 1998–2002 in various wards (N = 1308) Ward

1998

1999

2000

2001

2002

Total

%

36

110

123

180

219

668

51.1

neurology

0

6

35

27

41

109

8.3

nephrology

7

9

21

23

45

105

8.0

vascular disorders

12

13

6

17

7

55

4.2

gastroenterology

5

11

10

7

15

48

3.7

other

9

20

14

9

16

68

5.2

gastrointestinal surgery

3

14

18

34

22

91

7.0

neurosurgery

7

11

20

26

17

81

6.2

transplantation surgery

3

5

12

4

19

43

3.3

surgical ICU

0

3

8

1

7

19

1.4

Internal medicine haematology-oncology

Surgery

other Total

1

5

4

5

6

21

1.6

83

207

271

333

414

1308

100.0

Table III Detection of C. difficile toxin A (TcdA) in faecal samples of patients hospitalised in 1998–2002 (N = 847) Year

Total number of samples

Number of toxin A -positive samples

Percentage

1998

551

162

29.4

1999

702

123

17.5

2000

802

186

23.2

2001

1067

182

17.1

2002

1292

194

15.0

Total

4414

847

19.2

samples positive for C. difficile in culture predominated specimens from haematology-oncology (51.1%), neurology (8.3%), nephrology with dialysis unit (8.0%), gastrointestinal surgery (7.0%) and neurosurgery (6.2%) wards (Table II). There was an increase in the number of positive culture results over these years – 83, 207, 271, 333 and 414, respectively (Table II). This corresponded to the following percentages of culture-positive samples: 15.1%, 29.5%, 33.8%, 31.2% and 32.0% in the consecutive years of the analysed period. The results of testing for C. difficile toxin A done on 4414 samples are shown in Table III. In total there were 847 (19.2%) toxin A-positive samples and 3567 (80.8%) toxin A-negative results. Over five analysed years the percentage of toxin A-positive samples was 29.4% in 1998, 17.5% in 1999, 23.2% in 2000, 17.1% in 2001 and 15.0% in 2002. Discussion C. difficile is considered as the most frequent etiological agent of nosocomial diarrhoea occurring in hospitalised patients, spreading easily to the environment, the hands of the health care workers and subsequently to other patients, particularly in large hospitals. In the recent years there was a steady increase in the frequency of C. difficile-associated diseases, which accounted for up to 15% of outbreaks of hospitalacquired diarrhoea (Djuretic et al., 1999; Zadik and Moore, 1998). Between 1992–1997 in the UK there has been 2.6-fold rise in culture-positive reports, while the corresponding increase in toxin-positive reports was approximately 9-fold (Wilcox and Smyth, 1998). An increase in the number of culture-positive specimens has also been recorded in our institution (Table II). We observed nearly 5-fold increase in C. difficile culture-positive samples during the study period (83 in 1998 and 414 in 2002). Diseases caused by C. difficile are related to the increased morbidity and mortality of elderly patients, as well as patients hospitalised in the renal medicine and chest medicine wards (Wilcox and Smyth, 1998;

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Kyne et al., 1998; Wilcox et al., 1998; Zadik and Moore, 1998; Boswell et al., 1998). In our study strains of C. difficile were isolated mainly from patients in haematology-oncology ward, followed by nephrology/ renal unit patients, neurology, gastrointestinal surgery and neurosurgery wards (Table I). This points to high risk areas for nosocomial spread of C. difficile strains (Blot et al., 2003). In the wards included in our study most commonly used antimicrobial agents comprised cephalosporins of the 3rd generation (ceftriaxone, ceftazidime) and 4th generation (cefepime), carbapenems, amoxicillin/clavulanate, metronidazole and fluconazole. Standard laboratory methods for diagnosing these infections include stool culture and identification of bacterial isolate, faecal toxin detection and C. difficile antigen detection. PCR technique can also be used for the rapid identification of toxigenic C. difficile (Kato et al., 1991). It has been reported that culture for C. difficile was positive in 30% of stool samples from patients with nosocomial diarrhoea (Pituch et al., 2000). In our study the frequency was similar – 29.6% of positive specimens overall for the analysed period. We also observed a steady increase in the number of C. difficile culture-positive results from 1998 to 2002. This could be ascribed to emergence of C. difficile in hospital-acquired infections as well as increased awareness of the clinicians of this etiology of diarrhoea. The culture lacks however specificity due to the possible faecal carriage of non-toxigenic isolates, therefore many laboratories rely on toxin detection rather than culture for the diagnosis of C difficile infection (Wilcox et al., 1998). There have been reports that examined stool samples were positive for toxin A in 5.5% in community-acquired diarrhoea and up to 22% in nosocomial diarrhoea (Wilcox and Smyth, 1998; Pituch et al., 2000; Fedorko et al., 1999; Miller et al., 2002). In our study this value was 19.2% in samples from patients with possible CDAD. An immunoassay for the detection of toxin A of C. difficile is an easy and rapid method in comparison to other techniques (direct examination of the sample, culture and testing for C. difficile antigen) used for diagnosis of these infections (Fedorko et al., 1999). Detection of C. difficile toxin A has proved to be of diagnostic importance also in our study (Table III). Toxin A-producing C. difficile appears to be an emerging pathogen in patients hospitalised in our hospital, particularly in the haematology-oncology ward (Table II). The discrepancy between the increase in the number of culture-positive samples (Table II) and a relative (expressed in %) fall in the number of toxin A-positive results (Table III) may have resulted from the fact, that clinicians are more aware of this etiology of diarrhoea. Therefore, more patients were detected who were colonised with C. difficile in the gastrointestinal tract, while diarrhoea could be due to other reasons (other bacteria, viruses, fungi, protozoa). Mixed diarrhoeal infections are also observed (Rokosz et al., 2002). However, recent reports have shown that C. difficile strains negative for toxin A and positive for toxin B (A-B+), as well as strains producing binary toxin alone, may also be virulent and cause clinical symptoms (Alfa et al., 2000; Stubbs et al., 2000; Wilcox and Fawley, 2001). Brazier and coworkers reported the frequency of 3% of A-B+ strains in over 1300 isolates from 35 hospitals (Brazier et al., 1999). However, up to 28–31% of C. difficile strains may have a mutant toxin A gene (Al-Barrak et al., 1999; Pituch et al., 1999). Toxin A-negative isolates of C. difficile cultured from human stools usually contain a small deletion of 1.8 kb within the repetitive regions of the tcdA gene (van den Berg et al., 2004). Webb argues, that prevalence of A-B+ strains is highly variable, ranging in many reports from 0.2% up to 48% in a paediatric population (Webb, 2000). In our study some cases might be due to toxin A-negative toxin B-positive and/or binary toxin-positive strains, because we did not test for toxin B and binary toxin at the time the study was conducted. Therefore development of laboratory tests for routine use, which could rapidly detect three known C. difficile toxins becomes a necessity. At present results obtained by different methods should be used in conjunction with patient history when making a diagnosis of C. difficile infection. Control of C. difficile infections requires avoidance of unnecessary antibiotic use, especially clindamycin, third generation cephalosporins and other agents, which show the greatest association with C. difficile disease (Mylonakis et al., 2001, Zadik and Moore, 1998). A tight restriction of their use is therefore needed. We recorded a decrease in the hospital expenses on antibacterial agents during the study period, from 25% in 1998 to approximately 20% in 2002, calculated as a percentage of the total medical costs of the hospital. This might have also contributed to less cases of CDAD recorded recently, in comparison to the previous years. A change in antibiotic policy and implementation of standard infection control measures reduced the incidence of C. difficile symptomatic infections (Wilcox and Smyth, 1998; Wilcox et al., 1998; Boswell et al., 1998; Khan et al., 2003; Riley, 2004). Combined approach, involving effective infection control measures, the use of rapid and sensitive techniques for laboratory diagnosis, as well as prudent use of antibiotics, is necessary to reduce morbidity and mortality due to C. difficile-associated infections in hospitalised patients.

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Nosocomial diarrhoea due to Clostridium difficile. Curr. Opin. Infect. Dis. 17: 323–327. R o k o s z A., A. S a w i c k a - G r z e l a k, H. P i t u c h and M. L u c z a k. 2002. Cultivation of fungi from faecal specimens in cases of antibiotic-associated diarrhoea (AAD) (in Polish). Med. Doœw. Mikrobiol. 54: 371–377. S t u b b s S., M. R u p n i k, M. G i l b e r t, J. B r a z i e r, B. D u e r d e n and M. P o p o f f. 2000. Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile. FEMS Microbiol. Lett. 186: 307–312. V a n d e n B e r g R.J., E.C.J. C l a a s, D.H. O y i b, C.H.W. K l a a s s e n, L. D i j k s h o o r n, J.S. B r a z i e r and E.J. K u i j p e r. 2004. Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping. J. Clin. Microbiol. 42: 1035–1041. W e b b K.H. 2000. Toxin A negative/B positive Clostridium difficile strains. J. Hosp. Infect. 45: 331–332. W i l c o x M.H. and J. D a v e. 2000. The cost of hospital-acquired infection and the value of infection control. J. Hosp. Infect. 45: 81–84. W i l c o x M.H. and W.N. F a w l e y. 2001. Virulence of Clostridium difficile toxin A negative strains. J. Hosp. Infect. 48: 81. W i l c o x M.H., W.N. F a w l e y, C.D. S e t t l e and A. D a v i d s o n. 1998. Recurrence of symptoms in Clostridium difficile infection – relapse or reinfection? J. Hosp. Infect. 38: 93–100. W i l c o x M.H. and E.T.M. S m y t h. 1998. Incidence and impact of Clostridium difficile infection in the UK, 1993–1996. J. Hosp. Infect. 39: 181–187. Z a d i k P.M. and A.P. M o o r e. 1998. Antimicrobial associations of an outbreak of diarrhoea due to Clostridium difficile. J. Hosp. Infect. 39: 189–193.

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Slime Production and Cell Surface Hydrophobicity of Nasopharyngeal and Skin Staphylococci Isolated from Healthy People ANNA MALM, ANNA BIERNASIUK, RENATA £OŒ, URSZULA KOSIKOWSKA, MAREK JUDA, IZABELA KORONA-G£OWNIAK and GRZEGORZ GÓRNIEWSKI*

Department of Pharmaceutical Microbiology, Medical University of Lublin, ChodŸki 1, 20-093 Lublin, Poland *Department of Thoracic Surgery, Medical University of Lublin, Jaczewskiego 8, 20-954 Lublin, Poland Received 27 December 2004, received in revised form 1 March 2005, accepted 4 March 2005 Abstract The collection of 314 staphylococcal strains including Staphylococcus aureus and coagulase-negative staphylococci (CNS) was isolated from skin or nasopharynx of healthy people. It was found that the majority of staphylococci possessed the ability to produce slime intensively or moderately, irrespective of ecological niche-nose, throat or skin. Most of them showed the hydrophilic cell surface. However, among S. aureus skin isolates or CNS throat isolates predominated strains with hydrophobic cell surface. There was a slight correlation between slime production and the nature of cell surface among CNS isolates but not among S. aureus strains. It was found that most of slime-producing CNS strains showed hydrophilic cell surface, while slime-negative isolates usually possessed hydrophobic cell surface. Our data suggest that slime production but not cell surface hydrophobicity can be regarded as an essential colonization factor responsible for staphylococci adherence to skin or mucous membranes of upper respiratory tract. These data also suggest that slime production seems to be a general feature of staphylococci isolated from various niches of healthy people. K e y w o r d s: slime, cell surface hydrophobicity, staphylococci.

Introduction Adhesion of bacterial cells, i.e. their attachment to epithelial cells of skin or mucous membranes of the respiratory, alimentary or genitourinary tract is the first step of colonization. This is due to nonspecific or specific cell-cell interactions, involving several microbial and host factors. The cell surface hydrophobicity of bacteria is an important non-specific adhesion factor, while production of extracellular mucoid substances of polysaccharide nature, so-called slime or glycocalix, may enhance the ability of bacterial cells to adhere specifically to host tissue (Howard and Rees, 1994; Wilson et al., 1996). Staphylococci colonize several niches of the human body. These microorganisms usually existing as a resident or as a transient member of the normal flora of skin and upper respiratory tract, can be regarded as a potential reservoir of endogenous infections under predisposing conditions (Howard and Kloos, 1994; Wilson et al., 1996). Phage typing and antibiotic resistance patterns suggest that the colonizing and invading strains are usually identical (Cree et al., 1994). In the light of controversial or conflicting literature data regarding adhesion properties of staphylococcal strains (Baldassarri et al., 1997; Ammendolia et al., 1999), the aim of this paper was to compare the extent of slime production and the relative cell surface hydrophobicity of potentially pathogenic Staphylococcus aureus and coagulase-negative staphylococci (CNS) strains colonizing skin or mucous membranes of nasopharynx in healthy people. Experimental Materials and Methods Bacterial strains. Staphylococcal strains were isolated from January to March 2003 from skin (forehead), nasal or throat swabs of both children (aged from 3 to 10 years) and adults (aged from 21 to 60 years) with no clinical symptoms of skin or respiratory tract infections. Isolated species were identified by conventional methods (macroscopic, microscopic or biochemical assays) or by

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rapid commercial latex test – Slidex Staph-Kit (bioMerieux). API 20 STAPH was used to determine species of CNS strains. All isolates were classified as methicillin-sensitive staphylococci according to NCCLS standards (Cuny et al., 2002). Assay of slime production. Slime production by isolated staphylococcal strains was assesed by the visual method described by Freeman et al. (1989), using solid medium containing 5% sucrose and 0.08% Congo red. After inoculation, agar plates were incubated for 24 hrs at 37°C. The extent of slime production was assessed on the basis of colour of staphylococcal colonies, according to criteria presented by Freeman et al. (1989). Assay of cell surface hydrophobicity. The relative cell surface hydrophobicity of isolated staphylococcal strains was assessed using modified ammonium sulfate salt aggregation test (Lindahl et al., 1981). It was assumed that strains autoaggregated were described as very strong hydrophobic, aggregated at 0.4– 1.0 M (NH 4)2SO4 – as strong hydrophobic, at 1.2– 1.6 M (NH4)2SO4 – as hydrophobic, at >1.8 M (NH 4)2SO4 – as hydrophilic. Statistical analysis. Statistical analyses of adhesion properties of the isolated staphylococci were made using the nonparametric tests (Chi2 test, Chi2 test with Yates correction or V2 test), depending on the total (observed) or expected frequences. The difference at the 5% level was considered statistically significant. Correlation between slime production and cell surface hydrophobicity was assessed by the point-biserial coefficient (rpb).

Results The collection of 314 staphylococcal strains, including Staphylococcus aureus and coagulase-negative staphylococci (CNS), isolated from skin or nasopharynx in healthy people was presented in Table I. Table II shows data concerning slime production by the isolated staphylococcal species. The majority of them were able to produce slime intensively or moderately – 139/142 (97.9%) vs 158/172 (91.9%) among S. aureus or CNS isolates, respectively. However, slime-negative organisms were isolated more frequently among CNS strains than those belonging to S. aureus (p < 0.0190). It was also found that, irrespective of ecological niche, CNS strains showed more frequently ability to produce slime moderately than S. aureus isolates – 111/172 (64.54%) vs 51/142 (35.92%), respectively (p< 0.0000). Moreover, it was observed that intensive slime production was detected more frequently among skin S. aureus strains compared to nasopharyngeal isolates of this species – 24/26 (92.31%) vs 64/113 (56.64%), respectively (p< 0.0007). Generally, the majority of the isolated staphylococcal strains showed hydrophilic cell surface – 118/142 (83.1%) vs 125/172 (72.67%) among S. aureus or CNS isolates, respectively (Table III). However, strains with hydrophobic cell surface were isolated more frequently among CNS isolates than S. aureus isolates (p< 0.0280), particularly strains possessing strong hydrophobic cell surface – 20/172 (11.63%) vs 2/142 (1.41%), respectively (p< 0.0009). It was also found that specific situation regarding the nature of cell surface took place among S. aureus isolates from skin and CNS isolates from throat, since in both subcollections strains with hydrophobic cell surface predominated (p< 0.025 or p< 0.0042, respectively). Table I The collection of tested staphylococcal strains isolated from skin or mucous membranes of nasopharynx in healthy people Species

The group of staphylococci

Number of strains Nasal swabs

Throat swabs

Skin swabs

Coagulase-positive staphylococci S. aureus (n = 142)

77

38

27

Coagulase-negative staphylococci S. epidermidis (n = 67)

56

6

5

S. xylosus (n = 16)

9

4

3

S. hominis (n = 11)

7

2

2

(n =172)

S. haemolyticus (n = 14)

10

1

3

S. capitis (n = 2)

1

0

1

S. saprophiticus (n = 4)

2

2

–

S. sciuri (n = 3)

1

2

–

S. warneri (n = 9)

–

–

9

S. lentus (n = 2)

–

–

2

S. simulans (n = 1)

–

–

1

Novobiocin-sensitive Staphylococcus spp.* (n = 34)

15

1

18

Novobiocin-resistant Staphylococcus spp.* (n = 9)

3

5

1

* definitive identification of these CNS strains was impossible by using API 20 STAPH

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Characteristic of nasopharyngeal and skin staphylococci Table II Slime production by staphylococcal strains isolated from skin or mucous membranes of nasopharynx of healthy people Species (total number of strains)

Number (percent) of strains The extent of slime production Nasal swabs Throat swabs Skin swabs

S. aureus (n = 142)

Coagulase-negative staphylococci (n = 172)

+++

41 (53.3)

23 (60.5)

24 (88.9)

++

36 (46.7)

13 (34.2)

2 (7.4)

–

0 (0)

2 (5.3)

1 (3.7)

+++

29 (27.9)

8 (34.8)

10 (22.2)

++

69 (66.3)

14 (60.9)

28 (62.2)

–

6 (5.8)

1 (4.3)

7 (15.6)

+++ intensive slime production, ++ moderate slime production, – lack of slime production

Table III The relative cell surface hydrophobicity of staphylococcal strains isolated from skin or mucous membranes of nasopharynx of healthy people Species (total number of strains) S. aureus (n = 142)

The relative cell surface hydrophobicity

Number (percent) of strains Nasal swabs

Very strong hydrophobic

0 (0)

0 (0)

1 (1.3)

1 (2.6)

0 (0)

Hydrophobic

11 (14.3)

2 (5.3)

8 (29.6)

Hydrophilic

65 (84.4)

35 (92.1)

18 (66.7)

2 (1.9)

4 (17.4)

0 (0)

Strong hydrophobic

Coagulase-negative

Throat swabs Skin swabs

Very strong hydrophobic

staphylococci (n = 172) Strong hydrophobic

1 (3.7)

10 (9.6)

4 (17.4)

6 (13.3)

Hydrophobic

7 (6.7)

4 (17.4)

10 (22.2)

Hydrophilic

85 (81.7)

11 (47.8)

29 (64.5)

There was no correlation between the biochemical phenotype (API numerical code) and cell surface hydrophobicity or ability to slime production within individual species of isolated staphylococci (data not show). According to Figs. 1 A and B, most of slime-producing CNS strains showed hydrophilic cell surface – 120/158 (75.95%), while those with no ability to produce slime usually possessed hydrophobic cell surface – 9/14 (64.29%) (p < 0.0034). Besides, most of slime-producing, hydrophobic CNS strains were classified as moderately slime-producing organisms – 34/38 (89.47%) (p< 0.0030). Despite this, there was only a slight correlation between the extent of slime production and the nature of cell surface among isolated CNS (rpb = – 0.33); this correlation was comparable to nasopharyngeal isolates (rpb = – 0.38) and to skin strains (rpb = – 0.32). In contrast, there was no correlation between the extent of slime production and the nature of cell surface among S. aureus strains (rpb < – 0.1). Discussion Slime production appears to be one of virulence factors of staphylococci which provides not only a permanent binding to the host tissue, but also protects bacteria against phagocytosis interfering with specific acquired immune response and impairs an access of antibacterial agents to targets within bacterial cell. In addition, slime-producing bacteria, including staphylococci, have the ability to form in the host organism a structure, so-called biofilm, which consists of an elastic gel containing exopolysaccharide within which microcolonies can develop. Formation of this structure and its extreme inherent resistance to antimicrobial agents and to host defense mechanisms is the basis for many persistent and chronic bacterial infections, which causes difficulty in eradication of biofilm-producing bacteria (Costerton et al., 1999; Donlan and Costerton, 2002).

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Fig. 1. Correlation between slime production and cell surface hydrophobicity in Staphylococcus aureus strains (A) and coagulase-negative (CNS) strains (B) isolated from skin or mucous membranes of nasopharynx of healthy people. The extent of slime production: +++ intensive slime production; ++ moderate slime production; – lack of slime production

Production of slime plays an important role in staphylococcal diseases, especially in hospital-acquired infections caused by S. aureus or CNS associated with indwelling medical devices, since this allows bacteria to adhere to smooth surfaces of foreign body-biomaterials. The source of these microorganisms is usually the skin or mucosal surfaces of the patient or the hospital personel (von Eiff et al., 1999). Data obtained in this paper suggest that despite some differences in the extent of slime production by particular strains this phenomenon seems to be a general feature of staphylococci isolated from healthy people, irrespective of ecological niche. According to the literature (Cree et al., 1994), there is also no correlation between adhesion properties of staphylococci and their phage-type, plasmid profile or antibiotic resistance patterns. Among non-specific mechanisms of bacterial adherence to host tissues, hydrophobicity of cell surface of bacteria seems to be an essential adhesion factor. Our data suggest that in contrast to Gram-negative bacteria (Jankowski et al., 1997; Mikucka et al., 2000; Janicka et al., 2002; £oœ et al., 2004), the adherence of staphylococci to skin or mucous membranes of nose or throat in healthy people is rather not determined by cell surface hydrophobicity. However, it should be taken into account that slime production by staphylococci may be responsible for masking of the real nature of cell surface, since it may interfere with our ability to detect cell surface hydrophobicity in vitro. Our observations showed that a slight correlation between the extent of slime production and the nature of cell surface among isolated CNS is in agreement with data presented by other authors that slime production seems to be discriminative factor between hydro-

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phobic and hydrophilic CNS strains (Baldassarri et al., 1997). However, such correlation was not found among S. aureus strains. Extensive studies on non-specific or specific interactions between staphylococci and skin or airways mucous membranes provide information about microbial factors responsible for the carrier state, which may be a precursor to localized or invasive infections, including those associated with biomaterials (Drago et al., 2002; Wu et al., 2003). Our data suggest that slime production but not cell surface hydrophobicity can be regarded as an essential colonization factor responsible for staphylococci adherence to skin or mucous membranes of upper respiratory tract due to presence of specific extracellular matrix slime-reactive adhesions (Shuter et al., 1996; Baldassarri et al., 1997). Literature A m m e n d o l i a M.G., R. D i R o s a, L. M o n t a n a r o, C.R. A r c i o l a and L. B a l d a s s a r r i. 1999. Slime production and expression of the slime-associated antigen by staphylococcal clinical isolates. J. Clin. Microbiol. 37: 3235–3238. B a l d a s s a r r i L., G. D o n e l l i, A. G e l o s i a, A.W. S i m p s o n and G.D. C h r i s t e n s e n. 1997. Expression of slime interferes with in vitro detection of host protein receptors of Staphylococcus epidermidis. Infect. Immun. 65: 1522–1526. C o s t e r t o n J.W., P.S. S t e w a r t and E.P. G r e e n b e r g. 1999. Bacterial biofilms: a common cause of persistent infections. Science. 284: 1318–1322. C r e e R.G.A., P. A l e l j u n g, M. P a u l s s o n, W. W i t t e, W.C. N o b l e, A. L j u n g h and T. W a d s t r ö m. 1994. Cell surface hydrophobicity and adherence to extra-cellular matrix proteins in two collections of methicillin-resistant Staphylococcus aureus. Epidemiol. Infect. 112: 307–314. C u n y C., G. W e r n e r, C. B r a u k l e, I. K l a r e and W. W i t t e. 2002. Diagnostics of staphylococci with special reference to MRSA. J. Lab. Med. 26: 165–173. D o n l a n R.M. and J.W. C o s t e r t o n. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15: 167–193. D r a g o L., D e V e c c h i, M. V a l l i, L. N i c o l a and M.R. G i s m o n d o. 2002. Effect of linezolid in comparison with that of vancomycin on glycocalix production: in vitro study. Antimicrob. Agents Chemother. 46: 598–599. v o n E i f f C., C. H e i l m a n n and G. P e t e r s. 1999. New aspects in the molecular basis of polymer-associated infections due to staphylococci. Eur. J. Clin. Microbiol. Infect. Dis. 18: 843–846. F r e e m a n D.J., F.R. F a l k i n e r and C.T. K e a n c. 1989. New method for detecting slime production by coagulase negative staphylococci. J. Clin. Pathol. 42: 872–874. H o w a r d B.J. and W.E. K l o o s. 1994. Staphylococci, p. 243–256. In: Howard B.J., Keiser J.F., Smith T.F., Weissfeld A.S., Tilton R.C. (eds.), Clinical and pathogenic microbiology. Mosby, St. Louis. H o w a r d B.J. and J.C. R e e s. 1994. Host-parasite interactions: mechanisms of pathogenicity, p. 9–36. In: Howard B.J., Keiser J.F., Smith T.F., Weissfeld A.S., Tilton R.C. (eds.), Clinical and pathogenic microbiology. Mosby, St. Louis. J a n i c k a G., A. M i k u c k a, A. S ê k o w s k a, T. Z w i e r z c h l e w s k i and M. W r ó b l e w s k i. 2002. Autoaggregation, hydrophobic, and hydrophilic properties of Moraxella catarrhalis strains. Acta Microbiol. Polon. 51: 23–30. J a n k o w s k i S., J. S a r o w s k a, H. ¯ a r c z y ñ s k a and A. C i s o w s k a. 1997. Hydrophobic properties of Pseudomonas aeruginosa strains (in Polish). Med. Doœw. Mikrobiol. 49: 187–190. L i n d a h l M., A. F a r i s, T. W a d s t r ö m and S. H j e r t e n. 1981. A new test based on “salting out” to measure relative surface hydrophobicity of bacterial cells. Biochim. Biophys. Acta. 677: 471–476. £ o œ R., A. M a l m, A. B i e r n a s i u k, I. K o r o n a - G ³ o w n i a k and U. K o s i k o w s k a. 2004. Hydrophobic proporties of Gram-negative rods colonizing upper respiratory tract of healthy people (in Polish). Med. Doœw. Mikrobiol. 56: 57–65. M i k u c k a A., E. G o s p o d a r e k and B. U l a t o w s k a. 2000. Influence of growth conditions on cell surface hydrophobicity of rods of genus Serratia (in Polish). Med. Doœw. Mikrobiol. 52: 9–15. S h u t e r J., V.B. H a t c h e r and F.D. L o w y. 1996. Staphylococcus aureus binding to human nasal mucin. Infect. Immun. 64: 310–318. W i l s o n R., R.B. D o w l i n g and A.D. J a c k s o n. 1996. The biology of bacterial colonization and invasion of the respiratory mucosa. Eur. Respir. J. 9: 1523–1530. W u J.A., C. K u s u m a, J.J. M o n d and J.F. K o k a i - K u n. 2003. Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces. Antimicrob. Agents Chemother. 47: 3407–3414.

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Polish Journal of Microbiology 2005, Vol. 54, No 2, 123–135

Resistance Patterns of Streptococcus pneumoniae Strains Isolated in the West Pomerania Province in 2001–2003 MONIKA M. NOWOSIAD and STEFANIA T. GIEDRYS-KALEMBA

Department of Microbiology and Immunology, Pomeranian Medical University Powstañców Wielkopolskich 72 Street, 70-111 Szczecin, Poland Received 27 December 2004, received in revised form 11 March 2005, accepted 15 March 2005 Abstract An abrupt antimicrobial resistance increase among Streptococcus pneumoniae strains has become a serious therapeutic problem in the recent years. The aim of this study was to describe the resistance increase of S. pneumoniae strains isolated in the West Pomerania Province over three years (2001– 2003). Using E-tests method and NCCLS criteria for 80 pneumococal resistant strains the resistance degrees and patterns have been determined and analyzed in connection with their clinical origin. The majority of specimens of resistant strains isolated came from nasopharynx (80% strains) of infected ambulatory patients (81.3%), from children at nursery school age (65.7%), suffering from chronic upper respiratory tracts infection (86.7%). However, strains originated from older patients, hospitalized, in serious health condition showed higher resistance degrees. The greatest number of isolates (27.5%) showed resistance to 3 out of 9 tested drugs and over a half (53.8%) of the tested strains belonged to MDR strains, with increasing percentage over time: from 62.5% in 2001 to 69.8% in 2003. Resistance to 8 out of 9 determined antibiotics (except vancomycin) has occurred and domination of 4 resistance patterns: ELTS, S, TSH, PSI, present in 50.1% of the tested strains was observed. The phenomena observed in the study: growing resistance degree, increasing amount of MDR strains, emergence of new resistance patterns, testify to gradual pneumococcal resistance increase and give a picture of local trends in antibiotic therapy. Also the epidemiological data concerning patients, from whom the tested strains were isolated are adequate to risk factors of infection with resistant pneumococci. K e y w o r d s: Streptococcus pneumoniae, Poland, multidrug resistance, risk factors

Introduction S. pneumoniae causes a lot of serious diseases such as lobar pneumonia, meningoecephalitis and septicaemia belonging to main reasons of disease incidence and mortality regardless of age and part of the world (Feldman and Klugman, 1997; Appelbaum, 1992; Caputo et al., 1993). It is also the most frequent etiological factor of upper respiratory tracts infections, especially sinusitis and otitis media (Appelbaum, 1992), which being not so life-threatening but are the most often found in ambulatory diagnoses. Antibiotic therapy is not always supported with microbiological diagnostics and antimicrobial susceptibility determination, what more and more often leads to empirical therapy failures, especially in recent years. The source of these failures lies in abrupt antibiotic resistance increase among S. pneumoniae strains (Kaplan and Mason, 1998; Feikin et al., 2000; Lister, 1995; Dagan et al., 1996). The first penicillin resistant strain was described in Australia in 1967 (Hansman and Bullen, 1967). Since that time in many countries there have been reported pneumococci resistant to penicillin and later also to nearly all known groups of antibiotics (other than penicillin betalactams, macrolides, lincosamids, tetracyclines, cotrimoxasoles, chloramphenicol, fluorochinolons, aminoglycosides) and even strains showing tolerance to vancomycin in animals (Feldman and Klugman, 1997; Appelbaum, 1992; Dowson et al., 1994; Pallares et al., 1987; Carbon and Poole, 1999; Baguero, 1997; Appelbaum et al., 1996; Appelbaum, 1996; Baquero, 1997; Novak et al., 1999). The fact of co-occurrence of resistance to many antibiotics is especially distressing. Pneumococci resistant to 3 or more drug-groups are defined as multidrug resistant (MDR).

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In the recent years we could observe not only appearance and increase of pneumococcal resistance, but also abrupt spreading of multidrug resistant strains in many countries, including Poland (Trzciñski and Hryniewicz, 1997). National range of many isolated resistant clones has been described. A few of them have gained the name of pandemic strains: Spanish23F-1, Spanish6B- and Spanish14-5. The presence of the Spanish23F-1 clone and other its capsular variants has been documentary proved in 24 countries from all the inhabited continents excluding Australia (Appelbaum, 1992; Dowson and Trzciñski, 2001; Hermans et al., 1997; Reichmann et al., 1995; Caputo et al., 1993). In Poland in the 90s two of them (Spain 23F-1 and France9V-3) were detected and also two other epidemic national clones of Poland23F-16 and Poland6B-20 serotypes were described (Overweg et al., 1999). Microbial resistance is variable and unstable. It depends mainly on antibiotic policy in particular region. In certain countries multiresistance is very high e.g. in Korea it reaches 80% (Kim et al., 1996), in Spain and Hungary 40%, in France 20% (Grzesiowski et al., 1999). Also geographic variability of penicillin “nonsusceptibility” ranges from over 40% in 16 out of 60 countries analyzed by Dowson to below 5% in only three of them (Dowson and Trzciñski, 2001). Hence, there is a justified need for continuous and multicenter monitoring of local antibiotic resistance in order to build a more effective strategy of pneumococcal therapy. The aim of this study is to describe the changeability of the degree and patterns of resistance of S. pneumoniae strains isolated in the West Pomerania Province during three years (2001– 2003) in connection with their clinical origin. Experimental Materials and Methods Bacterial strains. In the research there has been applied a collection of 132 strains of S. pneumoniae showing lowered antibiotic sensitivity in routine testing, isolated from various specimens from infected patients in the Department of Microbiology and Immunology at the Pomeranian Medical Academy and in 5 microbiological laboratories in the West Pomerania Province from 2001 to 2003. Also data concerning patients’ sex and age, disease symptoms and kinds of specimens from which particular bacterial strains came were collected. The strains were preserved in Tryptic Soy Broth with addition of 15% glycerol at the temperature of – 70°C. Antimicrobial susceptibility testing. Initial determination of antibiotic sensitivity was carried out with disc-diffusion method according to NCCLS (NCCLS, 2000). The incubation was performed on Miller-Hinton agar with addition of 5% of sheep blood at the temperature of 35°C in air supplemented with 5% CO2. With disc-diffusion method using discs with oxacillin (1 µg), erythromycin (15 µg), clindamycin (2 µg), tetracycline (30 µg), cotrimoxazole (1.25/23.75 µg) (Beckton Dikinson) resistant strains and in case of oxacillin “nonsusceptible” strains (i.e. with growth inhibition zone below 20 mm) were determined. The macrolide resistance phenotype was defined with application of double-disc method with erythromycin and clindamycin discs. Then strains resistance was verified and subjected to extended antimicrobial susceptibility analysis with determination of the minimum inhibitory concentration (MIC) using E-tests for the following antimicrobial agents: benzylpenicillin (P), erythromycin (E), clindamycin (L), tetracycline (T), cotrimoxazole (S), ceftriaxone (C), chloramphenicol (H), vankomycin (W), imipenem (I) according to the producer’s directions (AB Biodisk, Solna, Sweden Jacobs et al., 1992). The obtained MICs were interpreted according the NCCLS criteria as resistant: for P≥ 2 µg/ml, E ≥ 2 µg/ml, L ≥ 1 µg/ml, T ≥ 8 µg/ml, S ≥ 4 µg/ml, C ≥ 2 µg/ml, H ≥ 8 µg/ml, I ≥ 1 µg/ml, and as intermediate: for P 0.12– 1 µg/ml, E 1 µg/ml, L 0,5 µg/ml, T 4 µg/ml, S 1– 2 µg/ml, C 1 µg/ml, I 0.25– 0.5 µg/ml (NCCLS, 2000). Subsequently, for every strain the resistance pattern was determined by qualifying intermediate and high resistance to particular antibiotics. The degree of their resistance (from 1- to 7-drug resistant strains) was determined and strains resistant to at least one of the antibiotics were subjected to further analysis. Percentage share of particular resistance patterns and degrees in years covered by study (2001–2003) were analyzed with relation to strain epidemiological data.

Results From previously collected 132 strains, after initial selection with disc-diffusion method and subsequent verification with E-tests in accordance with the above mentioned criteria, 80 strains resistant to at least one antibiotic were qualified for further analysis. These strains came from patients of different age: from 3 months to 68 years (average – 10.3 years, median: 5 years); in 54.7% from men. The patients suffered mainly form upper respiratory tract infections (59 out of 68 i.e. 86.7%): chronic pyogenic rhinitis (42), sinusitis (8), otitis media (6) or bronchitis (4). Single isolates came from people with conjunctivitis (2), vaginitis (1), fever of unknown origin (1). Other 5 strains were isolated from patients with serious infections: 3 from people with pneumonia and 2 from patients staying in intensive care units. These 5 strains were characterized by high degree of multidrug resistance (7-, 6-, 6-, 5-, 4- drug resistant). The majority of strains came from nosopharynx: from nose – in

2

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Resistance patterns of Streptococcus pneumoniae Pomeranian isolates

44 patients being tested (55%), from pharynx – in 8 (10%); both from nose and pharynx of the same patient in 12 cases (15%). The other materials from which mainly high resistance degree strains were isolated are the following: BAL (broncho – alveolar lavage) – two strains: 6-drug resistant and 7-drug resistant, sputum (5-drug resistant strain), swab from external acoustic duct (4-drug resistant strain), 2 swabs from conjunctival sac (4-drug resistant strains) and one swab from a five-year girl’s vagina (6-drug resistant strain). Two strains taken from sinus punctuates were resistant to three of tested antibiotics: highly cotrimoxazole resistant and intermediately penicillin and imipenem resistant. It is worth mentioning that strains of high resistance degree were isolated mainly in hospital wards: 2 out of 3 (66.7%) 7-drug resistant strains, 3 out of 5 (60%) 6-drug resistant strains and half (3 out of 6) 5-drug resistant strains. Whereas 13 out of 14 strains (92.9%) resistant to one antibiotic and all of two-drug resistant strains were obtained from ambulatory patients. Detailed description of epidemiological data together with the degree and patterns of resistance are presented in Table I. Among tested strains over a half (53.8%) constituted multidrug resistant strains MDR (43 out of 80), with growing tendency of their percentage in time: 62.5% in 2001, 61.5% in 2002, and 69.8% in 2003. The greatest number of isolates (22 out of 80 i.e. 27,5%) showed resistance to 3 out of 9 tested antibiotics, the smallest number showed resistance to 7 drugs (3 out of 80 i.e. 3,8%). The other percentage shares constituted 17.5% of strains 1-drug resistant, 16.3% of 2-drug resistant, 8.8% of 4-drug resistant, 7.5% of 5-drug resistant and 6.3% of 6-drug resistant (Fig. 1). The resistance degree analysis of isolates in given years and especially comparison of the most numerous occurrences in 2001 and 2003 points to the fact that the percentage of low degree resistance strains decreased: 1-drug resistant strains from 20.8% in 2001 to 14% in 2003 and 2-drug resistant strains from 16.7% to 16.3%. Instead, the percentage of multidrug resistant strains increased. From five 6-drug resistant strains four were isolated in 2003 and one in 2002; in reference to all isolates in a particular year it amounted

100%

Percentage of resistant strains

90% 80% 70% 60% 50% 40% 30%

12345678901 12345678901 7.5% 12345678901 123456789012345

123456789012 4.2% 123456789012 0% 123456789012 123456789012 123456789012 12.5% 123456789012 123456789012345 123456789012 123456789012345

12345678 12345678 12345678 12345678 12345678 12345678 27.5% 12345678 12345678 12345678 12345678 12345678

12345678 12345678 12345678 12345678 12345678 25.0% 12345678 12345678 12345678 12345678

123456789012345 3.8% 123456789012345 123456789012345 123456789012345 6.3% 12345678901 123456789012345 123456789012345 12345678901 123456789012345 123456789012345 123456789012345 123456789012345 8.8% 123456789012345 123456789012345

16.3%

123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 20.8% 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345

16.7%

7.7% 123456789012345 123456789012345 123456789012345 123456789012345 7.7% 123456789012345 123456789012345 123456789012345 0% 123456789012345 123456789012345 38.5% 123456789012345 123456789012345

12345678 12345678 12345678 12345678 12345678 12345678 12345678 12345678 12345678 38.5% 12345678 12345678 12345678 15.4%

0% 123 1237-drug resistant 123 12345 12345 12345 12346-drug resistant

12345-drug resistant

12345678901 12345678901 7.0% 123456789012345 12345678901 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 25.6% 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345 123456789012345

12345678 12345678 12345678 12345678 12345678 25.6% 12345678 12345678 12345678 12345678 16.3%

20% 10%

123456789012345 2.3% 123456789012345 123456789012345 123456789012345 123456789012345 9.3% 12345678901 123456789012345 123456789012345 12345678901

17.5%

20.8%

23.1%

14.0%

TOTAL

2001

2002

2003

3.8%

4.2%

7.7%

2.3%

6.3%

0.0%

7.7%

9,3%

123 1233-drug resistant

7.5%

12.5%

0.0%

7.0%

8.8%

20.8%

7.7%

25.6%

27.5%

25.0%

38.5%

25.6%

1-drug resistant

16.3%

16.7%

15.4%

16.3%

1-drug resistant

17.5%

20.8%

23.1%

14.9%

12345 12345 12345 12345 4-drug resistant

Year

Fig. 1. Percentage share of different resistance degrees of Streptococcus pneumoniae resistant strains isolated in 2001– 2003 period and in each year

126

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Nowosiad M. et al.

8 X 6 52 52 27 60 5 68 8 6 2 X 10 X 4 5 4 4 59 1 8 9 15 1 X 1 39 10 X 3 0 5 X 15 5 4 12 4 6

(b)* BAL C P BAL N P N V S N NP P X N X N N N E P N N N P N N N N N N N NP N X N N N C N P

Patient clinical diagnosis

years

K M K K K K K K M M K M X K X M M M M M K M M M K K M K K M M M K X K M K M K M

Specimen

M/K

( c)* P C U Rean Rean RU R V P R OR RB X R X S RS RS O S F B X U R X RU R RO P R OR X X R R RB C U U

Resistance degree

2003 2002 2001 2003 2003 2003 2002 2003 2003 2003 2003 2001 2001 2001 2003 2003 2003 2003 2001 2003 2003 2003 2003 2003 2003 2001 2003 2001 2001 2001 2002 2003 2001 2001 2001 2001 2002 2002 2002 2003

Resistance pattern

(a)*

Patient age

ICU NEPH AMB ICU ICU AMB AMB GIN HEM AMB AMB PED GIN AMB AMB LAR AMB AMB AMB AMB PED AMB AMB AMB AMB GIN AMB AMB AMB PED AMB AMB AMB AMB AMB AMB AMB AMB ICU AMB

Patient sex

Origin / hospital ward

80 128 93 29 32 56 125 129 27 81 24 98 106 69 15 23 41 72 78 1 10 11 30 44 49 66 82 95 110 111 120 46 68 83 87 99 114 124 127 86

The year of strain isolation

Strain no

Table I Epidemiological characterisic and resistance patterns of Streptococcus pneumoniae resistant strains isolated in West Pomerania Province in 2001–2003

(d)*

(e)*

PELTSCiIi PiELTSHIi PiELTSiHIi PiELSHIi PiELSHIi PiELSHIi PTSCiHIi PiELTSiIi PiELSH PiELTS PESCiIi PiTSHIi PTSHIi PTSiHIi ELTS ELTS ELTS ELTS ELTS ELTSi ELTSi ELTSi ELTSi ELTSi PiELT ELTSi PiELT ELTSi PiELT ELTSi ELTSi ELT TSH TSH TSH TSH TSH ELT ELT TSH

7 7 7 6 6 6 6 6 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3

(a)* AMB – ambulatory strain, ICU – intensive care unit NEPH – nephrology department, PED – pediatric department, HEM – hematology department, GIN – gynecology department, LAR – laryngology department SRG – surgery department; (b)* BAL-broncho-alveolar lavage C-conjunctival sac swab N-nasal swab S- sputum P- pharyngeal swab E- external acoustic duct swab NP- nasopharyngeal swab V- vaginal swab M-sinus punctuate X- unknown;

2

127

Resistance patterns of Streptococcus pneumoniae Pomeranian isolates

Patient age

Specimen

Patient clinical diagnosis

Resistance pattern

Resistance degree

(a)*

Patient sex

The year of strain isolation

Origin / hospital ward

Strain no

Table I continued

M/K

years

(b)*

( c)*

(d)*

(e)*

5 13

AMB AMB

2003 2003

K X

2 X

N X

U X

PiSH TSiH

3 3

14

AMB

2003

K

1

N

R

TSiH

3

112 115

AMB AMB

2001 2002

K K

26 4

P N

X R

TSiH ELSi

3 3

123 18

AMB AMB

2002 2003

M M

4 5

N M

R S

ELSi PiSIi

3 3

19

AMB

2003

M

2

N

R

PiSIi

3

52 55 61 79 3 28 48

AMB AMB PED AMB AMB AMB AMB

2003 2003 2001 2003 2003 2003 2003

M K M M M M M

7 7 5 7 5 3 9

N N M N N NP NP

R R S R R SUi U

PiSIi PiSIi PiSIi PiSIi PiSiIi SH SH

3 3 3 3 3 2 2

59 67 91 101 107

AMB AMB AMB AMB AMB

2001 2003 2001 2001 2003

M M X M K

2 2 X 7 2

N NP X N N

R RU X R R

TS TS TH SH TS

2 2 2 2 2

118 119 134 105 109 133 2 26 51 54 62 71 73 74 96 121 122 126 34 57

AMB AMB AMB AMB AMB AMB AMB AMB AMB AMB SRG AMB AMB AMB AMB AMB AMB AMB AMB AMB

2002 2002 2003 2003 2001 2003 2003 2003 2003 2003 2001 2001 2001 2001 2001 2002 2002 2002 2003 2003

M K M K X M K K K K M M K K X M M M K M

6 4 3 7 X 6 2 5 9 4 5 5 6 6 X 6 4 2 3 4

P NP N NP X N N NP N NP N N N N X NP NP N NP N

U RU R R X R R RSO R R U X RB RU X R R RU OR U

TH PiS TS PiS PiS PiSi E S T S S S T T S S S S Si Si

2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1

(c)* Rean- state after resuscitation P- pneumonia C- conjunctivitis O- otitis R-rhinitis S- sinusitis B- bronchitis U- upper respiratory tract infection V- vaginitis G- fever of unknown origin X- unknown; (d)* P- penicyllin E-erythromycin L-clindamycin T-tetracycline S-cotrimoxazole C-ceftriaxon H-chloramphenicol I-imipenem i-intermediate strain; ( e)* 7- 7-drug resistant strain etc.

128

18.0%

16.0%

12.0%

10.0%

Nowosiad M. et al.

Percentage of resistant strains

14.0%

8.0%

6.0%

4.0%

2.0%

0.0%

PELT PELT SCI SHI Serie 1 1.3% 2.5%

PELS PTSC PELT PELS PELT PESCI PTSHI ELTS PELT HI HI SI H S 3.8% 1.3% 1.3% 1.3% 1.3% 1.3% 3.8% 17.5% 3.8%

ELT

TSH

PSH

ELS

PSI

SH

3.8% 11.3% 1.3%

2.5%

8.8%

3.8%

TS

TH

5.0% 2.5%

PS 5.0%

E

S

1.3% 12.5%

T 3.8%

Resistance patterns

Fig. 2. Resistance patterns of resistant Streptococcus pneumoniae strains isolated in West Pomerania Province in 2001–2003

2

Percentage of resistant strains

PELT SHI 4.2%

7.7%

0.0%

PELT SCI 2001 0.0%

2002 0.0%

2003 2.3%

0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

7.0%

0.0%

0.0%

7.7%

PELS PTSC HI HI 0.0% 0.0%

2.3%

0.0%

PELT SI 0.0% 2.3%

0.0%

PELS H 0.0% 2.3%

0.0%

PELT S 0.0% 7.7%

0.0%

2.3%

15.4%

0.0%

ELT

PSH

7.0%

7.7%

0.0%

ELS

2.3%

0.0%

0.0% 15.4%

20.8% 0.0%

TSH

Resistance patterns

0.0% 20.9% 4.7%

0.0%

12.5% 16.7% 4.2%

PELT

14.0%

0.0%

4.2%

PSI

Fig. 3. Resistance patterns analysis in individual years.

2.3%

0.0%

0.0%

PESCI PTSHI ELTS

4.7%

0.0%

4.2%

SH

7.0%

0,0%

4.2%

TS

0.0%

7.7%

4.2%

TH

4.7%

7.7%

4.2%

PS

S

2.3%

9.3%

0.0% 23.1%

0.0% 12.5%

E

2.3%

0.0%

8.3%

T

2 Resistance patterns of Streptococcus pneumoniae Pomeranian isolates

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Nowosiad M. et al.

2

9.3% and 7.7% respectively. A similar growing tendency can be observed in case of percentage of 4-drug resistant strains: from 20.8% in 2001 to 25.6% in 2003, and 3-drug resistant strains: 25% in 2001, 38.5% in 2002 to 25.6% in 2003. One 7-drug resistant strain was found every year. All of the 80 tested resistant strains showed 23 various resistance patterns (Fig. 2). It is worth mentioning that resistance to 8 out of 9 tested antibiotics was noticed among tested strains: penicillin (P), erythromycin (E), clindamycin (L), tetracycline (T), cotrimoxazole (S), chloramphenicol (H), ceftriaxone (C) and imipenem (I). No vancomycin (W) resistant strains were observed. Resistance to ceftriaxone (Ci ) and imipenem (Ii) was intermediate. Four resistance patterns: ELTS, S, TSH, PSI, clearly dominated and were present in half (50.1%) of the tested strains. The ELTS resistance pattern was most frequent, present in 14 resistant strains out of 80 being tested (17.5%), the other patterns occurred in the following percentages: S – 12.5%, TSH – 11.3% a PSI – 8.8%. More rarely occurring TS resistance pattern was traced in 4 strains; PELSHI, PTSHI, PELT, ELT, SH, T resistance patterns in 3; PELTSHI, ELS and TH in 2 strains. The other 8 resistance patterns: PELTSCI, PTSCHI, PELTSI, PELSH, PELTS, PESCI, PSH, E were represented by single isolates. To this group belong mainly multidrug, 5-and-more-drug resistant strains out of which nearly all, with exception to the PTSCHI resistance pattern strain, appeared only in 2003. The other two strains of this group with rarely occurring M-phenotype of macrolide resistance – PESCI and E, were also determined for the first time in strains isolated only in 2003. The percentage of particular resistance patterns in years 2001–2003 is presented in Table I and Fig. 3. Discussion The study presented here aimed at analysis of resistance patterns and resistance degree of resistant strains collected in West Pomerania Province of Poland in years 2001–2003 with special attention paid to their clinical origin. The study covered only resistant strains, so the results cannot form a basis neither for conclusions concerning antimicrobial resistance of S. pneumoniae population in our region nor for comparison with epidemiological data from other regions. These results, however, enable us to follow the dynamics of changes in antimicrobial susceptibility. The epidemiological data concerning patients, from whom resistant S. pneumoniae strains were isolated, are consistent with risk factors of pneumococcal infection presented by others. The most important are: extreme age groups, staying in large communities, chronic diseases and dysfunction of local and systemic immunological system (Ussery et al., 1996; Block et al., 1995; Klugmann, 1996). Whereas protracting infections and long lasting antibiotic therapy are the factors predisposing to infection with multidrug resistant strain (Klugmann, 1996; Kristinsson, 1997; Cohen and Tartasky, 1997; Zenni et al., 1995; Dowell and Schwartz, 1997; Nava et al., 1994; Castillo et al., 1998; Jacobs and Appelbaum, 1995). In our study majority of strains were isolated from ambulatory patients (65 in 80; 81.3%), from children at nursery school age – below 7 years (46 in 70, 65.7%), patients suffering from chronic upper respiratory tracts infection. In such cases microbiological diagnostics is carried out only after a long-term antimicrobial therapy or therapy failures. This explains the fact that the resistance degree of certain strains reached even 7 drugs. The greatest number of resistant strains was isolated from nose or pharynx swabs, as it is the easiest, least invasive and therefore the most often performed testing in case of ambulatory diagnosed upper respiratory tracts infections. Majority of pneumococcal infections originate from either carriage that is present in nasopharynx in 20– 40% of children population and 5–10% (Grzesiowski et al., 1999) of adults, or droplet transmitted infection (Arnold et al., 1996). Due to their prevalence in our study, the strains isolated from ambulatory patients could be found in each group with differing resistance degree. While strains isolated from hospitalized patients belonged mainly to groups of the highest resistance degree: 2 out of 3 (67%) of 7-drug resistant, 3 out of 5 (60%) of 6-drug resistant, 3 out of 6 (50%) of 5-drug resistant, 4 out of 17 (23%) 4-drug resistant, 3 out of 22 (13.6%) of 3-drug resistant, none of 2-drug resistant and only one out of 14 (7.1%) 1-drug resistant strains came from hospital wards. The above mentioned regularity especially concerns the strains from patients with serious diseases like pneumonia or treated in intensive care unit, immunocompromised, exposed to various drugs including antimicrobial ones and therefore predisposed to multidrug resistant strains infection. The other group of patients susceptible to pneumococcal infections is constituted by people of old age. Insignificant number of 6 strains from patients of mature age (39–68 years of age) also showed high resistance degree: 3 of them are 6-drug resistant ( 60% of 6-drug resistant strains), 1 is 5-drug resistant (16,7% of

2

Resistance patterns of Streptococcus pneumoniae Pomeranian isolates

131

5-drug resistant strains) and 2 are 4-drug resistant (11.7% of 4-drug resistant strains). Half of the patients of this group belonged also to before mentioned group of people hospitalized because of serious health state. A similar percentage of resistant pneumococci was isolated from women (45.2%) and from men (54.7%). Slight prevalence of pneumococcal infections in men, though not clear, is often reported (Fenoll et al., 1998; Bennett et al., 2003; McKenzie et al., 2000). The analysis of resistance patterns presented here also demonstrates the gradual increase of resistance among S. pneumoniae strains. It is proved by increase in amount of MDR strains and resistance degree as well as by formation of new resistance patterns. Half of the isolates are multidrug resistant (53.8%). The percentage of MDR amounted 62.5% in 2001 and reached 69.8% in 2003. Abrupt growth in the amount of MDR strains was noted in many long-term studies of pneumoccoci resistance. In the USA the MDR percentage of all isolates grew from 9.1% in 1994–1995 to 22.4% in 1999–2000 (Doern et al., 2001). The only one among antibiotics tested in our study to which no resistance has been reported is vancomycin at maximum MIC 0.5 mg/ml. Occurrence of new resistance patterns over time, especially among the multidrug resistant strains, can also indirectly prove the increase of pneumococcal resistance. Practically all of singularly represented resistance patterns: PELTSCI, PELTSI, PELSH, PELTS, PESCI, PSH were identified in 2003 for the first time. Similarly, with reference to strains with high resistance degree – the first 6-drug resistant strain was reported in 2002 and other 4 strains of 2 new patterns appeared next year. Also 3 out of 4 patterns of resistance to 5 antibiotics were first reported in 2003. Though 7-drug resistant strains were present individually in every of the tested years, yet the isolate from 2003 showed different pattern. However, almost all other resistance patterns maintained throughout the analyzed years, patterns with resistance to tetracycline (T) and chloramphenicol (H) were an exception. The TH resistance pattern disappeared in 2003. Likewise, all other patterns with the TH resistance combination: PELTSHI, PTSCHI, PTSHI, TSH either disappeared till 2003, or, as in case of the TSH pattern, their percentage significantly decreased from 2001 to 2003 (from 28%, to 7.7% and finally to 7%). This fact can be connected with significant limitation of tetracycline and chloramphenicol consumption in the recent years, particularly in pediatrics. Besides, molecular testing proved that genes coding resistance to chloramphenicol (cat gene) and tetracycline (tetM gen) are carried by the same highly mobile transpozones: Tn5253 and Tn1545 and they tend to appear together (Ayoubi et al., 1991; Clewell et al., 1995). The another phenomenon observed in our study is the appearance of another macrolide resistance phenotype. There has been described two macrolide resistance phenotypes: cross MLSb resistance to macrolides of MIC > 64 mg/ml for erythromycin(CO2), lincosamides and streptogramines determined by the ermB gene and methylation of the drug target, ribosome, and M phenotype with only 14- and 15-membered-ring macrolides resistance with MIC within 1–32 mg/ml for erythromycin(CO 2) resulting from active drug elimination mediated mainly by the product of mefA gene (Johnston et al., 1998; Roberts et al., 1999; Leclercq and Courvalin, 1991; Tait-Kamradt et al., 1997; Pihlajamaki et al., 2003). Our strains showed distinct prevalence of the MLSb phenotype, which is typical for Europe (Johnston et al., 1998; Pihlajamaki et al., 2003) and for the majority of multiresistant clones (McGee et al., 2001), though Polish clone – Poland23F-16 is of an M phenotype and harbored neither the ermB nor the mefA gene (Overweg et al., 1999; McGee et al., 2001). The first strain showing resistance to macrolides with susceptibility to lincosamides noted in our study appeared in January 2003 as E-pattern. In February resistance to erythromycin in strain of PESCiIi pattern was reported. The lack of P, I or C resistance pattern despite numerous representation of multiresistant strains with these antibiotics (patterns: PELTSHI, PELSHI, PTSCHI, PESCI, PELST, PSI, PSH, PSH, PS) can be explained by phenomenon of coexistence of penicillin resistance with resistance to other antibiotics of betalactam group described in numerous reports (Bruggemann et al., 2001; Munoz et al., 1992) and reports about increased frequency of antimicrobial resistance to drugs from other groups among penicillin resistant strains (Baquero 1996; Doern et al., 2001; Markiewicz and Tomasz, 1989; Allen, 1991; Lister, 1995). Cross antibiotic resistance to drugs from betalactam group is connected with common grip point-penicillin binding protein (PBP) (Markiewicz and Tomasz, 1989; Barcus et al., 1995; Grebe and Hakenbeck, 1996). In Poland about 14% of S. pneumoniae strains are “nonsusceptible” to penincillin and half of them (6,8%) is highly resistant to penicillin and also to cephalosporines of the 2 nd and 3rd generation (Trzciñski and Hryniewicz, 1997; Hryniewicz et al., 2000). In our study 5 strains (6.2%) showed high resistance to penicillin and only one of them isolated only in 2003 revealed MIC over 2 µg/ml (4 µg/ml). All these strains were multidrug resistant: 5–7-drug resistant and showed cross intermediate resistance to other betalactams: ceftriaxone and imipenem. Much higher amount of strains – 24 (30%) indicated intermediate resistance to

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2

penicillin. In case of ceftriaxone only 3 strains with intermediate resistance and no highly resistant strains were described. Susceptibility to ceftriaxone among collected strains was relatively high in contradiction to data from other regions (Doern et al., 2001). Strains from the much more numerous group of 19 strains intermediately resistant to imipenem presented both high and intermediate resistance to penicillin. They were being isolated throughout the whole time of study and were already present in strains resistant to only one non-betalactam antibiotic. Such a significant amount of strains intermediately resistant to imipenem is surprising especially after consideration of the fact that this antibiotic is not used in ambulatory treatment and is not listed in any guidelines for treatment of pneumococcal diseases. Among few studies of pneumococcal resistance to imipenem that have been carried out in Europe a similar phenomenon was observed in Belgium, where the percentage of intermediately resistant strains amounted to 3.8% (Vanhoof et al., 2003) and in France where MIC for imipenem ranged from 0.03 to 0.25 (Barakett et al., 1992). In 304 strains from Hungary, 18.1% intermediately resistant and even 9.6% high resistant strains have been noted (Dobay et al., 2003). Whereas, reports from Island, Norway and even Italy present 100% susceptibility to imipenem (Michault and Simac, 2000; Marchese et al., 2000; Bergan et al., 1998). More reports about lowered susceptibility to imipenem come from Far East (Satoh et al., 2002; Hsueh et al., 1999) and the USA (Pallares etal., 1995; Frick et al., 1998) though no such prevalence of strains intermediately resistant to imipenem in relation to strains “nonsusceptible” to cephalosporines of 3 rd generation. In Taiwan, where there is one of the highest degree of antimicrobial resistance, among isolates from intensive care units the percentage of strains intermediately resistant to imipenem amounted to 21% and to cefotaxime to 33% (Hsueh et al., 2001). The percentage of strains “nonsusceptible” to ceftriaxone and imipenem in Japan was 28,9% and 8,9% respectively (Satoh et al., 2002). The tests carried out in the Washington State between 1995 and 1997 proved that among strains “nonsusceptible” to penicillin 28.6% were at the same time “nonsusceptible” to imipenem and 23,8% to ceftriaxone (Frick et al., 1998). In our study this percentage amounted to 79.1% for imipenem (19 out of 24 penicillin resistant) and to 12.5% for ceftriaxone (3 out of 24). The phenomenon observed here is worth of deeper insight, the more so because imipenem is a drug of final choice in many serious diseases. Coexistence of penicillin resistance with “nonsusceptibility” to antibiotics of other groups has been described since long time throughout the world. In collection of 10 000 strains tested from 1990 to 1996 in Spain, 72% of penicillin resistant strains were also resistant to other antibiotics, while among penicillin susceptible strains this percentage amounted 21.5% (Fenoll et al., 1998). In Poland the presence of strains “nonsusceptible” to penicillin and resistant to other antibiotics (tetracycline, cotrimoxazole, macrolides and cephalosporines of the 1st and 2nd generation) was already reported in 1996 (Trzciñski and Hryniewicz, 1997; Vanhoof et al., 2003). In our study we have noted 41 (out of 80 i.e. 51,2%) strains susceptible to penicillin and resistant to other antibiotics, whereas all the 29 penicillin “nonsusceptible” strains showed resistance to other groups antibiotics. Analysis of resistance patterns is of substantial practical meaning. Apart from providing directions for rational treatment it can also serve as an index of tendencies in antibiotic therapy applied in a given region. In our region the ELTS, S, TSH and PSI patterns occurred the most often. The amount of strains of the ELTS pattern during the period covered by study increased from 16.7% in 2001 to 20.9% in 2003. It can be connected with macrolide overdosing in ambulatory treatment not only in our country. Many sources, also studies within the Alexander project report alarming increase of macrolide resistance both among penicillin resistant and susceptible strains (16.5% and 10.4%, respectively, in 1996; while in 1997 it was 21.9% and 14.1%) (Schito et al., 2000). In our country the percentage of macrolide resistant pneumococci increased from 1,9% in 1992 to 12.3% in 1996 (Trzciñski and Hryniewicz, 1997). Similarly, cotrimoxasole resistance that as the S pattern is present in 12,5% strains also occurs almost in all of the described resistance patterns (with exception of two: ELT and PELT). Thereby it additionally covers 60 out of 66 (90.9%) of 2- and more- drug resistant strains. It can be explained by the fact that in our country cotrimoxasole belongs to relatively often applied drug, though it is not always justifiable. The phenomena presented in this study: growing resistance degree, increasing amount of multidrug resistant strains, emergence of new resistance patterns, prove gradual increasing resistance among S. pneumoniae strains in our region. The above changes have taken place in a relatively short time of 3 years. This testifies dynamics of this disturbing process and it more strongly justifies the necessity of constant monitoring pneumococcal resistance. We should be conscious that frequent therapeutic failures and exposition to further antibiotics can induce acquiring of resistance by bacteria and are a risk factor of infection with multidrug resistant strain.

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Polish Journal of Microbiology 2005, Vol. 54, No 2, 137–144

Whole Cell-derived Fatty Acid Profiles of Pseudomonas sp. JS150 during Naphthalene Degradation AGNIESZKA MROZIK1, SYLWIA £ABU¯EK 1 and ZOFIA PIOTROWSKA-SEGET2 1 Department

of Biochemistry, 2 Department of Microbiology, University of Silesia, Jagielloñska 28, 40-032 Katowice, Poland

Received 17 November 2004, received in revised form 4 March 2005, accepted 7 March 2005 Abstract Changes in cellular fatty acid composition during naphthalene degradation, at the concentrations of 0.5 g l –1 or 1.0 g l–1, by Pseudomonas sp. JS150 were investigated. In response to naphthalene exposure an increase in saturated/unsaturated ratio was observed. Additionally, the dynamic changes involved alterations in the contents of hydroxy, cyclopropane and branched fatty acids. Among the classes of fatty acids tested the most noticeable changes in the abundance of cyclopropane fatty acids were observed. Since day 4 of incubation these fatty acids were not dectected in bacterial cells growing on naphthalene. In contrast, markedly increased in the percentage of hydroxy fatty acids over time was observed. However, the proportions of saturated straight-chain and branched fatty acids did not change such significantly. K e y w o r d s: Pseudomonas sp. JS150, naphthalene degradation, fatty acid composition

Introduction Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental contaminants because many of them are toxic, mutagenic and resist biodegradation. The simplest of that class is naphthalene, a common component of industrial products and waste materials. Naphthalene is a dicyclic aromatic compound with molecular mass of 129.19, boiling point 218°C, melting point of 80.5°C, solubility (at 20°C) of 32 mg l –1 and specific gravity of 1.145. It is widely distributed in the environment because it is used as the starting material for the synthesis of moth repellent, soil fumigant, naphthylamines, anthranilic and phtalic acids, and syntetic resins (Vuchetich et al., 1996; Smith et al., 1997; Stohs et al., 2002). The fate of naphthalene is of great interest because its expossure might cause toxic effects on skin, lungs, eyes, kidney, liver and brain of animals and humans. Toxic manifestations depend on naphthalene dose, route of expossure and species involved (Stohs et al., 2002). Bacterial degradation represents a significant way for the removal of naphthalene (PAHs) from the environment. Numerous strains of microorganisms that are capable of degrading naphthalene have been isolated and identified. Considerable attention has focused on the metabolic pathways and their genetic regulation by gram-negative bacteria, particularly of the genera Pseudomonas (Fuenmayor et al., 1998; Filonov et al., 1999; Kozlova et al., 2004) and gram-positive bacteria of the genera Rhodococcus (Kulakov et al., 1998; Di Gennaro et al., 2001). The metabolism of naphthalene under aerobic conditions is different in grampositive and gram-negative bacteria. It is documented that naphthalene degradation in gram-negative bacteria proceeds through the formation of 1,2-dihydroxynaphthalene which is then dehydrogenated to the corresponding 1,2-dihydroxy derivative and further transformed into salicylic acid. The next step is salicylate oxidation to catechol, which can undergo either ortho or meta fission depending upon bacterial metabolism (Rossello-Mora et al., 1994; Mrozik et al., 2003). However, in naphthalene metabolism by gram-positive bacteria such as Rhodococcus, salicylate is converted to gentisic acid (Grund et al., 1992, Di Gennaro et al., 2001). A different naphthalene degradation pathway has been recently described in the thermophilic bacterium Bacillus thermoleovorans. Apart from typical metabolites known from mesophiles, intermediates such as 2,3-dihydroxynaphthalene, 2-carboxycinnamic acid, phthalic acid and benzoic acid in the pathway of this bacterium were identified (Annweiler et al., 2000).

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In the presence of naphthalene and other aromatic compounds crucial changes in the fatty acid composition of bacterial membrane lipids have been observed. Aromatic compounds disturb of membrane integrity and permeability, inhibit bacterial growth, respiration, nutrients transport or even cell death may occur. The toxic effects of these chemicals are based on their lipophilic properties, since they interact preferentially with cell membrane and change its fluidity (Sikkema et al., 1994; 1995; Mrozik et al., 2004b). Bacteria withstand these changes by altering the fatty acid composition and the degree of saturation of their membrane lipids. The saturation degree of fatty acids is known as a major adaptive response of the cells to keep the fluidity of their membranes at a constant value. This parameter changes when bacteria grow thus it can be a potential marker of toxicity only in living cells (Diefenbach et al., 1992; Loffhagen et al., 1995). Another fundamental mechanism enabling bacteria to adapt to presence of aromatic compounds is isomerization of cis to trans unsaturated fatty acids. This is a short-term response that does not depend on growth. Therefore, this parameter is a second potential indicator of the acute toxicity of these compounds (Heipieper et al., 1995; Loffhagen et al., 2001; Heipieper et al., 2003). For decreasing the deleterious effect of aromatic compounds on membrane, bacteria can also change the proportion between iso and anteiso branched fatty acids, content of cyclopropane fatty acids and membrane proteins, and the average acyl chains length (Heipieper et al., 1994; Sajbidor, 1997; Denich et al., 2003). These tolerance mechanisms enabling bacteria to stabilize of membrane fluidity and reduce the accumulation of toxic compounds in the membrane. Molecular and biochemical investigations performed to characterize these adaptive response systems were conducted using many strains of the genera Pseudomonas, including the major representatives P. putida and P. aeruginosa, and Vibrio and series of phenolic compounds such as phenol, p-cresol, toluene (Guckert et al., 1986; Heipieper et al., 1992; Weber et al., 1994; Mrozik et al., 2004a). However, there is still a little information about the effect of naphthalene on cellular fatty acids of these microorganisms. The aim of this study was to determine the changes in whole cell-derived fatty acids in Pseudomonas sp. JS150 during naphthalene degradation. Experimental Materials and Methods Strain and growth conditions. Pseudomonas sp. JS150 strain was kindly provided by Dr J. Spain from Air Force Civil Engeenering Support Agency, Tyndall Air Force Base, Florida, USA. Pseudomonas sp. JS150 is a nonencapsulated mutant of strain JS1 obtained after ethyl methanesulphonate mutagenesis (Haigler et al., 1992). Bacteria were grown in Kojima et al., (1962) minimal medium containing: 3.78 g of Na2HPO4 ×12H2O; 0.5 g of KH2PO4; 5.0 g of NH4Cl; 0.2 g of MgSO4 ×7H2O and 0.1 g of yeast extract in 1000 ml of deionized water. Naphthalene was added at concentrations of 0.5 g l–1 or 1.0 g l–1 as a sole carbon and energy source. Due to the low solubility of naphthalene in water it was dissolved in N,N-dimethylformamide (DMF) before addition to the medium. The final pH of the medium was 7.2–7.3. To show the impact of naphthalene on fatty acid composition, bacteria were also cultivated in medium without aromatic substrate. In this case sodium citrate at the concentration of 0.5 g l–1 was used. Naphthalene-dosed and control cultures were further incubated in the dark at 30°C with shaking at 125 rpm. Samples of the cultures were withdrawn periodically for analysis of cell density (OD) at 600 nm and naphthalene removal. Determination of naphthalene. For determination of naphthalene concentration triplicate samples of 10 ml cells culture were melted with 10 ml of hexane using magnetic stirrer for 15 min. After separation of hexane fraction, the lower phase was washed twice with 2 ml of hexane and hexane fractions were combined. The organic phase was filtrated through anhydrous sodium sulphate. Hexane was vacuum evaporated to the volume of 2 ml and each sample was transferred into GC vials and analysed by GC chromatography (Perkin Elmer) equipped with flame ionization detector and a capillary column (phenyl-methyl-polysiloxane 25 m × 0.25 mm in diameter) and helium as a carrier gas. Concentration of naphthalene was determined on the 4, 7 and 14 of incubation and calculated by comparison of the peak height of standard with the tested samples. Enzyme assay. For preparation of cell extract and enzyme activity assay the method of Feist and Hegeman (1969) was used. Enzyme activity was expresses as :mol of 2-hydroxymuconic semialdehyde formed per mg of protein per min. The protein content of the cell extract was estimated by the method of Bradford (1976) with bovine albumin as a standard. Catechol 2,3-dioxygenase activities were measured on 4, 7 and 14 day of culturing. Isolation and identification of fatty acids. The whole cell-derived fatty acids were extracted and determined on the 4, 7 and 14 day of incubation. Cellular fatty acids were extracted from both cells growing on naphthalene and sodium citrate. Bacteria were harvested by centrifugation (8000 g) at 4°C for 30 min. The cell pellets obtained from medium amended with naphthalene were washed with 1.0 ml of DMF to remove undegraded naphthalene and finally were washed twice with 0.85% NaCl to remove residue of the culture medium. To decrease the humidity of bacterial cell, pellets were left through 2h at room temperature. Next 55 mg of bacterial biomass was transferred in duplicate to reaction tubes (Pyrex) and 1 ml of first reagent (150 g NaOH in 1 litre of 50% methanol) for saponification was added. Samples were incubated for 30 min at 100°C in water bath. To methylate liberated fatty acids, 2 ml of reagent II (6N HCl in aqueous methanol) was added to each tube and incubated again for 10 min at 80°C in water bath. Fatty acid methyl esters (FAMEs) were extracted from the aqueous phase by addition of 1.15 ml of reagent III (hexane/methyl tert-butyl ether, 1:1, v/v) to each tube. Then samples were rotated end-over-end for 10 min. After removing aqueous (lower) phase,

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3 ml of 1.2% NaOH in H2O was added and the tubes were again rotated for 5 min (Sasser, 1990). Finally, the organic (upper) phase containing FAME was transferred to a gas chromatography vial (Hewlett-Packard). Fatty acids were analysed by gas chromatography (Hewlett-Packard 6890, USA) using capillary column Ultra 2-HP (cross-linked 5% phenyl-methyl silicone 25 m, 0.22 mm ID, thickness 0.33 :m) and hydrogen as a carrier gas. FAME were detected by a flame ionization detector (FID) and identified by MIS (Microbial Identification System) software, using the aerobe method and TSBA library version 3.9 (MIDI, USA). Fatty acids were designed by the number of carbon atoms, followed by a colon, the number of double bonds and then by a position of the first double bond from the methyl (T) end of the molecule. The prefixes c or t indicate cis or trans configuration of the double bond, cy – cyclopropane fatty acids, Me – the position of the methyl group from the acid end, and -OH indicates the position of the hydroxyl group from the acid end of the molecule. Branched fatty acids are designed as iso and anteiso, if the methyl branch is one or two carbon from the T end of acyl chain.

Results Cell growth and naphthalene biodegradation. Pseudomonas sp. JS150 was grown on naphthalene at the concentration of 0.5 g l–1 or 1.0 g l–1 as a sole carbon and energy source. The highest optical densities (OD) were observed on 4 day of incubation reaching the value 0.839 and 0.901 for the concentrations 0.5 g l–1 and 1.0 g l–1, respectively. These OD values were equivalent of bacterial cell numbers 5.6×108 and 4.1×109. After 4 days OD started to decrease and at the end of experimental period showed 0.496 and 0.399 for the lower and the higher dose of naphthalene.The increasing number of bacteria during the first 4 days of the incubation was accompanied with the highest degradation rate of naphthalene by strain used. In that time Pseudomonas sp. JS150 metabolized 60% and 66% of total naphthalene added to the medium at the concentrations of 0.5 g l–1 and 1.0 g l–1, respectively. In succesive days naphthalene was degraded much slowly. Cell growth and biodegradation rate of substrate by Pseudomonas JS150 is presented in Figure 1. The biodegradation ability of strain tested was correlated with an induction of catabolic enzymes involved in naphthalene metabolism. Catechol 2,3-dioxygenase activities in cell-free extracts are shown in Table I. The highest enzyme activities were observed for both naphthalene doses used on 4 day and showed value 0.70 and 0.63 :M min–1 mg–1 of protein. Then the activity of this enzyme was decreasing over time. When naphthalene was served as a substrate Pseudomonas sp. JS150 did not induce catechol 1,2-dioxygenase indicating that naphthalene degradation by this strain procceded via meta metabolic pathway. Changes in fatty acid composition. To determine the effect of naphthalene on whole cell-derived fatty acid profiles of Pseudomonas sp. JS150 cultured on naphthalene and sodium citrate were compared. Table II contains the percentage of total fatty acids and shows the compositional changes during naphthalene degradation by bacteria tested. For the interpretation of naphthalene impact on bacteria, identified fatty acids were grouped into two major classes. The first class included saturated fatty acids. It was additionaly divided into four sub-classes: straight-chain, hydroxy, cyclopropane and branched fatty acids. The second class comprises unsaturated fatty acids. 1

0,9

0,9

0,8

0,8

0,7

0,7

0,6

0,6

0,5

0,5

0,4

0,4

0,3

0,3

0,2

0,2

0,1

0,1

0

0 0

2

4

6

8

10

12

14

Time, days naphth. 1.0 g/l growth curve, naphth. 1.0 g/l

naphth. 0.5 g/l growth curve, naphth. 0.5 g/l

Fig. 1. Naphthalene degradation and growth curve of Pseudomonas sp. JS150.

Optical density, 600 nm

Naphthalene, g l-1

1

140

2

Mrozik A. et al. Table I Enzyme activities in cell-free extracts of Pseudomonas sp. JS150 grown on different naphthalene concentrations Bacterial strain

Naphthalene (g l–1)

Catechol 2,3-dioxygenase activity, µM min –1 mg–1 of protein 4 day

7 day

14 day

Pseudomonas sp.

0.50

0.70 ± 0.06

0.68 ± 0.05

0.45 ± 0.05

JS150

1.00

0.63 ± 0.02

0.58 ± 0.04

0.31 ± 0.01

Number of replicates, n = 3

As indicated in Table II the remarkable differences in contents of saturated fatty acids on day 14 were observed. For bacteria grown on naphthalene at the concentration of 0.5 g l–1 the content of this class of fatty acids was the lowest (80.0% of total saturated fatty acids) as compared to control (88.26%) and naphthalene treatment samples determined on 4 (83.55%) and 7 (84.30%) day. In contrast, in bacteria grown at the dose of 1.0 g l–1naphthalene the amount of saturated fatty acids was the highest (93.47%). As a consequence the changes in the saturated/unsaturated ratio were found. In the presence of lower naphthalene concentration this ratio was lower in comparison with control samples. It reached the value 5.85, 5.37 and 5.59 for 4, 7 and 14 day, respectively, whereas in control it was 7.52. In opposite, the saturated/unsaturated ratio significantly increased on day 7 (11.46) and 14 (14.31), when bacteria cultivated with 1.0 g l –1 of naphthalene. Naphthalene treatment caused changes in the distribution of straight-chain, hydroxy, cyclopropane and branched fatty acids in Pseudomonas sp. JS150. In a case of straight-chain fatty acids no significant differences in the amount of these fatty acids during naphthalene degradation were observed, with one exception. On the last sampling time in bacteria grown on the medium with 0.5 g l–1 of naphthalene the content of straight-chain saturated fatty acids was markedly lower (13.56%) as compared to the control (18.78%). In turn, essential changes in the distribution of hydroxy fatty acids over experimental period were detected. The addition of both concentrations of naphthalene increased the amounts of these fatty acids. The highest abundance was found at the naphthalene concentration of 0.5 g l–1 on 14 day and was 4 times higher than in the control, represented up 30.43% of the total saturated fatty acids. In remaining samples, the contents of hydroxy fatty acids were about 2–2.5-fold higher than in the control. In contrast, as a response to naphthalene exposure, in Pseudomonas sp. JS150 during the first 4 days of the incubation the amount of cyclopropane fatty acid 17:0 cy decreased as compared to the control. Interestingly, on the second and third sampling times this fatty acid either on the lower or higher naphthalene concentration was not detected. Similar trend was observed in a case of 18:0, 17:0 anteiso, 13:0 iso and one of unsataturated fatty acid MIDI-undistinguishable Day 4

Day 7

Day 14

100% 80% 60% 40% 20% 0%

straight-chain

hydroxy

cyclopropane

branched

unsaturated

Fig. 2. Proportions of fatty acids in Pseudomonas sp. JS150 growing on citrate (control) and naphthalene at the concentration 0.5 g l–1 (A) or 1.0 g l–1 (B) during naphthalene degradation. Class of hydroxy fatty acids contains additionally the branched hydroxy fatty acids. Methylated fatty acid 16:0 10 Me is included to branched fatty acids.

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Table II Percentage of total fatty acids from Pseudomonas sp. JS150 grown in the presence of citrate (0.5 g l–1) or different naphthalene concentrations during 14 days of incubation % of total fatty acids Fatty acids

4 day

7 day

14 day

Naphthalene g l

–1

Citrate g l –1

0.5

1.0

0.5

1.0

0.5

1.0

10:0

0.00

0.60

0.57

1.35

0.00

1.43

1.90

10:0 iso

0.00

0.00

0.00

0.00

0.00

0.00

9.30

10:0 3OH

0.00

0.39

0.37

0.00

0.00

1.50

0.00

11:0 iso

0.00

3.11

3.02

7.00

4.99

6.84

0.00

11:0 iso 3OH

0.00

2.96

3.06

0.00

5.57

6.59

0.00

12:0 3OH

2.95

5.66

5.54

0.00

0.00

12.16

0.00

12:0 iso 3OH

0.46

0.43

0.42

0.00

0.00

0.00

14.13

13:0 2OH

1.10

1.40

1.41

0.00

0.00

0.00

0.00

13:0 iso

0.00

0.78

0.58

0.00

0.00

0.00

0.00

13:0 iso 3OH

2.88

5.42

6.39

12.69

8.96

10.18

0.00

14:0

3.46

2.79

3.03

3.74

3.34

1.95

3.70

14:0 iso

1.73

1.22

1.36

0.00

0.00

0.00

0.00

15:0

1.13

0.62

0.56

0.00

0.00

0.00

0.00

15:0 iso

26.59

23.46

23.29

20.69

28.12

17.10

30.94

15:0 anteiso

19.36

12.76

15.33

14.51

13.84

7.57

10.52

16:0

Saturated

13.63

13.69

13.27

14.37

13.36

10.18

14.07

16:0 iso

3.52

2.00

2.80

3.00

3.20

2.14

5.22

16:0 anteiso

0.00

0.00

0.42

0.00

0.00

0.00

0.00

16:0 10Me

4.00

0.00

0.00

3.95

4.02

0.00

0.00

17:0 iso

3.00

3.37

2.86

3.00

3.10

2.36

3.41

17:0 anteiso

0.58

0.61

0.85

0.00

0.00

0.00

0.00

17:0 cy

3.31

1.24

1.68

0.00

0.00

0.00

0.00

18:0

0.56

1.04

1.44

0.00

0.00

0.00

0.00

19:0 iso

0.00

0.00

0.00

0.00

0.00

0.00

0.28

15:1 iso

0.75

0.75

0.80

0.00

0.00

0.00

0.00

16:1 T7c

5.72

4.20

3.02

5.38

5.14

4.33

4.40

16:1 T9c

2.73

2.35

2.10

2.32

2.44

1.49

0.00

17:1 T9c

0.00

0.00

3.51

0.00

0.00

2.54

0.00

17:1 iso

0.00

4.13

0.00

5.12

0.00

3.35

0.00

18:1 T9c

1.67

1.85

1.49

1.88

0.00

1.40

2.00

18:1 2OH

0.00

0.00

0.00

0.00

0.14

0.00

0.13

18:1 T7c/T9t/T12t

0.87

1.01

0.26

1.00

0.00

1.20

0.00

Other

0.00

2.16

0.00

0.00

3.78

5.61

0.00

Sat./unsat. ratio

7.52

5.85

7.84

5.37

11.46

5.59

14.31

Unsaturated

Abbreviations: T – methyl end of fatty acid, c or t indicate cis or trans configuration of the double bond, cy – cyclopropane fatty acid, Me – the position of the methyl group from the acid end, -OH indicates the position of hydroxyl group from the acid end, iso and anteiso – branched fatty acids.

18:1T7c/T9t/T12t. However, the last fatty acid was not detected only when bacteria grown on the medium amended with 1.0 g l–1 of naphthalene on 7 and 14 day of the incubation (Fig. 2, Table II). The impact of naphthalene on whole cell-derived fatty acids comprised also changes in the contents of branched fatty acids including iso, anteiso and methyl-fatty acids. During degradation of 0.5 g l–1 naphthalene

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Pseudomonas sp. JS150 decreased the abundance of branched fatty acids over time, whereas in the presence of 1.0 g l–1 of substrate used the abundance of these fatty acids was similar to those observed in the control. On day 4, the percentage of branched fatty acids isolated from bacteria grown on 0.5 g l–1 of naphthalene was 47.31% and declined to 36.01% on day 14, whereas in the control reached value 58.78%. (Fig. 2). The another reaction of tested strain to naphthalene stress was forming new fatty acids which were not found in the bacteria growing on citrate. During the period of the incubation Pseudomonas sp. JS150, depending on the sampling day and naphthalene concentrations, formed from 3 to 6 of new fatty acids. They were mainly represented by saturated short-chain 10:0, 10:0 iso, 10:0 3OH, 11:0 iso and 11:0 iso 3OH and 13:0 iso, as well as by unsaturated fatty acids such as 17:1T9c, 17:1 iso and 18:1 2OH. Surpraisingly, particulary high content (9.3%) of 10:0 iso fatty acid was detected only on 14 day in the presence of the higher naphthalene concentration. In turn, fatty acid 17:1 iso was present on each sampling day but only in bacteria cultured on the lower naphthalene doses. In the presence of 1.0 g l–1 of naphthalene Pseudomonas sp. JS150 did not form this fatty acid (Table II). Discussion Our results confirmed the ability of Pseudomonas sp. JS150 for biodegradation of naphthalene, wellknown from previous studies (Haigler et al., 1992). This genetic modified strain was able to metabolize not only naphthalene but also a range of aromatic compounds such as toluen, benzen, chlorobenzene, phenol, benzoate and salicylate. However, this strain did not metabolize of naphthalene so effectively and fast as compared to other wild-type strains from genus Pseudomonas. For example, P. vesicularis and P. stutzeri were more efficient degraders and utilized 0.5 g l–1 of naphthalene within 11 and 14 days of the incubation, respectively (Mrozik et al., 2004c), whereas Pseudomonas sp. JS150 characterized by much slower rate of naphthalene degradation and after 14 days of the incubation in the medium there was still about 26% of naphthalene added. Interestingly, Pseudomonas sp. JS150 degraded the higher dose of naphthalene more quickly and after 14-days experiment only 22% of naphthalene added was present in the culture medium. The biodegradation studies indicated that in Pseudomonas sp. JS150 naphthalene induced catechol 2,3-dioxygenase what evidenced that degradation of naphthalene proceeded via meta metabolic pathway (Dagley, 1971; Wiliams and Sayers, 1994). The obtained results shown that Pseudomonas sp. JS150 underwent crucial changes in cell-derived fatty acids when grown on naphthalene as a sole carbon and energy source. Structural changes involved alterations in distribution of the separated classes of fatty acids. Under naphthalene treatment the saturated/ unsaturated ratio depended on the dose of aromatic substrate. During degradation of 0.5 g l–1 of naphthalene this ratio was at the same level and was slightly lower as compared to control. In turn, this ratio in the presence of 1.0 g l–1 of naphthalene increased and at the end of the experiment was 2-fold higher in comparison with the control indicating an increase in degree of saturation of membrane fatty acids. The similar correlation between an increase in degree of membrane saturation and tolerance towards the toxic compounds has been observed in phenol-degrading strain Pseudomonas putida P8 (Heipieper et al., 1992), in Rhodoccocus sp. 33 in the presence of benzene (Gutierrez et al., 1999) and Ralstonia eutropha H850 in the presence of biphenyl (Kim et al., 2001). The increasing degree of membrane saturation is a major adaptive mechanism to the presence of many toxic substances, that enable bacterial cells to survive under aromatic hydrocarbons stress (Sikkema et al., 1994; 1995). Considering our results it may be suggested that naphthalene at the lower concentration did not influence on degree of fatty acid saturation indicating that this naphthalene dose was too low for Pseudomonas sp. JS150 to induce essential changes in the proportional contents of saturated and unsaturated fatty acids. Another mechanism for bacterial cells to adapt to membrane active compounds is to alter cyclopropane fatty acid composition of their membranes (Grogan and Cronan, 1997). In Pseudomonas sp. JS150 only one 17:0 cy fatty acid was detected and its amount significantly decreased at the begining of the experiment and was not found to the end of the incubation period. Similarly, the decrease in the cyclopropane fatty acid percentage was observed in our previous studies conducted with P. vesicularis and P. stutzeri. However, 17:0 cy was detected on each sampling days. The low level and the disappearance of cyclopropane fatty acids might indicate that cyclopropane ring were cleaved by a separate cellular system activated by enzymes involved in naphthalene biodegradation. Naphthalene exposure influenced also on the composition and content of branched fatty acids. In the presence of the lower naphthalene concentration the content of these fatty acids visibly decreased whereas

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in the presence of the higher dose of naphthalene did not change significantly. These obtained results for Pseudomonas sp. JS150 were different in comparison with our previous experiments when P. stutzeri, P. vesicularis and P. putida were used. In these strains during naphthalene treatment the content of branched fatty acids markedly increased. It should be point out that Pseudomonas sp. JS150, in contrast to other strains of Pseudomonas, under control conditions synthesized significant amounts of branched fatty acids. They constituted 62% of total saturated fatty acids and were mainly represented by two fatty acids 15:0 iso and 15:0 anteiso. In contrast, for example in P. vesicularis grown on glucose branched fatty acids constituted only 10% of the total saturated fatty acids (Mrozik et al., 2004c). An increase in branched fatty acids contents was also reported by Tsitko et al. (1999), who studied the impact of different aromatic hydrocarbons on cellular fatty acid composition of Rhodococcus opacus. The high proportion of branched fatty acids might be related with genetic manipulation of Pseudomonas sp. JS150 towards the resistance to aromatic compounds. Probably the high amount of branched fatty acids, found in this strain, is sufficient to keep the proper membrane stability, both under control and naphthalene stress conditions. Another reaction of Pseudomonas JS150 to naphthalene toxicity, in comparison with wild-strains from the genus Pseudomonas, were chnages in the percentage of hydroxy fatty acids. During naphthalene degradation by strain tested the content of this class of fatty acids increased average from 2 to 4-fold, while for example, in P. vesicularis the abundance of hydroxy fatty acids decreased upon naphthalene exposure (Mrozik et al., 2004c). This mechanism is not understood yet and seems to be an attribute of an individual strain. 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Adv. Microbiol. Physiol. 6: 1–46. D e n i c h T.J., L.A. B e a u d e t t e, H. L e e and J.T. T r e v o r s. 2003. Effect of selected environmental and physico-chemical factors on bacterial cytoplasmatic membranes. J. Microbiol. Meth. 52: 149–182. D i e f e n b a c h R., H.J. H e i p i e p e r and H. K e w e l o h. 1992. Conversion of cis to trans unsaturated fatty acids in Pseudomonas putida P8: evidence for a role in the regulation of membrane fluidity. Appl. Environ. Biotechnol. 38: 382–287. F e i s t C.F. and G.D. H e g e m a n. 1969. Regulation of the meta cleavage pathways for benzoate oxidation by Pseudomonas putida. J. Bacteriol. 100: 1121–1128. F i l o n o v A.E., I.F. P u n t u s, A.V. K a r p o v, R.R. G a i a z o v, I.A. K o s h e l e v a and A.M. B o r o n i n. 1999. Growth and survival of Pseudomonas putida strains degrading naphthalene in soil model systems with different moisture levels. Proc. Biochem. 34: 303–308. F u e n m a y o r S.L., M. W i l d, A.L. 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Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio proportions of cyclopropyl fatty acids. Appl. Environ. Microbiol. 52: 794–801. G u t i e r r e z J.A., P. N i c h o l s and I. C o u p e r w h i t e. 1999. Changes in whole cell-derived fatty acids induced by benzene and occurence of the unsual 16:1T6c in Rhodoccocus sp. 33. FEMS Microbiol. Lett. 176: 213–218. H a i g l e r B.E., C.A. P e t t i g r e w and J.C. S p a i n. 1992. Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150. Appl. Environ. Microbiol. 58: 2237–2244. H e i p i e p e r H.J., R. D i e f e n b a c h and H. K e w e l o h. 1992. Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity. Appl. Environ. Microbiol. 58: 1847–1852.

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Polish Journal of Microbiology 2005, Vol. 54, No 2, 145–151

A Potent Chitinolytic Activity of Alternaria alternata Isolated from Egyptian Black Sand EMAN FATHI SHARAF

Botany Department, Faculty of Science, Cairo University, Giza 12613, Egypt Received 6 October 2004, received in revised form 4 February 2005, accepted 7 February 2005 Abstract Eight fungal species characterized by chitinolytic activity were isolated from Egyptian black sand collected from Rosetta coast. Genus Aspergillus and Alternaria alternata exhibited the highest density (> 40% of the total count, each) on the isolation plates containing different treatments of native shrimp shell chitin. Genus Aspergillus was represented by A. flavus, A. niger, A. foetidus and A. ungius, with the former species being the most dominant. The other species were Cladosporium herbarum, Fusarium equisitum (5.71% of the total count, each) and Dendryphiella vinosa (3.21% of the total count). The isolated species were screened for chitinase production on agar plates containing 0.2% colloidal chitin. The chitinolytic activity of each individual was not always correlated with its density on the isolation plates. Alternaria alternata was the most promising species for chitinase excretion. The use of colloidal chitin (1.5%) as a sole carbon source was superior for the enzyme production by A. alternata. Maximum enzyme yield was obtained after 7 days incubation at 30°C with shaking (150 rev min –1), with an initial pH value of the growth medium at 5.0. Presence of NaNO3 (0.3%), the best nitrogen source, and CaCl2 (100 µg/ml) stimulated the induction of the enzyme. The crude A. alternata chitinase revealed a potential insecticidal effect on the larvae of fruitfly (82% mortality) and could degrade crude shrimp shell waste. K e y w o r d s: chitinase, Alternaria alternata, chitinolytic activity, Egyptian black sand

Introduction Egyptian black sand deposits occur in some locations along the mediterranean coast and extend from El Arish in the east to Abu Quir in the west. These black sands contain some radioactive elements like uranium, thorium and K40, in trace safe amounts, together with high salt content and some heavy minerals of economic value (Dabbour, 1995). Black sand habitats were extensively studied geologically, but rarely evaluated microbiologically. It is expected that microorganisms isolated from black sand will be highly active, especially in enzyme production and activity, due to their existence in weak radioactive environment. One of these enzymes is chitinase which is recently facing more attention in the field of biotechnology as it degrades chitin. Chitin is one of the most abundant polysaccharide on the earth. It is present in the cell walls of most fungi (Muzzarelli, 1977) and exoskeleton of arthropods. A lot of chitineous substances contained in shell of shrimp, crabs, lobsters and others are accounting for about 10% of global landings of the aquatic products (Nopakarn et al., 2002). However, these substances are discarded as wastes and its degradation is of great importance as it can contribute to both carbon and nitrogen cycles in the biosphere (Reguera and Leschine, 2003). Chitin is degraded by chitinase to N-acetyl glucosamine which can be utilized as a substrate in many industrial applications. Moreover, chitinase induces the bioconversion of chitineous wastes to cell, ethanol (Ferrer et al., 1996), fertilizer (Sakai et al., 1998) and production of chitoligomers for pharmaceutical or chemical purposes (Patil et al., 2000). Recently, chitinases could be exploided as a biocontrol agent for fungal phytopathogens (Palani and Lalithakumari, 1999; Giambattesta et al., 2001; Witkowska and Maj, 2002) and as an inhibitor for food spoiling moulds (Gad, 2003). Furthermore, the possible use of chitinase as insecticide was also elucidated ( Mendonsa et al., 1996; Ghaly, 2003).

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Sharaf E.F.

2

Accordingly, the present study aimed at isolation of chitinolytic fungi from Egyptian black sand. Emphasis was directed to chitinase production and factors which maximize the yield by the most potent producer Alternaria alternata. The chitinolytic activity of the crude enzyme was further tested on the larvae of fruitfly. Experimental Materials and Methods Samples of black sand. Ten black sand samples were collected, in clean plastic bags from Rosetta coast in Alexandria, and thoroughly mixed into one composite sample. Preparation of native shrimp shell chitin. Native shrimp shell wastes were collected from El-Uboor market. Chitin was prepared from the waste in three treatments: crude, treated or colloidal chitin. The crude chitin was prepared by washing part of the shrimp shell waste, drying up in sun and then grinding it (dehydrated ground shrimp shell chitin). Another part of the waste, after washing, was alternatively pretreated with NaOH and HCl (Jeuniaux, 1966) for several times, dried up and ground to obtain treated chitin. Colloidal chitin was prepared from treated chitin using phosphoric acid (H3PO4) according to Reid and Ogrydiazk (1981). Isolation of chitinolytic fungi. The soil dilution plate method (Johnson et al., 1960) was followed for isolation of chitinolytic fungal species from black sand. A basal mineral salt medium containing (g%) NaNO3, 0.2; KH2PO4, 0.1; MgSO4 ×7H2O, 0.05; KCl, 0.05; agar, 2 and streptomycin, 0.003 was supplied with the different forms of 0.5% chitin (crude, treated or colloidal, separately) as a sole carbon source. The pH was adjusted to ~5. One ml of black sand suspension, at the proper dilution, was placed on the plates (5 replicates, each). The plates were swirled and incubated at 28°C for 2 weeks. The developing fungal colonies were counted and identified according to Raper and Fennell (1965), Moubasher (1993) and Ellis (1971, 1976). The percentage of relative density (RD %) was calculated. Qualitative screening for chitinolytic activity. The isolated fungal species were screened for chitinolytic activity on chitin-agar medium containing 0.2% colloidal chitin in citrate phosphate buffer (pH 5.0). A fungal disc (6 mm) cut from the periphery of 7 days old culture, grown on Dox,s medium, were inoculated on the plates (triplicates). The plates were incubated for 5 days at 28°C. The diameters of clearing zones were measured (mm) and means were calculated and taken as an evidence for chitinolytic activity. Production of chitinase enzyme by Alternaria alternata. The fungal isolate Alternaria alternata which exhibited the greatest clearing zone on the agar plates was selected for further and more detailed studies. A basal growth medium containing (g%) colloidal chitin, 0.5; NaNO3, 0.2; KH2PO4, 0.1; Mg SO4 × 7H2O, 0.05 and KCl, 0.05 was used for chitinase production. The pH was adjusted to 5.0. The flasks (triplicates) were inoculated with 1 ml spore suspension (~106 ml–1) of 7-day old cultures. The inoculated flasks were incubated at 30°C under shaking (100 rev min –1) for 7 days. The culture broth was centrifuged, undercooling, and the clear supernatant was used as a source for crude chitinase. Enzyme assay. One ml of 0.5% colloidal chitin (suspended in citrate phosphate buffer, pH 5.0) was incubated with 1 ml of the crude chitinase at 40°C for 90 minutes. The reducing sugar was estimated according to Reissig et al. (1955) using a standard curve of N-acetyl glucosamine. One unit of enzyme activity is the amount of the enzyme which releases 1 mmol of N-acetyl glucosamine under the assay conditions. Cultural conditions controlling chitinase biosynthesis by Alternaria alternata a) Environmental conditions. The effect of incubation period (2– 9 days), incubation temperature (20 oC– 60oC), pH value of the growth medium (3–8) as well as shaking speed (100– 250 rev mn –1) on chitinase production were investigated. b) Nutritional conditions. The following nutrients were studied for their effect on chitinase production: different carbon sources (1 g%) as well as crude, treated and colloidal chitin (0.5 g%, each), colloidal chitin concentration (0.5– 3.0 g%), different inorganic N sources (at equimolecular bases), organic N sources (0.2 g%), concentration of NaNO 3 (0.1–0.4 g%) and salts of some microelements (100 µg/ml). Effect of crude chitinase on fruitfly larvae. Two µL of the crude enzyme was pipetted on the larvae (50) of the fruitfly. Control was treated with distilled water instead of the enzyme. The larvae were left for 24 hrs at room temperature in beakers covered with a piece of cloth. Percentage of mortality was calculated. Statistical analysis. Summary statistics were used to obtain the means and standard errors (SE). One way analysis of variance (ANOVA) was detected to evaluate the significant differences between means using SPSS statistical software (P< 0.01).

Results and Discussion The total count of chitinolytic fungi isolated from Egyptian black sand was 335 colonies/g dry soil (Table I). Fungi belonged to 8 species belonging to 5 genera. Genus Aspergillus and Alternaria alternata showed the highest density on the isolation plats (RD > 40%, each). Genus Aspergillus comprised A. flavus, A. foetidus, A. niger and A. ungius. A. flavus and A. alternata were developed on the plates containing colloidal, treated and crude chitin, exhibiting a strong chitinolytic activity. However, the other Aspergillus species were developed on plates which contained colloidal chitin only, showing less activity. Cladosporium herbarum and Fusarium equisitum came next in density (RD 5.7%, each). They were isolated from plates containing colloidal chitin, with the former species being more active as it could be also recovered from treated chitin. The least dominant Dendryphiella vinosa (RD 3.2%) appeared on colloidal chitin plates only, and showed low activity.

2

147

Chitinolytic activity of A. alternata Table I Total count (colonies/g dry soil) and relative densities (RD %) of chitinolytic fungi isolated from Egyptian black sand on media containing different treatments of native shrimp shell chitin Fungal species

Treatments of chitin

Total count Relative density (colonies/g) dry soil (RD%)

Crude

Treated

Colloidal

Aspergillus spp.

23

38

84

145

43.3

A. flavus

23

38

38

99

29.6

A. foetidus

–

–

8

8

2.4

A. niger

–

–

23

23

6.8

A. ungius

–

–

15

15

4.5

Alternaria alternata

19

80

42

141

42.1

Cladosporium herbarum

–

11

8

19

5.7

Dendryphiella vinosa

–

–

11

11

3.2

Fusarium equisitum

–

–

19

19

5.7

Total

57

91

187

335

100

The chitinolytic activity of fungal species isolated from Egyptian soil was demonstrated by El Naghy et al. (1985), Sherief et al. (1999), Nour El-Dein et al. (1999), Shindia et al. (2001), Ali and Ibrahim (2003) and Ghareib et al. (2004). They reported that the chitinolytic fungi showed great variations towards chitin degradation ranging from weak to strong activity, depending on inherited characters. It is evident that all the isolated species were recovered from medium containing colloidal chitin, i.e. they could degrade this form of chitin, whereas C. herbarum, A. flavus and A. alternata could also utilize the treated one (Table I). On the other hand, the last two species only were able to degrade the crude chitin, an observation which indicated the availability of the used form of chitin for degradation and/or the rate of chitinolytic activity of each individual. In this connection successive pretreatment of crude shrimp shell chitin with NaOH followed by HCl resulted in deproteinization (Chang and Tsai, 1997) and removal of most of CaCO3 from the shell (Cosio et al., 1982), respectively. This treated form of chitin is more available for degradation than the crude one. Moreover, when this treated chitin was converted to the colloidal form (chitodextrin) it became swollen and most available for attack (Monreal and Reese, 1969). The results of qualitative screening on agar plates revealed that the chitinolytic activity of each species was not always correlated with its density on the isolation plates (Table II). A. alternata followed by A. flavus exhibited the greatest clear zones (37 and 32 mm, respectively) which confirmed their higher chitinolytic activity. A. foetidus and F. equisitum showed a moderate activity and their clear zones measured 26 and 21 mm, respectively. The other species showed lower activity and achieved clear zones ranging from 10 to 18 mm. Therefore, A. alternata was selected for maximization of chitinase production. Chitinase biosynthesis by A. alternata was gradually increased by increasing the incubation period up to the 7th day (2.467 units/ml, Fig. 1a). However, prolonged incubation reduced the enzyme productivity. Similar result was obtained by Sherief et al. (1991) for chitinase of A. carneus. Neverthless, different Table II incubation periods were recorded for chitinase pro- Chitinolytic activity of fungal species isolated from Egyptian duction by filamentous fungi where it was 2–4 days black sand on agar plates as determined by diameter of clearing zone for Talaromyes emersonii (McCormack et al., 1991), 4 days for Chaetomium thermophilum and 8 days Diameter of clearing zone for Cunninghamella echinulata and Thermomyces Fungal species (mm ± SE) lanuginosus (Nour El-Dein et al., 1999 and Ali and Alternaria alternata 37 ± 2.8 Ibrahim, 2003, respectively). Aspergillus flavus 32 ± 3.0 Maximal chitinase production was obtained when the fungus was incubated at 30°C (Fig.1b). Any shift A. foetidus 26 ± 2.4 below or above this temperature was followed by A. niger 18 ± 1.7 retardation in chitinase excretion. Similar results A. ungius 14 ± 1.3 were obtained by Kabat et al. (1996) and Shindia Cladosporium herbarum 16 ± 1.2 et al. (2001). However, 45°C was found optimum Dendryphiella vinosa 10 ± 1.1 for the enzyme production by Ch. thermophilum (Ali Fusarium equisitum 21 ± 1.5 and Ibrahim, 2003).

148

2

(1a)

Chitinase production (units/ml)

Sharaf E.F. 3 2,5 2 1,5 1 0,5 0

(1b)

Chitinase production (units/ml)

2

3

4

Chitinase production (units/ml) Chitinase production (units/ml)

7

8

9

3 2 1,5 1 0,5 0 30

40

50

60

3 2,5 2 1,5 1 0,5 0 3

(1d)

6

2,5

20

(1c)

5

4

5

6

7

8

4 3 2 1 0 100

150

200

250

Fig.1. Influence of environmental factors affecting production of chitinase by A. alternata a) Incubation period (days), b) Incubation temperature (°C), c) Initial culture pH, d) Shaking speed of the culture medium (rev min–1)

In the present study, the pH of the growth medium exerted a significant effect on chitinase biosynthesis (P < 0.01). The optimum pH was 5 (2.478 units/ml), and any shift exerted remarkable reduction in enzyme productivity (Fig. 1c). This results were in complete accordance with those of Tweddell et al. (1994), Nour El-Dein et al. (1999) and Shindia et al. (2001). On the other hand, the optimum pH was found to be 7 for chitinase production by Stachybotrys elegans (Taylor et al., 2002), Aspergillus sp. S1-13 (Nopakarn et al., 2002) and Penicillium janthinellum (Giambattesta et al., 2001). Interestingly, increasing the shaking speed of the growth medium up to 150 rev min–1 was followed by a significant increase (P