Activities of synthetic peptides against human pathogenic bacteria

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Polish Journal of Microbiology 2004, Vol. 53, No 1, 5–6

POLSKIE TOWARZYSTWO MIKROBIOLOGÓW THE POLISH SOCIETY OF MICROBIOLOGISTS

Polish Journal of Microbiology formerly

Acta Microbiologica Polonica

2004 POLSKIE TOWARZYSTWO MIKROBIOLOGÓW

Polish Journal of Microbiology 2004, Vol. 53, No 1, 5–6

EDITORS K.I. Wolska (Editor in Chief)

A. Kraczkiewicz-Dowjat, A. Skorupska, L. Sedlaczek, E. Strzelczyk E.K. Jagusztyn-Krynicka (Scientific Secretary)

EDITORIAL BOARD President: Zdzis³aw Markiewicz (Warsaw, Poland) Ryszard Chróst (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)

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PolishJournal Journal of Microbiology Polish of Microbiology formerly 2004,Acta Vol. Microbiologica 53, No 1, 5–6 Polonica 2004, Vol. 53, No. 1

CONTENTS

5

IN MEMORIAM ORIGINAL PAPERS

Development of a multiplex PCR (m-PCR) test for rapid identification of genes encoding heat-labile (LTI) and heat-stable (STI and STII) toxins of enterotoxigenic Escherichia coli (ETEC) with internal control of amplification WEINER M., OSEK J.

Identification of OmpR protein and its role in the invasion properties of Yersinia enterocolitica RACZKOWSKA A., BRZOSTEK K.

7 11

Streptococcus group B (GBS) – characteristic, occurrence in children and adolescents with type 1 diabetes mellitus NOWAKOWSKA M., JAROSZ-CHOBOT P.

Molecular epidemiology of antibiotic-resistant Escherichia coli isolated from hospitalized patients with urinary tract infections in northern Palestine ADWAN K., ABU-HASAN N., ADWAN G., JARRAR N., ABU-SHANAB B., AL-MASRI M.

17

23

Incidence of extended-spectrum $-lactamases in clinical isolates of the family Enterobacteriaceae in a pediatric hospital RADOSZ-KOMONIEWSKA H., GNIADKOWSKI M., ROGALA-ZAWADA D., NOWAKOWSKA M., RUDY M., WIECHU£A B., MARTIROSIAN G.

27

Enterotoxigenic Bacteroides fragilis (ETBF) strains isolated in the Netherlands and Poland are genetically diverse OBUCH-WOSZCZATYÑSKI P., WINTERMANS R.G.F., VAN BELKUM A., ENDTZ H., PITUCH H., KREFT D., MEISEL-MIKO£AJCZYK F., £UCZAK M.

Activities of synthetic peptides against human pathogenic bacteria BOGUCKA K., KRÓLICKA A., KAMYSZ W., OSSOWSKI T., £UKASIAK J., £OJKOWSKA E.

Purification and characterization of extracellular Pseudomonas aeruginosa urate oxidase enzyme SAEED H.M., ABDEL-FATTAH Y.R., GOHAR Y.M., ELBAZ M.A.

Possible roles of nitrogen fixation and mineral uptake induced by rhizobacterial inoculation on salt tolerance of maize EL-KOMY H.M.A., ABDEL-SAMAD H.M., HETTA A.M.A., BARAKAT N.A.

Antigenicity of the Campylobacter coli CjaA protein produced by Escherichia coli RACZKO A., WYSZYÑSKA A., JAGUSZTYN-KRYNICKA E.K.

35 41 45 53 61

BOOK REVIEW

65

INSTRUCTIONS TO AUTHORS

67

Redakcja Polish Journal of Microbiology (formerly Acta Microbiologica Polonica) zawiadamia, ¿e realny Impact Factor w roku 2003 osi¹gn¹³ wartoœæ 0,286 Krystyna I. Wolska – Redaktor naczelny

Polish Journal of Microbiology 2004, Vol. 53, No 1, 5–6

Polish Journal of Microbiology 2004, Vol. 53, No 1, 5–6

IN MEMORIAM Prof. dr hab. Krystyna Kote³ko Professor emeritus of the University of £ódŸ (1920–2003)

Professor Krystyna Kote³ko was born on 7th November 1920 in Grodzisk Mazowiecki. After graduating from primary school and then Maria Konopnicka State Gymnasium in Warsaw (1938), for one year she studied at the Faculty of Biology and Mathematics of Warsaw University. After the war broke out, she took on the job of a voluntary worker at the City Institute of Hygiene, and later she worked as a laboratory technician in the Bacteriology Department. She was also a member of the Resistance Movement, first in the Scout Soldiers (Szare Szeregi), and next in the Sanitary Services of the Warsaw Headquarters of the Home Army. During the Warsaw Uprising she worked as a nurse in Karol and Maria’s Hospital, and after the fall, managed an analytical laboratory in W³odzimierzów near Sulejów. In 1947 she finished at the University of £ódŸ her biological studies interrupted by the war and started a job in the State Institute of Hygiene as a Head of the Section for Intestine Infections Diagnostics. In 1951 at the University of £ódŸ she obtained the doctor of science degree on the basis of the dissertation “Serological classification of hemolysing streptococci isolated from patients with scarlatine”, supervised by professor Bernard Zab³ocki. Then she started her job as an assistant professor at the Department of Bacteriology, and as a research worker of the University of £ódŸ, she obtained in 1961 the habilitation degree for the work “Chemical basis of immunological specificity of Salmonella O-antigens with a special focus on immunochemistry of group B O5 antigen”. In 1969 she was granted the scientific title of associate professor, and in 1976 she became full professor. In the first years of her scientific research, supervised by professor B. Zab³ocki, she focused on the biology of staphylococci, streptococci and Shigella rods. Later her interests were directed towards the structure and function of selected bacterial antigens, especially Proteus rods. The choice of this particular subject of research was inspired by two one-year studies at the Pasteur Institute in Paris, France and at the Max Planck Institute of Immunobiology in Freiburg, Germany. In 1961 professor Kote³ko created the Department of General Microbiology, and for 30 years she was the Head of it. She became also a leader of a very successful team of research workers in immunochemistry, well recognised both in Poland and abroad for initiating on a large scale team research concentrated on the chemical and biological investigation of surface antigens of facultatively pathogenic Proteus bacteria. Professor Kote³ko prepared together with coworkers 65 publications of experimental research, 10 review articles, 2 monographic articles on Proteus rods and 3 scientific books, which have had a few editions, as well as numerous communications at Polish and international congresses. The most significant results of the long lasting study on Proteus lipopolysacharides include: – detection of uronic acids, aminoacids and other sugar derivatives as non-typical lipopolysacharide constituents, – determination of LPS chemotypes for the whole genus Proteus, – obtaining and analysis of unique rough mutants from these microorganisms, necessary for structural and biological studies,

6

1

In memoriam

– determination of the full structure of complex LPS macromolecule of one Proteus strain and a few polysacharide structures of endotoxins from other serological Ptoteus groups, – description of the characteristics of the selected factors of Proteus rods pathogenicity. Those results could be obtained due to prof. Kote³ko’s fruitful cooperation with the major European centres in this field: Max Planck Institute of Immunobiology in Freiburg and Scientific Institute in Borstel (Germany) as well as the Institute of Organic Chemistry, the Russian Academy of Sciences in Moscow. Publications of these studies in leading scientific journals on the international scale made prof. Kote³ko’s team of workers recognised all over Europe as the “Proteus Group”. In recognition of prof. Kote³ko’s scientific achievements, she was many times appointed as a president of scientific sessions at international conferences and was invited by the leading scientific centres in Europe to give lecturs or conduct seminars. Prof. Kote³ko’s scientific work was always connected with didactics. She taught a wide range of classes to the students of microbiology where she proved to be an expert using the Polish language, erudition and thorough preparation in the discussed area. Her lectures and seminars were always received with delight and admiration by both students and young scientific staff. Her contribution to teaching and promoting the research workers is worth emphasising. She promoted 8 PhD’s, supervised 6 habilitation works and 4 of her disciples became professors. She was a reviewer of 45 doctor’s dissertations, 27 habilitation works and reviewed 23 professor’s applications. During 4 cadencies as a member of the Central Qualification Committee she prepared 40 opinions concerning habilitations or granting the professor’s degrees. Prof. Kote³ko’s unquestioned merit for the development of microbiology in Poland, establishing and promoting of the first Polish microbiological programme MR II.17 and its continuation PBP 04.02. This 10-year activity, in which all Polish universities were participating, contributed to the integration of the microbiological community, development of research and introduction of new significant scientific issues. The recognition of prof. Kote³ko’s merit for the University of £ódŸ and the whole country resulted her being awarded with the Gold Medal of the University of £ódŸ, Gold Cross of Merit, the Polonia Restituta – Cavalier and Commander Crosses, the City of £ódŸ Reward, the Meritorious Teacher of the Polish Republic, the Medal of the National Education Committee as well as many awards of the Rector, the Secretary of the Polish Academy Sciences, the Ministry of Science and Higher Education. In the Polish and world science prof. Kote³ko was distinguished with the title of the Member of Honour of the Polish Microbiological Society and elected a member of the International Endotoxin Society. As a human being professor Kote³ko was politically straight, scientifically excellent and socially wonderful. Prof. Kote³ko passed away after long lasting disease and suffering. It is very hard to express our grief and sorrow, but our memory about You, dear Professor, Master, Friend and Colleague will remain in our hearts for ever. Zygmunt Sidorczyk and Antoni Ró¿alski

Polish Journal of Microbiology 2004, Vol. 53, No 1, 7–10

Development of a Multiplex PCR (m-PCR) Test for Rapid Identification of Genes Encoding Heat-labile (LTI) and Heat-stable (STI and STII) toxins of Enterotoxigenic Escherichia coli (ETEC) with Internal Control of Amplification MARCIN WEINER and JACEK OSEK

Department of Hygiene of Food of Animal Origin, National Veterinary Research Institute, Partyzantów 57, 24-100 Pu³awy, Poland Received in revised form 2 December 2003 Abstract A multiplex PCR system was developed for specific identification of genes encoding heat-labile (LTI) and heat-stable (STI and STII) toxins of enterotoxigenic Escherichia coli (ETEC) strains. In addition, primers specific for the E. coli gene coding for 16S rRNA were used as an internal control of the DNA amplification. The specificity of the method was validated by single PCR tests performed with reference to E. coli strains as well as pig-isolated bacteria and 100% correlation was observed. The developed multiplex PCR allowed rapid and specific identification of enterotoxin-positive E. coli and may be used as a sensitive and specific method for a direct determination of ETEC and to differentiate them from other E. coli isolates. K e y w o r d s: ETEC, m-PCR, LTI, STI, STII enterotoxins

Introduction Most of Escherichia coli strains are harmless commensals in the gut but some of them are important enteric pathogen, causing diarrhoea in humans and animals (Holland, 1990; Nataro and Kaper, 1998). E. coli isolates associated with diarrhoea have been classified into six major groups, on the base of their distinct virulence properties: enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), diffusely adherent (DAEC) and enteroaggregative (EAEC) (Nataro and Kaper, 1998). Among them, ETEC is a predominant bacterial agent of diarrhoea in young animals as well as in infants and in adults travelling to developing countries (Nataro and Kaper, 1998; Nagy and Fekete, 1999). Bacteria of this group are defined as E. coli which are able to produce at least one enterotoxin: heat-labile I (LTI) heat-stable I (STI) or heat-stable II (STII) (Seriwatana et al., 1988; Celemin et. al., 1994; Sears and Kaper, 1996; Nair and Takeda, 1998). LTI and STI have been found in both human and animal ETEC whereas STII toxin is characteristic only for animal E. coli (Seriwatana et al., 1988; Celemin et al., 1994). ETEC have the ability to cause profuse, watery diarrhoea by releasing of either LT or ST enterotoxins or both. Differentiation between ETEC and other pathogenic and non-pathogenic E. coli bacteria as well as other enteric isolates requires detection of enterotoxins or their genes and it is essential for proper detection and control of diarrhoeal diseases. Conventional techniques of E. coli diagnosis depend on growth in pure cultures and subsequent biochemical identification of relevant bacterial colonies. These procedures are time-consuming and can take up to 3–5 days. Moreover, differentiation of pathogenic ETEC isolates needs additional toxin detection procedures (Ojeniyi et al., 1994). One of the approaches used for the toxin determination is polymerase chain reaction (PCR) which is a very sensitive and highly specific molecular biology tool widely used for diagnosis of ETEC. PCR performed with the use two or more primer pairs to amplify two or more target sequences, called multiplex PCR, has been applied for detection and differentiation of LTI, STI or LTI, STII and Shiga toxin genes (Nataro and Kaper, 1998; Tsen and Jian, 1998; Nagy and Fekete, 1999; Osek et al. 1999, Osek and Truszczyñski, 2000, Osek 2001). However, these

8

1

Weiner M., Osek J.

multiplex PCR systems do not allow the simultaneous detection of all enterotoxin genes and E. coli-specific 16S rRNA gene, used as an internal control of amplification. Although significant advancement has been made in the molecular identification of ETEC, there is still a need to improve the existing PCR assays for detection of genes encoding all enterotoxins, which are important in pathogenesis of ETEC diarrhoea and specific differentiation of ETEC from non-toxigenic E. coli bacteria. In the present study, a rapid and specific multiplex PCR for the simultaneous detection of the genes encoding LTI, STI, STII and E. coli-specific 16S rRNA gene used as an internal control has been described. Experimental Materials and Methods Bacterial strains. E. coli strains (n = 69) were isolated from weaned pigs (four- to six-weeks old) with diarrhoea characterized previously (Osek et al., 1999; Osek, 2000; Osek, 2001). For control purposes the following E. coli strains were used: 293 (LTI-STI-STII-positive), 256 (STI-STII-positive), 259 (LTI-STII-positive), 335 (STI-positive), 276 (STII-positive) and C600 (LTI-STI-STII-negative). Template DNA. Bacterial strains were grown on Luria-Bertani (LB) agar at 37°C for 18 h and one individual colony of each E. coli isolate was suspended in 50 µl of sterile, DNase, RNase-free deionized water (ICN Biomedicals, Costa Mesa, USA). The suspensions were centrifuged at 13 000× g for 1 min and the supernatant (5 µl) was subsequently used as a source of DNA template. PCR amplifications. PCR was used for the determination of the following genes encoding the E. coli enterotoxins: eltI (LTI toxin), estI (STI toxin), estII (STII toxin) as well as the gene responsible for expression of 16S rRNA of E. coli. All PCR primers (synthesized by IDT, Coralville, USA) used in the present study are listed in Table I. The primers flanking the 16S ribosomal RNA gene were included as an internal control to identify the presence of E. coli DNA in the PCR samples. Template DNA (5 µl) was added to the PCR mixture consisting of: 5 µl of PCR buffer (10×concentrated), 5 µl of each deoxynucleotide (dNTPs; final concentration 0.2 mM), 10 µl of 25 mM MgCl2 (final concentration 5 mM), 2.5 µl of 10 µM LT3 and LT4 primers (final concentration 0.5 µM each), 0.5 µl of 10 µM STA1 and STA2 primers (final concentration 0.1 µM each), 1.0 µl of 10 µM STIIB1 and STIIB2 primers (final concentration 0.2 µM each), 0.125 µl of 10 µM 16S-F and 16S-R primers (final concentration 0.025 µM each), 2 U of the Taq DNA polymerase (Fermentas, Vilnius, Lithuania) and water to final volume of 50 µl. The PCR program was as follows: after initial DNA denaturation at 94°C for 5 min, 30 cycles (94°C for 1 min, 72°C for 2 min., 55°C for 1 min.) were used. The amplified PCR products (10 µl aliquots) were visualized by gel electrophoresis in 2% agarose gel (Type 1, low EEO, Sigma Chemicals, St. Louis, USA) in Tris-Acetate-EDTA (TAE) buffer at 100 V. The gels were stained with ethidium bromide (5 µl ml–1) for 2 min, washed in distilled water, analyzed under UV light (300 nm) and photographed using the Gel Doc 2000 documentation system (Bio-Rad, Hercules, USA). The size of the obtained amplicons was compared to the 100 bp DNA ladder (Fermentas).

Table I Characteristics of the primers used in the study Primer code

→ 3’) Sequence (5’→

Target gene

LT3

TATCCTCTCTATATGCACAG

LT4

CTGTAGTGGAAGCTGTTATA

STA1

TCTTTCCCCTCTTTAGTCAG

STA2

ACAGGCCGGATTACAACAAAG

STIIB1

GCCTATGCATCTACACAATC

STIIB2

TGAGAAATGGACAATGTCCG

16S-F

AGAGTTTGATCATGGCTCAG

16S-R

GGACTACCAGGGTATCTAAT

eltI estI estII 16S rRNA

Gene product Heat-labile enterotoxin LTI Heat-stable enterotoxin STI Heat-stable enterotoxin STII 16S ribosomal RNA

PCR amplicon (bp)

Reference

480

Leong et al., 1985

166

Ojeniyi et al., 1994

278

Picken et al., 1983

798

Ehresmann et al., 1972

Results and Discussion The reaction conditions for the multiplex PCR assays were optimized to ensure that all the target gene sequences were satisfactorily amplified. Initially, the same concentrations (0.1 mM) of each set of primers were used but this approach resulted in uneven intensity or even lack of some of the amplified products. To overcome this problem the change in the proportions of the various primers in the reaction mixture was required to increase the concentrations of primers in the case of weak amplicons and to decrease the oligonucleotide concentration in the case of strong PCR bands. Moreover, the multiplex PCR test was

1

9

Multiplex PCR for E. coli enterotoxins M

1

2

3

4

5

6

7

798 bp → 480 bp → 278 bp → 166 bp → Fig. 1. Simultaneous identification of LTI, STI, and STII enterotoxin genes and E. coli-specific gene encoding 16S rRNA using a multiplex PCR system with LT3/LT4, STA1/STA2, STIIB1/STIIB2 and 16S-F/16S-R primers. Lane M: 100 bp ladder; lane 1: E. coli LTI/STI/STII-positive; lane 2: E. coli STI/STII-positive; lane 3: E. coli LTI/STII-positive; lane 4: E. coli STI-positive; lane 5: E. coli STII-positive; lane 6: E. coli LTI/STI/STII-negative; lane 7: H2O

simplified by the use of DNA templates prepared by a direct suspension of bacterial colonies in water instead of a time-consuming DNA extraction procedure. The specificity of the multiplex PCR system developed was tested with E. coli reference strains listed in the “Materials and Methods” section. Moreover, this test was also validated using E. coli isolates (n = 69) originated from weaned pigs with diarrhoea and characterized previously by PCR performed with the primer pairs specific for particular enterotoxin genes analysed (Osek et al., 1999; Osek, 2000). It was demonstrated that enterotoxin-positive E. coli isolates showed the specific 480 bp (LTI), 166 bp (STI) or 278 bp (STII) amplicons of the enterotoxin genes tested (Fig. 1, lanes 1–5). There was a 100% correlation between the results obtained with the multiplex PCR assay and with PCR analyses separately performed with single primer pairs specific for LTI, STI, and STII enterotoxin genes, respectively. The presence of the 798 bp amplified product (the 16S rRNA gene) was observed in all bacterial samples tested in the mPCR system indicating that all of them contained E. coli DNA. The E. coli enterotoxinnegative strain C600 generated only 798 bp amplification product of the 16S rRNA gene (Fig. 1, lane 6). Table II shows the gene profiles of 69 E. coli isolates tested in the present study. All isolates harboured the 16S rRNA gene. The most prevalent ETEC marker detected during this study was the estII gene that was found in 40 isolates (the 278 bp PCR amplicon). Among them, 20 isolates harboured the eltI gene only, 15 – estI (amplified DNA band corresponding to 480 and 166 bp, respectively) and 2 isolates had both of these genes. Moreover, 7 strains carried the estI gene and 3 isolates had the estII gene as the only marker of ETEC. The remaining 22 isolates possessed none of the ETEC markers that were sought. Table II Multiplex PCR results derived from 69 E. coli strains isolated from pigs with diarrhoea Gene profile

No of strains

16S rRNA

LTI

STI

STII

+

+

+

+

+

+

+

–

0

+

+

–

+

20

+

–

+

+

15

+

+

–

–

0

+

–

+

–

7

+

–

–

+

3

+

–

–

–

22

2

10

Weiner M., Osek J.

1

In the previous studies of Osek (Osek et al. 2000; Osek 2001), the primers specific for the universal stress protein A (UspA) of E. coli and enterotoxins genes were used and allowed a simultaneous amplification of the E. coli-specific uspA and the respective toxins genes. In the present study, the multiplex PCR for the simultaneous detection of the genes encoding LTI, STI, STII and E. coli 16S rRNA gene used as an internal control was developed. The m-PCR described in this study has been shown to be an efficient tool for simultaneous detection of three enterotoxin genes of ETEC strains and it also allows to identify potentially pathogenic (enterotoxinpositive) E. coli and to differentiate them from commensally (non-toxigenic) isolates. One of the most important advantages of the multiplex PCR described here is the specific detection of ETEC bacteria without any biochemical identification and subculturing of the isolates. As described earlier (Ehresmann et al. 1972), the 798 bp amplification product of 16S ribosomal RNA is presented in E. coli bacteria only and therefore, the primers specific for this genetic marker were used in the mPCR developed. This improvement would simplify the detection process and eliminate the requirement for confirmation procedures such as biochemical tests or toxin detection in vitro (Ojeniyi et al., 1994). PCR analysis of all reference E. coli strains as well as pig-isolated bacteria yielded consistent results with the PCR test separately performed with single primer pairs specific for particular three enterotoxin genes analysed. These results demonstrate high discriminatory power of the developed m-PCR assay and the potential of the test for rapid and specific identification and toxin profiling of ETEC. Literature C e l e m i n C., J. A n g u i t a, G. N a h a r r o, S. S u a r e z. 1994. Evidence that Escherichia coli isolated from healthy pigs intestine hybridize with LT-II, ST-Ib and SLTII DNA probes. Microb. Pathog. 16: 77–81. E h r e s m a n n C., P. S t i e g l e r, P. F e l l n e r, P. E b e l. 1972. The determination of the primary structure of the 16S ribosomal RNA of Escherichia coli. 2. Nucleotide sequence of products from partial enzymatic hydrolysis. Biochemie 54: 901–967. H o l l a n d R.E. 1990. Some infectious cases of diarrhoea in young farm animals. Clin. Microbiol. Rev. 3: 345–375. L e o n g J., A.C. V i n a l, W.S. D a l l a s. 1985. Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin. Infect. Immun. 48: 73–77. N a g y B., P.Z. F e k e t e. 1999. Enterotoxigenic Escherichia coli (ETEC) in farm animals. Vet. Res. 30: 259–284. N a i r G.B., Y. T a k e d a. 1998. The heat-stable enterotoxins. Microb. Pathog. 24: 123–131. N a t a r o J.P., J.B. K a p e r. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11: 142–201. O j e n i y i B., P. A h r e n s, A. M e y l i n g. 1994. Detection of fimbrial and toxin genes in Escherichia coli and their prevalence in piglets with diarrhoea. The application of colony hybridization assay, polymerase chain reaction and phenotypic assays. J. Vet. Med. B 41: 49–59. O s e k J., P. G a l l i e n, M. T r u s z c z y n s k i, D. P r o t z. 1999. The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland. Comp. Immunol. Microbiol. Infect. Dis. 22: 163–174. O s e k J., M. T r u s z c z y n s k i. 2000. Rapid and specific differentiation of enterotoxin-producing Escherichia coli strains from other Gram-negative enteric bacteria using multiplex PCR. Berl. Munch. Tierarztl. Wochenschr. 113: 265–270. O s e k J. 2000. Virulence factors and genetic relatedness of Escherichia coli strains isolated from pigs with post-weaning diarrhoea. Vet. Microbiol. 71: 211–222. O s e k J. 2001. Multiplex polymerase chain reaction assay for identification of enterotoxigenic Escherichia coli strains. J. Vet. Diagn. Invest. 13: 308–311. P i c k e n R.N., A.J. M a z a i t i s, W.K. M a a s. 1983. Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli. Infect. Immun. 42: 269–275. S e a r s C.L., J.B. K a p e r. 1996. Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol. Rev. 60: 167–215. S e r i w a t a n a J., J.E. B r o w n, P. E c h e v e r r i a, D.N. T a y l o r, O. S u t h i e n k u l, J. N e w l a n d. 1988. DNA probes to identify Shiga-like-toxins I and II producing bacterial pathogens isolated from patients with diarrhoea in Thailand. J. Clin. Microbiol. 26: 1614–1615. T s e n H.Y., L.Z. J i a n. 1998. Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water. J. Appl. Microbiol. 84: 585–592.

Polish Journal of Microbiology 2004, Vol. 53, No 1, 11–16

Identification of OmpR Protein and its Role in the Invasion Properties of Yersinia enterocolitica ADRIANNA RACZKOWSKA and KATARZYNA BRZOSTEK*

Institute of Microbiology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland Received 5 December 2003

Abstract Yersinia enterocolitica is a human pathogen that causes gastroenteric infections. Various environmental signals control the expression of the virulence factors in pathogenic Y. enterocolitica strains. OmpR, a global transcriptional regulator controls the expression of a wide spectrum of genes, some of which are required for virulence. In this study, we amplified, cloned and sequenced a Y. enterocolitica Ye9 ompR gene. Deduced amino acid sequence has been shown to have 98% homology to the Y. enterocolitica O:8, Y. pestis, S. typhi and S. enterica serovar Typhimurium OmpR proteins. Additional cell culture experiments was performed to investigate whether OmpR takes part in the virulence of Y. enterocolittica. We found that the Y. enterocolitica ompR mutant was unable to invade HeLa cells. In conclusion, we have shown that OmpR is a very highly conserved protein among enteric bacterial pathogens which plays an important role in the Y. enterocolitica virulence. K e y w o r d s: Y. enterocolitica, invasion, OmpR

Introduction Yersinia enterocolitica is a gram-negative intestinal rod that causes human diseases generally termed yersinioses. It is a facultative pathogen that is capable of infection and propagation in the host but is also able to grow as a saprophyte. Based on biochemical and genetical differences the Y. enterocolitica species has been divided into 40 serotypes and six biogroups. Two types of pathogenic strains of Y. enterocolitica have been defined, so called New World strains (serotypes O:8, O:4, O:20, O:13) and Old World strains (O:9, O:3). With respect to mouse virulence and ecology Y. enterocolitica New World strains are a high pathogenicity group (LD50 102 –103 compared to105 –106 for European strains, Bottone, 1997). According to16SrRNA sequence analysis these strains belong to two distinct phylogenetic groups. Pathogenesis determinants of Y. enterocolitica are coded chromosomally and by plasmid pYV (Yersinia virulence) (Cornelis et al., 1987; Cornelis et al., 1998). In early phases of infection chromosomal proteins responsible for the colonization and invasion of epithelial cells of the intestine are synthesized. These include flagellin, invasin, adhesin, proteins involved in iron metabolism and also toxin Yst that is produced by some strains (Pepe and Miller, 1993). Pathogenic bacteria residing in various ecological niches are constantly threatened by changes of physical and chemical environmental factors, such as osmolarity, pH, accessibility of nutrients, light intensity, viscosity of medium, etc. (Straley and Perry, 1995). The adaptation of bacteria to new conditions involves both rapid changes, e.g. of cell motility, as well as prolonged, global reorganization of gene expression. The mechanisms of molecular responses to signals from the outer environment are complex and depend, among others, on two-component regulatory systems (Albright et al., 1989; Barrett and Hoch, 1998). In two-component regulatory systems transmission of the signal occurs through a pair of proteins that communicate with each other via the conserved mechanism of phosphorylation (Stock et al., 1989; Bourret et al., 1991). The regulatory system OmpR-EnvZ participates in the bacterial response to changes in the * Corresponding author: e-mail [email protected]

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osmolarity of the external environment (Russo and Silhavy, 1991). It has best been studied in Escherichia coli, but is also found in other pathogens such as Salmonella or Shigella (Miller et al., 1989). The ompR and envZ genes of E. coli form part of the ompB operon. They code for the regulatory protein OmpR and the sensor protein EnvZ, respectively. They are involved in the osmoregulation of the transcription of the porin proteins OmpF and OmpC (Hall and Silhavy, 1981). The functions of protein OmpR include both the positive and negative regulation of the transcription of both proteins. OmpR of E. coli is a cytoplasmic protein (~28 kDa) consisting of 239 amino acid residues. A modulator domain in the N-terminal part of the protein and a DNA-binding effector domain, located in the C-terminal part, can be distinguished. The DNA-binding domain is capable of interacting with the promotor regions of genes, whereas the regulator domain is responsible for interactions with EnvZ and RNA polymerase (Itou and Tanaka, 2001). It has been proven that the two-component system OmpR/EnvZ plays an important role in controlling of the virulence of such enteric pathogens as Shigella and Salmonella. It has been found that in S. flexneri the deletion of a operon ompB that codes for the proteins OmpR-EnvZ, significantly reduces the ability of this bacterium to proliferate in epithelial cells and destroy them (Bernardini et al., 1990). It is also known that in Salmonella there is also a dependence between virulence and the two-component system OmpR-EnvZ (Dorman et al., 1989). Mutations in the regulator gene ompR have been shown to alter the pathogenicity of S. enterica serovar Typhimurium. Mutants of the virulent strain were attenuated in vivo. Moreover, it has been proven that S. enterica with mutations in the gene ompR are not able to infect murine cells and to induce the apoptosis of macrophages in vitro (Lindgren et al., 1996). We have previously demonstrated that the Y. enterocolitica Ye9 OmpR protein is involved in controlling the production of Yop virulence proteins and in the adaptation of the bacteria to multiple stresses. Furthermore, a ompR deletion mutant was impaired in survival and replication within macrophages (Brzostek et al., 2003). In this study, we performed a HeLa cell virulence assay and analyzed the predicted amino acid sequence of the Y. enterocolitica Ye9 OmpR protein. Experimental Material and Methods Bacterial strains, plasmids and growth conditions. Y. enterocolitica Ye9 is a serotype O:9 strain from the collection of the State Institute of Hygiene, Warsaw. The bacterial strains and plasmids used in this study are listed in Tables I and II. Bacteria were routinely grown in brain heart infusion (BHI) and Luria Bertani (LB) medium. Strains of Y. enterocolitica were cultivated with shaking at 25°C whereas strains of E. coli were grown at 37°C. The antibiotics used for the selection procedures were ampicillin (Ap, 200 :g/ml) and kanamycin (Km, 50 :g/ml). DNA manipulation and PCR conditions. The entire open reading frame of ompR gene of Y. enterocolitica Ye9 (0.720 kb) was obtained by the PCR amplification which was performed in an automated thermal cycler (MJ Research, Inc.) with TaqI

Table I Bacterial strains used in this study Comments

Strain Y. enterocolitica Ye9

serotype O:9 wild-type strain, pYVO9+

Source or Reference State Institute of Hygiene, Warsaw

Y. enterocolitica AR4 serotype O:9 ompR mutant, pYVO9+, KmR

(Brzostek et al., 2003)

E. coli Top10F’

Invitrogen

F’:{lacI Tn10 (Tet )}, mcrA, )(mrr-hsdRMS-mcrBC), M80)lacZ)M15, )lacX74, deoR, recA1, araD139, )(ara-leu), 7697galU, galK, rpsL (StrR), endA1, nupG q

R

Table II Plasmids used in this study Comments

Plasmid

Source or Reference

pBluescript II SK(+)

cloning vector, ApR

Stratagene

pYR9

pBluescript II SK(+) with 0.720 kb ompR gene of Y. enterocolitica Ye9 (from start to stop codon of translation)

This study

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DNA polymerase (Qiagen). The initial denaturation step (94°C, 5 min) was then followed by 30 cycles of denaturation (94°C, 1 min), annealing (55°C, 1 min) and extension (72°C, 2 min). The oligonucleotide primers used for PCR, forward pR1Bam (5’-CGCGGATCCATGCAAGAGAATCACAAGATTC-3’) and reverse pR2Sma (5’-TCCCCCGGGTCATGCTTTACTGCCGTCCGG-3’), were based on the known ompR sequence data for Y. enterocolitica serotype O:8. The ompR PCR product (0.720 kb) was cloned into the pBluescript II SK(+) vector in the BamHI/SmaI sites. The nucleotide sequence of ompR gene was determined using the universal primers of pBluescript II SK(+) (T3 and T7) and the ABI Prism BigDye terminator Cycle Sequencing System (PerkinElmer). This was subsequently read on an ABI Prism 377 DNA Sequencer (DNA Sequencing and Oligonucleotide Synthesis Laboratory, IBB PAS). The ompR sequence was analyzed using the Wisconsin Sequence Analysis Package (GCG, Madison, Wis.). The OmpR alignments were generated with program BESTFIT and PILEUP. HeLa cell infection. HeLa cells were cultured in 24-mm-diameter plastic wells containing minimal Eagle’s medium (MEM, Gibco BRL) supplemented with 5% heat-inactivated fetal bovine serum and 2 mM glutamine. HeLa cells were cultured until almost confluent and then were infected with the bacterial suspension at a multiplicity of 10. Cells were incubated at 37°C in a 5% CO 2 atmosphere with saturated humidity for 1 h. After this infection period, 100 µg of gentamicin per ml was added and then the plates were incubated for 2 h under the same conditions to kill adherent extracellular bacteria. After this period gentamicin was remov ed by two washes with PBS. Tissue culture cells were then lysed with 0.2 ml of 0.1% Triton X-100 to release intracellular bacteria . After 5 min, 0.8 ml of LB medium was added. The suspension was then diluted and plated on LB agar to determine viable counts. Viable counts of the initial bacterial culture were also determined.

Results and Discussion A variety of studies indicate that OmpR protein is required in E. coli for the osmodependent transcriptional regulation of ompC and ompF gene expression (Hall and Silhavy, 1981) but also plays an essential role in controlling flagellar expression (Shin and Park, 1995), cell division (Pruss, 1998), fatty acid transport (Higashitani et al., 1993) and acid tolerance response (Bang et al., 2002). It needs to be emphasized that OmpR protein is involved in the regulation of genes associated with the virulence of pathogenic bacteria like Salmonella or Shigella (Dorman et al., 1989; Bernardini et al., 1990; Chatfield et al., 1991; Lindgren et al., 1996; Lee et al., 2000). In view of these findings, the general regulator OmpR protein may contribute to the virulence of Y. enterocolitica Ye9. In this study, we contributed to knowledge about the role of OmpR in Y. enterocolitica Ye9 pathogenesis. The Y. enterocolitica Ye9 ompR gene was amplified by PCR with oligonucleotides pR1Bam and pR2Sma, designed from the ompR sequence of Y. enterocolitica serotype O:8. The primers pR1Bam and pR2Sma corresponded to nucleotides 1–22 of the sense strand and 720–699 of the antisense strand, respectively. This PCR product was then purified from the primers used for amplification, cloned into the pBluescript II SK (+) and sequenced. Sequence analysis using the Wisconsin Sequence Analysis package (GCG, Madison, Wis) indicated that the ompR gene encodes a protein of 239 amino acid residues. The predicted amino acid sequence was very similar to that of OmpR from other pathogens. OmpR has two distinctive domains: the receiver domain at the N-terminal portion (residue 1–125), and the C-terminal DNA-binding domain (residues 137–239). Analysis of OmpR predicted amino acid sequence has shown that observed 10 amino acid substitution in the protein sequence among chosen enteric bacterial pathogens did not affect function of OmpR (Fig. 1). The deduced amino acid sequence was 98% identical to OmpR of Y. enterocolitica O:8, Y. pestis, S. typhi and S. enterica serovar Typhimurium. This comparison revealed extensive amino acid identity, which confirmed the highly conserved structure of the OmpR regulator protein. The nucleotide sequence of the Y. enterocolitica Ye9 ompR gene has been submitted to the Gene Bank NCBI data base under accession number AY210888. A recent study has shown that the pleiotropic regulator OmpR controlling the coordinate expression of virulence determinants is required for bacteria to survive in the foreign host. It has been previously reported that ompR mutants of S. enterica serovar Typhimurium were avirulent in a mouse model and did not kill macrophages in vitro (Dorman et al., 1989, Chatfield et al., 1991; Lindgren et al., 1996). In S. typhi OmpR-EnvZ regulatory system was involved in the regulation of biosynthesis of the Vi polysaccharide capsular antigen which is associated with the virulence (Pickard et al., 1994). Furthermore, virulence of the S. flexneri ompR mutant was significantly decreased as a result of its inability to invade ephitelial cells (Bernardini et al., 1990). The ability of pathogenic Y. enterocolitica strains to invade HeLa cells is an important correlate of virulence (Lee et al., 1977). The pattern of invasion of the Y. enterocolitica Ye9 was compared with the AR4 strain, ompR mutant, which was constructed in the previous study by allelic exchange (Brzostek et al., 2003). The data have shown that the Y. enterocolitica ompR mutant invaded HeLa cells relatively poorly, at the levels of about 0.02% of the original recovered inoculum (Fig. 2). In contrast, the control strain, Ye9,

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Fig. 1. Amino acid sequence alignment of the Y. enterocolitica Ye9 OmpR with Y. enterocolitica serotype O:8 (Ye8), Y. pestis (Yp), S. enterica serovar Typhimurium (Stm), S. typhi (St) homologs. Protein sequences were aligned by the PILEUP program (Genetics Computer Group). Changes in amino acids are indicated in frames and by stars.

exhibited levels of invasion of 1%. All results are expressed as % invasion = 100 x (number of bacteria resistant to gentamicin/number of bacteria added). These experiments indicate that the AR4 strain (the ompR mutant) is weakly infective in comparison to wild type strain. It has been previously reported that ompB mutant of S. flexneri was impaired in intercellular spread and multiplied within the initially invaded HeLa cells (Bernardini et al., 1993). In addition other authors demonstrated that mutation in ompR and envZ genes of S. enterica serovar Typhimurium rendered these strains highly reduced for the induction of Sif formation during infection of HeLa cells (Mills et al., 1998). Sifs are tubular structures, which are somehow involved in bacteria’s ability to acquire nutrients and to replicate intracellularly. With regard to the absence of these specific structures in other known enteropathogenic bacteria, including Yersinia, we suppose that the global regulator, OmpR, could be involved in coordinating gene expression upon entry into the host cells in Y. enterocolitca Ye9. We also cannot exclude that genes submitted to OmpR regulation like ompC and ompF are involved in Y. enterocolitica virulence. It has been previously reported in Escherichia, Shigella and Salmonella that in the absence of OmpR protein neither porin is expressed (Russo and Silhavy, 1991; Bernardini et al., 1990; Dorman et al., 1989). It is possible that the lack of the major outer membrane proteins OmpC or OmpF in Y. enterocolitica ompR mutant (AR4) could cause a reduction of virulence. For example, in S. enterica

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OmpR and Y. enterocolitica invasion

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1,2

% invasion

1 0,8 0,6 0,4 0,2 0

Ye9

AR4

Fig. 2. Invasion of the wild-type strain Ye9 and the ompR mutant AR4 of Y. enterocolitica in HeLa cells.

Results shown are the means of duplicate determinations in two separate experiments with standard errors. ■ – Ye9 (wild type strain), o – AR4 (the ompR mutant).

serovar Typhimurium OmpC was a candidate ligand that potentially participates in host-cell recognition of bacteria by phagocytic cells (Negm and Pistole, 1999). On the contrary, in S. flexneri OmpC was involved in spreading from cell to cell and killing epithelial cells during infection (Bernardini et al., 1993). To help to clarify the nature of the Y. enterocolitica virulence network that is under the control of ompR gene we have yet to carry out further studies. Literature A l b r i g h t L.M., E. H u a l a and F.M. A u s u b e l. 1989. Prokaryotic signal transduction mediated by sensor and regulator protein pairs. Annu. Rev. Genet. 23: 311–336. B a n g I.S., J.P. A u d i a, Y.K. P a r k and J.W. F o s t e r. 2002. Autoinduction of the ompR response regulator by acid shock and control of the Salmonella enterica acid tolerance response. Mol. Microbiol. 44: 1235–50. B a r r e t t J.F. and J.A. H o c h. 1998. Two-component signal transduction as a target for microbial anti-infective therapy. Antimicrob. Agents and Chemother. 42: 1529–1536. B e r n a r d i n i M.L., A. F o n t a i n e, P.J. S a n s o n e t t i. 1990. The two-component regulatory system OmpR-EnvZ controls the virulence of Shigella flexneri. J. Bacteriol. 172: 6274–6281. B e r n a r d i n i M.L., M.G. S a n n a, A. F o n t a i n e, P.J. S a n s o n e t t i. 1993. OmpC is involved in invasion of epithelial cells by Shigella flexneri. Infect. Immun. 61: 3625–3635. B o t t o n e E.J. 1977. Yersinia enterocolitica: a panoramic view of a charismatic microorganism. Crit. Rev. Microbiol. 5: 211–241. B o u r r e t R.B., K.A. B r o k o v i c h and M.I. S i m o n. 1991. Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu. Rev. Biochem. 60: 401–441. B r z o s t e k K., A. R a c z k o w s k a and A. Z a s a d a. 2003. The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages. FEMS Microbiol. Lett. 228: 265–271. C h a t f i e l d S.N., Ch.J. D o r m a n, C. H a y w a r d and G. D o u g a n. 1991. Role of ompR-dependent genes in Salmonella typhimurium virulence: mutants deficient in both OmpC and OmpF are attenuated in vivo. Infect. Immun. 59: 449–452. C o r n e l i s G.R., A. B o l a n d, A.P. B o y d, C. G e u i j e n, M. I r i a r t e, C. N e y t, M.-P. S o r y and I. S t a i n i e r. 1998. The virulence plasmid of Yersinia, an antihost genome. Microbiol. Mol. Biol. Rev. 62: 1315–1352. C o r n e l i s G.R., Y. L a r o c h e, G. B a l l i g a n t, M.-P. S o r y and G. W a u t e r s. 1987. Yersinia enterocolitica, a primary model for bacterial invasiveness. Rev. Inf. Dis. 9: 64–87. D o r m a n Ch.J., S. C h a t f i e l d, Ch.F. H i g g i n s, C. H a y w a r d, G. D o u g a n. 1989. Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo. Infect. Immun. 57: 2136–2140. H a l l M.N. and T.J. S i l h a v y. 1981. Genetic analysis of the ompB locus in Escherichia coli K-12. J. Mol. Biol. 151: 1–15. H i g a s h i t a n i A., Y. N i s h i m u r a, H. H a r a, H. A i b a, T. M i z u n o and K. H o r i u c h i. 1993. Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli. Mol. Gen. Genet. 240: 339–47. I t o u H. and I. T a n a k a. 2001. The OmpR-family of proteins: insight into the tertiary structure and functions of two-component regulator proteins. J. Biochem. 129: 343–350. L e e A.K., C.S. D e t w e i l e r and S. F a l k o w. 2000. OmpR regulates the two-component system SsrA-SsrB in Salmonella pathogenicity island 2. J. Bacteriol. 182: 771–781.

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L e e W.H., P.P. M c G r a t c h, P.H. C a r t e r and E.L. E i d e. 1977. The ability of some Yersinia enterocolitica strains to invade HeLa cells. Can. J. Microbiol. 23: 1714–1722. L i n d g r e n S.W., I. S t o j i l j k o v i c and F. H e f f r o n. 1996. Macrophage killing is an essential virulence mechanism of Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 93: 4197–4201. M i l l e r J.F., J.J. M e k a l a n o s and S. F a l k o w. 1989. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science 243: 916–922. M i l l s S.D., S.R. R u s c h k o w s k i, M.A. S t e i n, B.B. Finlay. 1998. Trafficking of porin–deficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella – induced filaments. Infect. Immun. 66: 1806–1811. N e g m R.S. and T.G. P i s t o l e. 1990. The porin OmpC of Salmonella typhimurium mediates adherence to macrophages. Can. J. Microbiol. 45: 658–669. P e p e J. and V. M i l l e r. 1993. Yersinia enterocolitica invasin: a primary role in the initiation of infection. Proc. Natl. Acad. Sci. USA 90: 6473–6477. P i c k a r d D., J. Li, M. R o b e r t s, D. M a s k e l l, D. H o n e, M. L e v i n e, G. D o u g a n and S. C h a t f i e l d. 1994. Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression. Infect. Immun. 62: 3984–3993. P r u s s B.M. 1998. Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli. Arch. Microbiol. 170: 141–146. R u s s o F.D. and T.J. S i l h a v y. 1990. EnvZ controls the concentration of phosphoryled OmpR to mediate osmoregulation of the porin genes. J. Mol. Biol. 222: 567–580. S h i n S. and C. P a r k. 1995. Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR. J. Bacteriol. 177: 4696–702. S t o c k J.B., A.J. N i n f a and A.M. S t o c k. 1989. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol. Rev. 53: 450–490. S t r a l e y S.C. and R.D. P e r r y. 1995. Environmental modulation of gene expression and pathogenesis in Yersinia. Trends Microbiol. 3: 310–317.

Polish Journal of Microbiology 2004, Vol. 53, No 1, 17– 22

Streptococcus Group B (GBS) – Characteristic, Occurrence in Children and Adolescents with Type 1 Diabetes mellitus MARIA NOWAKOWSKA1* and PRZEMYS£AWA JAROSZ-CHOBOT2 2

1 Chair and Department of Microbiology, Silesian Medical University Clinic of Pediatrics, Endocrynology and Children Diabetology, Silesian Medical University, Poland

Received 3 November 2003 Abstract Group B streptococcus causes infections in woman during pregnancy and confinement, perinatal infections in newborns related to mothers carrier-state and in adults, mostly in the elderly, with one or more predisposing to infections conditions. Diabetes mellitus is the most common underlying condition. The aim of the study was to determine the frequency of GBS occurrence and GBS antibiotic susceptibility in children and adolescents with type 1 diabetes mellitus. In years 2000–2002 occurrence of GBS in some clinical materials (urine, swabs from pharynx and urogenital tract) taken from 161 diabetics: 90 girls and 71 boys, hospitalized for newly diagnosed diabetes or insufficient metabolic control/longer duration of diabetes and 37 children with hypostatura (control group) aged from 5– 17 years, was examined. Susceptibility of isolated GBS strains to ampicillin, erythromycin and clindamycin was determined. GBS were obtained from different materials from 36 (22.4%) diabetic children – 25 girls (27.8%), and 11 boys (15.5%). In all examined groups GBS was detected significantly in children with insufficient metabolic control/longer duration of diabetes (27 of 36 children; p= 0.029, P2 = 4.773). GBS in girls was isolated mainly from vestibule of vagina (25 cases) and in few cases (4) from the pharynx. GBS in boys was grown from materials from urethra (6 cases) and pharynx (5 cases). In the control group, GBS colonization was observed only in one case. All isolates (40 strains) were susceptible to penicillins, however lower susceptibility to erythromycin (3 resistant and 1 moderately sensitive) and clindamycin (3 resistant) were observed. High percentage of carriers of GBS both in girls and boys with diabetes mellitus is the potential risk factor of infection caused by GBS. K e y w o r d s: Diabetes mellitus, children, colonisation GBS, Streptococcus agalactiae

Introduction Streptococcus agalactiae, according to the Lancefield classification group B streptococcus (GBS), caused of bovine mastitis. First reports of infections in human date from the 30. of the XX century, for example: Frey described 3 cases of puerperal fever (Edwards and Baker, 2000). In the 70. in USA GBS was recognized as the main etiological factor of sepsis in newborns and infants up to the 3rd month of life, which was the main cause of death in infants (Edwards and Baker, 2000; Parker, 1984). In other countries the involvement of GBS in infections of newborns is not as big as in USA, but still many countries, including Poland, accepted the rules of prophylaxis of these infections that are in force in USA (Bevilacqua, 1999; Dzier¿anowska, 2000; Hryniewicz and Gonera, 1993; Jeljaszewicz and Meszaros, 2001; Lyytikainen et al., 2003; Poulain et al., 1997). The end part of the digestive tract (ano-rectal area) is the reservoir of GBS. Bacteria are observed in the anal region, urethra and vagina (Edwards and Baker, 2000; Honig et al., 2002; Jeljaszewicz and Meszaros, 2001). The carrier-state of GBS in vagina is not constant, has a changeable character and can be transitional or chronic. Most of the infections in newborns are gained from the mother during the intra-uterine life or labor (vertical infections). Therefore GBS is regarded as one of the main causes of morbidity and mortality * Corespondence author: dr Maria Nowakowska, Chair and Department of Microbiology, Silesian Medical University, 40-752 Katowice, Medyków 18, tel: +32 2088551; e-mail: [email protected]

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of newborns and infants up to the 3rd month of life, as well as of pregnant women. These bacteria cause also infections not connected with pregnancy, mainly in elderly people (Gardam et al., 1998; Jackson et al., 1998; Munoz et al., 1997; Opal et al., 1988; Trivalle et al., 1998). Two groups of infections in newborns and infants up to the 3rd month are distinguished – early-onset disease and late-onset disease. The early-onset disease evolves in the first week of life, usually until the 5th day after birth. The infection mostly develops in children with a low birth weight and in cases of premature outflow of amniotic fluid. As a consequence of a perinatal infection that is connected with colonization of genital passages by GBS, sepsis, more seldom pneumonia or meningitis develops (Edwards and Baker, 2000; Hryniewicz and Gonera, 1993; Jeljaszewicz and Meszaros, 2001; Parker, 1984). A late-onset disease develops between the 7th day and 3rd month after birth, usually between the 10 and th 20 day after birth and occurs mostly in healthy, mature newborns with a normal body weight. This infection is not connected with a carrier-state of GBS in the genital passages of the mother (Edwards and Baker, 2000). The most common clinical form of a late-onset disease is meningitis, more rarely other diseases as inflammation of bones and joints, necrotising fasciitis and bullous skin eruptions (Gardam et al., 1998). In comparison to the early-onset disease the late-onset disease had a better prognosis (Hryniewicz and Gonera, 1993). Infections of women connected with pregnancy and confinement: Massive infection of the vagina with GBS can lead to asymptomatic bacteriuria and in consequence to streptococcal infection of the urinary tract. Infection with GBS can concern the fetal membranes, mucus membrane of the uteri and as a result of an intra-uterine infection septic aborts can occur. In some of women after labor GBS cause puerperal fever (Edwards and Baker, 2000; Jeljaszewicz and Meszaros, 2001). Infections of adults not connected with pregnancy: These infections occur in women as well as in men and concern usually elderly people, who have at least one of the risk factors. These is e.g. diabetes mellitus, cancer disease, hepatocirrhosis, renal insufficiency, neurogenic bladder, trauma, decubitus ulcer and other (Edwards and Baker, 2000; Jackson et al., 1998; Munoz et al., 1997; Nolla et al., 2003; Opal et al., 1988; Ruoff et al., 1999). GBS belongs to microorganisms that cause hospital infections. The most commonly recognized infections are: infections of skin and soft tissue, inflammation of bones and joints, pneumonia, meningitis, endocarditis, bacteremia and sepsis (Edwards and Baker, 2000; Munoz et al., 1997; Opal et al., 1988; Parker, 1984; Trivalle et al., 1998; Tyrrell et al., 2000). Invasive GBS infections connected with the placement of a central venous line was also described (Jackson et al., 1998). Infections have often a mixed character, beside GBS Staphylococcus aureus is isolated. For prevention of infections in infants applying of ampicillin or penicillin G for women during labor is recommended. Women allergic to penicillin should be given erythromycin or clindamycin (Dzier¿anowska, 2000). Until now no penicillin resistant GBS were observed, but there were some strains resistant to erythromycin and clindamycin (Dzier¿anowska, 2000; Lin et al., 2000; Munoz et al., 1997; Murdoch and Reller, 2001; Pearlman et al., 1998). The aim of the study was to determine the frequency of occurrence of GBS in children and adolescents with type 1 diabetes mellitus and to estimate their susceptibility to antibiotics that are in practice used in prophylaxis of perinatal infections.

Experimental Material and Methods 161 children with type 1 diabetes mellitus (1–18 years old), hospitalized in the period form VIII 2000 to XII 2002 in the Department of Pediatrics, Endocrynology and Diabetology, Silesian University of Medicine in Katowice were examined during this study. The reason for hospitalization was new onset of diabetes (68 patients) or insufficient metabolic control (93 patients). Swabs taken from the pharynx, vestibule of vagina and urine from 90 girls as well as swabs from the pharynx, urethra and urine from 71 boys were examined. Nocturnal urine, obtained with the method of the middle stream, was cultured directly after collection on the transport-culture medium Uromedium or Uromedium PEG (BIOMED – Kraków). In none of the children clinical manifestations of infections of the urinary tract were observed; the biochemical analysis in all cases was correct. The material from the mucous membrane of the pharynx, ostium of urethra and vestibule of vagina was cultured on Columbia agar with 5% sheep blood. The $-hemolytic streptococci growing on this medium were serologically identified with the lateks agglutination test – Slidex Strepto-kit bioMérieux (France). Susceptibility of GBS to ampicillin, erythromycin, and clindamycin was performed on the Mueller-Hinton agar Oxoid (England) with addition of 5% sheep blood, according to the recommendations NCCLS (NCCLS, Villanova, 1997). The control group consisted of 37 children hospitalized for hypostatura. The statistic analysis was performed with the use of the test P2, contingency table 2×2.

1

19

GBS occurence in diabetic patients

Results From amongst 161 examined children GBS was cultured in 36 (22.4%): 25 girls (27.8%) and 11 boys (15.5%) with diabetes. The age of the children was 5 to 17 years; 15 children (11 girls and 4 boys) aged 13 and younger and 21 children (14 girls and 7 boys) older than 13 years. Most of the children, by whom GBS was isolated were hospitalized for insufficient metabolic control (27 patients), while the remaining 9 patients were hospitalized for new diagnosis of diabetes. Table I shows the frequency of GBS occurrence in children and adolescents with newly diagnosed diabetes and those with insufficient metabolic control. GBS colonization was found more often in children – both girls and boys – with insufficient metabolic control/a longer history of diabetes (33.9% girls and 21.6% boys), in comparison to newly diagnosed cases (17.7% girls and 8.82% boys). The analysis of the results forms the whole group of children with diabetes a statistically significant (p = 0.029; P2 = 4.773). Colonization was revealed in children with a longer history of the disease. Table II shows the materials, from where GBS was isolated. In girls, these bacteria were mostly found in the swab from the vestibule of vagina (25 cases) and more seldom from the mucus membrane of the pharynx (4 cases). Asymptomatic bacetriuria was revealed in 5 girls with a massive colonization of vagina, in 4 of them the number of bacteria in the urine was above 104 CFU/ml, in one above 103 CFU/ml. Table I Frequency of GBS occurrence in children and adolescents with diabetes Cause of hospitalization

Girls

Boys

Total

total

GBS + (%)

total

GBS + (%)

examined

GBS + (%)

New diagnose of diabetes

34

6 (17.65%)

34

3 (8.82%)

68

9 (14.1%)

Insufficient metabolic control

56

19 (33.9%)

37

8 (21.62%)

93

27 (29.0%) p = 0.029

Total

90

25 (27.8%)

71

11 (15.49%)

161

36 (22.4%)

Table II GBS occurrence in materials Examined material Swabs from pharynx Urine

Boys (71)

Girls (90) GBS + total

GBS %

GBS + total

GBS %

4

4.4

5

7.0

5

5.5

1

1.4

Swabs from vagina

25

27.8

–

–

Swabs from urethra

–

–

6

8.5

Presence of GBS in the region of ostium of urethra was stated in 6 boys, in one of them significant asymptomatic bacetriuria was found – above 105 bacteria in 1 ml of urine. From the mucus membrane of pharynx GBS was grown in 5 boys. In 21 children (10 girls and 11 boys) the microbiological examination was conducted two times (at an interval of few months) and in 4 cases three times. Within the group examined more than once, GBS was isolated in 11–6 girls and 5 boys. These bacteria were isolated two times in the same patient only in two cases (2 boys), in other children their presence was revealed only in one examination. In the control group – 37 children with hypostatura, GBS was grown neither from the swabs from the pharynx nor from the urine. Microorganisms were not well grown from the swabs from the region of the ostium of urethra of 18 boys from this group. Amongst the examined swabs taken from the vestibule of vagina from girls GBS was grown in one case. The girl was 14 years old. The susceptibility of the 40 strains of GBS to ampicillin, erythromycin and clindamycin is shown in Figure 1. All examined GBS strains were susceptible to ampicillin, however lower susceptibility to erythromycin (3 strains were resistant and 1 intermediate) as well as to clindamycin (3 strains resistant, which is 7.5%) was observed. Streptococci resistant to erythromycin were also resistant to clindamycin.

20

Nowakowska M., Jarosz-Chobot P.

1

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

ampicillin resistant

erythromycin intermediate

clindamycin susceptible

Fig. 1. Susceptibility of group B streptococci to chosen antibiotics

Discussion After recognizing the dominant role of GBS in perinatal infections of newborns, standards of prophylaxis and treatment of those infections were generated and introduced to hospital practice. Two schemes of procedures were elaborated depending on the presence of risk factors: GBS infection of the newborn from the former pregnancy, infection of the urine tract caused by GBS or asymptomatic bacteriuria and labor before the 37th week of pregnancy. The stated risk factors are an absolute indication for applying penicillin or ampicillin during the labor. From women, who do not have any risk factors, swabs from the vestibule of vagina and anus should be taken between the 35th and 37th week of pregnancy and examined for GBS. Presence of those bacteria in the material (positive result) is an indication for antibiotic prophylaxis during labor. Premature outflow of amniotic fluid above 18 hours before delivery and intrapartum fever (higher than 38°C) is an additional indication for antibiotic prophylaxis. The above mentioned recommendations were published also in Poland (Hryniewicz, 2002). Owing to the introduction of perinatal antibiotic prophylaxis the frequency of invasive early infections in newborns in USA has decreased (Baltimore et al., 2001; Schrag et al., 2000). The remaining problem is the late infections in newborns and infants and the more and more frequently occurring invasive infections of adults that are not connected with pregnancy. Authors from different countries emphasize the role of diabetes as one of the most common risk factors for GBS infections (Edwards and Baker, 2000; Jackson et al., 1998; Munoz et al., 1997; Nolla et al., 2003; Opal et al., 1988; Parker, 1984; Tyrrell et al., 2000). In women with diabetes more often than in those, who are healthy, GBS colonization of vagina is revealed and that is why these bacteria are the most frequent cause for their septic aborts (Jeljaszewicz and Meszaros, 2001). Although rules of GBS perinatal infections prophylaxis in women and newborns have been known in Poland for a long time (Hryniewicz and Gonera, 1993), not all doctors prescribe screening examinations or they are carried out incorrectly. The high percentage of colonization of vestibule of vagina by GBS strains in girls with diabetes showed in this study justifies the necessity of examinations of pregnant women with diabetes for GBS presence in vagina, anus and urine. In the available literature there are few studies concerning GBS occurrence in children and adolescents with diabetes. Liotta and coworkers (Liotta et al., 1987) while investigating the vestibule vagina flora in girls with diabetes mellitus type 1 revealed GBS in 28.5% of examined, which is comparable with our results – 27.8%. GBS colonization of mucus membrane of pharynx is observed seldom, it occurs less than in 10% people (Edwards and Baker, 2000; Parker, 1984); in our studies – in children with diabetes the results were similar.

1

GBS occurence in diabetic patients

21

GBS was grown in swabs from pharynx in 4.4% of girls and in 7% of boys (in total in 5.6% children). However GBS was not found in the oral cavity and pharynx of the children from the control group. All GBS strains isolated by us were susceptible to ampicillin, which is conformable to other authors (Dzier¿anowska, 2000; Lin et al., 2000; Pearlman et al., 1988). The percentage of strains resistant to erythromycin and clindamycin was 7.5% and was lower than the resistance of GBS strains isolated in other countries (Lin et al., 2000; Munoz et al., 1997; Murdoch and Reller, 2001; Pearlman et al., 1988). Strains resistant to erythromycin were also resistant to clindamycin, what suggests a type of resistance connected with a modification of the target place – phenotype MLSB, that manifestates itself by a cross resistance between macrolides, lincozamides and streptogramine B. Resistance of GBS to macrolides and lincozamides restricts the value of these antibiotics both in prophylaxis of perinatal infections and in empiric treatment of invasive GBS infections in adults. Conclusion The high percentage of GBS carrier-state in girls as well as in boys with diabetes is a risk factor for potential infections with this microorganism. Literature B a l t i m o r e R.S., S.M. H u i e, J.I. M e e k, A. S c h u c h a t and K.L. O’Brien. 2001. Early-onset neonatal sepsis in the era of group B streptococcal prevention. Pediatrics 108: 1094–1098. B e v i l a c q u a G. 1999. Prevention of perinatal infection caused by group B betahemolytic Streptococcus. Acta Biomed. Ateneo. Parmense. 70: 87–94. D z i e r ¿ a n o w s k a D. 2000. Order 3.3 Patogenicity and antibiotics susceptibility of the some main pathogen, Streptococcus agalactiae, p. 168–170. In: D. Dzier¿anowska (ed.), Practical antibiotic terapy. "-Medica Press, Bielsko-Bia³a. E d w a r d s M.S. and C.J. B a k e r. 2000. Chapter 190. Streptococcus agalactiae (group B streptococcus), p. 2156. In. G.L. Mandell, J.E. Bennett, R. Dolin. (eds), Principles and practice of infectious diseases, 5th ed. Churchill Livingstone. G a r d a m M.A., D.E. L o w, R. S i n g u r and M.A. M i l l e r. 1998. Group B streptococcal necrotising fasciitis and streptococcal shock-like syndrome in adults. Arch. Intern. Med. 158: 1704–1708. H o n i g E., W.J. M o u t o n and W.I. v a n d e r M e i j d e n. 2002. The epidemiology of vaginal colonisation with group B streptococci in a sexually transmitted disease clinic. Eur. J. Obstet. Gynecol. Reprod. Biol. 105: 177–180. H r y n i e w i c z W. and E. G o n e r a. 1993. Streptococcal infections, infections caused by Streptococcus agalactiae, p. 463. In: W. Magdzik (ed.), Infections and parasitic diseases. Prevention and control. 3rd ed. Vesalius, Kraków, (in Polish). H r y n i e w i c z W. 2002. Clinical microbiology, (in Polish). Medycyna Praktyczna – Pediatria. 2: 26–31. J a c k s o n L.A., R. H i l s d o n, M.M. F a r l e y, L.H. H a r r i s o n, A.L. R e i n g o l d, B.D. P l i k a y t i s, J.D. W e g n e r and A. S c h u c h a t. 1998. Risk factors for group B streptococcal disease in adults. Clin. Microbiol. Rev. 11: 497–513. J e l j a s z e w i c z J. and J. M e s z a r o s. 2001. Streptococcal infections, p. 497. In: W. Magdzik and D. Naruszewicz-Lesiuk (eds), Human infections and contagions. Epidemiology, prevention and control. (in Polish) 1sh ed. PZWL Warszawa. L i n F-Y.C., P.H. A z i m i, L.E. W e i s m a n, J.B. P h i l i p s I I I, J. R e g a n, P. C l a r k, G.G. R h o s d s, J. C l e m e n s, J. T r o e n d l e, E. P r a t t, R.A. B r e n n e r and V. G i l l. 2000. Antibiotic susceptibility profiles for group B streptococci isolated from neonates, 1995–1998. Clin. Infect. Dis. 31: 76–79. L i o t t a A., F. C a r d e l l a, D. F e r r a r a, D. N a t o l i, G. L i c a s t r o, F. M e l i and S. T e r e s i. 1987. Vaginal infections in a population of diabetic children and adolescents. Pediatr. Med. Chir. 9: 305–308. L y y t i k a i n e n O., J.P. N u o r t i, E. H a l m e s m a k i, P. C a r l s o n, J. U o t i l a, R. V u e n t o, T. Ranta, H. S a r k k i n e n, M. A m m a l a, A. K o s t i a l a and A.L. J a r v e n a a. 2003. Invasive group B streptococcal infections in Finland: a population – based study. Emerg. Infect. Dis. 9: 469–473. M u n o z P., A. L l a n c a q u e o, M. R o d r i g u e z - C r e i x e m s, T. P e l a e z, L. M a r t i n and E. B o u z a. 1997. Group B streptococcus bacteremia in nonpregnant adults. Arch. Intern. Med. 157: 213–216. M u r d o c h D.R. and L.B. R e l l e r. 2001. Antimicrobial susceptibilities of group B streptococci isolated from patients with invasive disease: 10-year perspective. Antimicrob. Agents Chemother. 45: 3623–3624. National Committee for Clinical Laboratory Standards (NCCLS): Performance standards for antimicrobial disc susceptibility tests 1997. 6th ed. Approved standard M2-A6. NCCLS, Villanova PA. 1997, vol. 17. N o l l a J.M., C. G o m e z - Va q u e r o, X. C o r b e l l a, S. O r d o n e s, C. G a r c i a - G o m e z, A. P e r e z, J. C a b o, J. Va l v e r d e and J. A r i z a. 2003. Group B streptococcus (Streptococcus agalactiae) pyogenic arthritis in nonpregnant adults. Medicine (Baltimore) 82: 119–128. O p a l S.M., A. C r o s s, M. P a l m e r and R. A l m a z a n. 1988. Group B streptococcal sepsis in adults and infants. Contrasts and comparisons. Arch. Intern. Med. 148: 641–645. P a r k e r M.T. 1984. Streptococcal diseases, p. 225–253. In: Smith G.R. (ed.), Topley and Wilson’s Principles of Bacteriology, Virology and Immunity, 7th edition, vol. 3, Bacterial diseases. Butler & Tanner, London. P e a r l m a n M.D., C.L. P i e r s o n and R.G. F a i x. 1998. Frequent resistance of clinical group B streptococci isolates to clindamycin and erythromycin. Obstet. Gynecol. 92: 258–61.

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1

P o u l a i n P., P. B e t r e m i e u x, P.Y. D o n n i o, J.F. P r o u d h o n, G. K a r e g e and J.R. G i r a u d. 1997. Selective intrapartum anti-bioprophylaxy of group B streptococci infection of neonates: a prospective study in 2454 subsequent deliveries. Eur. J. Obstet. Gynecol. Reprod. Biol. 72: 137–140. R u o f f K.L., R.A. W h i l e y and D. B e i g h t o n. 1999. Streptococcus, p. 283 – 305. Murray P.R., E.J. Baron, M.A. Pfaller, F.C. Tenover and R.H. Yolken (eds), Mannual of Clinical Microbiology, 7th edition. American Society for Microbiology, Washington. S c h r a g S.J., S. Z y w i c k i, M.M. F a r l e y, A.L. R e i n g o l d, L.H. H a r r i s o n, L.B. L e f k o w i t z, J.L. H a d l e r, R. D a n i l a, P.R. C i e s l a k and A. S c h u c h a t. 2000. Group B streptococcal disease in the era of intrapartum antibiotic prophylaxis. N. Engl. J. Med. 342: 15–20. T r i v a l l e C., E. M a r t i n, P. M a r t e l, B. J a c q u e, J.F. M e n a r d and J.F. L e m e l a n d. 1998. Group B streptococcal bacteraemia in the elderly. J. Med. Microbiol. 47: 649–652. T y r r e l l G.J., L.D. S e n z i l e t, J.S. S p i k a and D.A. K e r t e s z. 2000. Invasive disease due to group B streptococcal infection in adults: results from a Canadian, population-based, active laboratory surveillance study – 1996. J. Infect. Dis. 182: 168–173.

Polish Journal of Microbiology 2004, Vol. 53, No 1, 23–26

Molecular Epidemiology of Antibiotic-Resistant Escherichia coli Isolated from Hospitalized Patients with Urinary Tract Infections in Northern Palestine K. ADWAN*, N. ABU-HASAN, G. ADWAN, N. JARRAR, B. ABU-SHANAB and M. AL-MASRI Department of Biological Sciences, An-Najah N. University, Nablus, Palestine Received in revised form 31 October

Abstract Eighty isolates of Escherichia coli were collected in Northern Palestine throughout the 1996 to 2000 period from hospitalized patients with urinary tract infections (UTIs). Resistance rates were ampicillin, 65%; co-trimoxazole, 55%; cefuroxime, 10%; cefotaxime, 7.5%; ceftazidime, 2.5%; ciprofloxacin, 12.5%; gentamicin, 6.25% and amikacin, 1.25%. No imipenem-resistant isolates were identified. To determine whether this was due to intra-hospital transmission of resistant strains, clonal structure of 10 multiple-resistant isolates was examined by genomic DNA fingerprinting by enterobacterial repetitive intergenic concensus-polymerase chain reaction (ERIC-PCR) and all were clonally distinct. Thus, these strains are likely resistant due to convergent acquisition of resistance determinants by genetically unrelated uropathogenic strains rather than epidemic spread of resistant isolates. K e y w o r d s: E. coli, antibiotic-resistance, DNA fingerprinting, ERIC-PCR

Introduction Escherichia coli is recognized as one of the most important bacterial pathogens seriously contributing to the problem of hospital urinary tract infections (UTIs) all over the world. An increase in its resistance to numerous antibiotics has been noted in recent years. The $-lactams, sulphonamides and quinolones are drug families most concerned (Hsueh et al., 2002; Sotto et al., 2001; Okeke et al., 2000; Allen et al., 1999; Perrin et al., 1999; Gupta et al., 1999; Guyot et al., 1999). The present study aimed to obtain a snapshot of uropathogenic E.coli resistance in northern Palestine, a part of the world not previously surveyed for this type of resistance. To find out whether antibiotic resistance was related to intra-hospital transmission of resistant strains, ten multiple-resistant isolates were studied for genetic analysis by ERIC-PCR. Experimental Materials and Methods Escherichia coli isolates. A total of 80 E. coli strains isolated from hospitalized patients with urinary tract infections in Rafidya hospital in Nablus, Northern Palestine throughout the 1996 to 2000 period: 28 in 1996, 20 in 1997, 12 in 1998, 10 in 1998 and 10 in 2000 were studied. Reisolates of the same strain from the same patient were not included. The isolates were confirmed as E. coli by the API 20E system (bioMérieux, Marcy L’Etoile, France). Antimicrobial susceptibility. The susceptibility of isolates to antimicrobials was determined by disk diffusion (Bauer et al., 1966) in accordance with National Committee for Clinical Laboratory Standards (1993). The following antibiotics (:g) were used: ampicillin, cefuroxime, cefotaxime, ceftazidime, co-trimoxazole, imipenem, gentamicin, amikacin and ciprofloxacin. * Correspondence to: Dr. K.Adwan, Department of Biological Sciences, An-Najah N. University, P. O. Box (7)-Nablus, Palestine. Fax: 972 9 387 982; e-mail [email protected]

24

1

Adwan K. et al.

ERIC-PCR. Ten multiple-resistant isolates, i.e., resistant to ampicillin and two or more of the following antibiotics: co-trimoxazole, ciprofloxacin and aminoglycosides analayzed by ERIC-PCR. PCR was performed with primer ERIC2, 5/AAGTAAGTGAC TGGGGT GAGCG3/ (Dalla Costa et al., 1998) using crude heated isolates in 25-:l reaction mixtures and 5 :l of template DNA. Initial denaturation was carried out at 94°C for 5 min followed by 40 cycles of amplification (denaturation at 94°C for 60 sec, annealing at 25°C for 60 sec, and extension at 72°C for 90 sec) ending with a final extension at 72°C for 5 min. Separated PCR products in agarose gels were visualised as above and patterns that differed by one or more DNA bands were considered to be different ERIC-types. Electrophoresis. PCR products (25 :l) were mixed with 2 :l of agarose gel loading dye, separated on a 2% agarose gels containing 0.25 :g ethidium bromide per ml and run at 100V for 1 hr. A 100-bp DNA ladder was used as a molecular size marker. Gels were photographed on a 392-nm-wavelength transilluminator and band patterns were compared visually. Patterns that differed by one or more DNA bands were considered as different type.

Results The rates of resistance of Escherichia coli isolates to different antibiotics tested are reported in Table I. Among the antibiotic agents tested in our study, the highest rates of resistance were found for ampicillin (65%), co-trimoxazole (55%) and ciprofloxacin (12.5%). Broad-spectrum cephalosporins remained active, with the rate of resistance to these drugs ranging from 2.5 to 10%. Imipenem was always active. Rates of resistance to aminoglycosides were 6.25% to gentamicin, with amikacin having better activity (rate resistance to amikacin, 1.25%). Table I Rates of resistance to different antibiotics tested against 80 Escherichia coli strains isolated from urinary tract infections Antibiotic

No (%) of resistant isolates

Ampicillin

52 (65.0)

Cefuroxime

8 (10.0)

Cefotaxime

6 (7.50)

Ceftazidime

2 (2.50)

Imipenem

0 (000)

Co-trimoxazole

40 (50.0)

Gentamicin

5 (6.25)

Amikacin

1 (1.25)

Ciprofloxacin

10 (12.5)

Table II ERIC-PCR banding patterns obtained with multiple-resistant Escherichia coli isolates and their resistance patterns

a

Resistance patterna

ERIC-PCR Pattern

Strain no

Year

3

1996

Ampr, Sxtr, Ctxr, Cxmr, Genr

I

4

1996

Ampr, Ctxr, Cxmr, Genr

I

12

1996

Amp , Sxt , Cxm , Gen

38

1997

Ampr, Sxtr, Cipr

III

44

1997

Ampr, Sxtr, Cxmr, Cipr

VI

55

1998

Ampr, Sxtr, Cipr

V

58

1998

Ampr, Sxtr, Ctxr, Cxmr, Ctzr, Cipr

VI

64

1999

Ampr, Sxtr, Ctxr, Cxmr, Cipr

VII

68

1999

Ampr, Sxtr Ctxr, Cxmr, Ctzr, Amir Cipr

VIII

75

2000

Ampr, Sxtr, Ctxr, Cxmr, Genr, Cipr

r

r

r

r

II

IX

Amp: ampicillin, Sxt: Co-trimoxazole, Ctx: cefotaxime, Cxm: cefuroxime, Ctz: ceftazidime, Gen: gentamicin, Ami: amikacin, Cip: ciprofloxacin.

1

Antibiotic-resistant E. coli in Palestine

25

Fig. 1. ERIC-PCR profiles of DNA from Escherichia coli isolates. Lane 1, pattern I; Lane 2, pattern II; Lane 3, pattern III; Lane 4, pattern IV; Lane 5, pattern V; Lane 6, pattern VI; Lane 7, pattern VII; Lane 8, pattern VIII; Lane 9, pattern IX; Lane 10, Molecular sizes marker (100-bp ladder DNA).

Genomic fingerprinting by ERIC-PCR revealed diversity of the 10 multiple-resistant isolates analyzed. The isolates were allocated in 9 genotypes, 8 of which (80%) were represented by single strain. Isolates 3 and 4, although sharing very similar ERIC-PCR patterns were characterized by different antibiogram patterns (Table II and Figure 1). Discussion In this study, the Escherichia coli isolates showed high degrees of resistance to ampicillin and co-trimoxazole. Comparison of our results with the previous studies shows that there is a rising trend in incidence of resistance to penicillins, sulphonamides and also to the ciprofloxacin (Sotto et al., 2001; Lepelletier et al., 1999; Perrin et al., 1999; Gupta et al., 1999). This probably reflects the fact of the increased usage of these antimicrobial agents, particularly ciprofloxacin, in treating hospitalized patients with urinary tract infections in Rafidya hospital (personal communications). On the other hand, imipenem behaved as the most potent antibiotic. The highest activity of imipenem seems to be related to its stability against most $-lactamases and it is a rapid permeant (Livermore, 1995). While the resistance to ampicillin and co-trimoxazole is predictable, the high resistance to the ciprofloxacin gives cause for concern. In our study, the rate of resistance to ciprofloxacin was 12.5%. Reported rates of resistance to ciprofloxacin have steadily increased since its introduction and were frequently between 3 and 10% (Sotto et al., 2001; Perrin et al., 1999; Garau et al., 1999). However, rates differed widely from one study to another. For example, Gupta et al. (1999, 1994) investigated UTIs in young women who were outpatients and found resistance rates of 0 to 0.2%, whereas others investigators reported rates as high as 20.6% and that 20% of strains from hospitalized patients (Ena et al., 1998; Gruneberg, 1994). Genomic fingerprinting by ERIC-PCR revealed an extreme diversity of profiles among the multipleresistant isolates analyzed. Even within isolates sharing similar ERIC-PCR patterns no close relationships in antibiogram patterns were evident. The lack of correlation between antimicrobial resistance patterns and ERIC-PCR patterns of the strains suggests convergent acquisition of resistance determinants by genetically unrelated uropathogenic strains rather than intra-hospital transmission of resistant strains. This probably reflects the fact of the excessive and/or inappropriate usage of these antimicrobial agents, particularly broadspectrum agents, in treating E. coli responsible for urinary tract infections in these health centers.

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1

Literature A l l e n U.D., N. M a c D o n a l d, L. F u i t e, F. C h a n and D. S t e p h e n s. 1999. Risk factors for resistance to “first-line” antimicrobials among urinary tract isolates of Escherichia coli in children. Can. Med. Assoc. J. 160: 1436–1440. B a u e r A.W., W.M.M. K i r b y, J.C. S h e r r i s and M. T r u c k. 1966. Antibiotic susceptibility testing by a standardized single disk method. J. Gen. Microbiol. 45: 493–496. D a l l a C o s t a L.M., K. I r i n o, J. R o d r i g u e s, I.N.G. R i v e r a and L.R T r a b u l s i. 1998. Characterization of diarrhoeagenic Escherichia coli clones by ribotyping and ERIC-PCR. J. Med. Microbiol. 47: 227–234. E n a J., M.M. L o p e z - P e r e z a g u a, C. M a r t i n e z - P e i n a d o, M.A. C i a - B a r r i o and I. R u i z - L o p e z. 1998. Emergence of ciprofloxacin resistance in Escherichia coli isolates after widespread use of fluoroquinolones. Diagn. Microbiol. Infect. Dis. 30: 103–107. G a r a u J., M. X e r c a v i n s, M. R o d r i g u e z - C a r b a l l e i r a, J.R. G o m e z - Ve r a, I. C o l l, D. V i d a l, T. L l o v e t and A. R u i z - B r e m o n. 1999. Emergence and dissemination of quinolone-resistant Escherichia coli in the community. Antimicrob. Agents. Chemother. 43: 2736–2741. G r u n e b e r g R.N. 1994. Changes in urinary pathogens and their antibiotic sensitivities, 1971–1992. J. Antimicrob. Chemother. 33 (Suppl. A): 1–8. G u p t a K., T.M. H o o t o n, C.L. W o b b e and W.E. S t a m m. 1999. The prevalence of antimicrobial resistance among uropathogens causing acute uncomplicated cystitis in young women. Int. J. Antimicrob. Agents. 11: 305–308. G u p t a K., D. S c h o l e s and W.E. S t a m m. 1999. Increasing prevalence of antimicrobial resistance among uropathogens causing acute uncomplicated cystitis in women. J.A.M.A. 281: 736–738. G u y o t A., S.P. B a r r e t t, E.J. T h r e l f a l t, T. H a m p t o n and T. C h e a s t y. 1999. Molecular epidemiology of multiresistant Escherichia coli. J. Hosp. Infect. 43: 39–48. H s u e h P.R., M.L. C h e n, C.C. S u n, W.H. C h e n, H.J. P a n, L.S. Y a n g, S.C. C h a n g, S.W. H o, C.Y. L e e, W.C. H s i e h and K.T. L u h. 2002. Antimicrobial drug resistance in pathogens causing nosocomial infections at a university hospital in Taiwan, 1981–1999. Emerg. Infect. Dis. 8: 63–68. L e p e l l e t i e r D., N. C a r o f f, A. R e y n a u d and H. R i c h e t. 1999. Escherichia coli: epidemiology and analysis of risk factors for infections caused by resistant strains. Clin. Infect. Dis. 29: 548–552. L i v e r m o r e DM. 1995. $-lactamases in laboratory and clinical resistance. Clin. Microbiol. Rev. 8: 557–584. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disc susceptibility tests: tentative standards, vol. 3, No. 24, NCCLS document M2-A5. Villanova, Pa National Committee for Clinical Laboratory Standards, 1993. O k e k e I.N., S.T. F a y i n k a and A. L a m i k a n r a. 2000. Antibiotic resistance in Escherichia coli from Nigerian students, 1986–1998. Emerg. Infect. Dis. 6: 393–396. P e r r i n M., P.Y. D o n n i o, C. H e u r t i n - L e c o r r e, M.F. T r a v e r t and J-L. A v r i l. 1999. Comparative antimicrobial resistance and genomic of Escherichia coli isolated from urinary tract infections in the community and in hospitals. J. Hosp. Infect. 41: 273–279. S o t t o A., C.M.D. B o e v e r, P. F a b b r o - P e r a y, A. G o u b y, D. S i r o t and J. J o u r d a n i. 2001. Risk factors for antibiotic-resistant Escherichia coli isolated from hospitalized patients with urinary tract infections: a prospective study. J. Clin. Microbiol. 39: 438–444.

Polish Journal of Microbiology 2004, Vol. 53, No 1, 27–34

Incidence of Extended-Spectrum $-Lactamases in Clinical Isolates of the Family Enterobacteriaceae in a Pediatric Hospital RADOSZ-KOMONIEWSKA HALINA1, GNIADKOWSKI MAREK2, ROGALA-ZAWADA DANUTA1, NOWAKOWSKA MARIA1, RUDY MARIA1, WIECHU£A BARBARA1 and MARTIROSIAN GAYANE1 1 Department

of Microbiology, Medical University of Silesia Institute of Public Health in Warsaw

2 National

Received 19 December 2003

Abstract The incidence of extended-spectrum $-lactamases (ESBLs) was analyzed in Enterobacteriaceae population circulating in the Upper Silesian Child and Mother Health Center in Katowice (USC&MHC). Altogether 1164 clinical specimens, collected from children hospitalized in 8 different hospital units of USC&MHC were investigated. Five hundred and eighty-five clinical isolates of the family Enterobacteriaceae were identified in specimens collected from 403 patients. Two hundred and twenty-nine Enterobacteriaceae strains (39%) isolated from 162 patients were found to be putative ESBL producers as revealed by double-disc synergy (DDS) test. ESBL activity was the most prevalent in the population of Klebsiella pneumoniae (77%), followed by Klebsiella oxytoca (50%), Serratia marcescens (43%), Escherichia coli (30%), Enterobacter spp. (18%) and Proteus mirabilis (12%). ESBL producers demonstrated also wide resistance to the non-$-lactam antimicrobial co-trimoxazole (93%) and the aminoglycosides netilmicin (88%), gentamicin (84%) and amikacin (79%). K e y w o r d s: ESBL, Enterobacteriaceae, infections, epidemiology

Introduction Infections caused by organisms of the family Enterobacteriaceae nowadays pose a serious clinical problem, which, among others, is due to the spread of many new drug resistance mechanisms in these bacteria. The most common mechanism of their resistance to $-lactam antibiotics is the production of diverse $-lactamases (Livermore, 1995; Medeiros, 1997; Gniadkowski, 1997) out of which the so-called extended spectrum $-lactamases (ESBLs) belong to those of the highest clinical and epidemiological importance. They are able to hydrolyze almost all penicillins, cephalosporins (excluding cephamycins) and monobactams (Livermore, 1995; Medeiros, 1997; Gniadkowski, 1997; Bradford, 2001; Sirot, 1995). Even if ESBL-producing strains may demonstrate susceptibility in vitro to some of the ESBL substrates, such drugs have been often found unsuccessful in therapy (Livermore, 1995; Bradford, 2001; Paterson et al., 2001; Sanders et al., 1996). This requires clinical microbiology laboratories to routinely detect ESBLs in Enterobacteriaceae isolates and report them as resistant to all compounds that belong to the ESBL substrate spectrum (Livermore, 1995; Bradford, 2001; Paterson et al., 2001; Sanders et al., 1996; Steward et al., 2001). Therapeutic options include aminoglycosides, fluoroquinolones, co-trimoxazole, combinations of $-lactams with $-lactamase inhibitors and carbapenems. However, except for carbapenems, resistance to these drugs has been observed in more and more ESBL-producing strains in recent years (Livermore, 1995; Bradford, 2001). ESBL-encoding genes appear due to either mobilization of chromosomal genes coding for certain species-specific $-lactamases with ESBL activity (Navarro and Miró, 2002) or, more often, due to mutations in 1

Correspondence address: Halina Radosz-Komoniewska, Katedra i Zak³ad Mikrobiologii Œl¹skiej Akademii Medycznej, 40-752 Katowice, ul. Medyków18, tel./fax. 032/2526075, e-mail: [email protected]

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Radosz-Komoniewska H. et al.

1

plasmid genes encoding broad-spectrum penicillinases, such as TEM-1, TEM-2 or SHV-1 (Livermore, 1995; Bradford, 2001; Gniadkowski, 2001). The extremely wide distribution of these enzymes in nosocomial Enterobacteriaceae populations and the strong pressure of the use of newer generation $-lactams cause that ESBL-producing strains are frequently selected de novo in therapy (Livermore, 1995; Gniadkowski, 2001). Once selected, ESBL producers may quickly spread in a hospital environment by clonal dissemination. Moreover, since ESBL genes are usually plasmid-located, they may rapidly penetrate the enterobacterial population by the horizontal transfer of plasmids (Livermore, 1995; Bradford, 2001; Gniadkowski, 2001). Since 1983, when the first ESBL-producing strains were identified, these enzymes have been described in many gram-negative species, however, they are still mainly observed in Klebsiella pneumoniae and Escherichia coli (Livermore, 1995; Medeiros, 1997; Gniadkowski, 1997; Bradford, 2001; Sirot, 1995). The number of known ESBL variants has been constantly growing over the period of the last 20 years, exceeding the value of 150 within 10 different enzyme families, and resulting in their high structural and biochemical diversity (Bradford, 2001; Gniadkowski, 2001). The frequency of infections caused by ESBL producers varies depending on a group of examined patients and hospital unit specificity, which are factors that influence directly the level of antibiotic consumption (Livermore, 1995). Usually, the highest prevalence of ESBL-producing isolates within a hospital is observed in intensive care units (ICUs), neonatal and surgical wards (Gniadkowski, 2001). This study presents an analysis of Enterobacteriaceae isolates that were identified as putative ESBL producers in the Upper Silesian Child and Mother Health Center (USC&MHC) in Katowice in 1999–2000. Experimental Materials and Methods Clinical isolates. Altogether 1164 clinical specimens, collected from newborns and children hospitalized in eight different wards of USC&MHC in Katowice, were investigated in the Department of Microbiology of the Silesian Medical University between February 1999 and September 2000. Out of all 1356 clinical isolates recovered, 709 isolates were classified into different species of the family Enterobacteriaceae, which constituted 52% of all the organisms identified. They were collected from 630 clinical specimens. The isolates were recovered mostly from urine (585 isolates, 82.5%) and blood (53 isolates, 7.5%), collected mainly from patients in the nephrology outpatient clinic (222 isolates, 31%), neonatal intensive care unit (197 isolates, 28%) and the pediatrics department (90 isolates, 13%). The isolates cultured from urine, blood, cerebrospinal fluid and wound swabs (662 isolates, 93.4%) were assumed to represent etiologic agents of infections, and those recovered from other materials (e.g. pharyngeal and nasal swabs) were interpreted as colonisers. The 630 enterobacteria-positive clinical specimens were obtained from 403 patients, the majority of which were infected or colonized with a single enterobacterial species (338 patients). Two hundred and seventy three patients were examined only once; the remaining patients (130 patients) were examined two times or more, either in terms of different kinds of specimens (10 patients) or the sequential analysis of the same specimen type (120 patients) per patient. The change of the isolated species with time was observed in the cases of 50 patients. All repeated isolates of the same bacterium from the same patient and the same sampling site were excluded from statistical analysis. The microbiological analysis of the specimens collected was conducted in accordance with routine diagnostic procedures (ATB ID32E and ID32GN, bioMérieux). Detection of ESBL activity. ESBL activity was detected in Enterobacteriaceae isolates by the double disc synergy (DDS) test, using discs with amoxicillin and clavulanic acid (20/10 :g), cefotaxime (30 :g), ceftazidime (30 :g), and aztreonam (30 :g) (Becton Dickinson) (Jarlier et al., 1988; Gniadkowski et al., 1996). The cefotaxime, ceftazidime and aztreonam discs were placed in a distance of 20 mm (center-to-center) from the disc containing clavulanate. K. pneumoniae ATCC 700603 and E. coli ATCC 25922 was used as positive and negative controls. A DDS test result was regarded as positive when the inhibition zone around any of the cefotaxime, ceftazidime or aztreonam discs was expanded with amoxicillin and clavulanate. Selected isolates were also examined using E-Test ESBL strips (AB Biodisk), according to the manufacturer’s instructions (ETM, 2000). An isolate was regarded as ESBL positive when it was characterized by ceftazidime MIC of at least 1 :g ml–1 or cefotaxime MIC of at least 0.5 :g ml–1, and the ratio between cephalosporin MIC to the of its combination with clavulanate was at least 8 (Cormican et al., 1996; Livermore and Brown, 2001). Antimicrobial susceptibility testing. Antimicrobial susceptibility of putative ESBL-producing isolates was tested by the disc diffusion method in accordance with NCCLS guidelines (NCCLS, 2000). The following antimicrobial discs were tested: ampicillin (10 :g), amoxicillin with clavulanic acid (20/10 :g), piperacillin (100 :g), piperacillin with tazobactam (100/10 :g), cephalothin (30 :g), cefuroxime (30 :g), ceftazidime (30 :g), cefotaxime (30 :g), cefoxitin (30 :g), aztreonam (30 :g), imipenem (10 :g), gentamicin (10 :g), amikacin (30 :g), netilmicin (30 :g), tetracycline (30 :g), norfloxacin (10 :g), trimethoprim-sulfamethoxazole (1.25/23.75 :g) and nitrofurantoin (300 :g) ( Becton Dickinson, USA). E. coli ATCC 25922 and E. coli ATCC 35218 were used as reference strains. Results were interpreted according to the NCCLS criteria (NCCLS, 2000a). MICs of $-lactam antibiotics were also determined by the agar dilution method, in accordance with NCCLS guidelines ((NCCLS, 2000b). The following antibiotics were used: ampicillin (Polfa Tarchomin), cefuroxime (Glaxo Wellcome), ceftazidime (Glaxo Wellcome), cefotaxime (Polfa Tarchomin), cefoxitin (Sigma Chemical Company), aztreonam (Bristol Myers Squibb), imipenem (Merck Sharp & Dohme) and lithium clavulanate (SmithKline Beecham). In all clavulanate combinations the constant inhibitor concentration was 2 :g ml–1.

1

29

ESBLs in Enterobacteriaceae in pediatric hospital

Results Clinical isolates. Clinical data regarding the Enterobacteriaceae isolates are shown in Tables I and II. The results of species identification are presented in Table III. The most prevalent were isolates of E. coli (303 isolates, 52%), K. pneumoniae (127 isolates, 22%), Proteus mirabilis (41 isolates, 7%), K. oxytoca (32 isolates, 5,5%), Enterobacter spp. (28 isolates, 5%) and Serratia marcescens (21 isolates, 3,6%). Other Enterobacteriaceae species were identified less frequently. Interestingly, whereas the biggest number of E. coli isolates were collected from patients in the nephrology outpatient clinic (94 isolates, 31%), the majority K. pneumoniae isolates were recovered from patients in the neonatal intensive care unit (53 isolates, 42%). Putative ESBL-producing isolates and their clinical background. All 585 Enterobacteriaceae isolates were tested for the presence of ESBL activity and 229 isolates of these (39%), recovered from 162 patients, were identified as putative ESBL producers based on the positive result of the DDS test. The most frequent ESBL producer species was K. pneumoniae; 43% (n = 98) of all ESBL-producing isolates belonged to this species, and ESBL activity was found in 77% of all K. pneumoniae isolates collected. The high rate of DDS test positive results was also revealed among K. oxytoca (50%), S. marcescens (43%), E. coli (30%), Enterobacter spp. (18%) and P. mirabilis (12%) isolates. ESBL production was observed in four isolates of Citrobacter freundii (n = 11) and two isolates of Morganella morganii (n = 11). Table I The relationship of ESBL-positive/ESBL-negative strains of Enterobacteriaceae isolated in different wards of the USC& MHC Number of Enterobacteriaceae Hospital units

DDS test negative

Neonatal intensive care unit

61

Pediatrics Nephrology outpatient clinic Surgery department

DDS test positive (putative ESBL producers)

Total

101

162

31

49

80

153

14

167

20

14

34

7

15

22

Urology outpatient clinic

18

5

23

Department of daily diagnostics and therapy

11

6

17

4

7

11

Department of developmental age neurology

Department of infantile endocrinology and diabetology Other

51

18

69

Total

356

229

585

Table II The relationship of ESBL-positive/ESBL-negative strains of Enterobacteriaceae isolated in different clinical specimens Number of Enterobacteriaceae Material

DDS test negative

DDS test positive (putative ESBL producers)

Total

Urine

303

172

475

Blood

18

29

47

Cerebrospinal fluid

2

5

7

Pharyngeal swab

1

9

10

Nasal swab

1

1

2

10

1

11

Intravascular catheter

4

2

6

Wound swab

8

2

10

Vaginal swab

Other

9

8

17

Total

356

229

585

30

1

Radosz-Komoniewska H. et al. Table III Detection of putative ESBL-producing strains by DDS test among different species of Enterobacteriaceae Number of strains Species Klebsiella pneumoniae

DDS test negative

Number of examined strains/Percentage of DDS test positive (putative ESBL producers) ESBL-producing strains

29

98

127 / 77

Klebsiella oxytoca

16

16

32 / 50

Serratia spp.

12

9

21 / 43

Citrobacter freundii

7

4

11 / 36

Escherichia coli

213

90

303 / 30

Enterobacter spp.

23

5

28 / 18

Proteus mirabilis

36

5

41 / 12

Morganella morganii

9

2

11 / 18

Other

11

0

11 / –

Total

356

229

585 / 39

One hundred and seventy-two putative ESBL-producing isolates were recovered from urinary tract infections, which constituted 75% of all ESBL producers identified and 36% of all Enterobacteriaceae isolates collected from urine. The higher frequency of the ESBL-producing isolates regarding the infection site was observed in the case of isolates recovered from blood (62% of which /n = 47/ demonstrated positive DDS test). The ESBL prevalence was also very high (71%) among the isolates cultured from the CSF, however, the number of all isolates obtained from this source was low (n = 7). The majority of putative ESBL-producing isolates (101 isolates, 44%) were collected from patients hospitalized in the neonatal ICU of the USC&MHC, followed by the isolates from the pediatrics department (49 isolates, 21%). 90% and 96% of K. pneumoniae isolates were DDS test positive, respectively. These rates were also very high in the case of E. coli isolates, reaching values of 54% and 44%, respectively. Although the majority of all Enterobacteriaceae isolates were collected from patients in the nephrology outpatient clinic, a relatively low rate of ESBL production was observed among them (14 isolates, 6%). Thirty-nine percent of K. pneumoniae (n = 18) and 4% of E. coli (n = 94) isolates from this ward showed ESBL activity. Antimicrobial susceptibility testing of ESBL-producing isolates. All putative ESBL-producing isolates were resistant to ampicillin, piperacillin, cephalothin and cefuroxime. A high percentage of the isolates were resistant to co-trimoxazole (93%) and aminoglycosides, including netilmicin (88%), gentamicin (83%) and amikacin (79%). Resistance to tetracycline was demonstrated in 61% of the isolates, to piperacillin with tazobactam in 23% of the isolates, and to nitrofurantoin – in 33% of urine-derived isolates. A small fraction of the isolates were resistant to norfloxacin (2%) and none of them was resistant to imipenem. Resistance to cefotaxime and ceftazidime revealed a remarkable degree of diversity in the analyzed group of isolates, both in terms of resistance levels an patterns of resistance to the two cephalosporins. According to the standard NCCLS breakpoint criteria used in susceptibility testing, there were isolates resistant or susceptible in vitro to both compounds (45 and 4 isolates, respectively), as well as isolates representing almost all other possible combinations of resistance phenotypes. The most prevalent were isolates resistant to cefotaxime and susceptible to ceftazidime (69 isolates). In general resistance to cefotaxime was more widely spread among the isolates than that to ceftazidime. Detection of ESBL activity by E-Test. Sixteen K. pneumoniae and 4 E. coli DDS-positive isolates recovered from urine, blood and CSF samples from patients in the neonatal intensive care unit were selected for ESBL detection using E-Test ESBL. The results are shown in Table IV. All these isolates revealed high-level resistance to cefotaxime with MICs exceeding the highest antibiotic concentration available in the E-Test strip (MICs, >16 :g ml–1). In 14 isolates clavulanic acid reduced the cefotaxime MICs efficiently enough to indicate ESBL activity. In the remaining six isolates the results was non-interpretable, because cefotaxime with clavulanate MICs also exceeded the highest value in the test (MICs, > 1 :g ml–1). Ceftazidime MICs surpassed the concentration range of the test in the case of 11 isolates (MICs, >32 :g ml–1). In the remaining isolates MICs of ceftazidime were between 8 and 24 :g ml–1. Decrease of ceftazidime MICs in the presence of clavulanate indicated ESBL activity in 15 isolates and in five isolates the interpretation of results was not possible (ceftazidime with clavulanate MICs, >4 :g ml–1). The combination of

1

Table IV Detection of ESBL producers by E test ESBL among selected DDS positive strains K.p. ATCC E.c. ATCC 700603 32518 ESBL+ ESBL–

Compounda

E.c. 621

K.p. 1100

K.p. 1098

E.c. 956

K.p. 652

K.p. 797

K.p. 1457

K.p. 1367

E.c. 1337

E.c. 1158

K.p. 551

K.p. 552

K.p. 1085

K.p. 1135

K.p. 1308

K.p. 1141

K.p. 1331

K.p. 1326

K.p. 1049

K.p. 933

CT

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

>16

1.0

1

0.125 0.064

>1

1

>1

>1

0.25

>1

0.25

0.125 0.125

0.25

0.25

0.38

0.125

0.38

0.094

0.047

NDb

>128

>64

ND

>128 >250

ND

>16

ND

ND

>64

ND

>64

>128

>128

>64

>64

>42

>128

>42

>10

32

>32

8

>32

8

*

>32

>32

>32

>32

>32

>32

>32

>32

16

16

24

12

16

24

>32

4

>4

0.25

>4

0.5

0.38

2

1

>4

>4

0.5

1

1.5

1.5

0.75

1

0.75

0.25

0.5

1

0.38

0.094

TZ / TZL

ND

ND

32

ND

16

>8

>16

>32

ND

ND

>64

>32

>21

>21

21

16

32

48

32

24

>84

2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 1024

4

Cefuroxime

>2048 >2048 >2048 1024 >2048 1024 >2048 >2048 1024

4

1024 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048 >2048

512

32

Cefoxitin

256

8

8

256

8

2

8

16

128

128

16

16

8

8

8

8

16

8

8

128

16

2

Cefotaxime

32

128

128

16

128

16

128

256

16

16

128

256

256

256

256

256

256

256

256

16

–

–

Cefotaxime +clavulanate

8

0.5

0.5

8

0.5

0.06

0.5

0.5

8

8

4

2

0.5

0.5

0.5

0.5

0.5

0.5

0.5

4

0.25

0.06

Ceftazidime

32

16

32

32

16

16

16

32

32

32

256

128

16

32

16

16

32

32

32

32

32

0.125

Ceftazidime + clavulanate

16

2

2

16

2

0.25

2

2

16

16

2

2

2

4

2

2

4

2

2

16

1

0.125

Aztreonam

64

128

128

64

128

4

128

128

64

64

512

512

128

128

128

128

128

128

128

64

0.06

0.125

Aztreonam + clavulanate

32

0.5

0.5

16

0.5

≤0.03

0.5

0.5

16

16

0.5

1

0.5

0.5

0.5

0.5

0.5

0.5

16

32

0.125 0.125

Imipenem

0.5

0.5

0.5

0.125 0.125

0.5

0.125 0.125 0.125 0.125

0.125 0.125 0.125

0.25

0.125 0.125

0.25

0.125 0.125 0.125 0.25

ESBLs in Enterobacteriaceae in pediatric hospital

>1

CT/CTL

0.125

31

32

Radosz-Komoniewska H. et al.

1

cefotaxime and ceftazidime data demonstrated ESBL activity in 16 isolates. In four E. coli isolates the results were non-interpretable. Detection of MIC of $-lactam antibiotics among E-Test ESBL positive isolates. MICs of a representative set of $-lactam antibiotics were evaluated for the 20 ESBL-producing K. pneumoniae and E. coli isolates, which had been analyzed before by the E-Test ESBL. The results are shown in Table V. Fourteen isolates were resistant to cefotaxime (MICs, 64–512 :g ml–1), 13 isolates to ceftazidime (MICs, 32–256 :g ml–1) and 19 isolates to aztreonam (MICs, 64–512 :g ml–1). Clavulanic acid efficiently reduced MICs of the investigated antibiotics except for the four E. coli isolates studied that were non-interpretable in the E-Test ESBL. In the case of these isolates the decrease of cefotaxime, ceftazidime and aztreonam MICs in the presence of clavulanate was at the level of one or two consecutive dilutions. Five isolates were resistant to cefoxitin (MICs, 128–256 :g ml–1), and they included the four E. coli isolates. Discussion In this work the prevalence of ESBL-producing Enterobacteriaceae in the USC&MHC hospital in Katowice was demonstrated. The hospital is a new large tertiary medical center specializing in gynecology, obstetrics, neonatology and pediatrics. Due to several factors, including the high representation of patients with numerous infection risk factors and high consumption of antibiotics, such hospitals are usually exposed to the danger of efficient selection and rapid spread of microorganisms with diverse antimicrobial resistance mechanisms. The number of 229 Enterobacteriaceae isolates from 162 patients, including 98 K. pneumoniae isolates that were identified as putative ESBL producers over the period of 20 months seems to be very high. It may be compared only to a few cases described previously, like in a ~500 bed hospital in New York in the beginning of the 1990s, where 155 patients were infected or colonized with ESBL-producing K. pneumoniae over 19 months (Meyer et al., 1993). In 1993–95, 145 patients of a ~1,000 bed hospital in Barcelona were infected or colonized by ESBL-producing K. pneumoniae within a period of 25 months (Peña et al., 1998). The putative ESBL-producing strains have strongly contributed to nosocomial populations of almost all Enterobacteriaceae species identified. The highest rate of ESBL production, both in terms of the total number of ESBL producers as well as their percentage among all isolates of a species recovered at the time, was found in the case of K. pneumoniae. The major role of this species in ESBL production has been widely observed from the time of the first ESBL isolations (Livermore, 1995; Bradford, 2001; Gniadkowski, 2001, Bush, 1996). However, the rate of 77% of putative ESBL producers among all K. pneumoniae isolates in the USC&MHC at the time of the study seems to represent one of the more extreme cases ever reported and this value was even higher in certain wards (more than 90%), such as the neonatal intensive care unit and the pediatrics department. Similar data on the very high representation of ESBL-expressing K. pneumoniae isolates in hospitals or hospital wards may be found in other reports from Polish medical centers. For example, Kêdzierska et al. (1999) reported a number of 91% of ESBL producers among K. pneumoniae recovered from patients in the neonatal ICU and the prematurity department at the Neonatology University Hospital in Cracow. Lower, though still very high rates were documented by Andrzejewska et al. (1998) (40.4%) and Drulis – Kawa et al. (2000) (32.5%), whereas the value reported by Pa³ucha et al. (1999) in one hospital in Warsaw (16%) is located within ranges that are usually observed in Europe and the USA. In the study by Babini and Livermore (2000), who examined klebsiellae isolates from 21 ICU in Western and Southern Europe within the years 1997–98, the prevalence of ESBL-producing strains was estimated at the level of 25%. The representation exceeding 30% occurred sporadically in particular units in Turkey (even 83%), France (62%), Italy (52%), Belgium (37%) and Germany (34.5%). Also noteworthy is the very high prevalence of putative ESBL producers in the E. coli population in the USC&MHC, reaching the value of 30% of all isolates of the species analyzed. Although in general E. coli has been often observed as an important host of ESBL expression, it was usually due to high total number of this organism among ESBL producers in survey studies and not to the proportion of ESBL positive strains to all E. coli isolates. It is possible that a certain fraction of DDS test positive results among the USC&MHC E. coli isolates were false positive (see below), however, still the possible frequency of ESBL production in this species seems to be very high. In works of other authors, the frequency of ESBL occurrence in E. coli ranged from 1,69% to 10% (Janicka et al., 1997; Kêdzierska et al., 1999; Andrzejewska et al., 1998; DrulisKawa et al., 2000; Saurina et al., 2000). The prevalence of ESBL-producing P. mirabilis (12%) in the USC&MHC was significantly lower than that reported by Szymaniak et al. (1999) in a hospital in Szczecin (59%) and comparable to data obtained in a large survey conducted in France by De Champs et al. (2000)

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ESBLs in Enterobacteriaceae in pediatric hospital

33

(6.9%). In last years, the increasing role of AmpC producer species, such as Enterobacter spp., C. freundii, S. marcescens and M. morganii, has been observed among ESBL-expressing strains in different countries, e.g. Argentina and France (Gniadkowski, 2001). This phenomenon could also be noticed in the USC&MHC populations of Enterobacteriaceae, and, especially in S. marcescens, in which the prevalence of ESBL was estimated at the level of 43%. Similarly high values of the prevalence of ESBL-producing S. marcescens were reported in Poland before by Ulatowska et al. (2000) (57.7%), Pa³ucha et al. (1999) (32%), and Kêdzierska et al. (1999) (25%). The distribution of ESBL-producing isolates with respect to particular wards of the hospital is similar to the situation observed in other hospitals in many countries (Livermore, 1995; Gniadkowski, 2001). The majority of the isolates (44%) were recovered from patients in the neonatal ICU. Due to many factors, these patients form a group of a high risk of nosocomial infection and are intensively treated with antibiotics, which, moreover, creates an additional, strong infection risk factor. The very high incidence of putative ESBL producers in Enterobacteriaceae populations in the pediatrics department (96% of K. pneumoniae, 44% of E. coli) is striking, even if the total number of isolations was significantly lower in this ward than in the neonatal ICU (21% of all putative ESBL producers). This observation stimulates further epidemiological studies in this ward. What is also noteworthy is the high percentage of ESBL-producing strains among the isolates from blood. Sixty-two percent of Enterobacteriaceae isolates from blood showed ESBL activity. Moreover, 5 out of 7 Enterobacteriaceae isolates from CSF demonstrated the presence of these enzymes. In the work of Wojsyk-Banaszak et al. (2000) 62.9% of multi-resistant strains of K. pneumoniae with ESBL expression (17 of 27) were the etiologic agents of infections of the central nervous system in newborns. In the study by Babini and Livermore (2000) mentioned above on ESBL-producing klebsiellae from 21 intensive care units in Europe, 61% of such isolates were resistant to amikacin, 72% to gentamicin, and 31% to ciprofloxacin. In hospitals in Brooklyn, 78% of ESBL-producing K. pneumoniae demonstrated resistance to gentamicin, and 50% to amikacin and to ciprofloxacin (Saurina et al., 2000). This data, as well as the results from other laboratories demonstrate the increasing resistance to non-$-lactam antimicrobials among ESBL producer strains, more and more of which appear to be multi-resistant (Gniadkowski, 2001). Data presented here illustrate this phenomenon very well too. A high percentage (more than 80%) of examined strains were resistant to co-trimoxazole and to aminoglycosides, including amikacin, gentamicin and netilmicin. More than a half of the isolates were also resistant to tetracycline. The only low rate of resistance to fluoroquinolones (2%) may be explained by the fact that these drugs are usually not used in children. Twenty DDS test positive isolates were further analyzed in the study, by the E-Test ESBL and evaluation of MICs of selected $-lactam antibiotics. These were isolates from the neonatal intensive care unit and the majority of them, mostly K. pneumoniae isolates, were selected to the analysis based on their remarkably high-level resistance to cefotaxime and/or ceftazidime. The four E. coli isolates were characterized by the relatively weak inhibitor effect in the DDS test. The E-Test ESBL confirmed the ESBL production positive results in the case of all 16 K. pneumoniae isolates and gave non-interpretable results for the four E. coli isolates. MIC evaluation revealed the typical for ESBL producers pattern of $-lactam MICs in the K. pneumoniae isolates, but in the E. coli isolates it demonstrated the particularly high MICs of cefotaxime, ceftazidime and aztreonam combinations with clavulanate, which coincided with high cefoxitin MICs (MICs, 128–256 :g ml–1). The MICs of the high inhibitor combinations could not be evaluated with the E-Test and this was the reason of the non-interpretable results obtained with this method. It is very likely that these E. coli isolates did not express ESBLs but produced the species-specific AmpC $-lactamase at an elevated level. The DDS test positive result could be false-positive and reflect the weak inhibitory effect of clavulanate on the E. coli AmpC enzyme. E. coli strains with increased level of AmpC production, although rare, have been observed in different medical institutions (Livermore, 1995; Corvec et al., 2002) (1, 31). The routine clinical microbiology laboratory must have problems with the proper interpretation of such strains, however, classifying them as putative ESBL producers cannot be treated as an error from the most important, clinical point of view. Further analysis, including molecular typing of the isolates and the identification of the ESBL types is necessary in order to reveal the mechanism of the spread of ESBL producers in the hospital. Literature A n d r z e j e w s k a E., A. S z k a r a d k i e w i c z and M. K a n i a s t y. 1998. Sensitivity to selected $-lactam antibiotics of Escherichia coli and Klebsiella pneumoniae isolates (in Polish). Med. Doœw. Mikrobiol. 50: 197–205. B a b i n i G.S. and D.M. L i v e r m o r e. 2000. Antimicrobial resistance amongst Klebsiella spp. collected from intensive care units in Southern and Western Europe in 1997–1998. J. Antimicrob. Chemother. 45: 183–189.

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B r a d f o r d P.A. 2001. Extended-spectrum $-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14: 933–951. B u s h K. 1996. Is it important to identify extended-spectrum $-lactamase-producing isolates? Eur.J. Clin. Microbial. Infect. Dis. 15: 361–364. D e C h a m p s C., R. B o n n e t, D. S i r o t, C. C h a n a l and J. S i r o t. 2000. Clinical relevance of Proteus mirabilis in hospital patients: a two years survey. J. Antimicrob. Chemother. 45: 537–539. C o r m i c a n M.G., S.A. M a r s h a l l and R.N. J o n e s. 1996. Detection of Exrended-Spectrum $-lactamase (ESBL)-Producing Strains by the E-Test ESBL Screen. J. Clin. Microbiol. 34: 1880–1884. C o r v e c S., N. C a r o f f, E. E s p a z e, J. M a r r a i l l a c and A. R e y n a u d. 2002. –11 mutation in the ampC promoter increasing resistance to $-lactams in a clinical Escherichia coli strain. Antimicrob. Agents Chemother. 46: 3265–3267. D r u l i s - K a w a Z., E. L e w c z y k, S. J a n k o w s k i and W. D o r o s z k i e w i c z. 2000. Infectivity and resistance to antibiotics of uropathogenic Gram-negative rods (in Polish). Med. Doœw. Microbiol. 52:119–127. ETM, E-Test Technical Manual. 2000. AB BIODISK, Dalvägen 10, S-169 56 Solna, Sweden. G n i a d k o w s k i M. 1997. Beta-lactamases in Gram-negative rods (in Polish). Nowa Medycyna 4: 20–26. G n i a d k o w s k i M. 2001. Evolution and epidemiology of extended-spectrum $-lactamases (ESBLs) and ESBL-producing microorganisms. Clin. Microbiol. Infect. 7: 597–608. G n i a d k o w s k i M., K. T r z c i ñ s k i, A. P a ³ u c h a and W. H r y n i e w i c z. 1996. Detection of extended spectrum $-lactamases (ESBL) in clinical isolates of Klebsiella pneumoniae: a comparison of the double-disc and the ATB BLSE tests (in Polish). Diag. Lab. 32: 697–709. J a n i c k a G., D. W o j c i e c h o w s k a, K. H a r e ñ s k a, J. P o r a d a and C. K ³ y s z e j k o. 1997. The resistance to betalactam antibiotics of lactose-positive and lactose-negative strains of Escherichia coli. Acta Microbiol. Polon. 46: 339–403. J a r l i e r V., M.H. N i c o l a s, G. F o u r n i e r and A. P h i l i p p o n. 1988. Extended broad – spectrum $-lactam ases conferring transferable resistance to newer $-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev. Infect. Dis. 10: 867–878. K ê d z i e r s k a J., M. D o l e ¿ a l and P. K a c h l i k. 1999. Resistance patterns of clinical strains of Gram-negative rods in own material (in Polish). Med. Doœw. Mikrobiol. 51: 113–122. L i v e r m o r e D.M. 1995. $-lactamases in laboratory and clinical resistance. Clin. Microbiol. Rev. 8: 557–584. L i v e r m o r e D.M. and D.F.J. B r o w n. 2001. Detection of $-lactamase-mediated resistance. J. Antimicrob. Chemother. 48: Suppl. SI, 59–64. M e d e i r o s A.A. 1997. Evolution and dissemination of $-lactamases accelerated by generations of $-lactam antibiotics. Clin. Infect. Dis. 24 (Suppl 1): 19–45. M e y e r K.S., C. U r b a n, J.A. E a g a n, B.J. B e r g e r and J.J. R a h a l. 1993. Nosocomial outbreak of Klebsiella infection resistant to late-generation cephalosporins. Ann. Inter. Med. 119: 353–358. National Committee for Clinical Laboratory Standards. 2000a. Performance Standards for Antimicrobial Disc Susceptibility Tests; Approved Standard, 7th Edition, M2-A7. NCCLS, Wayne, PA. National Committee for Clinical Laboratory Standarts. 2000b. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, 5 th Edition, M7-A5. NCCLS, Wayne, PA. N a v a r r o F. and E. M i r ó. 2002. Update on CTX-M-type $-lactamases. Rev. Med. Microbiol. 13: 63–73. P a ³ u c h a A., B. M i k i e w i c z, H. H r y n i e w i c z and M. G n i a d k o w s k i. 1999. Concurrent-outbreaks of extended-spectrum $-lactamase-producing organisms of the family Enterobacteriaceae in a Warsaw hospital. J. Antimicrob. Chemother. 44: 489–499. P a t e r s o n D.L., W.-C. K o, A. v o n G o t t b e r g, J.M. C a s e l l a s, L. M u l a z i m o g l u, K.P. K l u g m a n, R.A. B o n o m o, L.B. R i c e, J.G. M c C o r m a c k and V.L. Yu. 2001. Outcome of cephalosporin treatment for serious infections due to apparently susceptible irganisms producing extended-spectrum $-lactamases: implications for the clinical microbiology laboratory. J. Clin. Microbiol. 39: 2206–2212. P e ñ a C., M. P u j o l, C. A r d a n u y, A. R i c a r t, R. P a l l a r e s, J. L i n a r e s, J. A r i z a and F. G u d i o l. 1998. Epidemiology and successful control of a large due to Klebsiella pneumoniae producing extended-spectrum $-lactamases. Antimicrob. Agents Chemother. 42: 53–58. S a n d e r s C.C., A.L. B a r r y, J.A. W a s h i n g t o n, C. S h u b e r t, E.S. M o l a n d, M.M. T r a c z e w s k i, C. K n a p p and R. M u l d e r. 1996. Detection of extended-spectrum-$-lactamase-producing members of the family Enterobacteriaceae with the Vitek ESBL test. J. Clin.Microbiol. 34: 2997–3001. S a u r i n a G., J.M. Q u a l e, V.M. M a n i k a l, E. O y d n a and D. L a n d m a n. 2000. Antimicrobial resistance in Enterobacteriaceae in Brooklyn, NY: epidemiology and relation to antibiotic usage patterns. J. Antimicrob. Chemother. 45: 895–898. S i r o t D. 1995. Extended-spectrum plasmid-mediated $-lactamases. J. Antimicrob. Chemother. 36: Suppl. A: 19–34. S t e w a r d C.D., J.K. R a s h e e d, S.K. H u b e r t, J.W. B i d d l e, P.M. R a n e y, G.J. A n d e r s o n, P.P. W i l l i a m s, K.L. B r i t t a i n, A. O l i v e r, J.E. M c G o w a n and F.C. T e n o v e r. 2001. Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extendedspectrum $-lactamase detection methods. J. Clin. Microbiol. 39: 2864–2872. S z y m a n i a k L., I. A l e k s a n d r o w i c z and S. G i e d r y s - K a l e m b a. 1999. Drug resistance and proticinogenic types of Proteus mirabilis strains isolated from urinary tract infections (in Polish). Med. Doœw. Mikrobiol. 51: 323–330. U l a t o w s k a B., H. C z a j k o w s k i, E. G o s p o d a r e k and A. M i k u c k a. 2000. Occurrence of ESBL in Serratia species. Med. Doœw. Mikrobiol. 52:17–24. W o j s y k - B a n a s z a k I. and J. S z c z a p a. 2000. Central nervous system infections in neonates caused by multiresistant Klebsiella pneumoniae (in Polish). Ginekol. Pol. 71: 975–978.

Polish Journal of Microbiology 2004, Vol. 53, No 1, 35–39

Enterotoxigenic Bacteroides fragilis (ETBF) Strains Isolated in the Netherlands and Poland are Genetically Diverse PIOTR OBUCH-WOSZCZATYÑSKI1*, ROB G.F. WINTERMANS2, ALEX VAN BELKUM3, HUBERT ENDTZ3, HANNA PITUCH1, DEBORAH KREFT3, FELICJA MEISEL-MIKO£AJCZYK1 and MIROS£AW £UCZAK1 1 Chair

and Department of Medical Microbiology, The Medical University of Warsaw, 5 Chalubinski Street, 02-004 Warsaw, Poland 2 Franciscusziekenhuis Medical Microbiology, Boerhavelaan 25, 4700 AE Roosendaal, The Netherlands 3 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands Received 5 December 2003 Abstract Gram-negative anaerobic rods isolated in The Netherlands and Poland from extraintestinal and intestinal sources were identified as Bacteroides fragilis (n = 210) on the basis of Gram staining, growth on selective Bacteroides Bile Esculine medium as black colonies, and biochemical characteristics. PCR-mediated assessment of the presence of the B. fragilis enterotoxin (fragilysin) gene in all strains identified 12 so-called enterotoxin-positive B. fragilis (ETBF) strains (15%) among the Dutch strains and 16 ETBF among the Polish strains (13%). NotI Pulsed Field Gel Electrophoresis (PFGE) analysis revealed that these strains are genetically heterogeneous. Among the Dutch strains an identical pair and a set of four indiscriminate strains were identified. This suggests that limited nosocomial spread of ETBF can be observed. However, there was no identity obeserved when strains from The Netherlands were compared to their Polish counterparts. The antimicrobial susceptibility testing revealed that one Polish strain isolated from a patient with antibiotic associated diarrhoeae (AAD) was simultaneously highly resistant to clindamycin and cefoxitin (MIC > 256 mg/L). Two other strains appeared to be clindamycin resistant. All resistant strains had different PFGE patterns, suggesting that resistance development occurred at independent occassions. K e y w o r d s: Bacteroides fragilis, enterotoxin, PFGE, antibiotic resistance

Introduction Bacteroides fragilis is a gram-negative anaerobic, asporulating, encapsulated rod. This bacterial species is bile stimulated and extremely saccharolytic. It inhabits the colon of healthy animals and humans in quantities amounting to 1% of the normal gut flora. It is the most frequently isolated anaerobe from clinical specimens. B. fragilis can be isolated as the etiological agent of endogenic suppurative soft tissue infections, abscesses or bacteremia (Finegold, 1995; Meisel-Miko³ajczyk, 1999). Virulence factors of this bacterium were described by several autors (Hofstad 1990, 1992; Botta et al., 1994; Sebald, 1996). The most important virulence factors of B. fragilis are, among other, its capsule, lipopolysaccharide (LPS), outer membrane protein (OMP), pili, short-chain fatty acids (Pruzzo et al., 1989; Pantosti et al., 1991; Poxton and Edmond, 1995; Gibson et al., 1998) . The capsule influences abscess formation, antiphagocytic activity and the mode of adhesion (Sebald, 1996). The structure and biological activity of B. fragilis LPS was described by many authors (Meisel-Miko³ajczyk and Didoszak, 1980; Miko³ajczyk et al., 1981; Beckmann et al., 1989; Delahooke et al., 1995). B. fragilis strains harbouring a recently described, new virulence factor called fragilysin (enterotoxin) were described by Myers and his group in 1984. The same laboratory reported on * Corresponding author: Piotr Obuch-Woszczatyñski, Chair and Department of Medical Microbiology, The Medical University of Warsaw, 5 Chalubinski Street, 02-004 Warsaw, Poland. Phone/fax +48 22 628-27-39. E-mail: Piotr.Obuch@ ib.amwaw.edu.pl

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1

the isolation of these enterotoxigenic B. fragilis strains (ETBF) from different animals (Myers et al., 1985; Myers and Shoop, 1987; Myers et al., 1987a). For the first time the isolation of ETBF from humans was reported by Myers et al. (1987b). Very soon in different countries ETBF strains were detected in human clinical material as well. Countries including the USA (Sack et al., 1992), France (Sebald and MeiselMiko³ajczyk, 1993), Italy (Pantosti et al., 1994), The Netherlands (Van Belkum et al., 1999), Japan (Kato et al., 1995), Poland (Meisel-Miko³ajczyk et al., 1994; 1996a; 1996b), Bangladesh (Sack et al., 1994), Sweden (Olsen et al., 1999) reported on the occurrence of ETBF. As isolation of enterotoxin producing strains was described from different countries, studies on the putative relatedness of these strains have to be undertaken to determine whether or not these strains share a common source (or not). The aim of the present study was to define whether among B. fragilis strains isolated in The Netherlands and Poland (from different specimens) the enterotoxin (fragilysin) producing strains can be found, whether in both countries the frequency of ETBF strains is similar and whether the strains are similar or different. This analysis is meant to shed light on strain clonality versus horizontal genetransfer as means of dissemination. Experimental Materials and Methods Strains. As reference strains various well known B. fragilis enterotoxigenic strains were used. These included NCTC 11295 (metronidazole-resistant), ATCC 43858, and ATCC 43859. Non-ETBF included was IPL E 323. For antimicrobial susceptibility testing B. fragilis ATCC 25285 (non-ETBF) and B. thetaiotaomicron ATCC 29741were used as reference strains. 210 B. fragilis strains were isolated from extraintestinal and intestinal clinical samples in The Netherlands and Poland. Samples were inoculated on a BBE (Bacteroides Bile Esculin agar, BioMérieux, France) and incubated in an anaerobic chamber (Glove box, Forma Scientific Inc., USA) at 37°C for 48 hours. Identification of bacterial strains was done according to growth on selective media, colony morphology, Gram and capsule staining. API 20 A test (BioMérieux, France) was used for the biochemical identification. PCR. DNA was isolated using the Genomic DNA PREP PLUS isolation kit manufactured by A&A Biotechnology (Gdynia, Poland). PCR was performed in a DNA thermal cycler (Techne, UK) employing the following primer pair: 404 (5’-GAG CCG AAG ACG GTG TAT GTG ATT TGT-3’) and 407 (5’-TGC TCA GCG CCC AGT ATA TGA CCT AGT-3’). PCRs were performed in incubation volumes of 25 :l containing approximately 50 ng of DNA and 0.08 U of Taq DNA Polymerase (Invitrogen Ltd, Paisley, United Kingdom). The cycling conditions for PCR were: 4 min at 94°C followed by 40 cycles of 1 min at 94°C, 1 min at 52°C, 1 min at 74°C. Amplification products were detected by electrophoresis in 1% agarose gel with ethidium bromide added. (Figure 1). Only strains positive in the PCR were used in the following experiments.

Fig. 1. Agarose gel electrophoresis of amplified DNA from selected B. fragilis strains. Lane 1: 123 bp DNA ladder. Lane 2: NCTC 11295 (ETBF) strain. Lane 3: IPLE 323 (NTBF) strain Lane 4 and 5: clinical NTBF strains. Lane 6 and 7: clinical ETBF strains.

Fig. 2. Pulsed field gel electrophoresis of NotI DNA macrorestriction fragments derived from Dutch and Polish ETBF strains. On top strain identification numbers are given. These correspond with the numbers in Table 1. 1: 2683/97; 2: 1504/2/98; 3: 1605/98; 4: 2455/98; 5: 640W; 6: 7H; 7: m1-D5; 8: 1393157. The first four strains are Polish (Warsaw), the latter Dutch (Roosendaal). On the left molecular size markers are given in kilobase pairs.

1

37

ETBF isolated in Netherlands and Poland

PFGE. According to Maslanka et al. (1999), bacteria were suspended in SE buffer (75 mM NaCl, 25 mM EDTA pH 8.0) and embedded in 0.5% agarose plugs. After solidification the plugs were immersed in lysis buffer (50 mM Tris HCl pH 8.0, 50 mM EDTA, 1% lauryl sarcosine, 1 mg/ml proteinase K). The mixture was incubated overnight at 55°C and plugs were washed 5 times in SE at room temperature afterwards. DNA in the plugs was digested using restriction endonuclease NotI (Boehringer-Mannheim, Mannheim, Germany). Electrophoresis was performed in a CHEF Mapper (BioRad, Veenendaal, The Netherlands). The voltage was 10 V/cm for 18 hours with linear sampling from 5 to 35 sek. at ± 60° angels (Figure 2). Antimicrobial susceptibility testing of B. fragilis isolates. Drug susceptibility of B. fragilis strains was determined with Etest (AB BIODISK, Solna, Sweden) – MICs for cefoxitine, amoxicillin/clavulanic acid, imipenem, clindamycin and metronidazole. MICs were estimated in accordance to the NCCLS recommendations (1997).

Results and Discussion Among the B. fragilis strains, 12/78 strains (15%) from The Netherlands and 16/132 Polish strains (13%) contained the fragilysin gene. The percentage of ETBF strains isolated in The Netherlands (strains isolated from extraintestinal materials) and Poland (strains isolated mainly from fecal samples of patients with AAD) is very similar. The PFGE analysis (Figure 2, Table I) revealed that these strains are genetically hetrogeneous. Table I Origin of the B. fragilis ETBF strains and PFGE results No.

Strain

Material

Country

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

074 075 2938723 086 3187323 1393157 m1-D5 7H 640W 14H 29 7

The Netherlands

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

W1 W2 P51 P131 2683/97 1504/2/98 1605/98 2455/98 2465/3/98 2785/98 210/2/99 1502/99 2/B 76/D 26 CD/2000 III

Poland2

29. 30. 31. 32.

NCTC 11295 ATCC 43858 ATCC43859 IPL E 323 (NTBF)

Reference strains

1 The

1

PFGE

Extraintestinal Extraintestinal Smear Douglas abscess Extraintestinal Blood Fluid ascites Extraintestinal Extraintestinal Extraintestinal Extraintestinal Clinical specimens Punctate of femur

E E E F G H I J J * AA AB

Child feces (without diarrhoea) Child feces (with diarrhoea) Fecal sample (AAD) Fecal sample (AAD) Fecal sample (AAD) Fecal sample (AAD) Fecal sample (AAD) Fecal sample (AAD) Fecal sample (AAD) Fecal sample (AAD) Fecal sample (AAD) Fecal sample (AAD) Pus Oral cavity (child) AAD infant feces Pus

AC AD K AG R L M N * O P Q * AF AH AI AE B A D

Netherlands Franciscusziekenhuis Medical Mikrobiology, Roosendaal. Strains nos 1–10, Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam. Strains nos 11 and 12. 2 Poland Department of Medical Microbiology, The Medical University of Warsaw, Warsaw. Strains nos 13–26, Department of Bacteriology, Hospital, P³ock. Strain no 27, Danuta Dzier¿anowska, Department of Microbiology and Immunobiology, The Children’s Memorial Health Institute, Warsaw. Strain no 28. * These strains are probably very rich in endogenous DNase, and maybe it can be a separate biotype.

38

1

Obuch-Woszczatyñski P. et al. Table II Susceptibility to antimicrobial agents of Bacteroides fragilis ETBF strains (MIC mg/l) No

Strain

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

074 075 2938723 086 3187323 1393157 m1-D5 7H 640W 14H 29 7

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

W1 W2 P51 P131 2683/97 1504/2/98 1605/98 2455/98 2465/3/98 2785/98 210/2/99 1502/99 2/B 76/D 26 CD/2000 III

29. 30. 31. 32.

NCTC 11295 IPLE 323 (NTBF) ATCC 25298 BT ATCC 297411

Cefoxitin (FX)

Amoxi./Clav. (XL)

Imipenem (IP)

Clindamycin (CM)

Metronidazole (MZ)

8 12 8 4 8 8 4 4 6 6 4 4

0.25 0.25 0.125 0.125 0.25 0.19 0.125 1.0 1.0 1.0 1.5 0.125

0.047 0.032 0.023 0.023 0.064 0.064 0.032 0.125 0.19 0.19 0.25 0.032

1.0 0.75 1.0 1.0 1.5 0.25 0.125 0.023 0.032 0.75 0.094 0.25

0.19 0.19 0.19 0.38 0.50 0.19 0.38 0.25 0.19 0.38 0.125 0.125

16 4 12 >256 4 16 8 4 6 4 4 8 16 6 6 8

0.125 0.125 0.25 2 0.25 0.5 0.19 0.5 0.5 0.38 0.25 0.19 1.0 0.19 0.5 0.5

0.032 0.023 0.032 1.5 0.064 0.25 0.032 0.5 0.5 0.125 0.047 0.047 0.125 0.047 0.125 0.094

0.125 0.5 1.5 >256 0.032 >256 0.75 0.094 0.19 0.125 0.50 0.75 >256 0.25 0.125 256 mg/L). Remaining strains were sensitive to clindamycin (MIC < 0,016–1,5 mg/L) and cefoxitin (MIC 4–16 mg/L). From the presented studies two general conclusions can be drown: 1. It is very important that all enterotoxigenic Bacteroides fragilis strains observed in this study are genetically nonhomogenous. 2. The most active (in vitro) antibacterials against ETBF strains under investigations are amoxicillin with clavulanic acid, imipenem and metronidazole.

1

ETBF isolated in Netherlands and Poland

39

Literature B e c k m a n n I., H.G. v a n E i j k, F. M e i s e l - M i k o ³ a j c z y k and H.C.S. W a l l e n b u r g. 1989. Detection of 2-keto-3deoxyoctonate in endotoxins isolated from six reference strains of the Bacteroides fragilis group. Int. J. Biochem. 21: 661–666. B o t t a G.A., A. A r z e s e, R. M i n i s i n i and G. T r a n i. 1994. Role of structural and extracellular virulence factors in gramnegative anaerobic bacteria. Clin. Infect. Dis. 18 (Suppl 4): 260–264. D e l a h o o k e D.M., G.R. B a r c l a y and I.R. P o x t o n. 1995. A re-appraisal of the biological activity of Bacteroides LPS. J. Med. Microbiol. 42: 102–112. F i n e g o l d S.M. 1995. Anaerobic infection in humans: an overview. Anaerobe 1: 3–9. G i b s o n F.C. III, A.B. O n d e r d o n k, D.L. K a s p e r and A.O. T z i a n a b o s. 1998. Cellular mechanism of intraabdominal abscess formation by Bacteroides fragilis. J. Immunol. 160: 5000–5006. H o f s t a d T. 1990. Microbiologic and structural aspects of obligate anaerobes. Current taxonomy of medically important nonsporulating anaerobes. Rev. Infect. Dis. 12 (Suppl. 2): 122–126. H o f s t a d T. 1992. Virulence factors in anaerobic bacteria. Eur. J. Clin. Microbiol. Infect. Dis. 11: 1044–1048. K a t o N., A. K a r u n i a w a t i, R. J o t w a n i, H. K a t o, K. W a t a n a b e and K. U e n o. 1995. Isolation of enterotoxigenic Bacteroides fragilis from extraintestinal sites by cell culture assay. Clin. Infect. Dis. 20 (Suppl. 2): 141. M a s l a n k a S.E., J.G. K e r r, G. W i l l i a m s, J.M. B a r b a r e e, L.A. C a r s o n, J.M. M i l l e r and B. S w a m i n a t h a n. 1999. Molecular subtyping of Clostridium perfringens by pulsed-field gel electrophoresis to facilitate food-borne-disease outbreak investigations. J. Clin. Microbiol. 37: 2209–2214. M e i s e l - M i k o ³ a j c z y k F. and A. D i d o s z a k. 1980. Immunochemical investigation on Bacteroides fragilis antigenic structure. Acta Microbiol. Pol. 29: 125–133. M i k o ³ a j c z y k E., A. R o k o s z, A. S a w i c k a - G r z e l a k and F. M e i s e l - M i k o ³ a j c z y k. 1981. The toxicity of Bacteroides fragilis endotoxins before and after removal of nucleic compounds. Acta Microbiol. Pol. 30: 97–100. M e i s e l - M i k o ³ a j c z y k F., M. S e b a l d, E. T o r b i c k a, K. R a f a ³ o w s k a and U. Z i e l i ñ s k a. 1994. Isolation of enterotoxigenic Bacteroides fragilis strains in Poland. Acta Microbiol. Pol. 43: 389–392. M e i s e l - M i k o ³ a j c z y k F., H. P i t u c h and G.S. R o u y a n. 1996a. Detection of enterotoxigenic Bacteroides fragilis (ETBF) among strains isolated between 1976 and 1995 in Poland. Acta Microbiol. Pol. 45: 187–192. M e i s e l - M i k o ³ a j c z y k F., E. P o d s i a d ³ y and G.S. R o u y a n. 1996b. Enterotoxigenic Bacteroides fragilis isolated from nondiarrheic adults. Acta Microbiol. Pol. 45: 181–186. M e i s e l - M i k o ³ a j c z y k F. 1999. Anaerobes today. Nova Acta Leopoldina. NF 80, Nr. 312: 207–217. M y e r s L.L., B.D. F i r e h a m m e r, D.S. S h o o p and M.M. B o r d e r. 1984. Bacteroides fragilis: a possible cause of acute diarrheal disease in newborn lambs. Infect. Immun. 44: 241–244. M y e r s L.L., D.S. S h o o p, B.D. F i r e h a m m e r and M.M. B o r d e r. 1985. Association of enterotoxigenic Bacteroides fragilis with diarrheal disease in calves. J. Infect. Dis. 152: 1344–1347. M y e r s L.L. and D.S. S h o o p. 1987. Association of enterotoxigenic Bacteroides fragilis with diarrheal disease in young pigs. Am. J. Vet. Res. 48: 774–775. M y e r s L.L., D.S. S h o o p and T.D. B y a r s. 1987a. Diarrhea associated with enterotoxigenic Bacteroides fragilis in foals. Am. J. Vet. Res. 48:1565–1567. M y e r s L.L., D.S. S h o o p, L.L. S t a c k h o u s e, F.S. N e w m a n, R.J. F l a h e r t y, G.W. L e t s o n and R.B. S a c k. 1987b. Isolation of enterotoxigenic Bacteroides fragilis from humans with diarrhea. J. Clin. Microbiol. 25: 2330–2333. N a t i o n a l C o m m i t t e e f o r C l i n i c a l L a b o r a t o r y S t a n d a r d s. 1997. Methods for antimicrobial susceptibility testing of anaerobic bacteria, 4th ed. Approved standard. NCCLS, Wayne, Pa. O l s e n I., C.O. S o l b e r g and S.M. F i n e g o l d. 1999. A primer on anaerobic bacteria and anaerobic infections for uninitiated. Infection 27: 159–165. P a n t o s t i A., A.O. T z i a n a b o s, A.B. O n d e r d o n k and D.L. K a s p e r. 1991. Immunochemical characterization of two surface polysaccharides of Bacteroides fragilis. Infect. Immun. 59: 2075–2082. P a n t o s t i A., M. C e r q u e t t i, R. C o l a n g e l i and F. D’ A m b r o s i o. 1994. Detection of intestinal and extra-intestinal strains of enterotoxigenic Bacteroides fragilis by the HT/29 cytotoxicity assay. J. Med. Microbiol. 41: 191–196. P o x t o n I.R. and D.M. E d m o n d. 1995. Biological activity of Bacteroides lipopolysaccharide – reappraisal. Clin. Infect. Dis. 20 (Suppl 2): S149–153. P r u z z o C., C.A. G u z m a n and B. D a i n e l l i. 1989. Incidence of hemagglutination activity among pathogenic and nonpathogenic Bacteroides fragilis strains and role of capsule and pili in HA and adherence. FEMS Microbiol. Lett. 50: 113–118. S a c k R.B., L.L. M y e r s, J. A l m e i d o - H i l l, D.S. S h o o p, W.C. B r a d b u r y and M. S a n t o s h a m. 1992. Enterotoxigenic Bacteroides fragilis: epidemiologic studies of its role as a human diarrhoeal pathogen. J. Diar. Dis. Res. 10: 4–9. S a c k R.B., M.J. A l b e r t, K. A l a m, P.K.B. N e o g i and M.S. A k b a r. 1994. Isolation of enterotoxigenic Bacteroides fragilis from Bangladeshi children with diarrhea: a controlled study. J. Clin. Microbiol. 32: 960–963. S e b a l d M. and F. M e i s e l - M i k o ³ a j c z y k. 1993. Final report on antigenic structure of enterotoxigenic strains of Bacteroides fragilis. Common action for cooperation in sciences and technology with Central, Eastern European Countries ERB-CIPA-CT-93 1563 (ERB 35 10PL 924454). S e b a l d M. 1996. Determinants de la pathogenicite de B. fragilis. Med. Mal. Infect. 26: 128–191. Va n B e l k u m A., P. L e s z c z y ñ s k i, M. Vo g e l, H. Ve r b r u g h and F. M e i s e l - M i k o ³ a j c z y k. 1999. An archival case of clinically significant infection in a surgical patient by enterotoxigenic Bacteroides fragilis PCR amplification of the metalloprotease enterotoxin gene from nonviable lyophilizates. Clin. Microbiol. Infect. 5: 117–118.

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Polish Journal of Microbiology 2004, Vol. 53, No 1, 41–44

Activities of Synthetic Peptides against Human Pathogenic Bacteria KRYSTYNA BOGUCKA1, ALEKSANDRA KRÓLICKA2, WOJCIECH KAMYSZ3, TADEUSZ OSSOWSKI4, JERZY £UKASIAK3 and EWA £OJKOWSKA2* 1 Department

of Bacteriology, Provincial Hospital of Gdañsk, Poland, of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdañsk and Medical University of Gdañsk, Poland, 3Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Gdañsk, Poland, 4Department of Supramolecular Chemistry, Faculty of Chemistry, University of Gdañsk, Poland 2 Department

Received 5 December 2003

Abstract The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. Synthesis and experimental application of the hybrids peptides may be one of the interesting possibilities in antimicrobial treatment. The aim of the present investigation is to determinate in vitro activities of two synthetic peptide amides: cecropin-melittin hybrid peptide (CAMEL) and protegrin analogue (IB-367) against control strains and multiresistant clinical isolates. Antimicrobial activities were measured by MIC and MBC. The tested strains were susceptible to the peptides at concentrations in the range of 1 to 32 :g ml–1. K e y w o r d s: Cecropin-melittin hybrid, protegrin analague

Introduction The introduction and increasing usage of antibiotics has initiated rapid development of antibiotic resistance in microorganisms, particularly in human pathogens (Berger-Bachi, 2002). Bacteria employ a variety of strategies to avoid the inhibitory effects of antibiotic agents and have evolved highly efficient means for the dissemination of resistance traits (Fraimow and Abrutyn, 1995). Resistance is found in numerous bacteria species, the most important of them are penicillin-resistant Streptococcus pneumoniae, vancomycinresistant Enterococcus spp., methicillin-resistant Staphylococcus aureus (MRSA) and multiresistant gramnegative bacilli, including strains producing an extended – spectrum beta-lactamase (ESBL) (Fraimow and Abrutyn, 1995; Bedenic and Zagar, 1998; Özkuyumcu, 1999;). Infections with such organisms may be particularly difficult to treat (Liu, 1999, Hand, 2000). Trials to introduce new antimicrobial drugs are being carried out in response to the constantly growing bacterial resistance to antibiotics. Recently a lot of attention has been paid towards cationic antibacterial peptides (CAPs), (Ganz and Lehrer, 1999; Oh et al., 2000; Osusly et al., 2000; Chmiel, 2001) Antibiotic peptides are a new group of antimicrobial agents with a unique mechanism of action (Gabay, 1994). Almost all antimicrobial peptides are cationic or amphiphilic and this feature determines the mode of their action (Kamysz et al., 2003). A characteristic feature of most of these compounds is the presence of basic amino acid residues (Lys, Arg). Cationic parts of the peptides are capable of interacting with negatively charged structures of the microbial cell wall and finally leads to its permeabilization (Hwang and Vogel, 1998; Ganz and Lehrer, 1999; Chmiel, 2001). CAPs are ubiquitous in nature and are thought to be an important component in innate host defences against infectious agents (Hancock et al., 1995). They are components of saliva, present on all surfaces exposed to the environment and are components of neutrophils as well. Often named “natural antibiotics” (produced by plants and animals), they are excellent templates for searching new antimicrobial agents. Based on the natural CAPs, * Corresponding author: [email protected]

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Bogucka K. et al.

synthetic analogues with greater antimicrobial activity can be synthesised. Since many CAPs possess a strong in vitro activity against microorganisms which are resistant to conventional antibiotics, they provide attractive templates for designing new antimicrobials agents which could be used for specific application. Some of antimicrobial peptides are already potential candidates for clinical applications (Zasloff, 2002). In the present study we tested antimicrobial activity of two synthetic peptide amides: cecropin-melittin hybrid peptide (CAMEL) and protegrin analogue (IB-367) in in vitro conditions. These peptides are analogues of naturally occurring compounds and are known as very effective agents against human pathogens (Mosca et al., 2000; Oh et al., 2000). In the present study we report high activity of two synthetic peptides against chosen strains from ATCC collection and against multiresistant clinical strains isolated in Poland. Experimental Materials and Methods Strains and growth conditions. The bacteria strains and their resistance to antibiotics, was determined by disc-diffusion susceptibility test (Table I). The disc-diffusion test was performed according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). The disc-diffusion tests were purchased from OXOID Co. Microorganisms used as control strains were obtained from American Type of Culture Collection (ATCC). Multiresistant microorganisms were clinical isolates obtained from Laboratory of Microbiology of the Provincial Hospital in Gdañsk. Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecium and Staphylococcus aureus MRSA were isolated from patient’s wounds and Klebsiella pneumoniae (ESBL +) from urine tract infection. All experiments were performed on strains grown in Mueller-Hinton II broth at 35°C for 24 h. Antibacterial compounds. CAMEL (KWKLFKKIGAVLKVL-NH2, cecropin – melittin hybrid peptide) and Iseganan IB-367 (RGGLCYCRGRFCVCVGR-NH2, protegrin analogue) were synthesized manually by the solid-phase method on TentaGel S RAM resin (0.22 mmol g–1; Rapp Polymere, Germany) using fluorenylmethoxycarbonyl (Fmoc) chemistry (Fields and Noble, 1990). The side-chain protecting groups of the amino acids were: t-butoxycarbonyl (Boc) for Lys and Trp, trityl for Cys, tert-butyl ether for Tyr, 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl (Pbf) for Arg. The peptides were synthesized using the following procedure: (i) 5 and 15 min deprotection steps using 20% piperidine in dimethylformamide (DMF) in the presence of 1% Triton; (ii) the

Table I Characteristics of bacteria strains used in this study

Species

Antibiotic agents Gram stain PRL TZP CIP CTX CAZ IPM AN OX GN Va

SXT

E

L

P

CN AMP 120

Escherichia coli ATCC 25922

(–)

26 (s)

30 (s)

35 (s)

34 (s)

30 (s)

31 (s)

21 (s)

–

–

–

–

–

–

–

–

–

Pseudomonas aeruginosa ATCC 27853

(–)

26 (s)

28 (s)

26 (s)

25 (s)

26 (s)

21 (s)

23 (s)

–

–

–

–

–

–

–

–

–

Enterococcus faecalis ATCC 29212

(+)

–

–

21 (s)

–

–

–

–

–

–

17 (s)

–

–

–

–

18 (s)

25 (s)

Staphylococcus aureus ATCC 25923

(+)

–

–

22 (s)

–

–

–

–

20 (s)

22 (s)

18 (s)

26 (s)

24 (s)

24 (s)

30 (s)

–

–

Acinetobacter baumannii

(–)

6 (r)

6 (r)

6 (r)

6 (r)

12 (r)

22 (s)

15 (i)

–

–

–

–

–

–

–

–

–

Klebsiella pneumoniae

(–)

6 (r)

18 (i)

34 (s)

6 (r)

15 (i)

22 (s)

6 (r)

–

–

–

–

–

–

–

–

–

Pseudomonas aeruginosa

(–)

6 (r)

10 (r)

21 (s)

6 (r)

6 (r)

6 (r)

15 (i)

–

–

–

–

–

–

–

–

–

Enterococcus faecium

(+)

–

–

16 (i)

–

–

–

–

–

–

22 (s)

–

–

–

–

6 (r)

6 (r)

Staphylococcus aureus MRSA

(+)

–

–

6 (r)

–

–

–

–

6 (r)

6 (r)

18 (s)

19 (s)

6 (r)

6 (r)

6 (r)

–

–

Inhibition zone diameter (mm) in disc-diffusion susceptibility test ; s – sensitive, i – intermediate, r – resistance PRL – Piperacillin, TZP – Piperacillin/Tazobactam, CIP – Ciprofloxacin, CTX – Cefotaxime, CAZ – Ceftazidime, IPM – Imipenem, AN – Amikacin, OX – Oxacilin, GN – Gentamicin, Va – Vancomycin, SXT – Sulphamethoxazole/Trimethoprim, E – Erythromycin, L – Lincomycin, P – Pennicilin G, CN 120 – Gentamicin, AMP – Ampicillin

1

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Activities of synthetic peptides

coupling reactions carried out with the protected amino acid diluted in a mixture of dimethylformamide and N-methyl-2-pyrrolidone (DMF/NMP), (1:1, v/v) in the presence of 1% Triton using N,N’-diisopropylcarbodiimide (DIC) as the coupling reagent in the presence of 1-hydroxybenzotriazole (HOBt); (Fmoc-AA:DIC:HOBt:1:1:1) for 1.5 h. The completeness of each coupling reaction was monitored with the chloranil test (Christensen, 1979). The peptides were cleaved from the solid support with trifluoroacetic acid (TFA) in the presence of water (2.5%), ethanedithiol (2,5%) and triisopropylsilane (2.5%) as scavengers. The cleaved peptides were precipitated with diethyl ether. IB-367 was cyclized by air oxidation as described (Chen et al., 2000). The peptides were purified by the solid-phase extraction (SPE) on a sorbent Kromasil C8 (5 :m particle size, 100 Å) using the protocol described previously (Kamysz et al., 2002). The resulting fractions with purity greater than 95–98% were tested by High performance Liquid Chromatography (HPLC). The peptides were analyzed with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Test with a commercial antibiotic: ciprofloxacin – fluoroquinolone (KRKA, d.d., Novo Mesto, Slovenia) was included for comparative purposes. MIC and MBC determinations. Solutions of CAPs freshly prepared on the day of the assay were applied to determine the lowest concentration inhibiting bacterial growth. The concentration range assayed for CAMEL, IB-367 and ciprofloxacin was from 0.06 to 256 :g ml–1. The minimal inhibitory concentration (MIC) of each compound was determined using the broth macrodilution method with Mueller-Hinton II broth and an initial inoculum of 5×10 5 cfu ml–1 (Thornsberry, 1991). The incubation was performed for 18 h at 35°C and followed by the determination of MIC. In order to establish the minimal bactericidal concentrations (MBC) 100 :l of the contents of the wells showing no visible growth of bacteria on Mueller-Hinton was plated out on agar plates, distributed evenly with sterile bent glass rods and incubated for 18 h at 35°C. The MBC was defined as the lowest concentration of the substrate that reduced the inoculum by 99.9% within 24 h (Thornsberry, 1991). All experiments were performed in triplicate.

Results and Discussion A large number of diverse natural antimicrobial peptides have been discovered the last two decades (Nicholas and Mor, 1995; Hancocock et al., 1995) These natural products vary greatly in their biological activity spectrum, killing bacteria at concentrations from 0.25 to 4 :g ml–1. Present study was performed to estimate the influence of two synthetic cationic peptides (CAMEL and Iseganan) on growth and multiplication of chosen ATCC strains and several multiresistant clinical isolates. MIC and MBC tests were used to compare antibacterial activity of CAMEL and Iseganan and commercial antibiotic – ciprofloxacin. The presented results indicated that both peptides exhibited antibacterial activity (Tab. II). CAMEL showed insignificantly lower activity inhibiting the growth of tested strains in higher concentrations (MIC range 2–32 :g ml–1, MBC range 4–32 :g ml–1) than Iseganan (MIC range 1–16 :g ml–1, MBC range 2–32 :g ml–1) (Tab. II). Both peptides appeared less active against resistant clinical isolate P. areuginosa (MIC = 16 :g ml–1, MBC = 32 :g ml–1) than ciprofloxacin. However, in case of A. baumannii and S. aureus MRSA (clinical isolates resistant to ciprofloxacin), CAMEL and Iseganan demonstrated good activities inhibiting their growth in concentrations from 2 to 4 :g ml–1, respectively. The level of the resistance to tested CAPs of the control strains and the multiresistant clinical isolates was not significantly different when tested in vitro. Antimicrobial activity of the tested peptides was similar in case of both gram-positive and gram-negative bacteria. Table II Comparison of minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of synthetic peptides and ciprofloxacin CAMEL (:g/ml)

Species Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853

Iseganan IB-367 (:g/ml)

MIC

MBC

MIC

4

4

8

8

MBC

Ciprofloxacin (:g/ml) MIC

MBC

< 0.06

< 0.06

8

16

4

32

1

2

Enterococcus faecalis ATCC 29212

32

32

4

8

0.5

2

Staphylococcus aureus ATCC 25923

4

8

2

2

0.5

8

Acinetobacter baumannii

2

2

2

4

> 256

>256

Klebsiella pneumoniae

8

8

8

16

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