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Antimicrobial Agents and Chemotherapy, January 2009, p. 287-291, Vol. 53, No. 1
0066-4804/09/$08.00+0     doi:10.1128/AAC.00746-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Nationwide Survey of CTX-M-Type Extended-Spectrum β-Lactamases among Klebsiella pneumoniae Isolates in Slovenian Hospitals {triangledown}

K. Mesko Meglic,1* S. Koren,1 M. F. I. Palepou,2 E. Karisik,2 D. M. Livermore,2 R. Pike,2 A. Andlovic,1 S. Jeverica,1 V. Krizan-Hergouth,1 M. Müller-Premru,1 K. Seme,1 the Slovenian ESBL Study Group {dagger} N. Woodford,2

Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Zaloska 4, 1105 Ljubljana, Slovenia,1 Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, United Kingdom2

Received 6 June 2008/ Returned for modification 20 August 2008/ Accepted 2 November 2008


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ABSTRACT
 
Among 177 extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates collected from 11 Slovenian hospitals in 2005 and 2006, 60 (34%), from eight hospitals, harbored genes for CTX-M enzymes, with blaCTX-M-15 detected by sequencing. These 60 isolates comprised 11 pulsed-field gel electrophoresis-defined strains, with several clusters of closely related isolates. Plasmids encoding CTX-M-15 enzyme were highly transmissible.


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INTRODUCTION
 
First reported in the late 1980s and sporadically found in different hosts for a decade, CTX-M-type enzymes have now become the most prevalent extended-spectrum β-lactamases (ESBLs) in much of the world, the most widespread variant being the CTX-M-15 enzyme (4, 9). Their species distribution differs from that of TEM- and SHV-derived ESBLs, occurring mainly in Escherichia coli but also in Klebsiella pneumoniae (2, 9). E. coli strains with CTX-M ESBLs have proliferated in the community in some regions, although it remains unclear to what extent direct transmission is occurring, insofar as many "community" cases involve a history of recent hospital exposure (3, 11). Because of the extensive use of antibiotics, hospitals remain a perfect environment for selecting resistance genes (1) and it is plausible that K. pneumoniae, a major source organism for ESBLs, increasingly including CTX-M types, is acting as a vector for their transfer into E. coli and other species (1, 9).

To date, only SHV-type ESBLs have been reported in K. pneumoniae in Slovenia, and then only in one hospital (7), with no record of CTX-M ESBLs in any species. The aim of the present nationwide survey was therefore to investigate whether CTX-M-type ESBLs have now appeared in K. pneumoniae in Slovenian hospitals.

(These results were presented in part at the 17th European Congress of Clinical Microbiology and Infectious Diseases and 25th International Congress of Chemotherapy, Munich, Germany, 2007, poster P676.)

We analyzed nonreplicate isolates of ESBL-producing K. pneumoniae from 177 patients with infections and/or colonization collected in 11 Slovenian hospitals from January 2005 to May 2006, covering almost all of the Slovenian patient population; just one microbiology department serving a small regional hospital did not participate. Identification and initial susceptibility testing results were confirmed with the Vitek 2 system (bioMérieux, Marcy l'Étoile, France) with the ID-GN and the AST-N041 test cards. ESBL production was confirmed by ESBL Etests (AB Biodisk, Solna, Sweden) with ceftazidime, cefotaxime, and cefepime strips, each with and without clavulanate. MICs were determined by Etest, with the results interpreted in accordance with CLSI guidelines (6).

CTX-M alleles were sought by a multiplex PCR assay which can detect all five major CTX-M lineages (14). blaCTX-M genes were sequenced on a CEQ 8000 (Beckmann-Coulter, High Wycombe, United Kingdom) with primers for ISEcp1 (12), with additional internal sequencing primers designed for this study (CTX-M-G1-IF [5'-AAA CTC TGC GGA ATC TGA CG-3'] and CTX-M-G1-IR [5'-TCG GTT CGC TTT CAC TTT TC-3']).

Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA with a CHEF DRII apparatus (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom) (13). Banding patterns were analyzed with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) with isolate clustering performed with Dice's coefficient in combination with the unweighted-pair group method using average linkages. Strains were defined as having PFGE profiles with ≥80% similarity.

To investigate whether plasmid transfer contributed to the spread of CTX-M ESBLs, conjugative transfer of blaCTX-M genes was studied by mating experiments on nutrient agar plates with single representatives of the major strains as plasmid donors and E. coli J53-2 pro Rifr as the recipient. Transconjugants were selected on nutrient agar plates supplemented with 2 µg/ml cefotaxime and 200 µg/ml rifampin (Sigma-Aldrich, Poole, United Kingdom) (8). Hybridization studies were performed on the same plasmids with a digoxigenin-labeled probe derived from the blaCTX-M gene sequence with the primer pair MA1/MA2 (8, 13).

Among the K. pneumoniae isolates collected in 2005 and in the first few months of 2006, we noted a statistically significant increase in the prevalence of ESBL production (P = 0.005) (Table 1). CTX-M-type β-lactamase genes were detected in 60 (34%) of the 177 ESBL-producing isolates analyzed, thus currently still representing a minority of the ESBL-producing K. pneumoniae strains in Slovenia. As in Italy and Poland (9, 10), only group 1 CTX-M enzymes were found. These were identified in 8 of the 11 participating hospitals, with a countrywide distribution (Table 1). The large difference in the prevalence of CTX-M enzymes among the eight affected hospitals (Table 1) may suggest that Slovenia is in an early stage of the dissemination of these ESBLs, as recently reported in Italy (10), or may reflect variation in the success of infection control at particular sites.


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  		TABLE 1. Details of ESBL-producing K. pneumoniae isolates from 11 Slovenian hospitals

In contrast to our neighboring country Italy, where CTX-M-15, CTX-M-1, and, less often, CTX-M-32 enzymes were detected (10), we identified only CTX-M-15. Each blaCTX-M-15 gene was linked to an upstream ISEcp1-like element, shown previously to play a key role in their mobilization (5).

All isolates with blaCTX-M genes showed substantial resistance to both cefotaxime and ceftazidime (Table 2) with uniform susceptibility to imipenem. Most were multiresistant, including to ciprofloxacin and gentamicin. Variable susceptibility to amoxicillin-clavulanic acid and piperacillin-tazobactam may have reflected the presence or absence of the OXA-1 inhibitor-resistant penicillinase, which is often associated with CTX-M-15 enzymes (8). Further analysis of resistance genes encoded by CTX-M-carrying plasmids is in progress.


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  		TABLE 2. Susceptibilities of major strains of K. pneumoniae producing CTX-M group 1 enzymes

Comparison by PFGE showed that the isolates with CTX-M ESBLs included 11 lineages (Fig. 1). Several clusters of related isolates were observed, some of them including isolates from more than one center. The largest cluster (strain A) comprised 26 clonally related producers of CTX-M group 1 ESBLs, 24 from one hospital (H9), along with 2 from further hospitals (H10 and H11), indicating possible spread with patients transferred from one hospital to another, reflecting common patient transfer patterns. Three other substantial clusters were noted (strains B, C, and D) (Fig. 1). The most complex epidemiology was found in larger hospitals (H9, H10, and H11); in hospital H11, strains A to E were all recognized (Table 1), with clusters of isolates belonging to strains B, C, and D. These clusters were from different clinical wards; e.g., the strain C isolates were from the urine of patients treated on urology wards, as well as on a nephrology ward and the infection ward, reflecting patient transfer patterns at the site. Smaller hospitals were mainly affected by only single clones with CTX-M ESBLs (e.g., H7 by strain B), which were always found also in larger centers (Table 1).


Figure 1
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  		FIG. 1. Dendrogram based on PFGE typing of 60 blaCTX-M-positive K. pneumoniae isolates, in comparison with 27 blaCTX-M-negative ESBL-producing isolates, from 11 Slovenian hospitals to illustrate their relatedness. The vertical line indicates the ≥80% similarity score adopted to assign isolates to the same strain (unweighted-pair group method using average linkages, Dice method).

Clearly, the clonal spread of K. pneumoniae strains producing CTX-M enzymes is an important route of dissemination of these enzymes in our hospitals; in addition, the blaCTX-M-carrying plasmids from the three major outbreak strains, A, B, and C, were highly conjugative, with transfer frequencies of 3.7 x 10–7, 2.8 x 10–3, and 1.1 x 10–5 per donor cell, respectively. Hybridization studies showed that blaCTX-M-15 was located on ~150-kb plasmids. The resistance phenotypes conferred by the transferred plasmids are shown in Table 3. Thus, spread of blaCTX-M-carrying plasmids to other strains of K. pneumoniae, and perhaps also to E. coli, seems likely, as reported for an epidemic CTX-M-3-encoding plasmid in Poland (1, 9).


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  		TABLE 3. MICs for representative isolates of the strains A, B, and C and their transconjugants

We have shown a complex epidemiology with endemic K. pneumoniae strains producing CTX-M-15 enzymes in multiple Slovenian hospitals and causing smaller clonal outbreaks in several. The ready transferability of the plasmids from the main outbreak strains increases the risk of their transfer to other bacterial species, like E. coli, and into the community.


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ACKNOWLEDGMENTS
 
We thank Ad Futura for supporting this work and Mediline d.o.o., in collaboration with AB Biodisk, for the gift of Etests.

We thank Petra Baranasic for technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Zaloska 4, 1105 Ljubljana, Slovenia. Phone: 00386 1 543 74 46. Fax: 00386 1 543 74 01. E-mail: karmen.mesko{at}mf.uni-lj.si Back

{triangledown} Published ahead of print on 10 November 2008. Back

{dagger} The members of the Slovenian ESBL Study Group (in alphabetic order) are Jerneja Ambrozic Avgustin, Department of Biology, Biotechnical Faculty, University of Ljubljana; Ingrid Berce, Public Health Care Center Nova Gorica; Marina Bujko, Institute of Public Health of the Republic of Slovenia; Jerneja Fiser, General Hospital Dr. Franc Derganc; Tatjana Harlander, Public Health Care Center Novo Mesto; Martina Kavcic, Public Health Care Center Koper; Slavica Lorencic-Robnik, Public Health Care Center Maribor; Irena Piltaver, General Hospital Slovenj Gradec; Helena Ribic, Public Health Care Center Kranj; Iztok Strumbelj, Public Health Care Center Murska Sobota; Viktorija Tomic, University Clinic for Pulmonary and Allergic Diseases Golnik; and Tjasa Zohar-Cretnik, Public Health Care Center Celje. Back


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REFERENCES
 
    1
  1. Baraniak, A., J. Fiett, A. Sulikowska, W. Hryniewicz, and M. Gniadkowski. 2002. Countrywide spread of CTX-M-3 extended-spectrum β-lactamase-producing microorganisms of the family Enterobacteriaceae in Poland. Antimicrob. Agents Chemother. 46:151-159.[Abstract/Free Full Text]
    2. 2
  2. Bonnet, R. 2004. Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 48:1-14.[Free Full Text]
    3. 3
  3. Brigante, G., F. Luzzaro, M. Perilli, G. Lombardi, A. Coli, G. M. Rossolini, G. Amicosante, and A. Toniolo. 2005. Evolution of CTX-M-type β-lactamases in isolates of Escherichia coli infecting hospital and community patients. Int. J. Antimicrob. Agents 25:157-162.[CrossRef][Medline]
    4. 4
  4. Cantón, R., and T. M. Coque. 2006. The CTX-M β-lactamase pandemic. Curr. Opin. Microbiol. 9:466-475.[CrossRef][Medline]
    5. 5
  5. Cao, V., T. Lambert, and P. Courvalin. 2002. ColE1-like plasmid pIP843 of Klebsiella pneumoniae encoding extended-spectrum β-lactamase CTX-M-17. Antimicrob. Agents Chemother. 46:1212-1217.[Abstract/Free Full Text]
    6. 6
  6. Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing. 17th informational supplement. M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA.
    7. 7
  7. Jutersek, B., A. Baraniak, T. Zohar-Cretnik, A. Storman, E. Sadowy, and M. Gniadkowski. 2003. Complex endemic situation regarding extended-spectrum β-lactamase producing Klebsiella pneumoniae in a hospital in Slovenia. Microb. Drug Resist. 9(Suppl. 1):S25-S33.
    8. 8
  8. Karisik, E., M. J. Ellington, R. Pike, R. E. Warren, D. M. Livermore, and N. Woodford. 2006. Molecular characterization of plasmids encoding CTX-M-15 β-lactamases from Escherichia coli strains in the United Kingdom. J. Antimicrob. Chemother. 58:665-668.[Abstract/Free Full Text]
    9. 9
  9. Livermore, D. M., R. Cantón, M. Gniadkowski, P. Nordmann, G. M. Rossolini, G. Arlet, J. Ayala, T. M. Coque, I. Kern-Zdanowicz, F. Luzzaro, L. Poirel, and N. Woodford. 2007. CTX-M: changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 59:165-174.[Abstract/Free Full Text]
    10. 10
  10. Mugnaioli, C., F. Luzzaro, F. De Luca, G. Brigante, M. Perilli, G. Amicosante, S. Stefani, A. Toniolo, and G. M. Rossolini. 2006. CTX-M-type extended-spectrum β-lactamases in Italy: molecular epidemiology of an emerging countrywide problem. Antimicrob. Agents Chemother. 50:2700-2706.[Abstract/Free Full Text]
    11. 11
  11. Rodríguez-Baño, J., M. D. Navarro, L. Romero, L. Martínez-Martínez, M. A. Muniain, E. J. Perea, R. Pérez-Cano, and A. Pascual. 2004. Epidemiology and clinical features of infections caused by extended-spectrum β-lactamase-producing Escherichia coli in nonhospitalized patients. J. Clin. Microbiol. 42:1089-1094.[Abstract/Free Full Text]
    12. 12
  12. Saladin, M., V. T. Cao, T. Lambert, J. L. Donay, J. L. Herrmann, Z. Ould-Hocine, C. Verdet, F. Delisle, A. Philippon, and G. Arlet. 2002. Diversity of CTX-M β-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol. Lett. 209:161-168.[Medline]
    13. 13
  13. Woodford, N., M. E. Ward, M. E. Kaufmann, J. Turton, E. J. Fagan, D. James, A. P. Johnson, R. Pike, M. Warner, T. Cheasty, A. Pearson, S. Harry, J. B. Leach, A. Loughrey, J. A. Lowes, R. E. Warren, and D. M. Livermore. 2004. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum β-lactamases in the UK. J. Antimicrob. Chemother. 54:735-743.[Abstract/Free Full Text]
    14. 14
  14. Woodford, N., E. J. Fagan, and M. J. Ellington. 2006. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases. J. Antimicrob. Chemother. 57:154-155.[Free Full Text]

Antimicrobial Agents and Chemotherapy, January 2009, p. 287-291, Vol. 53, No. 1
0066-4804/09/$08.00+0     doi:10.1128/AAC.00746-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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