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Molecular characterization of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates from hospitals in Lithuania

Vaida Šeputienė, Marius Linkevičius, Aurelija Bogdaitė, Justas Povilonis, Rita Plančiūnienė, Agnė Giedraitienė, Alvydas Pavilonis, Edita Sužiedėlienė

  • 1Department of Biochemistry and Biophysics, Faculty of Natural Sciences, Vilnius University, M. K. Čiurlionio 21, LT-03101 Vilnius, Lithuania
  • 2Department of Microbiology, Kaunas University of Medicine, A. Mickevičiaus 9, LT-44307 Kaunas, Lithuania
  • Correspondence:
    Edita Sužiedėlienė(
    edita.suziedeliene{at}gf.vu.lt
    )

    • Journal of Medical Microbiology 2010; 59(10):1263–1265 · https://doi.org/10.1099/jmm.0.021972-0

    View at publisher PubMed

    Abstract

    The production of extended-spectrum β-lactamases (ESBLs) is the major source of resistance to newer β-lactam antibiotics in Enterobacteriaceae (Bradford, 2001; Coque et al., 2008a). SHV-type and TEM-type ESBLs were the most prevalent enzymes in the world a decade ago, but now the epidemiology of dominant ESBL types has shifted to the CTX-M type β-lactamases (Livermore et al., 2007). A national spread of ESBL producers has been reported in many European countries in nosocomial and community settings (Arpin et al., 2009; Empel et al., 2008; Lau et al., 2008; Vinue et al., 2009). The European Antimicrobial Resistance Surveillance System (EARSS) data for 2006–2008 indicated that approximately 5.8 % of Escherichia coli and 30.2 % of Klebsiella pneumoniae invasive isolates in Lithuania were resistant to oxyimino-cephalosporins (EARSS, 2008). There were no screens for ESBLs in clinical laboratories in Lithuania until 2005 and no epidemiological surveys for ESBLs have been conducted. Here we report what is believed to be the first study involving molecular characterization of ESBL-producing clinical E. coli and K. pneumoniae isolates from hospitals in Lithuania.

    A total of 175 non-duplicate clinical isolates (62 E. coli and 113 K. pneumoniae) with reduced susceptibility to oxyimino-cephalosporins were collected from 2007 to 2009 in two regional tertiary-care medical centres (with 2000 and 998 beds) and one provincial secondary-care centre (with 419 beds). Most of the isolates were from bronchial specimens (n=87), followed by isolates from urine samples (n=40), surgical infection sites (n=36) and blood (n=12). The ESBL phenotypes were identified using ESBL confirmatory MIC plates (TREK Diagnostic Systems). β-Lactamase- and quinolone-resistance-encoding genes were amplified by PCR (Eckert et al., 2006; Fanget al., 2008; Machado et al., 2006). Multiplex PCR for plasmidic AmpC-type β-lactamase-encoding genes was carried out as described by Perez-Perez & Hanson (2002). Sequencing was performed to determine the subclass of blaCTX-M, blaTEM, blaSHV, blaOXA, blaCMY and qnr. The quinolone-modifying aminoglycoside acetyltransferase-encoding gene aac(6′)-Ib-cr was assessed by RFLP analysis of aac(6′)-Ib gene-specific amplicons (Park et al., 2006). The molecular typing of ESBL producers was performed by repetitive sequence-based PCR (rep-PCR) with Eric primers (Munday et al., 2004). The standard broth mating method was used for conjugation experiments (Yagi et al., 2000).

    A total of 47 of 62 (76 %) E. coli and 98 of 113 (87 %) K. pneumoniae isolates with reduced susceptibility to oxyimino-cephalosporins were identified as ESBL producers by phenotypic tests (Supplementary Table S1 available with the online journal). CTX-M-encoding genes were found in the majority of E. coli (96 %, 45/47) and K. pneumoniae (71 %, 70/98) isolates showing the ESBL phenotype, CTX-M-15 being the most frequent variant (36 % of E. coli and 37 % of K. pneumoniae isolates) (Table 1⇓). A new variant of CTX-M-2 (Ala205Thr), first observed in 2007 and designated CTX-M-92 (GenBank accession no. GU127598) was among the most prevalent CTX-M-encoding genes found in 17 % of E. coli and 15 % of K. pneumoniae isolates. E. coli transconjugants obtained from representative clinical E. coli isolates producing CTX-M-2 and CTX-M-92 had similar ESBL phenotypes (results not shown). Examination of other bla genes (blaSHV, blaOXA, blaTEM), showed that SHV-12 type enzymes constituted the second most prevalent ESBL group in K. pneumoniae (22 %, 22/98). Five K. pneumoniae isolates possessed SHV-12 together with CTX-M-15 or CTX-M-3, one E. coli isolate had both SHV-12 and AmpC-type cephalosporinase CMY-2. ESBL and plasmidic AmpC-encoding genes were not detected in one E. coli and six K. pneumoniae ESBL producers and also in those isolates with reduced susceptibility to oxyimino-cephalosporins, which did not show the ESBL phenotype. A gene encoding TEM-1 enzyme without ESBL activity was detected in 66 % (31/47) of E. coli and 93 % (91/98) of K. pneumoniae isolates. OXA-1 type β-lactamase was detected in 94 % (16/17) of E. coli and 83 % (30/36) of K. pneumoniae CTX-M-15 producers. All E. coli and K. pneumoniae CTX-M-2 and CTX-M-92 producers and one K. pneumoniae CTX-M-3 producer had the OXA-2 type β-lactamase.

    Characteristics of ESBL-producing E. coli and K. pneumoniae isolates

    Search for plasmid-borne quinolone resistance determinants among the clinical ESBL producers revealed a low frequency of qnr genes (qnrB1 and qnrB6 in two different K. pneumoniae isolates and qnrB19 in one E. coli isolate). In contrast, of 91 (63 %, 91/145) aac(6′)-Ib-positive ESBL producers, 50 (34 %, 50/145) harboured the aac(6′)-Ib-cr variant. All aac(6′)-Ib-cr carrying isolates had CTX-M-15, except for a single K. pneumoniae isolate with CTX-M-3 β-lactamase.

    Rep-PCR showed high diversity among E. coli and K. pneumoniae ESBL-positive isolates (30 and 31 unique rep-PCR types, respectively). Overall, clonal isolates contributed to 76 % of K. pneumoniae (70/92, 9 clone types) and to 50 % of E. coli (23/46, 7 clone types) ESBL producers.

    This study, believed to be the first characterizing ESBL-producing clinical E. coli and K. pneumoniae isolates in Lithuanian hospitals, showed that CTX-M type β-lactamases are dominant among ESBL producers, the CTX-M-15-like enzymes being the most common. The prevalence and types of ESBLs in Lithuania have not been studied previously; however, on the basis of our observations it can be argued that current ESBL epidemiology in the country is consistent with the general situation in Europe, where CTX-M producers, and particularly CTX-M-15 producers, are becoming prevalent (Coque et al., 2008a; Livermore et al., 2007). Interestingly, the distribution pattern of CTX-M types observed in this study differs from that reported in some neighbouring countries, such as Poland, where CTX-M-3 enzymes strongly prevailed among CTX-M-positive E. coli and K. pneumoniae isolates (Empel et al., 2008). In the present study, CTX-M-3, CTX-M-2, CTX-M-1 and CTX-M-14 type ESBLs were less common compared to CTX-M-15 and a novel CTX-M-92 type β-lactamase, which together accounted for 53 % of all E. coli and 52 % of all K. pneumoniae ESBL-positive isolates. The important observation is that CTX-M-92 appears to be a local ESBL variant, found among ESBL producers in two regional hospitals included in the study. Most of the CTX-M-92-producing E. coli and K. pneumoniae isolates were genetically diverse; however, three E. coli (3/8, 38 %) and nine K. pneumoniae (9/15, 60 %) isolates were assigned to two distinct rep-PCR types. Among E. coli and K. pneumoniae CTX-M-15 producers, clonal isolates constituted 76 % (13/17, 3 clone types) and 89 % (32/36, 3 clone types), respectively. All of them, except one, had OXA-1 and/or TEM-1 β-lactamase-encoding genes and the aac(6′)-Ib-cr gene. A similar genetic environment has been reported for epidemic strains harbouring multiresistance pC15-1a plasmids with CTX-M-15 alleles, firstly reported in Canada and later observed in other countries (Boyd et al., 2004; Coque et al., 2008b).

    In summary, our study shows that while some trends in the molecular epidemiology of clinical E. coli and K. pneumoniae ESBL producers are consistent with the general situation in Europe, local clones expressing CTX-M-92 β-lactamase substantially contributed to the population of ESBL producers in Lithuanian hospitals.

    • A table of antimicrobial susceptibility data is available as supplementary material with the online version of this paper.

    The production of extended-spectrum β-lactamases (ESBLs) is the major source of resistance to newer β-lactam antibiotics in Enterobacteriaceae (Bradford, 2001; Coque et al., 2008a). SHV-type and TEM-type ESBLs were the most prevalent enzymes in the world a decade ago, but now the epidemiology of dominant ESBL types has shifted to the CTX-M type β-lactamases (Livermore et al., 2007). A national spread of ESBL producers has been reported in many European countries in nosocomial and community settings (Arpin et al., 2009; Empel et al., 2008; Lau et al., 2008; Vinue et al., 2009). The European Antimicrobial Resistance Surveillance System (EARSS) data for 2006–2008 indicated that approximately 5.8 % of Escherichia coli and 30.2 % of Klebsiella pneumoniae invasive isolates in Lithuania were resistant to oxyimino-cephalosporins (EARSS, 2008). There were no screens for ESBLs in clinical laboratories in Lithuania until 2005 and no epidemiological surveys for ESBLs have been conducted. Here we report what is believed to be the first study involving molecular characterization of ESBL-producing clinical E. coli and K. pneumoniae isolates from hospitals in Lithuania.

    A total of 175 non-duplicate clinical isolates (62 E. coli and 113 K. pneumoniae) with reduced susceptibility to oxyimino-cephalosporins were collected from 2007 to 2009 in two regional tertiary-care medical centres (with 2000 and 998 beds) and one provincial secondary-care centre (with 419 beds). Most of the isolates were from bronchial specimens (n=87), followed by isolates from urine samples (n=40), surgical infection sites (n=36) and blood (n=12). The ESBL phenotypes were identified using ESBL confirmatory MIC plates (TREK Diagnostic Systems). β-Lactamase- and quinolone-resistance-encoding genes were amplified by PCR (Eckert et al., 2006; Fanget al., 2008; Machado et al., 2006). Multiplex PCR for plasmidic AmpC-type β-lactamase-encoding genes was carried out as described by Perez-Perez & Hanson (2002). Sequencing was performed to determine the subclass of blaCTX-M, blaTEM, blaSHV, blaOXA, blaCMY and qnr. The quinolone-modifying aminoglycoside acetyltransferase-encoding gene aac(6′)-Ib-cr was assessed by RFLP analysis of aac(6′)-Ib gene-specific amplicons (Park et al., 2006). The molecular typing of ESBL producers was performed by repetitive sequence-based PCR (rep-PCR) with Eric primers (Munday et al., 2004). The standard broth mating method was used for conjugation experiments (Yagi et al., 2000).

    A total of 47 of 62 (76 %) E. coli and 98 of 113 (87 %) K. pneumoniae isolates with reduced susceptibility to oxyimino-cephalosporins were identified as ESBL producers by phenotypic tests (Supplementary Table S1 available with the online journal). CTX-M-encoding genes were found in the majority of E. coli (96 %, 45/47) and K. pneumoniae (71 %, 70/98) isolates showing the ESBL phenotype, CTX-M-15 being the most frequent variant (36 % of E. coli and 37 % of K. pneumoniae isolates) (Table 1⇓). A new variant of CTX-M-2 (Ala205Thr), first observed in 2007 and designated CTX-M-92 (GenBank accession no. GU127598) was among the most prevalent CTX-M-encoding genes found in 17 % of E. coli and 15 % of K. pneumoniae isolates. E. coli transconjugants obtained from representative clinical E. coli isolates producing CTX-M-2 and CTX-M-92 had similar ESBL phenotypes (results not shown). Examination of other bla genes (blaSHV, blaOXA, blaTEM), showed that SHV-12 type enzymes constituted the second most prevalent ESBL group in K. pneumoniae (22 %, 22/98). Five K. pneumoniae isolates possessed SHV-12 together with CTX-M-15 or CTX-M-3, one E. coli isolate had both SHV-12 and AmpC-type cephalosporinase CMY-2. ESBL and plasmidic AmpC-encoding genes were not detected in one E. coli and six K. pneumoniae ESBL producers and also in those isolates with reduced susceptibility to oxyimino-cephalosporins, which did not show the ESBL phenotype. A gene encoding TEM-1 enzyme without ESBL activity was detected in 66 % (31/47) of E. coli and 93 % (91/98) of K. pneumoniae isolates. OXA-1 type β-lactamase was detected in 94 % (16/17) of E. coli and 83 % (30/36) of K. pneumoniae CTX-M-15 producers. All E. coli and K. pneumoniae CTX-M-2 and CTX-M-92 producers and one K. pneumoniae CTX-M-3 producer had the OXA-2 type β-lactamase.

    Table 1.

    Characteristics of ESBL-producing E. coli and K. pneumoniae isolates

    Search for plasmid-borne quinolone resistance determinants among the clinical ESBL producers revealed a low frequency of qnr genes (qnrB1 and qnrB6 in two different K. pneumoniae isolates and qnrB19 in one E. coli isolate). In contrast, of 91 (63 %, 91/145) aac(6′)-Ib-positive ESBL producers, 50 (34 %, 50/145) harboured the aac(6′)-Ib-cr variant. All aac(6′)-Ib-cr carrying isolates had CTX-M-15, except for a single K. pneumoniae isolate with CTX-M-3 β-lactamase.

    Rep-PCR showed high diversity among E. coli and K. pneumoniae ESBL-positive isolates (30 and 31 unique rep-PCR types, respectively). Overall, clonal isolates contributed to 76 % of K. pneumoniae (70/92, 9 clone types) and to 50 % of E. coli (23/46, 7 clone types) ESBL producers.

    This study, believed to be the first characterizing ESBL-producing clinical E. coli and K. pneumoniae isolates in Lithuanian hospitals, showed that CTX-M type β-lactamases are dominant among ESBL producers, the CTX-M-15-like enzymes being the most common. The prevalence and types of ESBLs in Lithuania have not been studied previously; however, on the basis of our observations it can be argued that current ESBL epidemiology in the country is consistent with the general situation in Europe, where CTX-M producers, and particularly CTX-M-15 producers, are becoming prevalent (Coque et al., 2008a; Livermore et al., 2007). Interestingly, the distribution pattern of CTX-M types observed in this study differs from that reported in some neighbouring countries, such as Poland, where CTX-M-3 enzymes strongly prevailed among CTX-M-positive E. coli and K. pneumoniae isolates (Empel et al., 2008). In the present study, CTX-M-3, CTX-M-2, CTX-M-1 and CTX-M-14 type ESBLs were less common compared to CTX-M-15 and a novel CTX-M-92 type β-lactamase, which together accounted for 53 % of all E. coli and 52 % of all K. pneumoniae ESBL-positive isolates. The important observation is that CTX-M-92 appears to be a local ESBL variant, found among ESBL producers in two regional hospitals included in the study. Most of the CTX-M-92-producing E. coli and K. pneumoniae isolates were genetically diverse; however, three E. coli (3/8, 38 %) and nine K. pneumoniae (9/15, 60 %) isolates were assigned to two distinct rep-PCR types. Among E. coli and K. pneumoniae CTX-M-15 producers, clonal isolates constituted 76 % (13/17, 3 clone types) and 89 % (32/36, 3 clone types), respectively. All of them, except one, had OXA-1 and/or TEM-1 β-lactamase-encoding genes and the aac(6′)-Ib-cr gene. A similar genetic environment has been reported for epidemic strains harbouring multiresistance pC15-1a plasmids with CTX-M-15 alleles, firstly reported in Canada and later observed in other countries (Boyd et al., 2004; Coque et al., 2008b).

    In summary, our study shows that while some trends in the molecular epidemiology of clinical E. coli and K. pneumoniae ESBL producers are consistent with the general situation in Europe, local clones expressing CTX-M-92 β-lactamase substantially contributed to the population of ESBL producers in Lithuanian hospitals.

    Acknowledgments

    The Lithuanian State and Sciences Foundation supported this work. M. L. and A. B. acknowledge Student Research Fellowship Awards from the Lithuanian Science Council.

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