Detection of three stable genetic clones of CTX-M-15-producing Klebsiella pneumoniae in the Barcelona metropolitan area, Spain

Sir, In recent years, extended-spectrum β-lactamases (ESBLs) have been detected extensively among Enterobacteriaceae isolates on all inhabited continents.1–4 Strains carrying the recently emerged CTX-M-15 enzyme have caused several reported outbreaks involving healthcare institutions in various countries.2,3 Within the last 2 years, concurrent with the worldwide detection of CTX-M-15-producing Escherichia coli clonal strains,1,4 CTX-M-15-producing Klebsiella pneumoniae clonal strains have also been associated with several major hospital outbreaks, such as those reported in Scandinavia and Hungary.2,3 In July 2008, an outbreak of K. pneumoniae producing CTX-M-15 was detected in the intensive care unit (ICU) of the Hospital General de l'Hospitalet (HGH) in the Barcelona metropolitan area, Spain. This outbreak prompted us to assess the clonality and persistence of CTX-M-15-producing K. pneumoniae isolates over the past several years. With this in mind, we conducted a retrospective study of ESBL-producing K. pneumoniae clinical samples stored from 2005 through 2008 at Hospital Sant Pau (HSP) and Hospital Vall d'Hebron (HVH), including 25 isolates from HSP obtained during 2005 and 72 isolates obtained at HVH from January 2007 to May 2008. Additionally, all the ESBL-producing K. pneumoniae isolates encountered at HGH during the outbreak period (July 2008), i.e. 10 from patients hospitalized in the ICU, 1 from a non-ICU patient and 3 environmental samples collected from the ICU, were included in the study. Susceptibility to antimicrobials was determined by disc diffusion, following CLSI recommendations.5 The MICs of tobramycin, amikacin, nalidixic acid, norfloxacin and ciprofloxacin were determined using the Etest (AB Biodisk, Solna, Sweden). Screening and sequence analysis of the blaCTX-M, aac(6′)-Ib, blaOXA-1, qnr and qepA genes, and the quinolone resistance-determining region (QRDR) of the gyrA and parC genes was carried out by PCR amplification and sequencing using specific primers.6 PFGE of XbaI-digested total DNA and dendrogram analysis were performed for all CTX-M-15-positive K. pneumoniae isolates as described previously.6 Several representatives of each pulsotype were also typed by multilocus sequence typing (MLST) using primers described elsewhere.7 Allele sequence and allelic profiles were verified at http://www.pasteur.fr/recherche/genopole/PF8/mlst. Among the 111 ESBL-producing K. pneumoniae isolates studied, a total of 37 isolates were found to carry the blaCTX-M-15 gene. Among them, eight different pulsotypes, for which there were three main clusters, were identified by PFGE analysis, corresponding with seven sequence types as determined by MLST [Figure S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. Sixteen isolates, including all isolates from the HGH outbreak and two from HVH, were assigned to the novel sequence type designated ST-326, and five isolates from HVH to the novel sequence type ST-327. The third clonal cluster, comprising five isolates from HSP and seven isolates from HVH, was identified as the ST-1 strain. Notably, isolates from ST-1 were found to persist at least from 2005 to 2007, and isolates from ST-326 and ST-327, from 2007 to 2008. Moreover, representatives of ST-1 and ST-326 were detected in two different hospitals each, with ST-326 causing an outbreak at one of these. The remaining four isolates represented the previously defined sequence types ST-147, ST-37, ST-14 and ST-321 (Table 1). Origins and characteristics of 37 CTX-M-15-producing K. pneumoniae isolates AMK, amikacin; bf, bifunctional enzyme; CIP, ciprofloxacin; NAL, nalidixic acid; NOR, norfloxacin; ST, sequence type; TOB, tobramycin; wt, wild-type enzyme. aThis group included 13 isolates (1H–13H). All experiments were carried out for isolates 1H–13H, except for the determination of MIC values, which was performed only for isolate 8H, the index isolate of this group. bStrain 14H is an environmental isolate. Origins and characteristics of 37 CTX-M-15-producing K. pneumoniae isolates AMK, amikacin; bf, bifunctional enzyme; CIP, ciprofloxacin; NAL, nalidixic acid; NOR, norfloxacin; ST, sequence type; TOB, tobramycin; wt, wild-type enzyme. aThis group included 13 isolates (1H–13H). All experiments were carried out for isolates 1H–13H, except for the determination of MIC values, which was performed only for isolate 8H, the index isolate of this group. bStrain 14H is an environmental isolate. The oxacillinase-encoding gene blaOXA-1 and the bifunctional acetyltransferase-encoding gene aac(6′)-Ib-cr have been detected previously in CTX-M-15-producing strains of E. coli and K. pneumoniae.2,4 In our study, the blaOXA-1 and aac(6′)-Ib-cr genes were detected in all ST-326 isolates (pulsotype I) and in all but three ST-1 isolates (pulsotypes VII and VIII). Additionally, all ST-326 isolates (pulsotype I) but one also carried the wild-type gene aac(6′)-Ib. In contrast, only one of the ST-327 isolates (pulsotype IV) carried the aac(6′)-Ib-cr gene (Table 1). Moreover, we found that all isolates from ST-326 and ST-1 exhibited high-level resistance to quinolones and fluoroquinolones. Those isolates showed quinolone resistance-associated predicted amino acid changes within the QRDR of both the gyrA and parC genes. Notably, within the total population, 26 of the 29 quinolone-resistant isolates carried the aac(6′)-Ib-cr gene, whereas this gene was absent from all (but one) of the isolates susceptible to quinolones and fluoroquinolones (Table 1). Since all study isolates were PCR-negative for the qnr and qepA genes, these results are consistent with the previously described involvement of the AAC(6′)-Ib-cr enzyme in the selection for QRDR chromosomal mutations, resulting in clinically relevant quinolone resistance levels.8 In conclusion, our findings document the dissemination and persistence of three K. pneumoniae clonal strains producing CTX-M-15 in different hospitals within the Barcelona metropolitan area. Two of these clones, i.e. ST-326 and ST-1, exhibited resistance to aminoglycosides and quinolones and were disseminated to at least two hospitals each. This report provides the first description of an outbreak caused by such strains within our country. These results may indicate that the observed seemingly high frequency of hospital outbreaks mediated by these strains may be a consequence of the strains' persistence within the affected locale. This work was supported by Fondo de Investigación Sanitaria (PI050289), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III – FEDER, Spanish Network for Research in Infectious Diseases (REIPI RD06/0008) and the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (J. R. J.). None to declare. We are grateful to Sylvain Brisse (Génotypage des pathogènes et Santé publique, Institut Pasteur, Paris, France) for validating and assigning designations to the new MLST profiles found in this study, and to P. Courvalin (Institut Pasteur, Paris, France) for providing E. coli TOP10/pΔT851 harbouring qepA.