Characterization of a large outbreak by CTX-M-1-producing Klebsiella pneumoniae and mechanisms leading to in vivo carbapenem resistance development

Abstract

All extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolates from patients admitted to and adult intensive care unit were prospectively documented from 2002 to 2005, when a large outbreak (51 patients affected) of multiresistant ESBL-producing Klebsiella pneumoniae infection was detected. The involvement of a single K. pneumoniae clone was demonstrated by pulsed-field gel electrophoresis. In addition to the ESBL-mediated resistance, the epidemic strain uniformly showed cross-resistance to ciprofloxacin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, and tetracycline, whereas resistance to the beta-lactam-beta-lactamase inhibitor combinations was variable. The ESBL involved was CTX-M-1, as demonstrated by isoelectric focusing, PCR amplification, and sequencing. CTX-M-1 as well as the aminoglycoside resistance determinants were encoded in a 50-kb plasmid that could be transferred to Escherichia coli only by transformation. In two of the infected patients, carbapenem resistance development (MICs of 8 to 12, 16, and >32 microg/ml for imipenem, meropenem, and ertapenem, respectively) was documented, both in clinical samples and in intestinal colonization studies. The analysis of the outer membrane proteins of the carbapenem-susceptible and -resistant isolates revealed that the former expressed only one of the two major porins, OmpK36, whereas the latter did not express either of them. In one of the cases, the lack of expression of OmpK36 was demonstrated to be mediated by the interruption of the coding sequence by the insertion sequence IS26. This is the first report of a large outbreak of CTX-M-1-producing Enterobacteriaceae and, curiously, the first documented description in the literature of CTX-M-1 in K. pneumoniae, despite the fact that this enzyme has been found in multiple species. Furthermore, we document and characterize for the first time carbapenem resistance development in CTX-M-1-producing Enterobacteriaceae.

FIG. 1.

A. Incidence of ESBL-producing Enterobacteriaceae (one isolate per patient) in the ICU during the period from 2002 to 2005. B. Distribution of new cases of isolation of ESBL-producing Enterobacteriaceae on a monthly basis during 2005.

FIG. 2.

XbaI PFGE patterns of ESBL-producing K. pneumoniae isolates from ICU patients. The single ESBL-producing K. pneumoniae isolates detected in 2003 and 2004 are represented in lanes 1 and 2, respectively, whereas lanes 3 to 22 contain representative isolates from the 2005 epidemic. MWM, molecular weight markers.

FIG. 3.

SDS-PAGE (A) and Western blotting with anti-OmpK36 serum (B) of OMP from K. pneumoniae LB1 (lane 1), the carbapenem-susceptible K. pneumoniae clinical isolates (lanes 2 and 3), and their respective in vivo-selected carbapenem mutants (lanes 4 and 5). The size of the molecular mass marker (in kilodaltons) is indicated on the left of panel A. Only the relevant parts of the gel and Western blot are shown.