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 ␤-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 crossresistance to ciprofloxacin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, and tetracycline, whereas resistance to the ␤-lactam-␤-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 g/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.

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

“…several studies have observed that there is a relationship between the balance of porin expression and β-lactam susceptibility in clinical K. pneumoniae isolates [24][25][26][27][28][29][30] . a similar phenomenon has been reported in a patient infected with Salmonella enterica subsp.…”

“…De-repression of marA in response to several chemical and antibiotic stresses triggers a cascade of events that results in global control of membrane permeability by the downregulation of porin synthesis and overexpression of efflux pump components [5][6][7][8]15,42 . In K. pneumoniae, several isolates have an insertion sequence (such as Is5 and Is26) in the OmpK36 and OmpK35 genes [43][44][45] . This insertion, which abolishes porin expression, effects an efficient bacterial response to β-lactam stress.…”

Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes in clinical Klebsiella pneumoniae and Escherichia coli strains

“…To further ascertain the efficiency of the new M-PCR system, each of the PCR products from the strains that tested positive for the bla group genes were subjected to direct bidirectional DNA sequencing using either the M-PCR primers or sequencing primers 24,25 (shown in Table 1). The DNA sequencing data were compared with known bla allele sequences (Lahey Clinic website: http://www.lahey.org/Studies/other.asp#table1) by multiple-sequence alignment using the ClustalW2 online program.…”

High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India

“…PCR Amplification of blaTEM, blaSHV, blaCTX-M, QnrA, QnrB and QnrS Genes Amplification of bla TEM , bla SHV , bla CTX-M , QNR A, QNR B and QNR S genes were performed in thermal cycler (Applied Biosystems, USA) using primers previously mentioned [13,14]. Briefly each reaction was carried out in 20 ll reaction volume using 1x PCR buffer (Fermentus, USA), 20 pmol of primers (Integrated DNA Technologies, USA), 1 mM of each dNTPs, 1 unit of Taq polymerase (Fermentus, USA) and 100 ng plasmid DNA.…”