An Ertapenem-Resistant Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae Clone Carries a Novel OmpK36 Porin Variant

Abstract

Carbapenem-resistant Klebsiella pneumoniae caused an outbreak in a hospital in Rome, Italy. The clinical isolates were tested by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, multilocus sequence typing, plasmid typing, and ␤-lactamase identification. The OmpK35 and OmpK36 porins were analyzed by SDS-PAGE, and their genes were amplified and sequenced. Complementation experiments were performed using a recombinant unrelated ompK36 gene. An ertapenem-resistant and imipenem-and meropenem-susceptible clone was identified and assigned to the sequence type 37 lineage by MLST; it carried SHV-12 and CTX-M-15 ESBLs, did not produce the OmpK35 due to a nonsense mutation, and expressed a novel OmpK36 variant (OmpK36V). This variant showed two additional amino acids located within the L3 internal loop, one of the highly conserved domains of the protein. Two isolates of the same clone also exhibited resistance to imipenem and meropenem, due to the loss of OmpK36 expression by a nonsense mutation occurring in the ompK36V variant gene. These were the first carbapenem-resistant K. pneumoniae isolates identified within the hospital. Screening for the ompK36V gene of unrelated K. pneumoniae isolates derived from patients from 2006 to 2009 demonstrated the high frequency of this gene variant as well as its association with ertapenem resistance, reduced susceptibility to meropenem, and susceptibility to imipenem.The susceptibility to carbapenems in the Enterobacteriaceae family is no longer guaranteed since the increasing occurrence of carbapenem-resistant isolates (26, 38). Carbapenem resistance can arise by acquisition of resistance genes encoding metallo-␤-lactamases (MBLs) and non-metallo-carbapenemases (of the KPC, GES, or OXA type) but has also been associated with the AmpC cephalosporinase or extended-spectrum ␤-lactamase (ESBL) production and alterations of the expression of the major nonspecific porins. The latter mechanism has been reported worldwide in clinical isolates of Klebsiella pneumoniae, Escherichia coli, Enterobacter spp., and others (11, 12, 14-18, 23, 24, 31, 35). The alteration of porin expression may be caused by several different events, including the disruption of the gene by insertion sequences, the termination of translation by nonsense mutations, and the downregulation of transcription by mutations occurring within the promoter (12,18,29). In particular, the effect of the alteration of OmpK35 and OmpK36 expression in ESBL-producing K. pneumoniae isolates has been demonstrated to confer a high level of ertapenem resistance, with the MICs of imipenem and meropenem being raised (11,12,14,16,17,24,39).Sequence analysis of the ompK36 genes of clinical isolates of Enterobacter aerogenes, E. coli, and K. pneumoniae demonstrated the importance of transmembrane ␤-strand loop 3 (L3) in the specific uptake of ␤-lactams (3,8,36). The L3 loop constitutes the channel eyelet of enterobacterial porins, extends inside the barrel, and constricts the pore (1). The mutations occurring in L3 drastic...

Outbreak Caused by an Ertapenem-Resistant, CTX-M-15-Producing Klebsiella pneumoniae Sequence Type 101 Clone Carrying an OmpK36 Porin Variant

“…In two clonal isolates obtained during this outbreak, the lack of expression of both OmpK35 and OmpK36 porins was associated with high-level resistance to carbapenems (13). Also, CTX-M-15-producing K. pneumoniae clones with modified OmpK36 porins were found to affect a few hospitalized patients in different hospital settings (14,15).…”

“…Pressure applied by treatment with carbapenems is considered to contribute to the selection of porindeficient mutants among susceptible ESBL-or AmpC-producing populations (10)(11)(12). However, the loss of only one of the involved porins is not commonly linked to a significant increase in carbapenem MICs, in contrast to simultaneous depletion of both porins, which is associated with higher levels of resistance (13,14).…”

“…However, reports presenting outbreaks or large dissemination of such strains are limited (14,15). In the present study, we investigated an outbreak caused by an ertapenem-resistant, CTX-M-15-producing K. pneumoniae porin-deficient strain in the intensive care unit (ICU) of a Greek hospital.…”

Klebsiella pneumoniae: Going on the Offense with a Strong Defense

“…OmpK35 and OmpK36 are porins that, interestingly, are often poorly or not expressed in antibiotic-resistant K. pneumoniae strains, including ESBL-producing and carbapenem-resistant strains (345)(346)(347)(348)(349)(350)(351). Downregulation of these porins appears to provide an advantage for these bacteria in the face of antibiotic selection, where the porins perhaps serve as a channel that allows the entry of antibiotics into the bacteria (352).…”

Reduced intracellular drug accumulation augments fluoroquinolone and -lactam drugs resistance in clinical Gram negative bacteria from Nigeria

“…Decreases in the amount of OmpF were found to be associated with decreased accumulation of fluoroquinolones in E. coli (Karczmarczyk et al, 2011). Similarly, Garcia-Fernandez et al (2010) in their study, also implicated loss of porins in increased resistance to β-lactams in comparison with their respective strains, and did not discountenance the importance of the efflux mechanism as a contributor to β-lactam resistance in K. pneumoniae. This has also been previously shown for P. aeruginosa (Farra et al, 2008).…”