A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated bla KPC-5 , was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of bla KPC-5 showed significant differences from the flanking regions of other bla KPC variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro 103 3Arg), while KPC-4 contained Pro 103 3Arg plus an additional amino acid change (Val 239 3Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus, KPC enzymes may be evolving through stepwise mutations to alter their spectra of activity.
Klebsiella pneumoniaecarbapenemase (KPC)-producing organisms are therapeutically and diagnostically challenging. It is possible thatblaKPCgene expression plays a role in the variability observed in clinical susceptibility testing.blaKPCtransformants together with 10 clinical isolates representing four genera were evaluated forblaKPCcopy number and gene expression and correlated with β-lactam MIC data. The data suggest that mechanisms other than gene copy number and expression ofblaKPCcontribute to variability in susceptibility when testing KPC-producing isolates.
These findings demonstrate that inc point mutations can be associated with increased copy number of a 100 kb IncI1 plasmid, and lead to increased bla(CMY-2) expression and piperacillin/tazobactam resistance.
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