Point mutations in the inc antisense RNA gene are associated with increased plasmid copy number, expression of blaCMY-2 and resistance to piperacillin/tazobactam in Escherichia coli.

BACKGROUND: High-level expression of AmpC ß-lactamase genes is associated with increased resistance to ß-lactam antibiotics. bla(CMY-2) is the most prevalent plasmid-encoded AmpC gene found in Escherichia coli worldwide, and the gene is often found on plasmids of the IncI1 replicon type. Replication of IncI1 plasmids is controlled by antisense RNA transcribed from the gene inc, and nucleotide changes in the hairpin loop region of inc have been associated with increased plasmid copy number of IncI1 mini-plasmid constructs. The objective of this study was to determine the mechanism(s) responsible for increased bla(CMY-2) expression in three piperacillin/tazobactam-selected E. coli mutant strains with bla(CMY-2) encoded on a 100 kb IncI1 plasmid. METHODS: Mutants were selected from a clinical E. coli strain by exposure to superinhibitory concentrations of piperacillin/tazobactam. ß-Lactam susceptibilities were measured by agar dilution. Relative bla(CMY-2) transcript levels, gene copy number and IncI1 plasmid copy number were measured by real-time PCR. The inc gene of all strains was sequenced. RESULTS: Piperacillin/tazobactam MICs were 16- to 128-fold higher for mutant strains than for their parent strain. This increase in MICs correlated with 3- to 13-fold increases in bla(CMY-2) gene expression, bla(CMY-2) copy number and IncI1 plasmid copy number. Two mutants with 8- and 13-fold increases in IncI1 copy number had single point mutations located within the hairpin loop region of inc. CONCLUSIONS: 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.

Association of IS5 with divergent tandem blaCMY-2 genes in clinical isolates of Escherichia coli.

OBJECTIVE: To characterize a unique tandem bla(CMY-2) gene arrangement found in two non-identical clinical strains of Escherichia coli. METHODS: Both plasmid and chromosomal DNA were evaluated using PFGE, restriction digest analysis, plasmid profiling and Southern hybridization. bla(CMY-2) gene expression and gene copy number were evaluated by real-time PCR. Susceptibilities to selected ß-lactam antibiotics were determined by agar dilution. RESULTS: A tandem arrangement for bla(CMY-2) was identified in both isolates and was the only arrangement for bla(CMY-2) observed. These isolates had distinct PFGE and plasmid profiles. Each strain exhibited 2-fold higher bla(CMY-2) mRNA expression and up to 8-fold lower ß-lactam susceptibility compared with a strain with a single copy of bla(CMY-2). CONCLUSION: This is the first report of IS5 being associated with tandem bla(CMY-2). IS5 has previously been associated with antibiotic resistance through tandem gene amplification. The unique tandem arrangement provides a mechanism for increased bla(CMY-2) expression.

bla(KPC) RNA expression correlates with two transcriptional start sites but not always with gene copy number in four genera of Gram-negative pathogens.

Klebsiella pneumoniae carbapenemase (KPC)-producing organisms are therapeutically and diagnostically challenging. It is possible that bla(KPC) gene expression plays a role in the variability observed in clinical susceptibility testing. bla(KPC) transformants together with 10 clinical isolates representing four genera were evaluated for bla(KPC) copy number and gene expression and correlated with ß-lactam MIC data. The data suggest that mechanisms other than gene copy number and expression of bla(KPC) contribute to variability in susceptibility when testing KPC-producing isolates.

Phenotypic and enzymatic comparative analysis of the novel KPC variant KPC-5 and its evolutionary variants, KPC-2 and KPC-4.

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)-->Arg), while KPC-4 contained Pro(103)-->Arg plus an additional amino acid change (Val(239)-->Gly). 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.