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

Abstract

High-level expression of AmpC β-lactamase genes is associated with increased resistance to β-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. Mutants were selected from a clinical E. coli strain by exposure to superinhibitory concentrations of piperacillin/tazobactam. β-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. 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. 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.