Mechanism and stability of hyperproduction of the extended-spectrum β-lactamase SHV-5 in Klebsiella pneumoniae

Abstract

Some isolates of SHV-5 β-lactamase-producing Klebsiella pneumoniae K2 from a single-strain outbreak of cross-infection produced approximately five-fold more β-lactamase than others. We investigated three possible genetic mechanisms of this hyperproduction: the presence of a more powerful promoter, an increase in plasmid copy number or an amplification of the gene on a plasmid. No differences between low and high β-lactamase producers were detected in the promoter region of the SHV-5 β-lactamase gene, which closely resembled that of SHV-2. SHV-5 β-lactamase production was encoded on a low copy number plasmid, but DNA-DNA hybridization with an SHV-specific probe detected a higher gene dose in hyperproducers. The β-lactamase hyperproduction was unstable on repeated subculture, with a reduction of about 75% after 100 generations. Hyperproducing mutants of a low-producing Klebsiella and its Escherichia coli K-12 transconjugants could be selected in vitro at a frequency of 10-5 to 10-6 and these variants had an increased SHV-5 β-lactamase gene copy number on low copy number plasmids. We conclude that hyperproduction of the extended-spectrum β-lactamase was caused by gene amplification that could be easily lost or gained in vitro. Since the change to hyperproduction occurred at a high frequency and hyperproducers showed increased resistance to many β-lactams and β-lactaml/β-lactamase inhibitor combinations, we suspect that these variants may readily be selected in patients during antibiotic therapy.