bla SHV Genes in Klebsiella pneumoniae : Different Allele Distributions Are Associated with Different Promoters within Individual Isolates

Abstract

ABSTRACT Extended-spectrum β-lactamases (ESBLs) emerge by point mutation from non-extended-spectrum precursors. The aims of this study were to reveal the basis for variations in resistance levels found in a collection of 21 Klebsiella pneumoniae clinical isolates from Brisbane, Australia. Previous studies have shown that 20 of these isolates possess bla SHV-11 , bla SHV-2a , and/or bla SHV-12 , and there is an association between the copy numbers of the ESBL-encoding genes and resistance levels. In this study, a real-time PCR method for interrogating the polymorphic sites at codons 238 and 240 was developed, and this confirmed the relationship between mutant gene copy numbers and resistance levels. The bla SHV promoter region was cloned from one of the ESBL-expressing isolates, and this showed that bla SHV genes exist downstream of two different promoters within this single isolate. These promoters have both been reported previously, and they differ by virtue of the presence or absence of an IS 26 insertion. The bla SHV copy numbers in cis with the different promoters were measured, and the copy number of the IS 26 promoter was correlated with resistance levels. Cloning and analysis of PCR products showed that different bla SHV variants existed in cis with individual promoters in individual isolates but that mutant genes were more abundant downstream of the IS 26 promoter. There were no ESBL-positive isolates without this promoter. It was concluded that bla SHV in cis with the IS 26 promoter is located on an amplifiable replicon, and the presence of the IS 26 insertion may facilitate the acquisition of an ESBL-positive phenotype.