Identification and Minisequencing-Based Discrimination of SHV β-Lactamases in Nosocomial Infection-AssociatedKlebsiella pneumoniaein Brisbane, Australia

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Abstract
Extended-spectrum β-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-oneKlebsiella pneumoniaeisolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV β-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried theblaSHV-2agene and seven strains carried theblaSHV-12gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBLblaSHV-11gene and one strain carried the non-ESBLblaSHV-1gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to theblaSHV-2aorblaSHV12gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed “first-nucleotide change,” involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carryingblaSHV-2afrom strains carryingblaSHV-12. In addition, this method was used to demonstrate an association between the relative copy numbers ofblaSHVgenes in individual strains and the levels of antibiotic resistance.

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