title: Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia.
abstract: Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the blaSHV-2a gene and seven strains carried the blaSHV-12 gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL blaSHV-11 gene and one strain carried the non-ESBL blaSHV-1 gene. 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 the blaSHV-2a or blaSHV12 gene. 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 carrying blaSHV-2a from strains carrying blaSHV-12. In addition, this method was used to demonstrate an association between the relative copy numbers of blaSHV genes in individual strains and the levels of antibiotic resistance.
authors.affiliation: Cooperative Research Centre for Diagnostics, Queensland University of Technology, Brisbane, Queensland, Australia.
authors.forename: Christopher
authors.initials: C
authors.lastname: Howard
authors.forename: Angela
authors.initials: A
authors.lastname: van Daal
authors.forename: Gregory
authors.initials: G
authors.lastname: Kelly
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Schooneveldt
authors.forename: Graeme
authors.initials: G
authors.lastname: Nimmo
authors.forename: Philip M
authors.initials: PM
authors.lastname: Giffard
mesh_terms: D001315:Australia; D001483:Base Sequence; D003428:Cross Infection / Q000382:microbiology*; D006801:Humans; D007525:Isoelectric Focusing; D007710:Klebsiella Infections / Q000382:microbiology*; D007711:Klebsiella pneumoniae / Q000235:genetics*; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001618:beta-Lactamases / Q000145:classification / Q000235:genetics*
chemical_list: C107197:beta-lactamase SHV-11; C107198:beta-lactamase SHV-12; D001618:beta-Lactamases