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Antimicrobial Agents and Chemotherapy, March 2002, p. 659-664, Vol. 46, No. 3
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.3.659-664.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification and Minisequencing-Based Discrimination of SHV ß-Lactamases in Nosocomial Infection-Associated Klebsiella pneumoniae in Brisbane, Australia

Christopher Howard,^1, Angela van Daal,¹ Gregory Kelly,¹ Jacqueline Schooneveldt,² Graeme Nimmo,² and Philip M. Giffard^1*

Cooperative Research Centre for Diagnostics, Queensland University of Technology, Brisbane, Queensland,¹ Queensland Health Pathology Services, Princess Alexandra Hospital, Brisbane, Queensland, Australia²

Received 2 May 2001/ Returned for modification 23 September 2001/ Accepted 26 November 2001

Extended-spectrum ß-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 ß-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla_SHV-2a gene and seven strains carried the bla_SHV-12 gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla_SHV-11 gene and one strain carried the non-ESBL bla_SHV-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 bla_SHV-2a or bla_SHV12 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 bla_SHV-2a from strains carrying bla_SHV-12. In addition, this method was used to demonstrate an association between the relative copy numbers of bla_SHV genes in individual strains and the levels of antibiotic resistance.

Present address: Centre for Molecular Biotechnology, Queensland University of Technology, Brisbane, Queensland 4001, Australia.

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