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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,{dagger} Angela van Daal,1 Gregory Kelly,1 Jacqueline Schooneveldt,2 Graeme Nimmo,2 and Philip M. Giffard1*

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 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.

* Corresponding author. Mailing address: CRC for Diagnostics, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia. Phone: 61 7 3864 2105. Fax: 61 7 3864 1534. E-mail: p.giffard{at}qut.edu.au.

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

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.



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