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Antimicrobial Agents and Chemotherapy, January 2005, p. 256-263, Vol. 49, No. 1
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.1.256-263.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

David S. Hammond,1 Jacqueline M. Schooneveldt,2 Graeme R. Nimmo,2 Flavia Huygens,1 and Philip M. Giffard1*

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

Received 1 May 2004/ Returned for modification 26 July 2004/ Accepted 26 September 2004

Extended-spectrum ß-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 blaSHV-11, blaSHV-2a, and/or blaSHV-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 blaSHV promoter region was cloned from one of the ESBL-expressing isolates, and this showed that blaSHV 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 IS26 insertion. The blaSHV copy numbers in cis with the different promoters were measured, and the copy number of the IS26 promoter was correlated with resistance levels. Cloning and analysis of PCR products showed that different blaSHV variants existed in cis with individual promoters in individual isolates but that mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL-positive isolates without this promoter. It was concluded that blaSHV in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL-positive phenotype.

* Corresponding author. Mailing address: CRC for Diagnostics, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia. Phone: 61 7 3864 2015. Fax: 61 7 3864 1534. E-mail: p.giffard{at}qut.edu.au.

Antimicrobial Agents and Chemotherapy, January 2005, p. 256-263, Vol. 49, No. 1
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.1.256-263.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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