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Antimicrobial Agents and Chemotherapy, October 2001, p. 2856-2861, Vol. 45, No. 10
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.10.2856-2861.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

SHV-1 beta -Lactamase Is Mainly a Chromosomally Encoded Species-Specific Enzyme in Klebsiella pneumoniae†

José Chaves,1,2 Margarita G. Ladona,1,* Concepción Segura,3 Amparo Coira,2 Roser Reig,2 and Coral Ampurdanés1

Department of Pharmacology1 and Department of Microbiology,2 Municipal Institute of Medical Investigation, 08003 Barcelona, and Laboratori de Rèferencia de Catalunya (Microbiology), Autovia de Castelldefels, 08907 L'Hospitalet de Llobregrat, Barcelona,3 Spain

Received 1 February 2001/Returned for modification 12 May 2001/Accepted 10 July 2001

The nature of the SHV-1 beta -lactamase gene was analyzed in 97 epidemiologically unrelated Klebsiella pneumoniae strains isolated from clinical samples. beta -Lactamase bands that focused at a pI of 7.6 (SHV-1-type) in 74 strains, at a pI of 7.1 (LEN-1-type) in 13 strains, and at a pI of 5.4 (TEM-1-type) in 10 strains were detected by analytical isoelectric focusing (IEF). Among the 74 SHV-1-producing strains, 40 had, in addition to the pI 7.6 band, an additional band on IEF: 20 had a band with a pI of 7.1 and 20 had a band with a pI of 5.4. Most of the 74 SHV-1-producing strains (76.7%) carried plasmids. Transfer of beta -lactam resistance by conjugation was possible in only 9.3% of the strains tested. SHV-1 gene-specific PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the chromosomal DNA was positive for 93 of the 97 strains and negative for only 4 of the 10 samples with K. pneumoniae TEM-1 producers. In an attempt to approximate the location of the SHV gene locus by endonuclease restriction analysis, RFLP analysis with Southern blotting of chromosomal DNA with a labeled SHV-1 fragment as a probe was used to study the 97 strains. A trial with EcoRI showed at least one positive hybridization band for 96 strains; two bands were detected for 8 strains. The hybridization was negative for only one TEM-1 beta -lactamase-producing strain. DNA sequence analysis showed no differences in promoter regions or extra stop-triplet sequences; only point mutations determined different allelic variants. The novel SHV-type variants are designated SHV-32 and SHV-33. As a result of the RFLP and sequencing analyses, it can be postulated that the loci for SHV-1 and LEN-1 genes are arranged in tandem. Our results strongly support the hypothesis that the ancestor of the SHV-1 beta -lactamase originated from the K. pneumoniae chromosome.

* Corresponding author. Mailing address: CID-CSIC, C/Jordi Girona, 18-26, 08034 Barcelona, Spain. Phone: 34-93-400 61 00. Fax: 34-93-204 59 04. E-mail: mglqob{at}cid.csic.es.

dagger Through this study, we wish to posthumously pay our last respects to Clara Roy, who was the head of the Microbiology Department for many years.

Antimicrobial Agents and Chemotherapy, October 2001, p. 2856-2861, Vol. 45, No. 10
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.10.2856-2861.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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