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Journal of Clinical Microbiology, April 2001, p. 1443-1448, Vol. 39, No. 4
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.4.1443-1448.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Use of a LightCycler gyrA Mutation Assay for Rapid Identification of Mutations Conferring Decreased Susceptibility to Ciprofloxacin in Multiresistant Salmonella enterica Serotype Typhimurium DT104 Isolates

R. A. Walker,1,* N. Saunders,2 A. J. Lawson,1 E. A. Lindsay,1 M. Dassama,1 L. R. Ward,1 M. J. Woodward,3 R. H. Davies,3 E. Liebana,3 and E. J. Threlfall1

Laboratory of Enteric Pathogens1 and Virus Reference Division,2 Central Public Health Laboratory, London NW9 5HT, and Veterinary Laboratories Agency, New Haw, Surrey KT15 3NB,3 United Kingdom

Received 27 November 2000/Returned for modification 20 January 2001/Accepted 3 February 2001

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GACright-arrowAAC) mutation, an Asp-87-to-Gly (GACright-arrowGGC) mutation, and a Ser-83-to-Phe (TCCright-arrowTTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GACright-arrowTAC) substitution and two animal strains had a Ser-83-to-Tyr (TCCright-arrowTAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

* Corresponding author. Mailing address: Laboratory of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Ave., London NW9 5HT, United Kingdom. Phone: 44 (0)20 8200 4400. Fax: 44 (0)20 8905 9929. E-mail: rwalker{at}phls.org.uk.

Journal of Clinical Microbiology, April 2001, p. 1443-1448, Vol. 39, No. 4
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.4.1443-1448.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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