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Journal of Clinical Microbiology, April 2001, p. 1443-1448, Vol. 39, No. 4
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 (GAC
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
AAC) mutation, an
Asp-87-to-Gly (GAC
GGC) mutation, and a Ser-83-to-Phe (TCC
TTC)
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 (GAC
TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC
TAC) 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.
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