Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistantSalmonella enterica serotype typhimurium DT104 isolates

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was develo ped to rapidly detect gyrA point mutations in multiresistant (MR) Salmonell a enterica serotype Typhimurium DT104 with decreased susceptibility to cipr ofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 an imal) were tested with three individual oligonucleotide probes directed aga inst an Asp-87-to-Asn (GAC --> 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 mutati ons by their probe-target melting temperatures. Thirty-seven human and 30 a nimal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isol ates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates ha d an Asp-87-to-Gly substitution. The remaining six strains all had mismatch es with the three probes and therefore different gyrA mutations. The sequen cing of gyrA from these six isolates showed that one human strain and two a nimal strains had an Asp-87-to-Tyr (GAC --> TAC) substitution and two anima l strains had a Ser-83-to-Tyr (TCC --> TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechani sm of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field ge l gel electrophoresis, and plasmid profiling, although they could be furthe r subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animal s in England and Wales may have arisen independently against a background o f clonal spread of MR DT104.