A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in Salmonella enterica serovars Typhi and Paratyphi A.
OBJECTIVE
Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers.


METHODS
By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (Nal(R)) and/or decreased susceptibility to fluoroquinolones.


RESULTS
This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (Nal(R) = 223 and Nal(S) = 69) and 106 isolates of Salmonella Paratyphi A (Nal(R) = 24 and Nal(S) = 82). All of the 247 Nal(R) Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143/223 for Salmonella Typhi and 18/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight Nal(S) Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies.


CONCLUSIONS
The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes.
Environmental Research Institute, University College Cork, Lee Road, Cork, Ireland.
Yajun
Y
Song
Philippe
P
Roumagnac
François-Xavier
FX
Weill
John
J
Wain
Christiane
C
Dolecek
Camila J
CJ
Mazzoni
Kathryn E
KE
Holt
Mark
M
Achtman
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics; D027101:DNA Topoisomerase IV / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024841:Fluoroquinolones / Q000494:pharmacology*; D005838:Genotype; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D020641:Polymorphism, Single Nucleotide; D012482:Salmonella paratyphi A / Q000187:drug effects* / Q000235:genetics*; D012485:Salmonella typhi / Q000187:drug effects* / Q000235:genetics*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase