JAC Advance Access originally published online on August 8, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):733-740; doi:10.1093/jac/dkl333
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The acquisition of full fluoroquinolone resistance in Salmonella Typhi by accumulation of point mutations in the topoisomerase targets
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus Hinxton, Cambridge CB10 1SA, UK
Received 27 April 2006; returned 2 July 2006; revised 18 July 2006; accepted 19 July 2006
*Corresponding author. Tel: +44-1223-834244; Fax: +44-1223-494919; E-mail: jw5{at}sanger.ac.uk
Objectives: To determine the contribution to fluoroquinolone resistance of point mutations in the gyrA and parC genes of Salmonella Typhi.
Methods: Point mutations that result in Ser-83
Phe, Ser-83Tyr and Asp-87Asn amino acid substitutions in GyrA and Glu-84Lys in ParC were introduced into a quinolone-susceptible, attenuated strain of Salmonella Typhi using suicide vector technology. This is the first time that this approach has been used in Salmonella and abrogates the need for selection with quinolone antibacterials in the investigation of resistance mutations.
Results: A panel of mutants was created using this methodology and tested for quinolone resistance. The ParC substitution alone made no difference to quinolone susceptibility. Any single GyrA substitution resulted in resistance to nalidixic acid (MIC 
512 mg/L) and increased by up to 23-fold the MIC of the fluoroquinolones ofloxacin (MIC 
2 mg/L) ciprofloxacin (MIC 1 mg/L) and gatifloxacin (MIC 0.38 mg/L). Among the double substitution mutants, those with a substitution in ParC were less prone to killing with ciprofloxacin. The triple substitution mutants (Ser-83Phe or Tyr and Asp-87Asn in GyrA with Glu-84Lys in ParC) showed high levels of resistance to all the fluoroquinolones tested (MICs: gatifloxacin, 3–4 mg/L; ofloxacin, 32 mg/L; ciprofloxacin, 32–64 mg/L).
Conclusions: In Salmonella Typhi the fluoroquinolones tested act on GyrA and, at higher concentrations, on ParC. The point mutations conferred reduced susceptibility to ofloxacin and ciprofloxacin, and also reduced susceptibility to gatifloxacin. Three mutations conferred resistance to ofloxacin (32 mg/L), ciprofloxacin (32 mg/L) and to the more active fluoroquinolone gatifloxacin (MIC 3 mg/L). These results predict that the use of ofloxacin or ciprofloxacin will select for resistance to gatifloxacin in nature.
Keywords: typhoid fever , antibiotic resistance , fluoroquinolones , nalidixic acid , ofloxacin , ciprofloxacin , gatifloxacin
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