Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges

Objective: To establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. Methods: Eight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NAL^S (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA, B, S; aac(6')-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci. Results: Eighty isolates with a nalidixic acid MIC of <32mg/L (NAL^S) and a ciprofloxacin MIC of >0.064mg/L CIP^I (ciprofloxacin reduced susceptibility) were found. In 36 NAL^S CIP^I isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n=34), mutation in gyrB at codon 464, NAL MIC of 3-12mg/L and CIP MIC of 0.064-0.5mg/L.; and Group C, mutation in gyrA at codon 83 (n=2) NAL MIC of 16mg/L and CIP MIC of 0.25-0.38mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. Conclusion: The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIP^I-NAL^S isolates, an established phenotype in India.