Novel restriction enzyme SSiI for the detection of mutation in GyrA gene of Salmonella enterica serovar Typhi.

ABSTRACT

Aim: Enteric fever is an ongoing problem in the developing nations. Resistance and reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. The present study was carried out with the objective of determining molecular basis of resistance to fluoroquinolone among the clinical isolates of Salmonella enterica serovar Typhi from different parts of India. Materials and Methods: A total of 60 S.Typhi clinical isolates were subjected to antimicrobial susceptibility testing and determination of minimum inhibitory concentration (MIC) to ciprofloxacin and nalidixic acid. Polymerase chain reaction (PCR) for GyrA gene followed by restriction fragment length polymorphism (RFLP) with restriction enzyme (RE) SSiI was performed to detect mutation at position Ser83. Further confirmation of mutation was done by nucleotide sequencing of GyrA gene. Results: Isolates showed 100% sensitivity to first-line drugs ampicillin, chloramphenicol, and cotrimoxazole. Twelve of the 60 isolates (18%) were susceptible to nalidixic acid (NASST) and the remaining 48 (82%) were resistant to nalidixic acid (NARST). Of these 48 NARST strains, 46 (97.5%) had reduced susceptibility to ciprofloxacin (MIC 0.25-1.0 μg/mL), whereas 2 strains (2.75%) were resistant to ciprofloxacin (MIC 4.0 μg/mL). In RFLP analysis, all the NASST strains showed 3 fragments, whereas all the NARST strains showed 2 fragments due to the loss of 1 restriction site as a result of mutation. All the NARST strains with reduced susceptibility to ciprofloxacin (n = 46) had a single mutation in gyrA gene (Ser 83→Tyr or Ser 83→Phe), whereas double mutations (Ser 83→Phe and Asp 87→Asn) were found in each of the 2 ciprofloxacin-resistant strains. None of the NASST strains (n = 12) revealed any mutation. Conclusion: Our study exemplifies the correlation between nalidixic acid screening test, MIC values, and the detection of mutation in GyrA gene by PCR-RFLP with a novel RE SSiI.This was further confirmed by nucleotide sequencing.