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


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 microg/mL), whereas 2 strains (2.75%) were resistant to ciprofloxacin (MIC 4.0 microg/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.


CONCLUSIONS
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.
Agrawal
Parul
P
Department of Microbiology, Armed Forces Medical College, Pune 411 040, India.
Kapila
Ketoki
K
Kumar
Satish
S
Ghosh
A N
AN
Maurya
Anil Kumar
AK
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002939:Ciprofloxacin / Q000494:pharmacology; D027081:DNA Gyrase / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D024841:Fluoroquinolones / Q000494:pharmacology*; D006801:Humans; D007194:India; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense; D009268:Nalidixic Acid / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012485:Salmonella typhi / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014435:Typhoid Fever / Q000382:microbiology
D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones; D009268:Nalidixic Acid; D002939:Ciprofloxacin; D004262:DNA Restriction Enzymes; D027081:DNA Gyrase