Induction of resistant mutants of Salmonella enterica serotype Typhi under ciprofloxacin selective pressure

Background & objectives: Infection with Salmonella enterica serovar Typhi (hereafter S. Typhi) is an important public health problem in India. There has been an increase in the number of reported clinical failures to ciprofloxacin treatment but the data on possible mechanism of failure are limited. One mechanism that has been widely reported and found associated with ciprofloxacin resistance, is the mutations in target genes in QRDR (quinolone resistance determining region). It is hypothesized that mutations in DNA gyrase or topoisomerase IV result in therapeutic failure under selective pressure of antibiotic while the patient is on treatment. We undertook in vitro sequential selection studies to expose the clinical isolates of S. Typhi to different concentration of ciprofloxacin to study the role of antibiotic selective pressure in the development of mutations in QRDR. Methods: Total 26 clinical isolates were divided in to two parts: part I included six isolates obtained from three patients with relapse of enteric fever and part II included 20 isolates with different ciprofloxacin MIC levels. For in vitro induction of mutation experiment, five S. Typhi isolates were selected which included three NAS (nalidixic acid sensitive) and 2 NAR (nalidixic acid resistant) S. Typhi. These isolates were grown under increasing concentrations of ciprofloxacin and mutations acquired in QRDR of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) were investigated by sequencing. Results: For the isolates included in the part I of the study, it was found that the MIC to ciprofloxacin increased in the isolates obtained during the relapse of enteric fever as compare to the first isolate. All isolates had single mutation in gyrA gene at S83 without additional mutation in the second isolate. In the second part of the study, the nine isolates with varying MICs to ciprofloxacin also had single mutation in gyrA gene at S83 and another six had triple mutations, two mutations in gyrA gene (at S83 and D87) and one mutation in parC gene (at S80). In in vitro induction of mutation experiment, all mutated isolates showed triple mutation (two mutation in gyrA and one in parC gene) while no mutations were found in wild isolates. Interpretation & conclusions: Upon exposure to the step-wise increased concentration of ciprofloxacin, isolates become more tolerant to the ciprofloxacin and showed 2-4 fold higher MICs without new mutation after 8 mu g/ml. So the accumulation of mutations under continuous ciprofloxacin pressure and tolerance of the mutant isolates led to the clinical failure. These results also suggested that there could be another mechanism responsible for resistance.