Ciprofloxacin Induced Antibiotic Resistance in Salmonella Typhimurium Mutants and Genome Analysis

Abstract

Salmonella species is an important food-borne pathogen. Ciprofloxacin is used for treatment of salmonellosis. The wild type of Salmonella Typhimurium was exposed to 10-fold of minimal inhibitory concentration (MIC) of ciprofloxacin. The survivors of the first exposure were regrown. This regrown population was exposed to similar ciprofloxacin treatment. After 20 repetitions of the antibiotic exposure, the survival population was evaluated for antibiotic resistance. The mutants had 100-fold more of the initial MIC. The mutants were of smaller size (0.7 ± 0.5 mm) with a lag phase of 70 min. These mutants were resistant to other antibiotics belonging to different classes but were sensitive to heat and osmotic stress. The whole genome sequencing (WGS) analysis of antibiotic resistant mutants revealed interesting mutation pattern. Total 40513 mutations were observed across the genome (total 3843 annotated genes). Relatively small percentage (5.2%) of mutations were non-synonymous. Four-fold more transitions were observed than transversions. Ratio of <1 transition vs transversion shows a positive selection for antibiotic resistant trait. Seven genes with over 50 mutations were observed. However, mutation distribution across the genome was relatively uniform. The important genes like dnaE, gyrA, iroC, metH and rpoB involved in antibiotic resistance had point mutations. The genome analysis showed that most affected pathways were transcription regulation, transmembrane transport, cell adhesion, pathogenesis, pilus assembly, oxidation-reduction mechanisms, phosphor-relay signal transduction and LPS biosynthesis. We propose that development resistance to ciprofloxacin is because of the mutations in these genes.