Mutation rate of bacteria is often affected by environmental conditions. Various stress such starvation, oxidative or radiating stress can result in increased frequency of mutations leading to antibiotic resistance. The aim of this work was to determine the mutation frequency leading to ciprofloxacin resistance induced by depleted media and hydrogen peroxide and to find molecular determinants of ciprofloxacin resistance in selected mutants. Methods: Oxidative stress was evoked by 3h cultivation of S. typhimurium with hydrogen peroxide in 3 concentrations (0.4, 0.8, 2, 4 mM). Starvation was studied after incubation (3h) of bacteria in control Luria-Bertani medium (LB) and in nutritionally depleted media 10%LB and Nutrient broth No. 1. Resistant strains were counted on agar plates supplemented with ciprofloxacin (0.06 mg/ml) after 72 h of incubation at 37?C. The frequency of resistant mutants (resistance index RI) represents mean number of resistant cells divided by the total number of viable cells per culture. Data represent the mean of three independent experiments; each experiment was made in five parallels and statistically evaluated by Students t-test. Mutations in gyrA were determined using AS-PCR-RFLP method. Levels of outer membrane porin F were detected with SDS-PAGE. Results: Short-term cultivation in 10% LB caused 430 fold increase of RI while in Nutrient broth No.1 it was only 57 fold rise. In ciprofloxacin-resistant strains generated by long-term starvation were detected decreased levels of OmpF protein. With rising dose of H2O2 was RI increasing up to 33-fold of spontaneous mutation frequency to ciprofloxacin resistance. In nutritionally depleted medium with H2O2 has mutation frequency increased more than 103-time. 80% of resistant strains had mutation in gyrA. 37% of them had mutation in codon Ser-83 and 63% in codon Asp-87. Conclusions: 1) Starvation increases mutagenesis leading to ciprofloxacin resistance. 2) Short-term treatment of S.typhimurium in nutritionally depleted media caused higher increase of ciprofloxacin RI than long-term incubation.3) Long-term starvation is leading to decreased levels of OmpF. 4) Oxidative stress induced by H2O2 in conjunction with lack of nutrients in environment increases mutation frequency to ciprofloxacin resistance. 5) Majority of ciprofloxacin-resistant strains generated by H2O2 has mutation in gyrA gene.

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