Stationary phase selects predominantly ciprofloxacin resistant strains of Salmonella Typhimurium with mutation in gyrA

Abstract

This study was focused on determination the mutation frequency leading to ciprofloxacin resistance as well as mechanism of resistance to ciprofloxacin and changes in the ability to form a biofilm at various growth stages of S. Typhimurium.

The mutation frequency leading to resistance (resistance index RI) was expressed as the mean number of resistant colonies divided by the mean of the total number of viable cells per culture. The resistance index (RI) was increased linearly over time and it had the highest value in the death phase. The mutations in the Quinolone Resistance Determining sources Regions (QRDR) of gyrA in S. Typhimurium strain were identified. All mutations identified in this study were substitutions in the QRDR of the gyrA gene, Serine 83 (Ser 83) and Aspartate 87 (Asp 87). The Ser 83 mutations were found already in exponential phase and their occurrence increased at other growth stages. Point mutations in Asp-87 were found only in later growth stages. The Ser 83 mutations occurred in all resistant strain obtained from stationary phase, with substitutions by Phenylalanine (Phe). Detection of gyrA mutations was carried out by AS-PCR-RFLP assay. This occurrence correlated with the increased expression of efflux pumps in mutant strains. The highest over-expression of efflux pump was found in resistant strains obtained from 96 h of cultivation (death phase). Ciprofloxacin resistant mutants formed a biofilm to a greater extent compared to the parental strain. Our data strengthen opinion that stressful environment conditions selects for antibiotic resistant mutants and in case of Salmonella spp. could contribute to improved ability to form biofilm.