Plasmid copy number and gene expression in selection of fluoroquinolone-resistant .

Fluoroquinolone resistance in Enterobacteriales is developed by chromosomal and plasmid-mediated mechanisms. Plasmids play an important role in dissemination of resistant genes and they carry genes that protect bacteria in different stress-induced situations. In this study, we studied strains, each carried one plasmid-mediated quinolone resistance determinant namely, , and We exposed 0.5 McFarland density of each strain to 0.5 mg/L ciprofloxacin from the period of 30, 60, 90, and 120 min over 24 h. All treated strains were further exposed to a constantly increasing 1, 2, 4, and 8 mg/L ciprofloxacin solution through 24, 48, and 120 h. In given timepoints, RNA was extracted from all treated strains. Expression of , and was investigated by quantitative PCR. Mutations in and genes were analyzed by PCR and nucleic acid sequencing. In this study, during 0.5 mg/L ciprofloxacin exposition, the following expression levels were detected: 1.2 for , 1.47 for , 12.44 for , and 80.63 for . In case of long-term study, we selected a resistant strain in -positive , and its expression increased from 105.91 to 212.31. On the contrary, plasmid copy number increased in time from 1 to 4.13. No mutations in or in chromosomal genes of treated strains were detected. Our results show that -positive strain was able to develop fluoroquinolone resistance by upregulated expression that was linked to a minor increase in plasmid copy number but no mutations occurred in or