Contribution of target alteration, protection and efflux pump in achieving high ciprofloxacin resistance in Enterobacteriaceae.

The study aims at revealing the comprehensive contribution of target alteration, target protection and efflux pump to the development of high level of ciprofloxacin (CIP) resistance in Enterobacteriaceae bacteria of environmental, clinical and poultry origins. Antibiotic susceptibility test was used to detect CIP resistant (CIPR) isolates and MICCIP was determined by broth microdilution method. The presence of qnrS gene was identified by PCR and Southern blot hybridization (SBH) confirmed their location. Checkerboard titration demonstrated the effect of NMP on CIP action. PCR followed by sequencing and in silico analysis revealed the contribution of mutations in acrR, marR and gyrA to CIPR development. Out of 152 isolates, 101 were detected as CIPR. Randomly selected 53 isolates (MICCIP 4-512 µg/mL) were identified as Escherichia spp. (26), Enterobacter spp. (7), Klebsiella spp. (5) and Salmonella spp. (15) and of them 31 isolates carried qnrS. qnrS harboring 18 highly CIPR isolates (MICCIP: 256-512 µg/mL) were selected for further study. SBH confirmed 7 isolates harbored qnrS gene in plasmids. The acrA, acrB and tolC were present in all 18 isolates and NMP had an additive (12-isolates) or synergistic (6-isolates) effect on CIP action. Most isolates contained double amino acid (aa) substitutions (S83L and D87N) in QRDR of GyrA resulting in an altered conformation of putative CIP binding pocket. However, some isolates contained single (S83L or S83Y) or no aa substitution but showed high CIPR implicating that the concerted action of three mechanisms is responsible for high CIPR with the most significant role of efflux pump.