Abstract
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0294-9) contains supplementary material, which is available to authorized users.
Highlights
Ciprofloxacin (CIP) is a second generation fluoroquinolone and extensively used in the treatment of a wide range of infections caused by Enterobacteriaceae, and Pseudomonas aeruginosa (Kaplan et al 2013; Oliphant and Green 2002)
minimum inhibitory concentration (MIC) of ciprofloxacin and presence of Quinolone resistance encoding gene (qnrS) gene within Enterobacteriaceae isolates MICs of the ciprofloxacin (MICCIP) for the selected 53 isolates were in the range of 4–512 μg/mL; among which 18 Escherichia spp., 4 Enterobacter spp., 5 Klebsiella spp. and 3 Salmonella spp. (3 out of 15 isolates) showed very high resistance to CIP (MICCIP: 128-512 μg/mL) (Table 2)
In contrast to our current knowledge that efflux pump is usually responsible for low level of CIP resistance (Hooper 2001; Jacoby 2005), this investigation demonstrated that efflux pump can contribute to a high resistance phenotype even in the absence of any mutation in the DNA gyrase subunit A
Summary
Ciprofloxacin (CIP) is a second generation fluoroquinolone and extensively used in the treatment of a wide range of infections caused by Enterobacteriaceae, and Pseudomonas aeruginosa (Kaplan et al 2013; Oliphant and Green 2002). Chakrabarty et al AMB Expr (2016) 6:126 studies on CIP resistance mechanisms within highly resistant isolates would impede the attempts to increase the potency and decrease the resistance emergence by modifying the existing current drug or designing new one. This investigation focused on addressing this fundamental gap in our knowledge by unveiling the contribution of different prevailing mechanisms to the development of high level of CIP resistance among multidrug resistant Enterobacteriaceae bacteria isolated from clinical waste water (CWW), urinary tract infection (UTI) and cloacal swabs of poultry (CSP) origins in Bangladesh for public health interest
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