Plasmid-Mediated Fluoroquinolone Resistance in Pseudomonas aeruginosa and Acinetobacter baumannii.

Introduction Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens in health care-associated infections. Fluoroquinolone resistance has emerged in these pathogens. In this study, we aimed to determine the occurrence of plasmid-mediated quinolone resistance (PMQR) determinants ( qnrA , qnrB , qnrS , aac(6')-Ib-cr , oqxAB , and qepA ) by polymerase chain reaction (PCR) and the transmissibility of plasmid-borne resistance determinants in clinical isolates of P. aeruginosa and A. baumannii . Materials and Methods The study included P. aeruginosa (85) and A. baumannii (45) which were nonduplicate, clinically significant, and ciprofloxacin resistant. Antibiotic susceptibility testing was done by disk diffusion method for other antimicrobial agents, namely amikacin, ceftazidime, piperacillin/tazobactam, ofloxacin, levofloxacin, and imipenem. Minimum inhibitory concentration of ciprofloxacin was determined. Efflux pump activity was evaluated using carbonyl-cyanide m-chlorophenylhydrazone (CCCP). The presence of PMQR genes was screened by PCR amplification. Transferability of PMQR genes was determined by conjugation experiment, and plasmid-based replicon typing was performed. Results Resistance to other classes of antimicrobial agents was as follows: ceftazidime (86.9%), piperacillin/tazobactam (73.8%), imipenem (69.2%), and amikacin (63.8%). The minimal inhibitory concentration (MIC)50 and MIC90 for ciprofloxacin were 64 and greater than or equal to 256 µg/mL, respectively. There was a reduction in MIC for 37 (28.4%) isolates with CCCP. In P. aeruginosa , 12 (14.1%) isolates harbored qnrB , 12 (14.1%) qnrS , 9 (10.5%) both qnrB and qnrS , 66 (77.6%) aac(6')-Ib-cr , and 3 (3.5%) oqxAB gene. In A. baumannii , qnrB was detected in 2 (4.4%), 1 (2.2%) harbored both the qnrA and qnrS , 1 isolate harbored qnrB and qnrS , 21 (46.6%) aac(6')-Ib-cr , and 1 (2.2%) isolate harbored oqxAB gene. Notably, qepA gene was not detected in any of the study isolates. Conjugation experiments revealed that 12 (9.2%) were transferable. Of the transconjugants, seven (58.3%) belonged to IncFII type plasmid replicon, followed by four (33.3%) IncA/C and one (8.3%) IncFIC type. Conclusion The plasmid-mediated resistance aac(6')-Ib-cr gene is primarily responsible for mediating fluoroquinolone resistance in clinical isolates of P . aeruginosa and A. baumannii . The predominant plasmid type is IncFII.

Molecular Characterization of High-Level Aminoglycoside Resistance among Enterococcus Species.

Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminoglycoside by producing aminoglycoside modifying enzymes (AMEs). In enterococci, high level resistance to aminoglycosides is mediated by acquisition of plasmid mediated genes encoding for aminoglycoside modifying enzymes (AMEs). High level gentamicin resistance (MIC ≥ 500µg /mL) is predominantly mediated by aac(6')-Ie-aph(2″)-Ia, encoding the bifunctional aminoglycoside modifying enzyme AAC(6')-APH(2″). This enzyme eliminates the synergistic activity of gentamicin when combined with a cell wall active agent. Other AME genes such as aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id and ant(4')-1a have also been detected in enterococci. Objective This study was carried out to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of Enterococci . Materials and Methods A total number of 150 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion. Minimum Inhibitory Concentration (MIC) was ascertained by agar dilution method. Polymerase chain reaction was done to screen the following AMEs (aac(6')-Ie-aph(2″)-Ia; aph(2″)-Ib; aph(2″)-Ic; aph(2″)-Id and aph(3')- IIIa genes) . Results 51.3% of the study isolates exhibited high level gentamicin resistance. Polymerase chain reaction revealed that aph(3')-111a is the most prevalent AME, followed by aac(6')-1e-aph(2″)-1a . The combination of both the genes were detected in 44.1% of the study isolates. The rest of the AMEs and their combinations were not encountered in this study. 8.6% of the study isolates did not harbour any AME genes screened for, but was phenotypically resistant to gentamicin. In contrast 31.3% anchored the AME genes but phenotypically appeared susceptible to gentamicin. Conclusion This study indicates the high- level aminoglycoside resistance disseminated among Enterococci in our geographical region. It also emphasizes the detection of AMEs by PCR is mandatory because strains that appear susceptible by disc diffusion and/or MIC method may harbour one or more AMEs genes leading to therapeutic failure.