MOLECULAR CHARACTERISATION OF MULTIDRUG-RESISTANT PSEUDOMONAS AERUGINOSA IN SOUTHWESTERN NIGERIA

Abstract

Multidrug-Resistant (MDR) associated nosocomial infection is a global problem resulting in treatment failure particularly with respect to Pseudomonas aeruginosa. There is paucity of information on the molecular mechanisms of multidrug-resistant P. aeruginosa in Nigeria. Knowledge of the genetic basis of resistance of the organism to available antimicrobial agents will further improve empirical treatment. This study was undertaken to genetically characterise the multiple antibiotic resistance determinants in P. aeruginosa and describe the genetic locations of the resistance genes. Antimicrobial susceptibility of 54 clinical isolates of P. aeruginosa obtained from 5 hospitals in 3 southwestern states of Nigeria, to 21 antibiotics representing nine classes of antimicrobial agents was determined using the antibiotic disk-diffusion method. Minimum inhibitory concentrations were determined by Etest. Plasmid DNA for the isolates were extracted by alkaline lysis while plasmid curing was carried out using acridine orange, ethidium bromide and sodium dodecyl sulphate. The Extended-Spectrum Beta-Lactamase (ESBL) phenotypic detection was carried out using double-disk synergy method. Twenty clinical isolates with resistance to more than three "anti-pseudomonas" drugs were randomly selected for molecular studies. Genetic characterisation of ESBL and other drug resistant genes were achieved by polymerase chain reaction with specifically designed primers and direct sequencing of the amplicons. Significant trends in the association of plasmid counts and antimicrobial resistance among P. aeruginosa strains was achieved using Fisher's Exact Test. All the strains of P. aeruginosa were found to be resistant to ampicillin, tetracycline and amoxicillin-clavulanic acid, while 53.7%, 63.0%, 79.6% and 87.0% were resistant to ceftriaxone, carbenicillin, kanamycin, and ticarcillin-clavulanic acid respectively. Plasmid profile of the 54 isolates revealed the presence of 1-4 resistance plasmids varying in sizes from 2.3 to 210.0 kb. Highest curing activity was achieved with ethidium bromide on 81% of the isolates at 40 μg/mL. The MDR P. aeruginosa strains harboured significantly more plasmids (?3) compared to their non-MDR counterparts, which carried < 2 plasmids (p<0.01). Out of the 20 isolates randomly selected for molecular studies, 80% harboured blaOXA-10 that were plasmid encoded. Chromosomally encoded AmpC ?-lactamase was found in 85%, while blaSHV and blaCTXM-1 were detected in one isolate each. Efflux pump regulators: mexR and nfxB were found in 45%, aac (6?) � I was detected in 50% and ant (2??) � IV in 45% while both genes coding for aminoglycoside modifying enzymes were harboured in 35%. The class 1 integrons harbouring gene cassette array aaA6-orfD and aaA13, were also detected in the chromosomes of the isolates. The presence of resistance plasmids, class 1 integrons, extended-spectrum beta-lactamase, aminoglycoside modifying enzymes and efflux pump regulator genes among the population of P. aeruginosa tested indicated a high prevalence of multidrug resistance