Isolation and characterisation of multi-drug resistant pseudomonas aeruginosa from clinical, environmental and poultry litter sources in Ashanti Region of Ghana.

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Antibiotic resistance in bacteria is now a major global health challenge. The increase and indiscriminate use of antibiotics is pivotal in the selection of resistant bacteria strains and the spread of resistance genes and resistance determining factors. The occurrence of Pseudomonas aeruginosa, a commonly implicated organism in nosocomial infections as well as poultry diseases has been found to be on the increase in samples in Ghana. This study therefore sought to determine the prevalence, susceptibility pattern, resistance mechanisms, resistance determining factors and the clonal relatedness of P. aeruginosa isolates obtained from stool, urine, blood, poultry litter and the environment in the Ashanti Region of Ghana. The P. aeruginosa isolates were identified using their biochemical characteristics and genotypically confirmed through PCR amplification of specific outer membrane lipoprotein (oprL) genes. Kirby-Bauer disc diffusion method was used to determine the susceptibility of the isolates to commonly used antipseudomonal agents. Plasmid sizes and resistance determining factors present in the isolates were detected using alkaline lysis method and PCR, respectively. Out of 900 samples screened, 87(9.7%) P. aeruginosa isolates were obtained. 75% of the P. aeruginosa isolates from the various sources were identified to be resistant to more than a single antipseudomonal agent and 38(43.6%) of the isolates were multidrug resistant (resistant to antibiotics from three or more antipseudomonal classes). The most common resistance pattern was observed with ciprofloxacin (62%), gentamicin (69%) and ticarcillin (56%). High prevalence of extended spectrum β-lactamases (84.2%), metallo- β-lactamases (34.1%) and AmpC inducible cephalosporinases (50%) were observed in the MDR xiv isolates. However, no strain produced KPC type carbapenemase. Among the MDR strains, 57.8% displayed moderate to very high efflux capacity and 65.7% of the MDR isolates haboured one to five plasmids with sizes ranging from 2.0kb to 116.8kb. While common β- lactamase encoding genes (blaSHV, blaTEM, blaCTX-M, blaVIM and blaIMP) were not detected in any MDR isolates, class 1 integrons were detected in 89.4% of the MDR isolates with 15.7% and 13.1% respectively carrying quinolone resistance gene mutations in gyrA and parC subunits of DNA gyrase and topoisomerase IV. Antibiogram typing was found to be discriminatory (D=0.9502), differentiating the MDR isolates into 24 antibiogram types with 19 distinct susceptibility patterns and 5 antibiogroups. Genotypic relatedness of the strains from the various sources generated through ERIC-PCR identified all the P. aeruginosa isolates to belong to two groups at a similarity of 62%. Dendrogram generated using Pearson coefficient as a similarity index and UPGMA as a distance measure revealed 27 P. aeruginosa genotypes. All the clinical strains of P. aeruginosa were closely related. From this study, there is the possibility of MDR P. aeruginosa transfer from the environment to patients as well as among patients in the same hospital. P. aeruginosa strains in humans and poultry may develop extensive antipseudomonal resistance which could be disseminated between patients and the environment.
Thesis submitted in fulfillment of the requirements for the degree of Master of Philosophy at the Kwame Nkrumah University of Science and Technology, Kumasi- Ghana,