Molecular Characterization of Multi-Drug Resistant Clinical Isolates of Pseudomonas Aeruginosa in Nsukka Environs, Nigeria

Abstract

Pseudomonas aeruginosa is a frequent colonizer of hospitalized patients. It is responsible for serious infections such as meningitis, ear infections, urinary tract infections, wound infections and pneumonia. This study was performed to determine the prevalence, antimicrobial resistance patterns and clonal variations of multi-drug resistant Pseudomonas aeruginosa (MDRPA) from clinical samples. A total of 1000 samples comprising 950 clinical samples and 50 samples from hospital fomites were collected from various hospitals in Nsukka environs after obtaining informed consent and ethical approval for the study. The sample sources were wound / pus swabs, ear swabs, sputum, mid-stream urine and hospital fomites. These samples were inoculated on brain heart infusion broth and Pseudomonas cetrimide agar which was supplemented with 10 ml/L of glycerol and identified using standard microbiological techniques. Antibiotic resistance profiles of the isolates were determined using Kirby Bauer disk diffusion method. The minimum inhibitory concentrations (MIC) of the multi-drug resistant isolates were determined using macro broth dilution and E-test strip methods. Polymerase chain Reaction (PCR) was used to detect both the Pseudomonas aeruginosa consensus region of 16S rRNA and PstS genes with amplicon sizes of 1499 bp and 606 bp respectively. Genetic relatedness of the isolates was determined using plasmid DNA profiles, restriction patterns from restriction fragment length polymorphism (RFLP) using ALU 1 digest; random amplified polymorphic DNA using three oligonucleotide primers (B-01, B-11 and M13) and sequencing analyses. A total of 192 (19.20%) out of 1000 samples yielded Pseudomonas aeruginosa. The percentage occurrence of Pseudomonas aeruginosa from different clinical samples was as follows: wound / pus, 32.86%; urine, 5.33%; ear swabs 22.50%; sputum 12.00%; and hospital fomites 8.00%. The differences in prevalence of Pseudomonas aeruginosa among the different sample sources were significant (χ2 = 88.052, P = 0.001). A high degree of antibiotic resistance was observed among the Pseudomonas aeruginosa isolates. Out of the 192 Pseudomonas aeruginosa isolates, 136 (78.83%) were multi-drug resistant. The most resisted antibiotics were cefotaxime (88.02%) and Ticarcillin (87.50%), while the least resisted antibiotics were meropenem (24.48%); imipenem (12.50%) and polymyxin B (4.68%). The analysis of multiple antibiotic resistances (MAR) index of the MDRPA isolates showed MAR index values ranging from 0.27 to 0.91. The percentage occurrence of MDRPA among the various clinical samples was highest among isolates from wound / pus (80.87%) followed by isolates from hospital fomites (75.00%). There was a significant relationship between sample sources and MDRPA (χ2 = 15.235, P = 0.004). Minimum inhibitory concentration values of 4 different antibiotics (meropenem, ceftazidime, ciprofloxacin and gentamicin) against MDRPA were 0.12 to 32μg/ml; 2 to 64μg/ml; 8 to 128μg/ml and 8 to 64μg/ml, respectively. Out of 88 presumptive Pseudomonas aeruginosa, 71 (80.68%) were confirmed by sequencing and PCR detection of the 1499 bp amplicon of the 16S rRNA gene. Of these, 50% possessed the PstS gene for multidrug resistance. The sequence analysis revealed that all the isolates shared homology with each other and showed sequence similarity to known strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 27853; KT315654 Pseudomonas aeruginosa; KU321274 Pseudomonas aeruginosa and KT894767 Pseudomonas aeruginosa). The plasmid profile of the MDRPA revealed that all the strains were found to possess plasmid DNA bands of approximately 24.5 kb and 30.5 kb which showed relative homogeneity among the isolates. The PCR – RFLP analysis also revealed that there was a lot of genetic relatedness among the isolates. Fifteen restriction patterns that were grouped into 7 clones were detected. In RAPD analysis only the M13 primer was able to detect polymorphism while the other two B–01 and B-11 primers did not detect any polymorphism among the isolates. This study shows that there is high prevalence of MDRPA among the clinical isolates in Nsukka and there is a lot of genetic relatedness among the MDRPA isolates circulating in Nsukka area.