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Title: Genetic Diversity and Molecular Mechanisms of Antibiotic Resistance and Virulence in Clinical Isolates of Pseudomonas Aeruginosa
Authors: Ali, Amjad
Keywords: Biological & Medical Sciences
Issue Date: 2020
Publisher: University of Peshawar, Peshawar.
Abstract: Pseudomonas aeruginosa is pathogenic bacterium and leading cause of nosocomial infections around the world. The large and complex genome of P. aeruginosa demonstrates an evolutionary adaptation and enables the bacterium to thrive in diverse environmental conditions and resist variety of antimicrobials. Current study aimed at investigation of multi-drug resistant (MDR) P. aeruginosa isolated from different clinical specimens. The isolates were analayzed phenotypically for occurrence of extended spectrum beta-lactamases (ESBL) and metallo beta lactamases (MBL). Molecular analysis of extended spectrum beta-lactamases (blaCTX-M, blaOXA-10, blaPER-1, blaSHV and blaTEM) and metallo beta lactamases (bla-IMP, bla-VIM, bla-NDM1 and bla-SPM1) was performed in the isolates. Virulence genes (algD, lasB, toxA, plcH, plcN and exoS) of the isolates were analyzed. The study also focused on estimation of genetic diversity among the clinical isolates of P. aeruginosa. A total of 187 P. aeruginosa isolates were recovered from different clinical specimens collected from tertiary care hospitals of Peshawar, Pakistan. Antibiotic susceptibility was evaluated by Kirby Bauer disc diffusion method using nineteen different antibiotics and multiple antibiotic resistance (MAR) index was determined. Phenotypic analysis of extended spectrum β-lactamases and metallo beta-lactamases were studied by double disc synergy test and combine disc test respectively. Molecular analyses of ESBL, MBL and virulence genes were performed by PCR amplification of the target genes. Genetic diversity among the isolates was determined through RAPD-PCR. A total of 36.89% (n=69) isolates showed multi drug resistance. Imipenem and meropenem were most effective antibiotics with 85.02% and 82.88% susceptibility respectively. All imipenem resistant isolates were susceptible to colistin. The multiple antibiotic resistance (MAR) index of 34.22% (n=64) isolates was higher than 0.2. Phenotypically, 21.39% (n=40) isolates showed ESBL production and 76% (n=19) imipenem resistant isolates showed MBL production. Prevalence of ESBL genes blaOXA-10, blaCTX-M, blaTEM and blaSHV was 36.89% (n=69), 20.85% (n=39), 5.34% (n=10) and 3.2% (n=6) respectively. The occurrence of MBL genes blaIMP, blaNDM-1 and blaVIM was 68% (n=17), 48% (n=12) and 4% (n=1) respectively among imipenem resistant isolates. The blaPER-1 and blaSPM-1 genes were not detected. The occurrence of virulence xvi genes algD, lasB, plcH, exoS, plcN and toxA was 98.53% (n=179), 98.53% (n=179), 97.8% (n=178), 96.15% (n=175), 93.4% (n=170) and 85.71% (n=156) respectively. RAPD typing showed high genetic diversity among the isolates. Out of 182 isolates of P. aeruginosa, 171 isolates showed different RAPD profile (93.95% polymorphism), 160 were unique RAPD strains and based on similarity coefficient ≥ 80% 22 isolates were clustered into 11 distinct clones. In conclusion, resistance to antibiotics is increasing in P. aeruginosa which is a matter of concern and needs proper management. Spread of MDR, ESBL, MBL and virulence genes among the clinical isolates of P. aeruginosa in the regional hospitals is shocking. Genotypic surveillance studies must be performed on regular basis in order to control the spread of infectious diseases. Good infection control policies, strict hygienic conditions and appropriate use of antibiotics are vital to halt further spread of resistant, virulent and genetically diverse strains of P. aeruginosa.
Gov't Doc #: 21630
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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