Muhammad Bilal Habib ¹ Naseer Ali Shah ^1* AFREENISH AMIR ^2* HUDA AHMED ALGHAMDI ^3* MUHAMMAD HASEEB TARIQ ⁴ KIRAN NISA ^1* Marium Ammoun ^4*

• ¹ Department of Biosciences, COMSATS University, Islamabad, 44000, Pakistan., ISLAMABAD, Pakistan
• ² National Institute of Health (Pakistan), Islamabad, Islamabad, Pakistan
• ³ Department of Biology, College of Sciences, King Khalid University, Abha, Saudi Arabia, ABHA, Saudi Arabia
• ⁴ Department of Pathology, Viva Health Laboratories, New Lodge Drift Road, Windsor SL4 4RR United Kingdom, WINDSOR, United Kingdom

Emerging drug resistance in Pseudomonas aeruginosa is of great concern in clinical settings. P. aeruginosa activates its efflux-pump system in order to evade the effect of antibiotics. The current investigation aims to detect MexB genes in P. aeruginosa, their structural and molecular analysis and their impact on antimicrobial susceptibility profiling. A total of 42 clinical specimens were aseptically collected from hospitalized patients who had underlying infections related to medical implants. Matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) were used for the identification of isolates. The methods used in this study were antibiotic susceptibility profiling, minimum inhibitory concentration (MIC), polymerase chain reaction (PCR), sanger sequencing, phylogenetic analysis, MolProbity score, Ramachandran plot analysis and multiple sequence alignment. The highest resistance was shown by P. aeruginosa against cefoperazone (67%), gentamycin and amikacin (66%) each, followed by cefotaxime (64%). The prevalence of multi-drug resistant (MDR) and extensively drug resistant (XDR) was 57% and 12%, respectively. The presence of an active efflux-pump system was indicated by the MexB genes found in most of the resistant isolates (p<0.05). Following addition of efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a significant decrease (p<0.05) in MIC was observed in resistance, that revealed the presence of active efflux pump system. Phylogenetic analysis revealed evolutionary relationships with the P. aeruginosa strains isolated in Switzerland, Denmark and Germany. Protein domain architecture revealed that MexB gene proteins were involved in particular efflux pump function. Protein sequences aligned by multiple sequence alignment revealed conserved regions and sequence variants, which suggested antibiotic translocation and evolutionary divergence. These highly conserved regions could be used for diagnostic purposes of efflux pump MexB genes. To avoid their spread in hospital settings, responsible authorities ought to begin rigorous initiatives in order to reduce the prevalence of multi-drug resistant, extensively drug resistant, and efflux pump carrying isolates in clinical settings.