Sirna-Mediated Gene Silencing of Efflux Pump Genes in Multidrug-Resistant Pseudomonas Aeruginosa Isolated from Iraqi Patients

Abstract

Background: Pseudomonas aeruginosa is an important opportunistic bacterium responsible for multidrug-resistant infections in hospitalised patients. The MexAB-OprM efflux pump system is a major contributor to antibiotic resistance by the expulsion of antimicrobial drugs from bacterial cells. Therefore, siRNA-mediated gene silencing is being developed as a promising method for resistance-related gene silencing and improving therapeutic efficacy.

Objective: To explore the prevalence and antibiotic resistance profile of Pseudomonas aeruginosa infection clinical isolates and assess the silencing of efflux pump genes (mexB and oprM) that are linked to multidrug resistance and virulence by siRNA.

Methods: Twenty-nine P. aeruginosa strains were recovered from 200 clinical specimens. The identification was achieved by morphological characteristics, biochemical tests, and with the aid of the Vitek 2 Compact system. The antimicrobial susceptibility was tested using 13 antibiotics by the Kirby-Bauer disk diffusion method. Production of AmpC β-lactamase was phenotypically determined, and efflux pump activity was determined by the ethidium bromide cartwheel method. The genes, mexB and oprM, were detected by conventional PCR. A 21-bp siRNA duplex was designed that targets these efflux pump genes, and quantitative RT-PCR was used to assess the level of gene expression prior to and following siRNA treatment.

Results: The highest level of antibiotic resistance was seen with ticarcillin-clavulanate and gentamicin (89.7%), and the lowest was with piperacillin-tazobactam (24.1%) of the 29 isolates. Sixty-seven percent (12/12) of the isolates produced AmpC β-lactamase. None of the 9 (45%) isolates that were found to be positive by phenotypic testing for efflux pump activity were characterized as multidrug resistant. The 9 isolates (45%) identified as efflux pump positive, none were classified as MDR. All the selected isolates were positive for the mexB and oprM genes by conventional PCR. After siRNA transfection, qPCR showed a dramatic down-regulation of the expression of both genes when compared to the control. The fold change of mexB was found to be 2.107 to 0.0006, and that of oprM was 1.521 to 0.0032, respectively, which means the gene silencing was effective.

Conclusion: This study shows the high prevalence of antibiotic resistance and virulence mechanisms associated with efflux pumps in clinical isolates of P. aeruginosa. siRNA-mediated knockdown of mRNA expression of mexB and oprM genes was found to be highly efficient, suggesting that siRNA-based therapy could be an effective approach for the treatment of multidrug-resistant P. aeruginosa infections.