Molecular Detection and Quantification Gene Expression of Efflux Pump Antibiotic Resistance Genes in Extensive Drug Resistance Pseudomonas Aeruginosa Isolated from Clinical Infection Patients in Al Diwaniyah City of Iraq

Abstract

The study was included investigated 16 extensive drug resistance pseudomonas aeruginosa isolateswhich previously isolated from sputum, burns, urine, and ear swab clinical patients samples. The reversetranscription Real Time PCR (RT-qPCR) was performed for molecular detection and quantification of geneexpression levels for efflux pump antibiotic resistance gene (mexA, mexB, mexC, mexE, and mexY) andchromosomal lactamase (AmpC) gene, as well as outer membrane prion protein (OprD) gene in extensivedrug pseudomonas aeruginosa isolates. The relative expression analysis was done by used the 2-??CT (Livak)method, this method was used for normalization of expression levels (mRNA transcript levels) in targetgenes with suitable housekeeping gene. In this study 30S ribosomal protein S12 (rpsL) housekeeping genewas used in normalization of antibiotic resistance genes expression levels in extensive drug pseudomonasaeruginosa isolates. The efflux pump resistance gene expression were by RT-qPCR technique. The relativegene expression analysis were showed highest frequency of overexpression at (100%, 100%, 87.5%,81.25%, 56.25%) for (mexB, mexE, mexA,mexC, and mexY) with fold change ranged (2.16- 184.82) foldchange compared to wild type PA control isolate. The relative gene expression analysis of chromosomallactamase AmpC gene was showed highest frequency of overexpression (100%) in XDR isolates withexpression levels ranged (6.77- 73.1) fold change compared to wild type PA control isolate. Whereas, therelative gene expression analysis of OprD gene was showed (68.75%) frequency of down expression inXDR Pseudomonas aeruginosa isolates with expression levels ranged (0.01-0.16) fold change compared towild type PA control isolate. In conclusion, The XDR Pseudomonas aeruginosa isolates can ability to overexpression production of inducible chromosomal lactamase AmpC gene, efflux pumps protein genes, andlow expression outer membrane prion protein (OprD) gene as important antimicrobial mechanism againstCephalosporins and Carbapenems antibiotic classes.