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 isolates
which previously isolated from sputum, burns, urine, and ear swab clinical patients samples. The reverse
transcription Real Time PCR (RT-qPCR) was performed for molecular detection and quantification of gene
expression levels for efflux pump antibiotic resistance gene (mexA, mexB, mexC, mexE, and mexY) and
chromosomal lactamase (AmpC) gene, as well as outer membrane prion protein (OprD) gene in extensive
drug 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 target
genes with suitable housekeeping gene. In this study 30S ribosomal protein S12 (rpsL) housekeeping gene
was used in normalization of antibiotic resistance genes expression levels in extensive drug pseudomonas
aeruginosa isolates. The efflux pump resistance gene expression were by RT-qPCR technique. The relative
gene 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) fold
change compared to wild type PA control isolate. The relative gene expression analysis of chromosomal
lactamase AmpC gene was showed highest frequency of overexpression (100%) in XDR isolates with
expression levels ranged (6.77- 73.1) fold change compared to wild type PA control isolate. Whereas, the
relative gene expression analysis of OprD gene was showed (68.75%) frequency of down expression in
XDR Pseudomonas aeruginosa isolates with expression levels ranged (0.01-0.16) fold change compared to
wild type PA control isolate. In conclusion, The XDR Pseudomonas aeruginosa isolates can ability to over
expression production of inducible chromosomal lactamase AmpC gene, efflux pumps protein genes, and
low expression outer membrane prion protein (OprD) gene as important antimicrobial mechanism against
Cephalosporins and Carbapenems antibiotic classes.