Molecular detection and Expression of some Virulence Genes in Pseudomonas aeruginosa Isolated from Wounds

Abstract

Pseudomonas aeruginosa considered to be toxigenic and an invasive, that induces infections in abnormal host defenses patients. Besides, it is very important pathogenic nosocomial which produces infection in wounds, giving rise to blue-green pus. In this study 61 swabs were collected from wounds infection and cultured, the isolated bacteria have been identified by biochemical tests, and VITEK-2 compact System. Drug susceptibility tests for the 30 isolates of P. aeruginosa were done. Out of 30(49.2%) P. aeruginosa isolates were 20 (32.8%) Resistant isolates and 10 (16.4%) Sensitive isolates were found. The DNA has been isolated from 30 P. aeruginosa isolates and have been extracted by extraction kit of DNA. For 1µl of DNA ,199 µl of diluted Quanty Flour Dye was mixed. DNA concentration values has been estimated after 5min incubation at room temperature. In order to screening the virulence genes, Polymerase Chain Reaction has been utilized , (exoU, exoS, lasB) and resistance genes (ampC, oprM, oprN, rpoS). Result revealed that, isolates were positive for exoU gene 33.3%, exo S gene was 56%, lasB gene 93.3%, oprM and oprN genes were100%, ampC gene was 100% and rpoS gene were 100% in wound swabs. The technique of (RT-qPCR) was used for the transcription of the RNA to cDNA, followed by their amplification. The result showed that the exoU gene expression was increase (2.00,40.39) in resistance isolates when used Imipenem and Ciprofloxacin, respectively, compared with sensitive isolates. Expression of oprM gene increase (3.66) in resistance isolates while used Ciprofloxacin more than in sensitive isolates.    Expression of lasB gene increase in resistance isolates when used Erythromycin (30.91) more than in sensitive isolates. While expression of oprM and rpoS genes, in sensitive isolates when used Gentamycin and Ciprofloxacin respectively, increased in able this bacterium to become resistant to those antibiotics compared with already resistance isolates to those antibiotics.  In rpoS, oprM and ampC genes, fold change expression was reduced while treated isolates by Imipenem and Ceftazidime respectively.  In addition, we found that tow genes (rpoS and exoS) have not contribution in Ciprofloxacin antibiotic resistance process.