Molecular detection of Virulence Genes oprI,ToxA and nan-1 of Pseudomonas aeruginosa Isolated from Different Clinical Specimens by Multiplex PCR, Khartoum, Sudan

Abstract

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, was estimated to be involved in 10% to 22.5% of the hospital-acquired infections (HAI) as well in adults as in children. Furthermore is frequently life-threatening and often challenging to treat because it expresses a combination of bacteria-associated factors (intrinsic and acquired antimicrobial resistance, expression of different virulence factors. This study attempt to determine the virulence genes of P.aeruginosa which had correlation with their antibiotic resistance using multiplex Polymerase Chain Reaction. Total of 50 Pseudomonas aeruginosa clinical isolates were collected from different hospitals, the samples were isolated on CLED and identified by gram stain, biochemical test and sensitivity test was carried, and were confirmed by Gene detection. The purity of the extracted DNA was determined by running the DNA sample on 1.5% agarose gel, All outcome data were analyzed by using Statistical Package for Social Sciences. The oprI genes were detected in 48 isolates (96%) and negative in 2 isolate (4%). They were recovered from urine 17 (94%), wound 10 (90%), ear swab 3 (100%), sputum 8(100%), blood 6(100%) high vaginal swabs 2 (100%) and body fluid 2(100%).The ToxA gene was detected in 47(94%) and negative in 3 isolate. nan1 gene was detected in 6(12%) and were negative in 44(88). There was significant difference in the prevalence of virulence genes among the different sites. The gene ToxA gene was harbored in all .samples, especially in urine samples which was significantly higher than wounds specimen. Comparing to other genes, Nan1 gene was higher percent in sputum 2 (25 %) and wound samples 2 (18 %). All 50 isolates were cultured in Mueller-hinton medium using 0.5 Mcfarland turbidity standard for antibiotic susceptility testing against Amikacin (30mcg), colistin (10 mcg), aztreonam (30mcg), ciprofloxacin (5mcg), gentamicin (10mcg), imipenem (10mcg) and ceftazidime (30mcg), piperacillin /tazobactam (100/10 mcg) discs. The overall results revealed that there was significant strong association between the presence of (oprI, toxA and nan1) genes and sensitive to Ciprofloxacin (p-value 0.4- 0.3 and 0.1 respectively). This study showed that the oprI and Toxa genes are commonly disseminated among the P. aeruginosa. The differences in the distributions of virulence genes in the isolated strains need further studies to find out the actual role of these genes of P. aeruginosa in their resistance to antibiotics. PCR showed that all P. aeruginosa strains do not necessarily have similar virulence genes. It seems that simultaneous use of oprI genes provides more confident detection of P. aeruginosa by PCR .