Molecular Detection of Some Virulence Traits among Pseudomonas aeruginosa Isolates , Hilla-Iraq

Published by Oriental Scientific Publishing Company © 2018 This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted Non Commercial use, distribution and reproduction in any medium, provided the original work is properly cited. Molecular Detection of Some Virulence Traits among Pseudomonas aeruginosa Isolates, Hilla-Iraq

Pseudomonas aeruginosa is a momentous life-intimidating, hospital pathogen that play a noticeable role in wound infections of burned patients [1,2].It is success can be attributed to wide arrays of virulence factors which leads to adaptation and withstand for different inconvenient niches.Burn injuries stays one of the utmost public forms of trauma that push a major community health problem internationally.Skin compile a physical barricade against invasive microbes and the infections resulted from burn is common when this barrier breached.It is the supreme cause of burn wound infections [3,4,5,6].The notable aptitude of P. aeruginosa to acclimatize and flourish among varied environments due to its extensive inherited adaptability and its intrinsic and acquired resistance to different antibiotics, which contributes meaningfully to its possible pathogenicity [7,8].Wound-associated infections are frequently hard to treat due to various bacterial pathogens [9,10,11,12,13].
P. aeruginosa have an arrays of virulence factors that frustrate host defenses and make direct tissue's harm or rise bacterium's affordability.The most important virulence factors of P. aeruginosa includes Exotoxin A (ETA), outer membrane associated protein L and I and quorum-sensing determinant system [14].Exotoxin A represent the main dangerous virulence factor produced by P. aeruginosa [15].OprL and OprI are peptidoglycanrelated outer membrane proteins that fetter the outer membrane to the principal peptidoglycan.Lipoprotein of outer membrane lipoprotein (OprL) concerned in purge transporting systems leading to disturbing cell penetrability [16].They are accountable for intrinsic resistance to antiseptics and antibiotics.Quorum-sensing (QS) system permits bacteria to respond to their community mass by harmonized adaptable their gene expression patterns.It is vital for the countenance of a series of virulence traits in addition to biofilm development [17,18,19,20].
The current study aimed to investigate the occurrence of exoA, oprL, oprI, lasI and lasB gene among P. aeruginosa isolated from wound infection in Hlla-Iraq.

Samples
Wounds of 114 burned patients were swabbed from patients admitted to Al-Hilla teaching hospitals, Hilla-Iraq during a period of 6 months.All swabs were firstly cultivated on Pseudomonas chromogenic agar plate (Condalab/ Spain) and then confirmed as Pseudomonas aeruginosa by amplification of 16S rDNA gene using specific primer pairs.

DNA Extraction
Preparation of bacterial samples were performed according to Al-Dahmoshi (2017) 6 and then the steps of protocol of Mini DNA extraction kit (Favorgen/Taiwan) were followed.The extracted DNA checked using gel electrophoresis (0.7%) and then stained with safe DNA stain, RedSafe (IntronBio/Korea) and then visualized using Gel Documentation (Vilber/France).

Preparation of Primer and PCR
A working solution of primer pair were prepared by addition of nuclease free water (New England Biolabs/UK) to the lyophilized primer pairs (Realgene/China) to get 10pmole/¼l concentration.A reaction mixture of 25 µl were prepared using OneTaq Quick-Load 2X Master  Mix.The primer sequence and PCR conditions were mentioned in the table 1 and 2 respectively.

RESULTS AND DISCUSSION
Results of isolation revealed that 39 isolate were Pseudomonas spp.while amplification  of 16S rDNA gene showed that only 26 (22.8%) isolates were Pseudomonas aeruginosa (figure 1).The PCR product was 956 bp (figure 2) and this results was in accordance with many local and international studies who found that the isolation percentage of P. aeruginosa were 22-32% 1,8,12,25,26] .
The highest isolation percentage may be due to the fact that it is the 3 rd popular pathogen related with hospital-acquired.infections [25,26] .
ExoA responsible for toxigenesis trait of P. aeruginosa while invasiveness achieved by LasB and so the coexistence of ExoA, LasI and LasB let both of mechanism of infection available and increase the degree of wound worseness [27,28,29,30] .ExoA had distinct role in hindrance of wound contraction and remedial [15] .Both of OprL and OprI have a role in antibiotic resistance via efflux mechanism and alterations of membrane permeability.OprL, OprI and LasI were engaged in antibiotic resistance and biofilm creation leads to many problems concerning treatment of P. aeruginosa infections and make the infection hard to cured [31,32] .Bacteria attachment and immune system disruption can be facilitated by LasB.It is a protease (Elastase) that splits collagen, immunoglobulin G and A and complement moreover to destruction of fibronectin to uncover ligands for bacterial adhesion [27] .Nikbin et al., (2012) found that all isolates carried oprI, oprL and lasB genes [16] .The presence of ExoA, OprL, OprI, LasI and LasB among P. aeruginosa isolates suppose their linking with different levels of intrinsic virulence and pathogenicity [33,34] .The presence of toxins and enzymes genes make them important clinically and environmtally [35] .
Results of this study can lead us to conclude that P. aeruginosa have an arrays of virulence traits via which can adapt to different conditions and so cause a wide range of hard to cured infections and the delay in healing and worseness degree may be attributed to owning multivirulence factors.

Table 1 .
Show primer pairs sequence and amplicon size

Table 2 .
Show the PCR.conditions Fig. 1.Percentage of isolate recovery of wound swabs

Table 3 .
Show the coexisted virulence factors among isolates