{"id":20508,"date":"2018-06-25T11:06:27","date_gmt":"2018-06-25T11:06:27","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=20508"},"modified":"2020-04-23T07:24:39","modified_gmt":"2020-04-23T07:24:39","slug":"molecular-detection-of-some-virulence-traits-among-pseudomonas-aeruginosa-isolates-hilla-iraq","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol11no2\/molecular-detection-of-some-virulence-traits-among-pseudomonas-aeruginosa-isolates-hilla-iraq\/","title":{"rendered":"Molecular Detection of Some Virulence Traits Among Pseudomonas aeruginosa Isolates, Hilla-Iraq"},"content":{"rendered":"

Intriduction<\/strong><\/p>\n

Pseudomonas aeruginosa<\/em> is a momentous life-intimidating, hospital pathogen that play a noticeable role in wound infections of burned patients.1,2<\/sup>\u00a0It 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\u00a0 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 \u00a0the supreme cause of burn wound infections.3,4,5,6\u00a0\u00a0<\/sup>The notable aptitude of P. aeruginosa<\/em> 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]<\/sup>. Wound-associated infections are frequently hard to treat due to various bacterial pathogens.9,10,11,12,13<\/sup><\/p>\n

P. aeruginosa<\/em> 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<\/em> includes Exotoxin A (ETA), outer membrane associated protein L and I and quorum-sensing determinant system.14\u00a0<\/sup>Exotoxin A represent\u00a0 the main dangerous virulence factor produced by P. aeruginosa.<\/em>15\u00a0<\/sup>OprL and OprI are peptidoglycan-related 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\u00a0<\/sup>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<\/sup><\/p>\n

The current study aimed to investigate the occurrence of\u00a0 exoA, oprL, oprI, lasI <\/em>and lasB<\/em> gene among P. aeruginosa isolated from wound infection in Hilla-Iraq.<\/p>\n

Materials And Methods<\/strong><\/p>\n

Samples<\/strong><\/p>\n

Wounds of\u00a0 114 burned\u00a0 patients were \u00a0swabbed 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\u00a0 16S rDNA gene using specific primer pairs.<\/p>\n

DNA Extraction<\/strong><\/p>\n

Preparation of bacterial samples were performed according to Al-Dahmoshi (2017)[6]<\/sup> 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).<\/p>\n

Preparation of Primer and PCR<\/strong><\/p>\n

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\/\u03bcl concentration. A reaction mixture of 25 \u03bcl were prepared using OneTaq Quick-Load 2X Master Mix. The primer sequence and PCR conditions were mentioned in the table 1 and 2 respectively.<\/p>\n

Table 1: Show primer pairs sequence and amplicon size.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Primer Name<\/strong><\/td>\nSequence (5\u055b-3\u055b) <\/strong><\/td>\nAmplicon\u00a0<\/strong>(bp)<\/strong><\/td>\nReferences<\/strong><\/td>\n<\/tr>\n
PA-SS-F<\/td>\nGGGGGATCTTCGGACCTCA<\/td>\n956<\/td>\n[21]<\/td>\n<\/tr>\n
PA-SS-R<\/td>\nTCCTTAGAGTGCCCACCCG<\/td>\n<\/tr>\n
ETA-F<\/td>\nGACAACGCCCTCAGCATCACCA<\/td>\n397<\/td>\n[22]<\/td>\n<\/tr>\n
ETA-R<\/td>\nCGCTGGCCCATTCGCTCCAGCG<\/td>\n<\/tr>\n
oprL-F<\/td>\nATG GAAATGCTGAAATTCGGC<\/td>\n504<\/td>\n[23]<\/td>\n<\/tr>\n
oprL-R<\/td>\nCTTCTTCAGCTCGACGCGACG<\/td>\n<\/tr>\n
oprI-F<\/td>\nATGAACAACGTTCTGAAATTCTCTGCT<\/td>\n249<\/td>\n[23]<\/td>\n<\/tr>\n
oprI-R<\/td>\nCTTGCGGCTGGCTTTTTCCAG<\/td>\n<\/tr>\n
LasI-F<\/td>\nCGTGCTCAAGTGTTCAAGG<\/td>\n295<\/td>\n[24]<\/td>\n<\/tr>\n
LasI-R<\/td>\nTACAGTCGGAAAAGCCCAG<\/td>\n<\/tr>\n
lasB-F<\/td>\nTTCTACCCGAAGGACTGATAC<\/td>\n153<\/td>\n[24]<\/td>\n<\/tr>\n
lasB-R<\/td>\nAACACCCATGATCGCAAC<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/strong><\/p>\n

Table 2: Show the PCR. conditions.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Gene<\/strong><\/td>\nIni. Denat.<\/strong><\/td>\nDenat.<\/strong><\/td>\nAnneal.<\/strong><\/td>\nExten.<\/strong><\/td>\nFinal. Exten.<\/strong><\/td>\nReference<\/strong><\/td>\n<\/tr>\n
16S rDNA<\/td>\n95\u00b0C.<\/p>\n

2 min.<\/td>\n

95\u00b0C.<\/p>\n

30 sec.<\/td>\n

61\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

100 sec.<\/td>\n

72\u00b0C.<\/p>\n

5 min.<\/td>\n

Current study<\/td>\n<\/tr>\n
# of Cycles<\/td>\n1<\/td>\n30<\/td>\n1<\/td>\n<\/tr>\n
exoA<\/td>\n95\u00b0C.<\/p>\n

2 min.<\/td>\n

95\u00b0C.<\/p>\n

30 sec.<\/td>\n

64.6\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

40 sec.<\/td>\n

72\u00b0C.<\/p>\n

5 min.<\/td>\n

Current study<\/td>\n<\/tr>\n
# of Cycles<\/td>\n1<\/td>\n30<\/td>\n1<\/td>\n<\/tr>\n
oprL<\/td>\n95\u00b0C.<\/p>\n

2 min.<\/td>\n

95\u00b0C.<\/p>\n

30 sec.<\/td>\n

60.7\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

5 min.<\/td>\n

Current study<\/td>\n<\/tr>\n
# of Cycles<\/td>\n1<\/td>\n30<\/td>\n1<\/td>\n<\/tr>\n
oprI<\/td>\n95\u00b0C.<\/p>\n

2min.<\/td>\n

95\u00b0C.<\/p>\n

30 sec.<\/td>\n

60.7\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

5 min.<\/td>\n

Current study<\/td>\n<\/tr>\n
# of Cycles<\/td>\n1<\/td>\n30<\/td>\n1<\/td>\n<\/tr>\n
lasI<\/td>\n95\u00b0C.<\/p>\n

2 min.<\/td>\n

95\u00b0C.<\/p>\n

30 sec.<\/td>\n

56.7\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

5 min.<\/td>\n

Current study<\/td>\n<\/tr>\n
# of Cycles<\/td>\n1<\/td>\n30<\/td>\n1<\/td>\n<\/tr>\n
lasB<\/td>\n95\u00b0C.<\/p>\n

2 min.<\/td>\n

95\u00b0C.<\/p>\n

30 sec.<\/td>\n

54.6\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

30 sec.<\/td>\n

72\u00b0C.<\/p>\n

5 min.<\/td>\n

Current study<\/td>\n<\/tr>\n
# of Cycles<\/td>\n1<\/td>\n30<\/td>\n1<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/strong><\/p>\n

Results and Discussion<\/strong><\/p>\n

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 \u00a0P. aeruginosa<\/em> were 22-32%.1,8,12,25,26<\/sup> The highest isolation percentage may be due to the fact that it is the 3rd<\/sup> \u00a0popular pathogen related with hospital-acquired. infections.25,26<\/sup><\/p>\n

The PCR results of virulence factor occurrence showed that exoA was present among 12(46.15%), oprL was 11(42.3%), oprI was 22(84.61%), lasI was 14(53.84%) and lasB was 18(69.23%) (figure 3). Amplicon of exoA was 397 bp, 504 bp for oprL, 249 bp for oprI, 295 bp for lasI and 153 bp for lasB (figure 4,5,6,7,8)<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 1: Percentage of isolate recovery of wound swabs.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 2: Agarose electrophoresis (0.7%) of amplicon (956 bp) of\u00a0 16S rDNA gene of Pseudomonas aeruginosa.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

M lane represent 100 bp DNA Marker while the rest lanes represents samples.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 3: Percentage of P. aeruginosa<\/em> isolation among wound swabs.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/a><\/td>\nFigure 4: Agarose electrophoresis (0.7%) of amplicon (397 bp) of\u00a0 exoA gene of Pseudomonas aeruginosa.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

M lane represent 100 bp DNA Marker while the rest lanes represents samples.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 5: Agarose electrophoresis (0.7%) of amplicon (504 bp) of\u00a0 oprL gene of P. aeruginosa<\/em>.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

M lane represent 100 bp DNA Marker while the rest lanes represents samples.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 6: Agarose electrophoresis (0.7%) of amplicon (249 bp) of\u00a0 oprI gene of P. aeruginosa<\/em>.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

M lane represent 100 bp DNA Marker while the rest lanes represents samples.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 7: Agarose electrophoresis (0.7%) of amplicon (295 bp) of\u00a0 lasI gene of P. aeruginosa<\/em>.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

M lane represent 100 bp DNA Marker while the rest lanes represents samples.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 8: Agarose electrophoresis (0.7%) of amplicon (153 bp) of\u00a0 lasB gene of P. aeruginosa<\/em>.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

M lane represent 100 bp DNA Marker while the rest lanes represents samples.<\/p>\n

Coexistence of more than one virulence factor within the same isolate were recorder and the results displayed that 8\/26 have all five virulence factors, 4\/26 have four virulence factors, 1\/26 have three virulence factors and 5\/26 have only two virulence factors (table 3).<\/p>\n

Table 3: Show the coexisted virulence factors among isolates.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
Virulence Factor<\/strong><\/td>\nNo. of Isolates <\/strong><\/td>\n<\/tr>\n
ExoA, OprL, OprI, lasI, LasB<\/td>\n8<\/td>\n<\/tr>\n
ExoA, OprI, lasI, LasB<\/td>\n2<\/td>\n<\/tr>\n
\u00a0OprL, OprI, lasI, LasB<\/td>\n2<\/td>\n<\/tr>\n
OprI, LasI, LasB<\/td>\n1<\/td>\n<\/tr>\n
OprI, LasB<\/td>\n2<\/td>\n<\/tr>\n
OprI, LasI<\/td>\n2<\/td>\n<\/tr>\n
OprL, OprI<\/td>\n1<\/td>\n<\/tr>\n
Total<\/td>\n18<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

ExoA responsible for toxigenesis\u00a0 trait of P. aeruginosa<\/em> 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<\/sup>\u00a0ExoA had distinct role in hindrance of wound contraction and remedial.15\u00a0<\/sup>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\u00a0<\/sup>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]<\/sup>. Nikbin et al., (2012) found that all\u00a0 isolates carried\u00a0oprI<\/em>,\u00a0oprL<\/em>\u00a0and\u00a0lasB<\/em>\u00a0genes.16<\/sup>\u00a0The presence of ExoA, OprL, OprI, LasI and LasB among P. aeruginosa<\/em>\u00a0isolates suppose \u00a0their linking with different levels of intrinsic virulence and pathogenicity.33,34\u00a0<\/sup>The presence of toxins and enzymes genes make them important clinically and environmtally.35<\/sup><\/p>\n

Results of this study can lead us to conclude that P. aeruginosa<\/em> 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\u00a0 multivirulence factors.<\/p>\n

Acknowledgment<\/strong><\/p>\n

I would like to show our gratitude to the staff of advanced microbiology laboratory-College of science, University of Babylon for their assistance.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

There is no conflict of interest<\/p>\n

Funding Source<\/strong><\/p>\n

The financial support is fully personal.<\/p>\n

References<\/strong><\/p>\n

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