Hussein O. M. Al-Dahmoshi¹, Noor S. Al-Khafaji², Ahmed Abdulzahra Jeyad³, Hasanain Khaleel Shareef⁴ and Rafah F. Al-Jebori⁵  

^1,2Biology Dept., College of Science-University of Babylon, Hilla-Iraq.

³Al-Sadiq Teaching Hospital, Babylon Health Directorate-Ministry of Health-Iraq.

⁴Biology Dept., College of Science for Women -University of Babylon, Hilla-Iraq.

⁵Laboratory Devision, Babylon Health Directorate-Ministry of Health-Iraq.

Corresponding Author E-mail: dr.dahmoshi83@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/1439

Abstract

Wound infections regards one of the most common infections encountered in hospital records. Pseudomonas aeruginosa regard the 3^rd  common pathogen among healthcare-related infections. Their ability to adapt to different conditions and presence of pool of virulence factors may render their infections delay in healing. During a period of  six months 114 wound swabs were collected and inoculated on Pseudomonas chromogenic agar and then Pseudomonas aeruginosa isolated confirmed by PCR using specific primer for 16S rDNA gene of Pseudomonas aeruginosa. Molecular investigation of some virulence factor like ExoA, OprL, OprI, LasI and LasB were performed using a sets of specific primer pairs. The results revealed that only 26 (22.8%) isolates were Pseudomonas aeruginosa and the coexistence of more than one  virulence factors within the same isolates was also recorder. OprI and LasB were most common followed by LasI, ExoA and OprL. Occurrence of virulence factor genes were 12(46.15%) for exoA, oprL was 11(42.3%), oprI was 22(84.61%), lasI was 14(53.84%) and lasB was 18(69.23%). 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-ranging of hard to cured infections and the delay in healing and worseness degree may be attributed to owning  multivirulence factors.

Keywords

 Aeruginosa; ExoA;  LasI; LasB  P. Virulence;

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Copy the following to cite this article: Al-Dahmoshi H. O. M, Al-Khafaji N. S, Jeyad A. A, Shareef H. K, Al-Jebori R. F. Molecular Detection of Some Virulence Traits Among Pseudomonas aeruginosa Isolates, Hilla-Iraq. Biomed Pharmacol J 2018;11(2).

Copy the following to cite this URL: Al-Dahmoshi H. O. M, Al-Khafaji N. S, Jeyad A. A, Shareef H. K, Al-Jebori R. F. Molecular Detection of Some Virulence Traits Among Pseudomonas aeruginosa Isolates, Hilla-Iraq. Biomed Pharmacol J 2018;11(2). Available from: http://biomedpharmajournal.org/?p=20508

Intriduction

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 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 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 Hilla-Iraq.

Materials And Methods

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/μl 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.

Table 1: Show primer pairs sequence and amplicon size.

Primer Name	Sequence (5՛-3՛)	Amplicon (bp)	References
PA-SS-F	GGGGGATCTTCGGACCTCA	956	[21]
PA-SS-R	TCCTTAGAGTGCCCACCCG
ETA-F	GACAACGCCCTCAGCATCACCA	397	[22]
ETA-R	CGCTGGCCCATTCGCTCCAGCG
oprL-F	ATG GAAATGCTGAAATTCGGC	504	[23]
oprL-R	CTTCTTCAGCTCGACGCGACG
oprI-F	ATGAACAACGTTCTGAAATTCTCTGCT	249	[23]
oprI-R	CTTGCGGCTGGCTTTTTCCAG
LasI-F	CGTGCTCAAGTGTTCAAGG	295	[24]
LasI-R	TACAGTCGGAAAAGCCCAG
lasB-F	TTCTACCCGAAGGACTGATAC	153	[24]
lasB-R	AACACCCATGATCGCAAC

 

Table 2: Show the PCR. conditions.

Gene	Ini. Denat.	Denat.	Anneal.			Exten.	Final. Exten.	Reference
16S rDNA	95°C. 2 min.	95°C. 30 sec.	61°C. 30 sec.			72°C. 100 sec.	72°C. 5 min.	Current study
# of Cycles	1	30					1
exoA	95°C. 2 min.	95°C. 30 sec.		64.6°C. 30 sec.	72°C. 40 sec.		72°C. 5 min.	Current study
# of Cycles	1	30					1
oprL	95°C. 2 min.	95°C. 30 sec.		60.7°C. 30 sec.	72°C. 30 sec.		72°C. 5 min.	Current study
# of Cycles	1	30					1
oprI	95°C. 2min.	95°C. 30 sec.		60.7°C. 30 sec.	72°C. 30 sec.		72°C. 5 min.	Current study
# of Cycles	1	30					1
lasI	95°C. 2 min.	95°C. 30 sec.		56.7°C. 30 sec.	72°C. 30 sec.		72°C. 5 min.	Current study
# of Cycles	1	30					1
lasB	95°C. 2 min.	95°C. 30 sec.		54.6°C. 30 sec.	72°C. 30 sec.		72°C. 5 min.	Current study
# of Cycles	1	30					1

 

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

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)

Figure 1: Percentage of isolate recovery of wound swabs.   Click here to View figure

 

Figure 2: Agarose electrophoresis (0.7%) of amplicon (956 bp) of  16S rDNA gene of Pseudomonas aeruginosa.   Click here to View figure

 

M lane represent 100 bp DNA Marker while the rest lanes represents samples.

Figure 3: Percentage of P. aeruginosa isolation among wound swabs.   Click here to View figure

 

Figure 4: Agarose electrophoresis (0.7%) of amplicon (397 bp) of  exoA gene of Pseudomonas aeruginosa.   Click here to View figure

 

M lane represent 100 bp DNA Marker while the rest lanes represents samples.

Figure 5: Agarose electrophoresis (0.7%) of amplicon (504 bp) of  oprL gene of P. aeruginosa.   Click here to View figure

 

M lane represent 100 bp DNA Marker while the rest lanes represents samples.

Figure 6: Agarose electrophoresis (0.7%) of amplicon (249 bp) of  oprI gene of P. aeruginosa.   Click here to View figure

 

M lane represent 100 bp DNA Marker while the rest lanes represents samples.

Figure 7: Agarose electrophoresis (0.7%) of amplicon (295 bp) of  lasI gene of P. aeruginosa.   Click here to View figure

 

M lane represent 100 bp DNA Marker while the rest lanes represents samples.

Figure 8: Agarose electrophoresis (0.7%) of amplicon (153 bp) of  lasB gene of P. aeruginosa.   Click here to View figure

 

M lane represent 100 bp DNA Marker while the rest lanes represents samples.

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).

Table 3: Show the coexisted virulence factors among isolates.

Virulence Factor	No. of Isolates
ExoA, OprL, OprI, lasI, LasB	8
ExoA, OprI, lasI, LasB	2
OprL, OprI, lasI, LasB	2
OprI, LasI, LasB	1
OprI, LasB	2
OprI, LasI	2
OprL, OprI	1
Total	18

 

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.

Acknowledgment

I would like to show our gratitude to the staff of advanced microbiology laboratory-College of science, University of Babylon for their assistance.

Conflict of Interest

There is no conflict of interest

Funding Source

The financial support is fully personal.

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