The present study aimed to identify two virulence genes in P. aeruginosa isolated from infected burn wound. Out of 70 burn swabs, 54 (77%) showed positive growth on enriched media which obtained from burn wound at Al-Mussan Teaching Hospital in Mussan province, Iraq. Only 13 isolates (35%) identified as P. aeruginosa, and those isolates examined using Polymerase Chain Reaction (PCR) for detection the exoS and phzS genes. The results recorded that 46% and 77% of isolates have exoS, phzS genes, respectively. We concluded a P. aeruginosa harbor diverse percentages of both  genes as virulence factors, and those factors must be increased the pathogenicity of this pathogen

Ahmed A. Nasir

Alaa Methqal Nassar

Haydar Khamis Al-Malk

Zainab Dakhil Degaim

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University of Thi-Qar Journal Of Science (UTsci) Website: jsci.utq.edu.iq                                                                                                 Email: jsci@utq.edu.iq Volume 7, Number 1, June 2019  26   Molecular detection of two virulence factors of Pseudomonas  aeruginosa isolated from burn patients  1Zainab Dakhil Degaim*    2Haydar Khamis Al-Malk   3Alaa Methqal Nassar  4Ahmed A. Nasir   1*Microbiology department-College of Medicine-University of Thi-Qar, Iraq.  2Microbiology department-College of Veterinary Medicine- University of Thi-Qar. 3Private Mazaiea College.  4Medical Physical and physiology department-College of Medicine-University of Thi-Qar.  *Email: haydarkhamis@yahoo.com  *Mobile: 07807605134  Abstract:- The present study aimed to identify two virulence genes in P. aeruginosa isolated from infected burn wound. Out of 70 burn swabs, 54 (77%) showed positive growth on enriched media which obtained from burn wound at Al-Mussan Teaching Hospital in Mussan province, Iraq. Only 13 isolates (35%) identified as P. aeruginosa, and those isolates examined using Polymerase Chain Reaction (PCR) for detection the exoS and phzS genes. The results recorded that 46% and 77% of isolates have exoS, phzS genes, respectively. We concluded a P. aeruginosa harbor diverse percentages of both  genes as virulence factors, and those factors must be increased the pathogenicity of this pathogen. Keywords: P.  aeruginosa, burn, exoS, phzS genes, virulence factors.  Material and Methods:-  Samples collection:- Seventy swabs were collected from different locations of burns patients in burn unit of AL-Mussan Teaching Hospital of Mussan province/Iraq in the period from July to November, 2014 by moistened sterile swabs with normal saline. Swabs were transported for the laboratory for diagnosis, these swabs directly inoculated on Blood agar and MacConkey agar (LAB/ United Kingdom) and incubated at 37ºC for 24 hours.                                                                                                                    Identification of P. aeruginosa:- P. aeruginosa was identified based on the morphological properties in culture media and biochemical tests included (catalase test, oxidase test, indol test, methyl red test, voges-proskaure test) which done according to Bergeys manual (MacFaddin, 2000; Murray et al., 2003). VITEK 2 system (BioMerieux, France) was done to confirmed identification of  P. aeruginosa.  Preparation of bacterial DNA:-  All P. aeruginosa isolates were inoculated aerobically on Brain Heart Infusion broth (LAB/ United Kingdom) and incubated for 18–24 h at 37° C. Bacterial DNA extraction was performed using Genomic DNA Extraction kit (Geneaid/Korea).The exoS and phzS genes were identified using primers described in Table (1).  The final volume of PCR reaction tubes is 20μl, consist of  5μl Master Mix (Bioneer/Korea), 1μl of each forward and reverse of the primers specific for each gene, 5μl of DNA template and the volume was completed by adding nuclease free water. The thermocycling conditions of exoS gene were set at 94 °C for 3 min followed by 30 cycles of 94 °C for 30 sec, 55 °C for 60 sec, and 72 °C for 90 sec, and final extension at 72 °C for 5 min (Fazeli and Momtaz, 2014), while the thermocycling program of phzS gene was set at 96°C for 5 min, followed by 30 cycles of 94 University of Thi-Qar Journal Of Science (UTsci) Website: jsci.utq.edu.iq                                                                                                 Email: jsci@utq.edu.iq Volume 7, Number 1, June 2019  26  °C for 30 sec, 63 °C for 30 sec, 72°C for 1 min, and final extension at 72 °C for 4 min.            Results and Discussion:- Out of 70 burn swabs, 54 (77%) showed positive growth on blood agar, only 13 isolates (35%) were P. aeruginosa, P. aeruginosa is the common frequent bacterial agent of burn wound. The results of current study are comparable to other studies, such as the results of Alkaabi, (2013) reported a P. aeruginosa was the commonest pathogen, with a percentages of 48.14%, also Kanagapriya et al., (2015) documented the predominant bacteria isolated from the infected wounds were P. aeruginosa (28%).  The results showed that the prevalence of exoS  gene harbored in about 46% of all isolates. The band size was 444 bp corresponds to amplification of  exoS gene, Fig. (1). The exoS gene may be associated with increasing the pathogenesis of this bacteria, (Olson et al., 1999) revealed that the exoS gene is directly translocated into eukaryotic cells by the contact-dependent type III secretory process, it provides the bacterium with a mechanism for manipulating the eukaryotic cells it encounters, exoS gene contributing to pathogenicity of  this bacteria, bacterial translocation of exoS into epithelial cells results in a general inactivation of cellular function, as known by the inhibition of DNA synthesis, loss of focal adhesion, microvillus effacement  and cell rounding. The percentage of exoS gene in present study showed slightly percentage in comparison with the results of other studies which documented a high percentages such as Fazeli and Momtaz, (2014) recorded that 67.6% of human clinical samples which isolated from burn, respiratory and urinary tract infections had exoS gene as the most commonly detected virulence genes. Afterward, Wolfgang et al., (2003) showed that 13 out of 19 strains of P. aeruginosa contained exoS gene, also Finnan et al., (2004) revealed that the massive majority of P. aeruginosa isolates contain exoS gene, because this genes are involved in phagocytosis and lung injury in the human host. The exoS gene must be related to some diseases, Sun et al., (2012) recorded that the exoS gene contributed to dissemination in burn, keratitis and lung diseases. Exotoxin S and other exotoxin such as exoU, and exoT genes were importance in the pathogenicity of lung and other diseases (Shaver and Hauser, 2004). Some studies like Feltman et al., (2001) suggested that all P. aeruginosa isolates harbor some genes which encoding the type III secretion apparatus but that three of the four type III effector proteins (ExoT, ExoS, ExoU, and ExoY) are variable and that the huge majority of  isolates contain either the exoS or the exoU gene but not both.  The results of PCR assay showed that 77% of P. aeruginosa isolates ported phzS gene, the band was 1752 bp size corresponds to amplification of goal gene. The results of present study was agreed with study of Fazeli and Momtaz, (2014) depicted that a high percentage of clinical isolates of P. aeruginosa had phzS gene. On other hand, the most of the clinical isolates carried all the genes involved in phenazine production, with the exception of CF194, which lacked both phzI and phzS, and CF242, which lacked phzS  (Finnan.et.al.,2004).     Table (1): Oligonucleotide primers sequences for PCR amplified of phzS and exoS genes 7 Fig. (1): Agarose gel electrophoresis of exoS  gene amplification,  M: ladder, 1-8: positive results University of Thi-Qar Journal Of Science (UTsci) Website: jsci.utq.edu.iq                                                                                                 Email: jsci@utq.edu.iq Volume 7, Number 1, June 2019  26       Conclusions:- We concluded a P. aeruginosa have diverse percentage of exoS and phzS genes as virulence factors, and those factors must be increased the pathogenicity of this pathogen.  References:- Alkaabi, S A. (2013). Bacterial isolates and their antibiograms of burn wound infections in burns specialist hospital in Baghdad. J.  Bagh. Sci., 10 (2):331-340. Deng, Q.; Zhang, Y. and Barbieri, J T. (2007). Intracellular trafficking of ExoS, a type III cytotoxin. J. Traffic., 8: 1331-1345. 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Molecular detection of two virulence factors of Pseudomonas  aeruginosa isolated from burn patients

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Molecular detection of two virulence factors of Pseudomonas aeruginosa isolated from burn patients

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