Microsoft Word - 26-30 IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/10.30526/2017.IHSCICONF.1772 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/  www.ihsciconf.org  Biology|26    Detection of tox A gene in Pseudomonas aeruginosa that isolates from different clinical cases by using PCR Rana M. Abdullah Al-Shwaikh dr.rana_alshwaikh@yahoo.com Abbas Falih Alornaaouti Dept. of Biology / College of Education) Ibn-Al Haitham)for pure science, University of Baghdad. Abstract Current study obtained (75) isolate of Pseudomonas aeruginosa collected from different cases included: 28 isolates from otitis media, 23 isolates from burn infections, 10 isolates from wound infections, 8 isolates from urinary tract infections and 6 isolates from blood, during the period between 1/9/2014 to 1/11/2014 The result revealed that the tox A gene was present in 54 isolates (72%) of Pseudomonas aeruginosa. The gel electrophoresis showed that the molecular weight of tox A gene was 352 bp. The result shows 17 isolates (60.71%) from otitis media has tox A gene, 18 isolates (78.26%) from burn followed by 8 isolate (80%) from wound infection and 5 isolates (62.5%) from urinary tract infection, finally 6 isolates (100%) from blood have this gene. Key words: - Pseudomonas aeruginosa, tox A gene. IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/10.30526/2017.IHSCICONF.1772 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/  www.ihsciconf.org  Biology|27    1. Introduction Pseudomonas aeruginosa are one of the opportunistic pathogens that infect humans [1]. cause many diseases that lead to the death of a person so it is necessary to follow the accurate and rapid methods in the diagnosis of this type of infection [2]. Pseudomonas aeruginosa cause many infections, especially in patients with an immune compromised [3] As well as get infected by respiratory infections, which have a role in the spread of P. aeruginosa and transmission from one person to another by tissue and hands of patients and then transmitted directly by skin and the patient's clothing, as well as injuries digestive system and water and food pollution by carriers of the pathogen [4]. P. aeruginosa increased resistance to a variety of chemical compounds, including antibiotics, detergents and disinfectants [5]. These bacteria can be stay in disinfectants, liquid medicines, as well as on the floor of the hospitals and this has helped to increase the infection of bacteria in patients which are asleep in hospitals, especially in intensive care unit [6]. This bacterium has many virulence factors including external toxin type A is a highly toxic protein, which is one of the factors which are too severe and cause some deaths when excreted by the bacteria P. aeruginosa which cause topical and systemic disease and is linked to the occurrence of necrosis at the site of injury bacterial [7]. Exotoxin A in the inhibition of the process of protein synthesis in the host cells through the formation of the complex 5-Diphosphate Ribosyl with one elongation factors EF2 through the transfer of ADP-ribosyl of NAD to factor EF2 and thus prevents the protein chain elongation on the ribosome therefore exotoxic a mechanism similar to the work of the diphtheria toxin. But the exotoxic are present on the cell surface are different from the diphtheria toxin receptor [8]. And also to the role of this toxin to penetrate tissue, especially in patients with cystic fibrosis it found that strains that are unproductive for external toxin be less virulent strains producing [9]. tox A gene encodes Exotoxin A, this toxin when produced by the bacterial cell is linked to specific receptors on the surface of the host cell, which allowed to enter [10]. The aims of study to detection of gene tox A in pseudomonas aeruginosa that isolated from different clinical cases. 2. Experimental 2.1. Material and method 2.1.1.Collection of bacteria:- Collection the bacteria from different infection cases include (burn and wound infections, otitis media, urinary tract infection and blood samples) during the period between 1/9/2014 to 1/11/2014 2.1.2. Identification of bacteria: - Identification the isolates by culturing on media culture include:- MacConkey agar, Blood agar, Cetrimide agar, Pseudomonas agar and CHROMagar Orientation then identified by using biochemical tests including (oxidase and catalase test) and further identification by using a API20E system [11]. 2.1.3. Isolate of DNA: - Use DNA kit to extract DNA of bacteria isolates (Geneaid Biotech kit system, UK) according to the manufacturer's instructions. 2.1.4. Detection of tox A gene: -To detection tox A gene of P. aeruginosa isolates by using the primer in this study:-Primer sequence(5′–3′)F (GGTAACCAGCTCAGCCACAT) R (TGATGTCCAGGTCATGCTTC) size 352bp, Lanotte et al. (2004) IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/10.30526/2017.IHSCICONF.1772 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/  www.ihsciconf.org  Biology|28    Detection of gene tox A attended the solution concentration of 10 pekmole / µl (by taking 10 µl from Stock solution and addition of 90 µl of distilled water Alloaona) save the Stock solution under - 20 ° C . Using polymerase chain PCR interactive and conditions described below interaction. Program step 1 (Initial denaturation at 94ºC for 3 min 1 cycle), step 2 (30 cycles) A:- Denaturation DNA template at 94 ° C for 30 sec, B:- Annealing at 55 ºC for 1 min, C:- Extension at 72 ºC for 1 min . step 3 (A final extension at 72 ºC for 5 min 1 cycle) [12] 2.1.5. Separation of DNA bands: - PCR products were separated on a 2% agarose with 5µl Ethidium bromide, at 50 volt for 2 hour. Using 100 bp Ladder. The DNA bands were visualized and photographed under UV light [13]. 3. Result and discussion We obtained (75) isolate of Pseudomonas aeruginosa collected from different cases included : 28 isolates from otitis media, 23 isolates from burn infections, 10 isolates from wound infections, 8 isolates from urinary tract infections and 6 isolates from blood . The result revealed that the tox A gene was present in 54 isolates (72%) of Pseudomonas aeruginosa. The gel electrophoresis showed that the molecular weight of tox A gene was 352 bp. The result shows 17 isolates (60.71%) from otitis media has tox A gene , 18 isolates (78.26%) from burn followed by 8 isolates (80%) from wound infection and 5 isolates (62.5%) from urinary tract infection , finally 6 isolates (100%) from blood has this gene is shown in table (1) and figure(1). This study was similar with [14] who showed 73% of her isolated was tox A and does not have an agreement with 15 who found 81.5% of P . aeruginosa that isolated from wound swab and cystic fibrosis patent has tox A gene .As well as Nikbin et al. [16] found 90% of the isolates of this bacteria own tox A gene This high rate is mostly due to the isolates obtained source was one of the swabs burns and this result agrees with the results of the current study that isolate 23 from burns swabs and all of the isolates has tox A 100%. The tox A gene encodes Exotoxin A, a highly toxic proteins and has a lot to do with the occurrence of Necrosis at the site of infection with the bacterium The danger of this toxin in the inhibition of the process of protein synthesis [8] Also, for this toxin role to penetrate the tissue process, particularly in patients with cystic [9]. IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/10.30526/2017.IHSCICONF.1772 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/  www.ihsciconf.org  Biology|29    Figure (1): Agarose gel electrophoresis (2%) of PCR products of tox A gene (352bp). Line M: DNA marker (100bp -2000 bp ladder, Promega, USA); Lanes 51, 53,54,56,59,60,61, 64, 65,66,67,68,69,70,71,72,73 Pseudomonas aeruginosa PCR-positive isolates. 4. Conclusions The results of detection of virulence genes tox A was found in 72 % of Pseudomonas aeruginosa isolates. This gene encodes Exotoxin A, a highly toxic proteins and has a lot to do with the occurrence of Necrosis at the site of bacterium infection. References [1] S.Bentzmann, and P. Plesiat, The Pseudomonas aeruginosa opportunistic pathogen and human infections. J. Applied Micribiology. 13(7): 1655-1665. 2011 [2] J.Overhage,; M. Bains,; M.D. Brazas, and R.E. Hancock, Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factor and antibiotic resistance. J. Bacteriology190(8): 2671-2679. 2008 [3] S.Hassan,; O. Parviz, and Y. Bagher, Drug susceptibility and molecular epidemiology of Pseudomonas aeruginosa isolated in a burn unit. American J. Infectious Diseases. 10: 301-306. 2009 [4] L.Martinez-Solano,; M.D. Macia,; A. Fajardo,; A. Oliver, and J.L. Martinez, Chronic Pseudomonas aeruginosa infection in chronic obstructive pulmonary disease. Clinical infection Disease. 47: 1526-1533. 2008 Table (1): Number and percentage of tox A gene of P. aeruginosa (%) tox A No. of tox A positive isolates Sources No. 17 (60.71) 28 Otitis media 1 18 (78.26) 23 Burn 2 8 (80) 10 Wound 3 5 (62.5) 8 UTI 4 6 (100) 6 Blood 5 54 (72) 75 Total 352 bp  2000  1000  800  600  500  100  400  200  700  900  300  1600  1200        M  51  52  53  54   55  56  57 58   59  60  61 62 63    64  65  66  67  68  69  70  71  72  73   IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/10.30526/2017.IHSCICONF.1772 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/  www.ihsciconf.org  Biology|30    [5] M. Mitiku,; S. Ali, and G. Kibru, Antimicrobial drug resistance and disinfectants susceptibility of Pseudomonas aeruginosa isolates from clinical and enviromental samples in jimma university specialized hospital, southwest ethiopia . Ame. J. Biom. Life Sci., 2(2): 40-45. 2014 [6] S.A. Ochoa; F. Lopez-Montiel, ; G. Escalona, ; A.Cruz-Cordova, ; L.B. Davila; B.Lopez-Martinez, ; Y. Jimenez-Tapia, ; S. Giono, ; C. Eslava ; R. Hernandez-Castro, and Xicohtencati-Cortes, J. Pathgenic characteristics of Pseudomonas aeruginosa strians resistance to carbapenems associated with biofilm formation. J. Bol. Med. Hosp. Infant. 70(2): 133-144. 2013 [7] B. Baradaran,; S. Farajnia,; J. Majidi,; Y. Omidi, and N. Saeedi, Recombinant expression and purification of Pseudomonas aeruginosa truncated exotoxin A in Escherichia coli. Pharmaceutical Sciences. 19(1): 31-34. 2013 [8] D. Xing,; R. Youle,; D. Fitzgerald, and I. Pastan, Pseudomonas exotoxin A mediated apoptosis is bak dependent and preceded by the degradation of Mci-1. J. PMC. 1: 1-9. 2010 [9] M. Davinic,; N.L. Carty,; J.A. Colmer-Hamood,; M.S. Francisco, and Hamood, A.N. Role of Vfr in regulation exotoxin A production by Pseudomonas aeruginosa . J. Microbiology. 155: 2265-2273. 2009 [10] J. Morlon-Guyot,; J. Mere; A. Bonhoure, and B. Beaumellem Processing of Pseudomonas aeruginosa exotoxin A is dispensable for cell intoxication. J. Infec. Imm. 77(7): 3090-3099. 2009 [11] E. J. Baron,; S. M. Finegold, and . L. R. Peterson, IBailey and Scott،s Diagnostic Microbiology. 9th ed. Mosby Company. Missouri. 2007. [12] P. Lanotte; S. Watt; L. Mereghetti; N. Dartiguelongue,; A. Rastegar-Lari,; A. Goudeau, and R. Quentin, Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patients compared with those of isolates from others origins. J. Medical Microbiology. 53: 73-81. 2004 [13] J. Sambrook, and D. W. Rusell, Molecular cloning a laboratory manual. Cold spring Harbor, NY: cold spring Harbor Laboratory press. 2001. [14] E.T. Garallah, Molecular analysis of some virulence genes of Pseudomonas aeruginosa isolated from cystic fibrosis and non-cystic fibrosis sources. M.Sc. Thesis. College of Science. AL-Mustansiryia University. 2015. [15] S.R. Taee; B.Khansarinejad,; H. Abtahi,; M. Najafimosleh, and Ghaznavi-Rad, E. Detection of alg D, opr L and exo A genes by new specific primers as an efficient, rapid and accurate procedure for direct diagnosis of Pseudomonas aeruginosa strains in clinical samples. Jundishapur J. Microbiol. 7(10): 1-10. 2014 [16] V.S. Nikbin; Z. M.M.Aslani; Sharafi; M. Hashemipour,; F. Shahcheraghi, and Ebrahimipour, G. H. Molecular Identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins.Iranian J. Microbiology., 4(3):118-123. 2012