ژنوتایپینگ جدایه های سودوموناس آئروژینوسای جدا شده از عفونت های بیمارستانی
محورهای موضوعی : میکروب شناسی مولکولیغلامرضا بنی شریف 1 , حسن ممتاز 2
1 - کارشناس ارشد، گروه میکروب شناسی، واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد
2 - دانشیار، گروه میکروب شناسی، واحد شهرکرد،
دانشگاه آزاد اسلامی، شهرکرد
کلید واژه: rep-PCR, RAPD-PCR, عفونت های بیمارستانی, سودوموناس آئروژینوسا, ERIC-PCR,
چکیده مقاله :
سابقه و هدف: سودوموناس آئروژینوسا یک پاتوژن فرصت طلب شایع بیمارستانی است که مسئول طیف وسیعی از عفونت‌های انسانی می‌باشد. مطالعه حاضر با هدف دسته بندی ژنتیکی جدایه های سودوموناس آئروژینوسا جدا شده از عفونت های بیمارستانی انجام شد. مواد و روش ها: این مطالعه مقطعی توصیفی بر روی 18 جدایه سودوموناس آئروژینوسا جداسازی شده از بیماران بستری در بیمارستان های سطح شهرستان شهرکرد انجام گرفت. به منظور ژنوتایپینگ آن ها از سه روش RAPD-PCR، ERIC-PCR و Rep-PCR استفاده گردید.یافته ها: در آزمون RAPD-PCR، 86 باند مختلف DNA از حدوداً 300 تا 10000 جفت باز در جدایه های مورد بررسی ایجاد شد. از این میان تعداد 74 باند از آن ها پلی مورفیک بود. در آنالیز جدایه های سودوموناس آئروژینوسا با روش ERIC-PCR تعداد 98 باند مختلف با حدود 150 تا 8000 جفت باز مشاهده شد که الگوی باندینگ 14 جدایه از 18 جدایه مورد بررسی پلی مورفیک بود. در آزمایش Rep-PCR، 16پروفایل ژنومی حاصل گردید که در مجموع شباهتی بین حدود 30 تا 86 درصد بین جدایه ها مشاهده شد و تعدادی از نمونه ها دارای شباهت 100 درصد بودند. نتیجه گیری: به طور کلی تمام جدایه های مورد بررسی دارای الگوی باندی پلی مورفیک بودند. هیچ باند مونوفورمیکی در جدایه ها مشاهده نشد. وجود الگوی باندی پلی مورفیک در استفاده از تکنیک های مورد بررسی نشان می دهد که سرعت بالایی از پلی مورفیسم در ژنوم سودوموناس آئروژینوسا وجود دارد و روش های مورد استفاده در این مطالعه هر کدام ابزارهایی قدرتمند در دسته بندی سودوموناس آئروژینوسا می باشند.
Background & Objectives: Pseudomonas aeruginosa is a common opportunist pathogen in hospitals and the etiologic agent of the majority of infections in human beings. This study aimed to genotyping of P. aeruginosa isolated from hospital infections. Materials & Methods: This cross-sectional study was performed on 18 isolates of P. aeruginosa isolated from hospitalized patients in Shahrekord hospitals. We applied three different methods, RAPD-PCR, Rep-PCR and ERIC-PCR for genotyping of the isolates. Results: Based on RAPD-PCR method, overall 86 different bands of DNA with a range of 300 to 1000 bps were obtained from the under study isolates and among them, 74 bands were polymorphic. Analysis of P. aeruginosa isolates based on ERIC-PCR produced 98 bands with a range of 150-8000 bps. Overall 16 genomic profile with 30 to 86% and for a few strains, 100% similarities were produced based on Rep-PCR. Conclusion: Overall, all isolates showed polymorphic band patterns and no monochromic band was observed for the isolates. The presence of polymorphic band patterns in these techniques shows high rates of polymorphism in the genome of P. aeruginosa. Furthermore, the techniques used in this study are reliable approaches for genotyping of P. aeruginosa.
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2. Ahmadi K, Hashemian AM, Bolvardi E, Khadem Hosseini P. Vancomycin-resistant Pseudomonas aeroginosa in the cases of trauma. Med Arch. 2016; 70(1): 57-60.
3. El-Bialy AA, El-Shennawy GA, Mosaad AA, Bendary LA. Phenotyping and genotyping of Pseudomonas aeruginosa urine Isolates in Zagazig University Hospitals. Egyp J Med Microbiol. 2008; 17(4): 615-626.
4. Nanvazadeh F, Dokht Khosravi A, Zolfaghary MR, Parhizgari N. Genotyping of Pseudomonas aeruginosa strains isolated from two wards of Taleghani Hospital by RAPD-PCR in Ahvaz city of Iran. Jentashapir Sci Med J. 2013; 1: 49-58.
5. Brisse S, Milatovic D, Fluit AC, Kusters K, Toelstra A, Verhoef J, Schmitz FJ. Molecular surveillance of European quinolone-resistant clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. using automated ribotyping. J Clin Microbiol. 2000; 38(10): 3636-3645.
6. Speijer H, Savelkoul PH, Bonten MJ, Stobberingh EE, Tjhie JH. Application of different genotyping methods for Pseudomonas aeruginosa in a setting of endemicity in anintensive care unit. J Clin Microbiol. 1999; 37(11): 3654-3661.
7. Singh A, Goering RV, Simjee S, Foley SL, Zervos MJ. Application of molecular techniques to the study of hospital infection. Clin Microbiol Rev. 2006; 19(3): 512-530.
8. Thangaraj M, Prem V, Ramesh T, Lipton AP. RAPD fingerprinting and demonstration of genetic variation in three pathogens isolated from Mangrove environment. Asian J Biotechnol. 2011; 3(3): 269-274.
9. Tang YW, Stratton CW. Advanced techniques in diagnostic microbiology. 2th edition, Springer US, New York, 2013; pp: 91-246.
10. Stehling EG, Leite DS, Silveira WD. Molecular typing and biological characteristics of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Brazil. Braz J Infect Dis. 2010; 14(5): 462-467.
11. Salimi H, Owlia P, Yakhchali B, Rastegar Lari A. Characterization of Pseudomonas aeruginosa in burn patients using PCR– restriction fragment length polymorphism and random amplified polymorphic DNA analysis. Iran J Med Sci. 2010; 35(3): 236-241.
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13. Wolska K, Kot B, Jakubczak A. Phenotypic and genotypic diversity of Pseudomonas aeruginosa strains isolated from hospitals in siedlce (Poland). Braz J Microbiol. 2012; 43(1): 274-282.
14. Strateva T. 2008. Microbiological and molecular-genetic investigations on the resistance mechanisms and virulence factors in clinical strains of Pseudomonas aeruginosa. MS.c. thesis. Medical University of Sofia, Bulgaria.
15. Mahenthiralingam E, Campbell ME, Foster J, Lam JS, Speert DP. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients withcystic fibrosis. J Clin Microbiol. 1996; 34(5): 1129-1135.
16. Wolska K, Szweda P. A comparative evaluation of PCR ribotyping and ERIC PCR for determining the diversity of clinical Pseudomonas aeruginosa isolates. Pol J Microbiol. 2008; 57(2): 157-163.
17. Hassan KI, Rafik SA, Mussum K. Molecular identification of Pseudomonas aeruginosa isolated from hospitals in Kurdistan region. J Adv Med Res. 2012; 2(3): 90-98.
18. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 7th edition, Saunders, Washington. 2012; 425-432.
19. Tsukayama DT, van Loon HJ, Cartwright C, Chmielewski B, Fluit AC, van der Werken C, Verhoef J. RADAR trial. The evolution of Pseudomonas aeruginosa during antibiotic rotation in a medical intensive care unit: theRADAR-trial. Int J Antimicrob Agents. 2004; 24(4): 339-345.
20. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature. 2000; 406(6799): 959-964.
21. Wiegand I, Marr AK, Breidenstein EB, Schurek KN, Taylor P, Hancock RE. Mutator genes giving rise to decreased antibiotic susceptibility in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2008; 52(10): 3810-3813.
22. O'Carroll MR, Bell SC, Coulter C, Wainwright CE, Nissen M, Sloots T, Syrmis M. Rapid genotyping of Pseudomonas aeruginosa using repetitive element based PCR assays. In: Thompson PJ, Respirology. TSANZ 2003 Annual Scientific Meeting, Adelaide, Australia, (A47). 4-9 April, 2003.
23. Syrmis MW, O'Carroll MR, Sloots TP, Coulter C, Wainwright CE, Bell SC, Nissen MD. Rapid genotyping of Pseudomonas aeruginosa isolates harbored by adult and pediatric patients with cystic fibrosis using repetitive-element-based PCR assays. J Med Microbiol. 2004; 53(11): 1089-1096.
_||_1. Wolska K, Kot B. Twitching motility activity, biofilm formation, and genetic typing for clinical isolates of Pseudomonas aeruginosa by random amplified DNA PCR. Acta Microbiol Immunol Hung. 2013; 60(3): 313-328.
2. Ahmadi K, Hashemian AM, Bolvardi E, Khadem Hosseini P. Vancomycin-resistant Pseudomonas aeroginosa in the cases of trauma. Med Arch. 2016; 70(1): 57-60.
3. El-Bialy AA, El-Shennawy GA, Mosaad AA, Bendary LA. Phenotyping and genotyping of Pseudomonas aeruginosa urine Isolates in Zagazig University Hospitals. Egyp J Med Microbiol. 2008; 17(4): 615-626.
4. Nanvazadeh F, Dokht Khosravi A, Zolfaghary MR, Parhizgari N. Genotyping of Pseudomonas aeruginosa strains isolated from two wards of Taleghani Hospital by RAPD-PCR in Ahvaz city of Iran. Jentashapir Sci Med J. 2013; 1: 49-58.
5. Brisse S, Milatovic D, Fluit AC, Kusters K, Toelstra A, Verhoef J, Schmitz FJ. Molecular surveillance of European quinolone-resistant clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. using automated ribotyping. J Clin Microbiol. 2000; 38(10): 3636-3645.
6. Speijer H, Savelkoul PH, Bonten MJ, Stobberingh EE, Tjhie JH. Application of different genotyping methods for Pseudomonas aeruginosa in a setting of endemicity in anintensive care unit. J Clin Microbiol. 1999; 37(11): 3654-3661.
7. Singh A, Goering RV, Simjee S, Foley SL, Zervos MJ. Application of molecular techniques to the study of hospital infection. Clin Microbiol Rev. 2006; 19(3): 512-530.
8. Thangaraj M, Prem V, Ramesh T, Lipton AP. RAPD fingerprinting and demonstration of genetic variation in three pathogens isolated from Mangrove environment. Asian J Biotechnol. 2011; 3(3): 269-274.
9. Tang YW, Stratton CW. Advanced techniques in diagnostic microbiology. 2th edition, Springer US, New York, 2013; pp: 91-246.
10. Stehling EG, Leite DS, Silveira WD. Molecular typing and biological characteristics of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Brazil. Braz J Infect Dis. 2010; 14(5): 462-467.
11. Salimi H, Owlia P, Yakhchali B, Rastegar Lari A. Characterization of Pseudomonas aeruginosa in burn patients using PCR– restriction fragment length polymorphism and random amplified polymorphic DNA analysis. Iran J Med Sci. 2010; 35(3): 236-241.
12. Wolska K, Kot B, Jakubczak A, Rymuza K. BOX-PCR is an adequate tool for typing of clinical Pseudomonas aeruginosa isolates. Folia Histochem Cytobiol. 2011; 49(4): 734-738.
13. Wolska K, Kot B, Jakubczak A. Phenotypic and genotypic diversity of Pseudomonas aeruginosa strains isolated from hospitals in siedlce (Poland). Braz J Microbiol. 2012; 43(1): 274-282.
14. Strateva T. 2008. Microbiological and molecular-genetic investigations on the resistance mechanisms and virulence factors in clinical strains of Pseudomonas aeruginosa. MS.c. thesis. Medical University of Sofia, Bulgaria.
15. Mahenthiralingam E, Campbell ME, Foster J, Lam JS, Speert DP. Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients withcystic fibrosis. J Clin Microbiol. 1996; 34(5): 1129-1135.
16. Wolska K, Szweda P. A comparative evaluation of PCR ribotyping and ERIC PCR for determining the diversity of clinical Pseudomonas aeruginosa isolates. Pol J Microbiol. 2008; 57(2): 157-163.
17. Hassan KI, Rafik SA, Mussum K. Molecular identification of Pseudomonas aeruginosa isolated from hospitals in Kurdistan region. J Adv Med Res. 2012; 2(3): 90-98.
18. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 7th edition, Saunders, Washington. 2012; 425-432.
19. Tsukayama DT, van Loon HJ, Cartwright C, Chmielewski B, Fluit AC, van der Werken C, Verhoef J. RADAR trial. The evolution of Pseudomonas aeruginosa during antibiotic rotation in a medical intensive care unit: theRADAR-trial. Int J Antimicrob Agents. 2004; 24(4): 339-345.
20. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature. 2000; 406(6799): 959-964.
21. Wiegand I, Marr AK, Breidenstein EB, Schurek KN, Taylor P, Hancock RE. Mutator genes giving rise to decreased antibiotic susceptibility in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2008; 52(10): 3810-3813.
22. O'Carroll MR, Bell SC, Coulter C, Wainwright CE, Nissen M, Sloots T, Syrmis M. Rapid genotyping of Pseudomonas aeruginosa using repetitive element based PCR assays. In: Thompson PJ, Respirology. TSANZ 2003 Annual Scientific Meeting, Adelaide, Australia, (A47). 4-9 April, 2003.
23. Syrmis MW, O'Carroll MR, Sloots TP, Coulter C, Wainwright CE, Bell SC, Nissen MD. Rapid genotyping of Pseudomonas aeruginosa isolates harbored by adult and pediatric patients with cystic fibrosis using repetitive-element-based PCR assays. J Med Microbiol. 2004; 53(11): 1089-1096.
