ارزیابی میزان بقای فرم های غیر قابل کشت هلیکوباکتر پیلوری در نمونه های آب
محورهای موضوعی : میکروب شناسی مولکولی
پرستو چمن رخ
1
,
محمد حسن شاه حسینی
2
,
مهناز مظاهری اسدی
3
,
طاهر نژاد ستاری
4
,
داوود اسماعیلی
5
1 - دکتری، گروه زیست شناسی، دانشگاه آزاد اسلامی، واحد علوم و تحقیقات تهران
2 - دانشیار، دانشگاه آزاد اسلامی، واحد شهر قدس،
گروه میکروب شناسی
3 - استاد، سازمان پژوهش های علمی و صنعتی ایران، گروه بیوتکنولوژی محیط زیست
4 - دانشیار، دانشگاه آزاد اسلامی، واحد علوم و تحقیقات تهران، گروه زیست شناسی
5 - استادیار، دانشگاه بقیه الله، دانشکده علوم پزشکی، گروه میکروب شناسی
کلید واژه: کشت, واکنش زنجیره ای پلی مراز, هلیکوباکترپیلوری, کوکوئید, ژن glmM,
چکیده مقاله :
هلیکوباکتر پیلوری عامل مهمی در ایجاد زخم های پپتیک و سرطان معده در انسان است. هنگامی که این باکتری در درون آب قرار می گیرد به فرم زنده اما غیرقابل کشت کوکوئید در می آید. این امر می تواند یکی از عوامل مهم انتقال آلودگی محسوب گردد. این مطالعه با هدف ارزیابی میزان بقای فرم های کوکوئید هلیکوباکتر پیلوری در نمونه های آب توسط روش های کشت و PCR انجام شد. این پژوهش به صورت تجربی بر روی 10 سویه هلیکوباکتر پیلوری جدا شده از نمونه های بالینی انجام گرفت. سویه های جدا شده به آب اضافه و در دماهای 4، 22 و 37 درجه سلیسیوس و به فواصل زمانی 1 و 2 ماه گرماگذاری شدند. در هر بار، نمونه ها بر روی محیط بروسلا بلاد آگار کشت داده شدند. پس از استخراج DNA، به منظور تکثیر ژن glmM از روش PCR استفاده گردید. همچنین حساسیت و اختصاصیت روش PCR مورد ارزیابی قرار گرفت. در مطالعه حاضر با استفاده از روش کشت در دمای 4 درجه در ماه اول هیچ فرم کوکوئیدی از باکتری در محیط کشت مشاهده نشد. اما درصد شناسایی باکتری در دمای 22 و 37 درجه سلیسیوس برابر با 10% و 20% بود. در ماه دوم نتیجه مثبتی تشخیص داده نشد. با استفاده از روش PCR مشخص گردید که این روش با حساسیت بیشتر نسبت به کشت در ماه اول و در دماهای 22، 4 و 37 درجهسلیسیوس به ترتیب قادر به شناسایی فرم های کوکوئید در 10%، 30% و 40% موارد هلیکوباکتر پیلوری بود. این گزارش در ماه دوم به صورت 0%، 20% و 30% بود. یافته های این مطالعه نشان می دهد که فرم های کوکوئید غیرقابل کشت هلیکوباکترپیلوری در آب، توسط روش های حساس، دقیق و اختصاصی مانند PCR قابل شناسایی می باشند.
H. Pylori is an important cause of human peptic ulcers and stomach cancers. Once the bacterium is placed in the water, it changes into a viable, but non-cultivable coccoid form, which is considered as an important factor to spread out the infection. The aim of this study was to evaluate the survival rate of coccoid forms of H. pylori in water samples, using PCR and culture methods. This experimental study was performed on 10 strains of H. pylori isolated from clinical specimens. Isolates were added to water at the temperatures of 4°C, 22°C, and 37°C and incubated at intervals of 1 and 2 months. Each time, the samples were cultured on Brucella blood Agar medium. Following DNA extraction, the presence of glmM gene was confirmed using PCR method. Furthermore, the sensitivity and specificity of PCR method were evaluated. While culturing in first month at 4°C showed no H. pylori coccoid growth on medium, some positive results (growth rate of 10% and 20%, respectively) were detected at 22°C and 37°C during the same month. No positive result was obtained during the second month. Performing PCR, with more sensitivity as compared to culturing method, identified H. pylori coccoid growth of 10%, 30%, and 40%, for the first month, and 0%, 20%, and 30% for the second month at 4°C, 22°C, and 37°C, respectively. The results of this study showed that the non-cultivable cocoid forms of H. pylori in water can be detected by non-culturing methods such as PCR which is sensitive, specific, and accurate.
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2. Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Bordovsky M, Rappuoli R, Covacci A. cag a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease associated virulence factors. Proc Natl Acad Sci USA. 1996; 93(25): 14648-14653.
3. Park SR, Mackay WG, Reid DC. Helicobacter sp. recovered from drinking water biofilms sampled from a water distribution system. Pergamon. 2001; 35(6): 1624-1626.
4. Massoudian SH, Ghane M, Golijanimoghadam R, Ghorbanimoein F. Absence of Helicobacter pylori in the river waters in the north of Iran. Afr J Microbiol. 2012; 6(8):1790-1795 .
5. Azevedo NF, Almeida C, Fernandes I, Cerqueira L, Dias S, Keevil CW. Survival of gastric and enterohepatic Helicobacter spp in water implications for transmission. Appl Environ Microbiol. 2008; 74(6): 1805-1811.
6. Chen TSH. Is the coccoid form of Helicobacter pylori viable and transmissible? Med Assoc. 2004; 67: 547-548.
7. Catrenich CE, Makin KM. Characterization of the morphologic conversion of Helicobacter pylori from bacillary to coccoid forms. Scand J Gastroenterol. 1991; 26(181): 58-64.
8. Cellini L, Allocati N, Angelucci D, lezzi T, Campli ED, Marzio L, Dainelli B. Coccoid Helicobacter pylori not cultivable in vitro reverts in mice. Microbiol Immunol. 1994; 38: 843-850.
9. Bode G, Mauch F, Malfertheiner P. The coccoid forms of Helicobacter pylori. Criteria for their viability. Epidemiol Infect. 1993; 111: 483-490.
10. Fei-Fei Sh, Jian-Yin L, Jun-Yan L, Cheng H, Dong-Hui S. Virulence of water-induced coccoid Helicobacter pylori and its experimental infection in mice. World J Gastroenterol. 2003; 9(3): 516-520.
11. Benson JA, Fode-Vaughan KA, Collins MLP. Detection of Helicobacter pylori in water by direct PCR. Letters Appl Microbiol. 2004; 39: 221-225.
12. Bunn JE, MacKay WG, Thomas JE, Reid DC, Weaver LT. Detection of Helicobacter pylori DNA in drinking water biofilms implications for transmission in early life. Letters Appl Microbiol. 2002; 34: 450-454.
13. Ahmed KS, Khan AA, Ahmed I, Tiwari SK, Habeeb A, Ahi JD, Abid Z, Ahmed N, Habibullah CM. Impact of household hygiene and water source on the prevalence and transmission of Helicobacter pylori: a South Indian perspective. Singapore Med J. 2007; 48(6): 543-549.
14. Guadalupe Co´rdova Espinoza M, Gonza´lez Vazquez R, Morales Mendez I, Ruelas Vargas C, Giono Cerezo S. Detection of the glmM gene in Helicobacter pylori isolates with a novel primer by PCR. J Clin Microbiol. 2011; 49(4): 1650-1652.
15. Shahamat M , Alavi M, Watts JEM, Gonzalez M, Sowers KR, Maeder DW, Robb FT. Development of two PCR-based techniques for detection helical and coccoid forms of Helicobacter pylori. J Clin Microbiol. 2004; 42(8): 3613-3619.
16. Esmaeili D, Mohabati Mobarez A, Hatef Salmanian A, Zavaran HA. Optimization of Helicobacter pylori culture in order to prepare favorable antigens. J Bacteriol Research. 2009; 1(9): 101-104.
17. Jang-jih L, Cherng-lih P, Rong-yaun SH, Chi-hsiang CH, Qinyuan L, Sonny KF, Chong, Chao-hung Lee. Comparison of five PCR methods for detection of Helicobacter pylori DNA in gastric tissues. J Clin Microbiol. 1999; 37(3): 772.
18. Smith SI, Oyedeji KS, Arigbabu AO, Cantet F, Megraud F, Ojo OO. Comparison of three PCR method for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens. World J Gastroenterol. 2004; 10: 1958-1960.
19. Hazeleger W, Arkesteijn C, Toorop-Bouma A, Beumer RR. Detection of the coccoid form of Campylobacter jejuni in chicken products with the use of the polymerase chain reaction. Int J Food Microbiol. 1994; 24: 273-281.
20. Chan WY, Hui PK, Leung KM, Chow J, Kwok F, Ng CS. Coccoid forms of Helicobacter pylori in the human stomach. Am J Clin Pathol. 1994; 102: 503-507.
21. Krogfelt KA, Lehours P, Megraud F. Diagnosis of Helicobacter pylori infection. Helicobacter. 2005; 10(1): 5-13.
22. Khanolkar-Gaitone SS, Reubish GK, Lee CK. Isolation of bacteria other than Helicobacter pylori from stomachs of squirrel monkeys (Saimiri spp) with gastritis. Dig Dis Sci. 2000; 45: 272-280.
23. Kisa O, Albay A, Mas MR. The evaluation of diagnostic methods for the detection of Helicobacter pylori in gastric biopsy specimens. Diagn Microbiol Infect Dis. 2002; 43: 251-255.
24. Brooks HJ, Ahmed D, McConnell MA, Barbezat GO. Diagnosis of Helicobacter pylori infection by polymerase chain reaction: is it worth it? Diagn Microbiol Infect Dis. 2004; 50: 1-5.
25. Watson CL, Owen RJ, Said B, Lai S, Lee JV, Surman-Lee S, Nichols G. Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England. J Appl Microbiol. 2004; 97: 690-698.
26. Janzon A1, Sjöling A, Lothigius A, Ahmed D, Qadri F, Svennerholm AM. Failure to detect Helicobacter pylori DNA in drinking and environmental water in Dhaka, Bangladesh, using highly sensitive real-time PCR assays. Appl Environ Microbiol. 2009; 75(10): 3039-3044.
_||_1. Steven L, Thomas P. Transmission of Helicobacter pylori and the role of water and biofilms. J Water Health. 2009; 7: 469-477.
2. Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Bordovsky M, Rappuoli R, Covacci A. cag a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease associated virulence factors. Proc Natl Acad Sci USA. 1996; 93(25): 14648-14653.
3. Park SR, Mackay WG, Reid DC. Helicobacter sp. recovered from drinking water biofilms sampled from a water distribution system. Pergamon. 2001; 35(6): 1624-1626.
4. Massoudian SH, Ghane M, Golijanimoghadam R, Ghorbanimoein F. Absence of Helicobacter pylori in the river waters in the north of Iran. Afr J Microbiol. 2012; 6(8):1790-1795 .
5. Azevedo NF, Almeida C, Fernandes I, Cerqueira L, Dias S, Keevil CW. Survival of gastric and enterohepatic Helicobacter spp in water implications for transmission. Appl Environ Microbiol. 2008; 74(6): 1805-1811.
6. Chen TSH. Is the coccoid form of Helicobacter pylori viable and transmissible? Med Assoc. 2004; 67: 547-548.
7. Catrenich CE, Makin KM. Characterization of the morphologic conversion of Helicobacter pylori from bacillary to coccoid forms. Scand J Gastroenterol. 1991; 26(181): 58-64.
8. Cellini L, Allocati N, Angelucci D, lezzi T, Campli ED, Marzio L, Dainelli B. Coccoid Helicobacter pylori not cultivable in vitro reverts in mice. Microbiol Immunol. 1994; 38: 843-850.
9. Bode G, Mauch F, Malfertheiner P. The coccoid forms of Helicobacter pylori. Criteria for their viability. Epidemiol Infect. 1993; 111: 483-490.
10. Fei-Fei Sh, Jian-Yin L, Jun-Yan L, Cheng H, Dong-Hui S. Virulence of water-induced coccoid Helicobacter pylori and its experimental infection in mice. World J Gastroenterol. 2003; 9(3): 516-520.
11. Benson JA, Fode-Vaughan KA, Collins MLP. Detection of Helicobacter pylori in water by direct PCR. Letters Appl Microbiol. 2004; 39: 221-225.
12. Bunn JE, MacKay WG, Thomas JE, Reid DC, Weaver LT. Detection of Helicobacter pylori DNA in drinking water biofilms implications for transmission in early life. Letters Appl Microbiol. 2002; 34: 450-454.
13. Ahmed KS, Khan AA, Ahmed I, Tiwari SK, Habeeb A, Ahi JD, Abid Z, Ahmed N, Habibullah CM. Impact of household hygiene and water source on the prevalence and transmission of Helicobacter pylori: a South Indian perspective. Singapore Med J. 2007; 48(6): 543-549.
14. Guadalupe Co´rdova Espinoza M, Gonza´lez Vazquez R, Morales Mendez I, Ruelas Vargas C, Giono Cerezo S. Detection of the glmM gene in Helicobacter pylori isolates with a novel primer by PCR. J Clin Microbiol. 2011; 49(4): 1650-1652.
15. Shahamat M , Alavi M, Watts JEM, Gonzalez M, Sowers KR, Maeder DW, Robb FT. Development of two PCR-based techniques for detection helical and coccoid forms of Helicobacter pylori. J Clin Microbiol. 2004; 42(8): 3613-3619.
16. Esmaeili D, Mohabati Mobarez A, Hatef Salmanian A, Zavaran HA. Optimization of Helicobacter pylori culture in order to prepare favorable antigens. J Bacteriol Research. 2009; 1(9): 101-104.
17. Jang-jih L, Cherng-lih P, Rong-yaun SH, Chi-hsiang CH, Qinyuan L, Sonny KF, Chong, Chao-hung Lee. Comparison of five PCR methods for detection of Helicobacter pylori DNA in gastric tissues. J Clin Microbiol. 1999; 37(3): 772.
18. Smith SI, Oyedeji KS, Arigbabu AO, Cantet F, Megraud F, Ojo OO. Comparison of three PCR method for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens. World J Gastroenterol. 2004; 10: 1958-1960.
18. Moran AP, Upton ME. Factors affecting production of coccoid forms by Campylobacter jejuni on solid media during incubation. J Appl Bacteriol. 1987; 62: 527-537.
19. Hazeleger W, Arkesteijn C, Toorop-Bouma A, Beumer RR. Detection of the coccoid form of Campylobacter jejuni in chicken products with the use of the polymerase chain reaction. Int J Food Microbiol. 1994; 24: 273-281.
20. Chan WY, Hui PK, Leung KM, Chow J, Kwok F, Ng CS. Coccoid forms of Helicobacter pylori in the human stomach. Am J Clin Pathol. 1994; 102: 503-507.
21. Krogfelt KA, Lehours P, Megraud F. Diagnosis of Helicobacter pylori infection. Helicobacter. 2005; 10(1): 5-13.
22. Khanolkar-Gaitone SS, Reubish GK, Lee CK. Isolation of bacteria other than Helicobacter pylori from stomachs of squirrel monkeys (Saimiri spp) with gastritis. Dig Dis Sci. 2000; 45: 272-280.
23. Kisa O, Albay A, Mas MR. The evaluation of diagnostic methods for the detection of Helicobacter pylori in gastric biopsy specimens. Diagn Microbiol Infect Dis. 2002; 43: 251-255.
24. Brooks HJ, Ahmed D, McConnell MA, Barbezat GO. Diagnosis of Helicobacter pylori infection by polymerase chain reaction: is it worth it? Diagn Microbiol Infect Dis. 2004; 50: 1-5.
25. Watson CL, Owen RJ, Said B, Lai S, Lee JV, Surman-Lee S, Nichols G. Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England. J Appl Microbiol. 2004; 97: 690-698.
26. Janzon A1, Sjöling A, Lothigius A, Ahmed D, Qadri F, Svennerholm AM. Failure to detect Helicobacter pylori DNA in drinking and environmental water in Dhaka, Bangladesh, using highly sensitive real-time PCR assays. Appl Environ Microbiol. 2009; 75(10): 3039-3044.
