تشخیص باکتریهای کلی‌فرمی، تعیین گروه‌های فیلوژنی و الگوی مقاومت آنتی‌بیوتیکی اشریشیا کولای در چشمه‌ها و قنات‌های آلوده استان آذربایجان‌شرقی
محورهای موضوعی :
علوم و صنایع غذایی
نعیمه شعبانی لکرانی
1
(
کارشناسی ارشد گروه میکروبیولوژی، واحد اهر، دانشگاه آزاد اسلامی، اهر، ایران
)
جلال شایق
2
(
استادیار گروه میکروبیولوژی دامپزشکی، واحد شبستر، دانشگاه آزاد اسلامی، شبستر، ایران
)
جاوید صادقی
3
(
استادیار گروه میکروب‌شناسی، دانشکده پزشکی، دانشگاه علوم پزشکی، تبریز، ایران
)
زهره موسوی
4
(
کارشناس ارشد ژنتیک، دانشکده پزشکی، دانشگاه علوم پزشکی، تبریز، ایران
)
تاریخ دریافت : 1396/03/10
تاریخ پذیرش : 1396/03/10
تاریخ انتشار : 1396/03/01
کلید واژه:
قنات,
مقاومت آنتی بیوتیکی,
چشمه,
گروه‌های فیلوژنی,
<,
i>,
اشریشیا کولای<,
/i>,
,
چکیده مقاله :
اشریشیا کولای به‌عنوان باکتری شاخص آلودگی مدفوعی آب حائز اهمیت است. هدف از این مطالعه تعیین نوع و فراوانی باکتری های کلی‌فرمی و تنوع باکتری های اشریشیا کولای و تعیین میزان مقاومت آنتی‌بیوتیکی اشریشیا کولای جداشده از منابع قنات ها و چشمه های آذربایجان شرقی می باشد. بدین منظور 118 چشمه و قنات انتخاب و به روش MPN مورد بررسی قرار گرفت. نمونه های کلی‌فرم مثبت به روش‌های فنوتیپی و ژنوتیپی شناسایی شدند؛ در مرحله بعد جهت تعیین تنوع ژنتیکی اشریشیا کولای های جداشده از روش تعیین تیپ فیلوژنی به روش multiplex PCR استفاده شد. جهت تعیین الگوی حساسیت آنتی بیوتیکی از دیسک های آنتی بیوتیکی نالیدیکسیک اسید، کوتریماکسازول، آموکسی سیلین، جنتامایسین، سیپیروفلوکساسین، کلرامفنیکل، ایمیپنم، سفوتاکسیم و سفتازیدیم آنتی بیوگرام استفاده شد طبق نتایج مطالعه. 48% از نمونه ها توسط multiplex PCR از نظر کلی‌فرمی مثبت ارزیابی شدند که 40% اشریشیا کولای و 19% کلبسیلا تشخیص داده شد. تعلق 23 جدایه به گونه باکتری اشریشیا کولای تأیید شد. به استناد تعیین گروه‌های فیلوژنی 44% از سویه‌های مورد آزمایش متعلق به گروه B2وD و 56% از سویه ها متعلق به گروه A وB1 بودند. نتایج آنتی بیوگرام، مقاومت مربوط به آموکسی سیلین را معادل 92% نشان داد. همه سویه‌ها نسبت به ایمیپینم حساسیت داشتند. حضور سویه‌های بیماری‌زای اشریشیا کولای با مقاومت بالای آنتی‌بیوتیکی در منابع آبی می‌تواند به‌عنوان یک مخاطره بهداشتی برای انسان مطرح باشد.
چکیده انگلیسی:
Escherichia coli as a fecal contamination and is considered as an index in water. The aim of this study was to determine the phenotypic and genotypic characteristics of E. coli and antibiotic resistance of the isolates collected from qanats and springs in East-Azerbaijan province. For this purpose, 118 samples were selected from above mentioned area and examined by MPN method. The positive coliform samples were identified by phenotypic and genotypic methods. Afterwards, to determine the genetic diversity of E. coli isolates, phylogenetic typing we conducted by means of multiplex PCR. To determine the antibiotic resistance profile, antibiotic discs of Nalidixic Acid, Co-trimoxazol, Amoxicillin, Gentamaicin Ciprofloxacin, Chloramphenicol, Imipenem, Cefotaxime and Ceftazidime antibiogram were used. Based on results, 48% of the samples were evaluated as positive for coliform including 40% for E. coli and 19% for Klebsiella. Amongst 23 isolates confirmed as E. coli by PCR. Phylogenetic typing revealed that 44% of E. coli strains belonged to type D and B2 and 56% belonged to A and B1 phylotypes. Antimicrobial susceptibility pattern showed that 92% of E. coli isolates were resistant to Amoxicillin. All E. coli isolates were sensitive to Imipenem. It was concluded that presence of pathogenic E. coli with high rate of antibacterial resistance in waters source could be considered as a human health hazard.
منابع و مأخذ:
· Aragones, L., López, I., Palazon, A., Lopez-Ubeda, R. and Garcia, C. (2016). Evaluation of the quality of coastal bathing waters in Spain through fecal bacteria Escherichia coli and Enterococcus. Applied and Environmental Microbiology, 82: 288-297.
· Arbeit, R. D. (1999). Manual of Clinical Microbiology. ASM Press, Washington, DC, USA, pp. 116–137.
· Bouchet, V., Huot, H., Goldstein, R. (2008). Molecular Genetic Basis of Ribotyping. Comprehensive Microbial Resource, 21: 262–273.
· Chander Y., Gupta, S.C., Kumar, K., Goyal, S.M. and Murray, H. (2008). Antibiotic use and the prevalence of antibiotic resistant bacteria on turkey farms. Journal of the Science of Food and Agriculture, 88: 714–719.
· Chander, Y., Kumar, K., Goyal, S.M. and Gupta, S.C. (2005). Antibacterial activity of soil-bound antibiotics. Journal of Environmental Quality. 34:1952- 1957.
· Cheesbrough, M. (2005). District laboratory practice in tropical countries (part 2). Second edition, The Edinburgh Building Cambridg United Kingdom, pp. 136-142.
· Chiueh, L.C and Shiang W.H. (2001). Characterization of Escherichia coli serotype O157 strains isolated in Taiwan by PCR and moltilocus enzyme analysis. Journal of Food and Drug Analysis, 9(1): 12-19.
· Clerment, O., Constantinou, N. and Frankel, G. (2012). Infection strategies of enteric pathogenic Escherichia coli. Journals Gut Microbes, 3(2): 71–87.
· Clermont, O., Bonacorsi, S. and Bingen, E. (2000). Rapid and simple determination of Escherichia coli phylogenetic group. Applied and Environmental Microbiology Journal, 66(10): 4555-4558.
· Cowan, S.T. (1979). Cowan and Steel’s Manual for the Identification of Medical Bacteria. Third Edition, London, Public Health Laboratory Service England and Wales, pp.134-137.
· Diarra, M.S., Silversides, F.G., Diarrassouba, F., Pritchard, J., Masson, L., Brousseau, R. et al., (2007). Impact of feed supplementation with antimicrobial agents on growth performance of broiler chickens, Clostridium perfringens and Enterococcus counts, and antibiotic resistance phenotypes and distribution of antimicrobial resistance determinants in Escherichia coli isolates. Applied and Environmental Microbiology, 73: 6566–6576.
· Doumith, A.D., Hope, R., Wai, J. and Woodford, N. (2012). Improved Multiplex PCR Strategy for Rapid Assignment of the Four Major Escherichia coli phylogenetic groups. Journal of Clinical Microbiology, 50(9): 3108-3110.
· Eaton, AD and Franson, M.A. (2005). Standard Methods for the Examination of Water and Wastewater. Washington DC, American Public Health Association, pp.1809-1810.
· Ewers, C,. Antao, E.M,. Diehl, I., Philipp, H.C. and Wieler, L.H. (2009). Intestine and environment of the chicken as reservoirs for extraintestinal pathogenic Escherichia coli strains with zoonotic potential. Applied and Environmental Microbiology, 75: 184–192.
· Gordon, D. (2010). Strain typing and the ecological structure of Escherichia coli. Association of Analytical Communities International, 93(3): 974-984.
· Jawetz, M and Adelbergs, M. (2013). Medical Microbiology. Review of Medical Microbiology and Immunology, Lange Medical Books, pp. 68-72.
· Kelly, A., Reynolds, K. and Charles, P. (2008). Reviews of Enviromental Contamination and Toxicology. Reviews of Environmental Contamination and Toxicology, 192: 124-150.
· Lecointre, G., Rachdi, F., Darlu, P. and Denamur, E. (1998) Escherichia coli molecular phylogeny using the incongruence length difference test. Institut National de la Santé et de la Recherche Medicale, 155: 1685-1695.
· Malekzadeh, F. (2004). Microbiology. Tehran University. pp. 218-251
· Matthew, A., Croxen R.J.L., Roland, S., Kristie, M., Keeney, M. and Wlodarska, B. (2013). Recent advances in understanding enteric pathogenic Escherichia coli. Clinical Microbiology Reviews, 26(4): 822–880.
· Noller, A.C., McEllistrem, M.C., Stine, O.C., Morris, J.G. and Boxrud, D.J. (2003). Multilocus sequence typing reveals a lack of diversity among Escherichia coli O157:H7 isolates that are distinct by pulsed-field gel electrophoresis. Journal of Clinical Microbiology, 41(2): 675–679.
· Pitout, J.D and Laupland, K.B. (2008). Extended-spectrum -lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Diseases, 8(3):159-166.
· Renato, H., Orsi, N.C., Stoppe, M. (2007). Identification of Escherichia coli from groups A, B1, B2 and D in drinking water in Brazil. Journal of Water and Health, 05(2): 323-327.
· Rooklidge, S.J. (2004). Environmental antimicrobial contamination from terraccumulation and diffuse pollution pathways. Science of the Total Environment, 325: 1–13.
· Smith, J.L., Drum, Y., Dai, J.M., Kim, S. and Sanchez, J. (2007). Impact of antimicrobial usage on antimicrobial resistance in commensal Escherichia coli strains colonizing broiler chickens. Applied and Environmental Microbiology, 73(5): 1404-1414.
· Soltan Dallal, M.M. and Shmkani, F. (2012). Detection of ESBLs (type TEM) of E. coli by using phenotypic and genotypic tests in clinical isolates. Medical Journal of Tabriz University, 34(1): 62-56.
· Souza, V., Rocha M., Valera, A., and Eguiarte, L. (1999). Genetic structure of natural populations of Escherichia coli in wild hosts on different continents. Applied and Environmental Microbiology, 65(8): 3373-3385.
· Tenover, F.C. (2006). Mechanisms of Antimicrobial Resistance in Bacteria. American Journal of Medicine, 119 (6): 62–70.
· Van Soolingen, D., de Haas P.E., Hermans, P.W. and van Embden, J.D. (1994). DNA fingerprinting of Mycobacterium tuberculosis. Methods in Enzymology, 235: 196-205.
· World Health Organization. (1996). Guidelines for drinking-water quality. Health criteria and other supporting information, 2nd Edition. WHO, Geneva.
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· Aragones, L., López, I., Palazon, A., Lopez-Ubeda, R. and Garcia, C. (2016). Evaluation of the quality of coastal bathing waters in Spain through fecal bacteria Escherichia coli and Enterococcus. Applied and Environmental Microbiology, 82: 288-297.
· Arbeit, R. D. (1999). Manual of Clinical Microbiology. ASM Press, Washington, DC, USA, pp. 116–137.
· Bouchet, V., Huot, H., Goldstein, R. (2008). Molecular Genetic Basis of Ribotyping. Comprehensive Microbial Resource, 21: 262–273.
· Chander Y., Gupta, S.C., Kumar, K., Goyal, S.M. and Murray, H. (2008). Antibiotic use and the prevalence of antibiotic resistant bacteria on turkey farms. Journal of the Science of Food and Agriculture, 88: 714–719.
· Chander, Y., Kumar, K., Goyal, S.M. and Gupta, S.C. (2005). Antibacterial activity of soil-bound antibiotics. Journal of Environmental Quality. 34:1952- 1957.
· Cheesbrough, M. (2005). District laboratory practice in tropical countries (part 2). Second edition, The Edinburgh Building Cambridg United Kingdom, pp. 136-142.
· Chiueh, L.C and Shiang W.H. (2001). Characterization of Escherichia coli serotype O157 strains isolated in Taiwan by PCR and moltilocus enzyme analysis. Journal of Food and Drug Analysis, 9(1): 12-19.
· Clerment, O., Constantinou, N. and Frankel, G. (2012). Infection strategies of enteric pathogenic Escherichia coli. Journals Gut Microbes, 3(2): 71–87.
· Clermont, O., Bonacorsi, S. and Bingen, E. (2000). Rapid and simple determination of Escherichia coli phylogenetic group. Applied and Environmental Microbiology Journal, 66(10): 4555-4558.
· Cowan, S.T. (1979). Cowan and Steel’s Manual for the Identification of Medical Bacteria. Third Edition, London, Public Health Laboratory Service England and Wales, pp.134-137.
· Diarra, M.S., Silversides, F.G., Diarrassouba, F., Pritchard, J., Masson, L., Brousseau, R. et al., (2007). Impact of feed supplementation with antimicrobial agents on growth performance of broiler chickens, Clostridium perfringens and Enterococcus counts, and antibiotic resistance phenotypes and distribution of antimicrobial resistance determinants in Escherichia coli isolates. Applied and Environmental Microbiology, 73: 6566–6576.
· Doumith, A.D., Hope, R., Wai, J. and Woodford, N. (2012). Improved Multiplex PCR Strategy for Rapid Assignment of the Four Major Escherichia coli phylogenetic groups. Journal of Clinical Microbiology, 50(9): 3108-3110.
· Eaton, AD and Franson, M.A. (2005). Standard Methods for the Examination of Water and Wastewater. Washington DC, American Public Health Association, pp.1809-1810.
· Ewers, C,. Antao, E.M,. Diehl, I., Philipp, H.C. and Wieler, L.H. (2009). Intestine and environment of the chicken as reservoirs for extraintestinal pathogenic Escherichia coli strains with zoonotic potential. Applied and Environmental Microbiology, 75: 184–192.
· Gordon, D. (2010). Strain typing and the ecological structure of Escherichia coli. Association of Analytical Communities International, 93(3): 974-984.
· Jawetz, M and Adelbergs, M. (2013). Medical Microbiology. Review of Medical Microbiology and Immunology, Lange Medical Books, pp. 68-72.
· Kelly, A., Reynolds, K. and Charles, P. (2008). Reviews of Enviromental Contamination and Toxicology. Reviews of Environmental Contamination and Toxicology, 192: 124-150.
· Lecointre, G., Rachdi, F., Darlu, P. and Denamur, E. (1998) Escherichia coli molecular phylogeny using the incongruence length difference test. Institut National de la Santé et de la Recherche Medicale, 155: 1685-1695.
· Malekzadeh, F. (2004). Microbiology. Tehran University. pp. 218-251
· Matthew, A., Croxen R.J.L., Roland, S., Kristie, M., Keeney, M. and Wlodarska, B. (2013). Recent advances in understanding enteric pathogenic Escherichia coli. Clinical Microbiology Reviews, 26(4): 822–880.
· Noller, A.C., McEllistrem, M.C., Stine, O.C., Morris, J.G. and Boxrud, D.J. (2003). Multilocus sequence typing reveals a lack of diversity among Escherichia coli O157:H7 isolates that are distinct by pulsed-field gel electrophoresis. Journal of Clinical Microbiology, 41(2): 675–679.
· Pitout, J.D and Laupland, K.B. (2008). Extended-spectrum -lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Diseases, 8(3):159-166.
· Renato, H., Orsi, N.C., Stoppe, M. (2007). Identification of Escherichia coli from groups A, B1, B2 and D in drinking water in Brazil. Journal of Water and Health, 05(2): 323-327.
· Rooklidge, S.J. (2004). Environmental antimicrobial contamination from terraccumulation and diffuse pollution pathways. Science of the Total Environment, 325: 1–13.
· Smith, J.L., Drum, Y., Dai, J.M., Kim, S. and Sanchez, J. (2007). Impact of antimicrobial usage on antimicrobial resistance in commensal Escherichia coli strains colonizing broiler chickens. Applied and Environmental Microbiology, 73(5): 1404-1414.
· Soltan Dallal, M.M. and Shmkani, F. (2012). Detection of ESBLs (type TEM) of E. coli by using phenotypic and genotypic tests in clinical isolates. Medical Journal of Tabriz University, 34(1): 62-56.
· Souza, V., Rocha M., Valera, A., and Eguiarte, L. (1999). Genetic structure of natural populations of Escherichia coli in wild hosts on different continents. Applied and Environmental Microbiology, 65(8): 3373-3385.
· Tenover, F.C. (2006). Mechanisms of Antimicrobial Resistance in Bacteria. American Journal of Medicine, 119 (6): 62–70.
· Van Soolingen, D., de Haas P.E., Hermans, P.W. and van Embden, J.D. (1994). DNA fingerprinting of Mycobacterium tuberculosis. Methods in Enzymology, 235: 196-205.
· World Health Organization. (1996). Guidelines for drinking-water quality. Health criteria and other supporting information, 2nd Edition. WHO, Geneva.
