• صفحه اصلی
  • تایپینگ مولکولی و تعیین روابط ژنتیکی جدایه های E.coli O157:H7 با تکنیکRAPD-PCR و ارتباط الگوی مقاومت آنتی بیوتیکی و ژنتیکی آن‌ها

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آدرس مقاله


کد مقاله : JMW-2210-2041 (R1) بازدید : 176 صفحه: 282 - 293

10.30495/jmw.2022.1969083.2041

20.1001.1.20083068.1401.15.53.4.2

نوع مقاله: پژوهشی

تایپینگ مولکولی و تعیین روابط ژنتیکی جدایه های E.coli O157:H7 با تکنیکRAPD-PCR و ارتباط الگوی مقاومت آنتی بیوتیکی و ژنتیکی آن‌ها

محورهای موضوعی : میکروب شناسی غذایی

صدیقه مختاری 1 , یحیی تهمتن 2 , محمد کارگر 3 , کیوان تدین 4 , الهام معظمیان 5

1 - گروه میکروبیولوژی، واحد شیراز، دانشگاه آزاد اسلامی، شیراز
2 - بخش باکتری شناسی موسسه تحقیقات واکسن و سرم سازی رازی شیراز
3 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
4 - قسمت واکسن های باکتریایی دامبزشکی سازمان تحقیقات آموزش و ترویح کشاورزی موسسه رازی
5 - گروه میکروبیولوژی، واحد شیراز، دانشگاه آزاد اسلامی ، شیراز

تاریخ دریافت : 1401/05/30 تاریخ پذیرش : 1401/10/12 تاریخ انتشار : 1401/12/15

کلید واژه: RAPD, مقاومت آنتی بیوتیکی, E.coli O157:H7, تایپینگ مولکولی,

چکیده مقاله :

سابقه و هدف: اشریشیاکلی O157:H7 پاتوژن مهم غذایی است که منجر به بیماری‌ های شدید گوارشی و مرگ می‌ شود. گاو مخزن اصلی این باکتری می باشد و طی مراحل کشتار وارد زنجیره غذایی می شود. شناسایی منبع آلودگی نقش مهمی در کنترل باکتری ایفا می کند. هدف از این مطالعه، تایپینگ مولکولی و تعیین روابط ژنتیکی جدایه های E.coli O157:H7 با تکنیکRAPD-PCR و بررسی ارتباط الگوی مقاومت آنتی بیوتیکی و ژنتیکی آن‌ ها می باشد.مواد و روش ها: بهمنظور تعیین روابط ژنتیکی جدایه ها از تکنیک RAPDاستفاده شد. باند های حاصل از واکنش، ردیابی و پروفایل‌ های متعددی از DNA ترسیم شد. نتایج با نرم افزار NTSYSpc آنالیز و ارتباط ژنتیکی جدایه‌ ها بر اساس ضریب تشابه تعیین گردید. الگوی مقاومت آنتی بیوتیکی با تکنیک دیسک دیفیوژن بررسی شد.یافته ها: اکثر جدایه ها به هفت آنتی بیوتیک حساسیت نشان دادند و در شش سویه مقاومت مشاهده شد. انگشت نگاریDNA ، 10الگوی ژنتیکی را نشان داد. جدایه ها از منابع مختلف بر اساس شباهت در الگوی RAPD خوشه بندی شدند. سویه ها حاوی الگوی DNA متفاوت و فاصله ژنتیکی بالا، الگوهای متفاوتی از حساسیت آنتی بیوتیکی را نشان دادند.نتیجه گیری: تنوع ژنومی متعدد، پروفایل های متمایزی از DNA را در سویه های با روابط کلونال یکسان نشان داد و الگو های متفاوت از مقاومت آنتی بیوتیکی آشکار شد. از این رو استفاده همزمان از الگوهای مقاومت آنتیب یوتیکی و تایپینگ مولکولی با تکنیک RAPD-PCR می تواند در ردیابی جدایه ها و کنترل عفونت موثر باشد.

چکیده انگلیسی:

Background & Objectives: Escherichia coli O157:H7 is one of the main foodborne pathogen that cause severe gastrointestinal issues and death. Cattle are the major reservoir for E. coli O157:H7. During the slaughtering process, E. coli O157:H7 enter to food chain. Identifying the source of contamination plays important role in the control of bacteria. The purpose of this study is molecular typing and determining of genetic relationships among E.coli O157:H7 isolates using RAPD-PCR technique and investigating the relationship between their genetic and antibiotic  resistance patterns.Materials& methods: The Random amplified polymorphic DNA (RAPD) analysis was used to evaluate genetic relationship among the isolates and the results was analyzed by NTSYSpc software. Antibiotic susceptibility of the isolates was examined through disc diffusion method.Results: The most isolates showed sensitivity to seven antibiotics and resistance were observed in six strains. DNA fingerprinting showed 10 genetic patterns. The isolates from different sources were clustered based on the similarity in RAPD pattern. The strains contain different DNA pattern and high genetic distance, showed different patterns of antibiotic susceptibility.Conclusion: The genomic diversity showed distinct profiles of DNA in strains with the same clonal relationships and different patterns of antibiotic resistance were revealed. Therefor determining the clonal relationships of isolates with RAPD PCR technique, simultaneous using of antibiotic resistance patterns and molecular typing can be effective in detecting isolates and infection control. 

منابع و مأخذ:

References

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  4. Ndegwa E.O. Brien D. Matthew K. Wang Z. Kim J. Shiga toxin subtypes, serogroups, phylogroups, RAPD genotypic diversity and select virulence markers of Shiga-Toxigenic Escherichia coli strains from goats in Mid-Atlantic US. Microorganisms. 2022; 10(9):1842
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  6. Iwu C.D. du Plessis E. Korsten L. Okoh A.I. Prevalence of E. coli O157: H7 strains in irrigation water and agricultural soil in two district municipalities in South Africa. Int J Environ Stud. 2021; 78(3): 474-483.
    7.
  7. Mir R.A. Brunelle B.W. Alt D.P. Arthur T.M. Kudva I.T. Supershed Escherichia coli O157: H7 Has potential for increased persistence on the rectoanal junction squamous epithelial cells and antibiotic resistance. Int.J. Microbiol. 2020; 2020.
    8.
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  1. Al-Ajmi D. Rahman S. Banu S. Occurrence virulence genes and antimicrobial profiles of Escherichia coli O157 isolated from ruminants slaughtered in Al Ain, United Arab Emirates. BMC Microbiol. 2020; 20(1): 1-10.
    2.
  2. Haidari N. Tahamtan Y. Molaee H. The effect of adjuvants on the efficacy and safety of anti-diarrhea Escherichia coli O157: H7 vaccine. JMW. 2020;13(1):36-46 [persian].
    3.
  3. Moran R.A. Anantham S. Holt K.E. Hall R.M. Prediction of antibiotic resistance from antibiotic resistance genes detected in antibiotic-resistant commensal Escherichia coli using PCR or WGS. J. Antimicrob. Chemother. 2017; 72(3): 700-704.
    4.
  4. Dehkharghani A.D. Haghighat S. Farzami M.R. Douraghi M. Rahbar M. Subtyping β-lactamase-producing Escherichia coli strains isolated from patients with UTI by MLVA and PFGE methods. IJBMS. 2021; 24(4): 437.
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  5. Raeispour M and Ranjbar R. Antibiotic resistance, virulence factors and genotyping of Uropathogenic Escherichia coli strains. Antimicrob. Resist. Infect. Control. 2018; 7(1): 1-9.
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    7.
  7. Nguyen T.T. Van Giau V. Vo T.K. Multiplex PCR for simultaneous identification of E. coli O157: H7, Salmonella spp. and L. monocytogenes in food. 3 Biotech. 2016; 6 (2):1-9.
    8.
  8. Franck S.M. Bosworth B.T. Moon H.W. Multiplex PCR for enterotoxigenic, attaching and effacing and Shiga toxin-producing Escherichia coli strains from calves. J. Clin. Microbiol. 1998; 36 (6):1795-1797.
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  9. Rashid R.A. Tabata T.A. Oatley M.J. Besser T.E. Tarr P.I. Moseley S.L. Expression of putative virulence factors of Escherichia coli O157: H7 differs in bovine and human infections. Infect. Immun. 2006; 74 (7):4142-4148.
    10.
  10. Hopkins K.L. Hilton A.C. Restriction endonuclease analysis of RAPD-PCR amplicons derived from Shiga-like toxin-producing Escherichia coli O157 isolates. J. Med. Microbiol. 2001; 50 (1):90-95.
    11.
  11. Madico G. Akopyants N.S. Berg D.E. Arbitrarily primed PCR DNA fingerprinting of Escherichia coli O157: H7 strains by using templates from boiled cultures. J. Clin. Microbiol. 1995; 33 (6):1534-1536.
    12.
  12. Vidovic S. Korber D.R. Prevalence of Escherichia coli O157 in Saskatchewan cattle: characterization of isolates by using random amplified polymorphic DNA PCR, antibiotic resistance profiles and pathogenicity determinants. Appl. Environ. Microbiol. 2006; 72(6): 4347-4355.
    13.
  13. Bodendoerfer E. Marchesi M. Imkamp F. Courvalin P. Böttger E. Mancini S. Co-occurrence of aminoglycoside and β-lactam resistance mechanisms in aminoglycoside-non-susceptible Escherichia coli isolated in the Zurich area, Switzerland. Int. J.Antimicrob. Agent. 2020; 56(1): 106- 109.
    14.
  14. Weinstein MP. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute. 2021.
    15.
  15. Tayh G. Boubaker SM. Khedher RB. Jbeli M, Chehida FB. Mamlouk A. Daaloul-Jedidi M. Messadi L. Prevalence virulence genes and antimicrobial profiles of Escherichia coli O157: H7 isolated from healthy cattle in Tunisia. JIDC. 2022;16(08):1308-16.
    16.
  16. Mir R.A. Weppelmann T.A. Kang M. Bliss T.M. DiLorenzo N. Lamb G.C. Ahn S. Jeong K.C. Association between animal age and the prevalence of Shiga toxin-producing Escherichia coli in a cohort of beef cattle. Vet. Microbiol. 2015; 175(2-4): 325-331.
    17.
  17. Afshari A. Rad M. Seifi H.A. Ghazvini K. Genetic variation among Escherichia coli isolates from human and calves by using RAPD PCR. Iran. J. Vet. Res. 2016; 10(1): 33-40.
    18.
  18. Suardana I.W. Artama W.T. Widiasih D.A. Mahardika I. Genetic diversity of Escherichia coli O157: H7 strains using random amplified polymorphic DNA (RAPD). Int Res J Microbiol. 2013; 4 (72): 8
    19.
  19. Williams J.G.K. Kubelik A.R. Livak K.J. Rafalski J.A. Tingey S.V.'DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. Spec. Publ. 1990; 18 (22):6531-6535.
    20.
  20. Radu, S. Ling O.W. Rusul G. Karim M.I.A. Nishibuchi M. Detection of Escherichia coli O157: H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses. J. Microbiol. Methods. 2001; 46 (2):131-139.
    21.
  21. Saadatian Farivar A. Nowroozi J. Eslami G. Sabokbar A. RAPD PCR profile, antibiotic resistance, prevalence of armA Gene, and detection of KPC enzyme in Klebsiella pneumoniae isolates. Can. J. Infect. Dis. Med. Microbiol. 2018;2018.
    22.
  22. Chansiripornchai N. Ramasoota P. Sasipreeyajan J. Svenson S. Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplifed polymorphic DNA (RAPD) analysis. Vet. Microbiol. 2001; 80:75-83.
    23.
  23. Nielsen K. Dynesen P. Larsen P. Frimodt-Moller N. Faecal Escherichia coli from patients with E. coli urinary tract infection and healthy controls who have never had a urinary tract infection. J of Med Micro. 2014; 63:582–589.
    24.
  24. Zhang S. Huang Y. Chen M. Yang G. Zhang J. Wu Q. Wang J. Ding Y. Ye Q. Lei T. Su Y. Characterization of Escherichia coli O157: non-H7 isolated from retail food in China and first report of mcr-1/IncI2-carrying colistin-resistant E. coli O157: H26 and E. coli O157: H4. Int. J. Food Microbiol. 2022; 378:109805.
    25.
  25. Marialouis X.A. Santhanam A. Antibiotic resistance, RAPD-PCR typing of multiple drug resistant strains of Escherichia coli from urinary tract infection (UTI). JCDR. 2016; 10(3): pDC05.
    26.
  26. Cobbaut K. Houf K. Boyen F. Haesebrouck F.De Zutter L. Genotyping and antimicrobial resistance patterns of Escherichia coli O157 originating from cattle farms.  Foodborne Pathog. Dis. 2011; 8(6):719-724.
    27.
  27. Kim H.H. Samadpour M. Grimm L. Clausen C.R. Besser T.E. Baylor M. Kobayashi J.M. Neill M.A. Schoenknecht F.D. Tarr P.I. Characteristics of antibiotic-resistant Escherichia coli O157: H7 in Washington State, 1984–1991. J. Infect. Dis. 1994; 170(6):1606-1609.
    28.
  28. Fazel F. Jamshidi A. Khoramian B. Phenotypic and genotypic study on antimicrobial resistance patterns of E. coli isolates from bovine mastitis. Microb. Pathog. 2019; 132:355-361.
    29.

 

  1. Khan A and Yamasaki S. Prevalence and genetic profiling of virulence determinates of Non-O157 STEC isolate from cattle, beef and humans Calcutta India. Emerg Inf Dis. 2002; 8 (1): 54-62.
    2.
  2. Oluborode O.B. Smith S.I. Seriki T.A. Fowora M. Ajayi A. Coker A.O. Antibiotic susceptibility pattern and molecular typing by PCR-RAPD analysis of clinical and environmental isolates of Pseudomonas aeruginosa. Microbiol. Biotechnol. Lett. 2018; 46(4): 434-437.
    3.
_||_

References

  1. Elsayed M.S.A.E. Eldsouky S.M. RoshdyT. Bayoume A.M.A. Nasr G.M. Salama A.S. Akl B.A. Hasan A.S. Shahat A.K. Khashaba R.A. Abdelhalim W.A. Genetic and antimicrobial resistance profiles of non-O157 Shiga toxin-producing Escherichia coli from different sources in Egypt. BMC Microbiol. 2021; 21(1): 1-19.
    2.
  2. Bumunang EW. Zaheer R. Stanford K. Laing C. Niu D. Guan LL. Chui L. Tarr GA. McAllister TA. Genomic analysis of shiga toxin-producing E. coli O157 cattle and clinical isolates from Alberta, Canada. Toxins. 2022 ; 31:14(9):603.
    3.
  3. Haile A.F. Alonso S. Berhe N. Atoma T.B. Boyaka P.N. Grace D. Prevalence, Antibiogram, and multidrug-resistant profile of E. coli O157: H7 in retail raw beef in Addis Ababa, Ethiopia. Front. Vet. Sci. 2022;9.
    4.
  4. Ndegwa E.O. Brien D. Matthew K. Wang Z. Kim J. Shiga toxin subtypes, serogroups, phylogroups, RAPD genotypic diversity and select virulence markers of Shiga-Toxigenic Escherichia coli strains from goats in Mid-Atlantic US. Microorganisms. 2022; 10(9):1842
    5.
  5. Fadel H.M. Afifi R. Al-Qabili D.M. Characterization and zoonotic impact of Shiga toxin producing Escherichia coli in some wild bird species. Vet. World. 2017; 10(9): 1118.
    6.
  6. Iwu C.D. du Plessis E. Korsten L. Okoh A.I. Prevalence of E. coli O157: H7 strains in irrigation water and agricultural soil in two district municipalities in South Africa. Int J Environ Stud. 2021; 78(3): 474-483.
    7.
  7. Mir R.A. Brunelle B.W. Alt D.P. Arthur T.M. Kudva I.T. Supershed Escherichia coli O157: H7 Has potential for increased persistence on the rectoanal junction squamous epithelial cells and antibiotic resistance. Int.J. Microbiol. 2020; 2020.
    8.
  8. Kolodziejek AM. Minnich SA. Hovde CJ. Escherichia coli 0157: H7 virulence factors and the ruminant reservoir. Curr. Opin. Infect. Dis. 2022;35(3):205-14.
    9.

 

  1. Al-Ajmi D. Rahman S. Banu S. Occurrence virulence genes and antimicrobial profiles of Escherichia coli O157 isolated from ruminants slaughtered in Al Ain, United Arab Emirates. BMC Microbiol. 2020; 20(1): 1-10.
    2.
  2. Haidari N. Tahamtan Y. Molaee H. The effect of adjuvants on the efficacy and safety of anti-diarrhea Escherichia coli O157: H7 vaccine. JMW. 2020;13(1):36-46 [persian].
    3.
  3. Moran R.A. Anantham S. Holt K.E. Hall R.M. Prediction of antibiotic resistance from antibiotic resistance genes detected in antibiotic-resistant commensal Escherichia coli using PCR or WGS. J. Antimicrob. Chemother. 2017; 72(3): 700-704.
    4.
  4. Dehkharghani A.D. Haghighat S. Farzami M.R. Douraghi M. Rahbar M. Subtyping β-lactamase-producing Escherichia coli strains isolated from patients with UTI by MLVA and PFGE methods. IJBMS. 2021; 24(4): 437.
    5.
  5. Raeispour M and Ranjbar R. Antibiotic resistance, virulence factors and genotyping of Uropathogenic Escherichia coli strains. Antimicrob. Resist. Infect. Control. 2018; 7(1): 1-9.
    6.
  6. Tahamtan Y. Hayati M. Namavari MM. Moazeni GR. Improved sorbitol macConkey agar medium containing cefixime and potassium tellurite for isolation and diagnosis of E. coli O157: H7 from clinical case. JMW. 2009; 2(1):37-48 [Persian].
    7.
  7. Nguyen T.T. Van Giau V. Vo T.K. Multiplex PCR for simultaneous identification of E. coli O157: H7, Salmonella spp. and L. monocytogenes in food. 3 Biotech. 2016; 6 (2):1-9.
    8.
  8. Franck S.M. Bosworth B.T. Moon H.W. Multiplex PCR for enterotoxigenic, attaching and effacing and Shiga toxin-producing Escherichia coli strains from calves. J. Clin. Microbiol. 1998; 36 (6):1795-1797.
    9.
  9. Rashid R.A. Tabata T.A. Oatley M.J. Besser T.E. Tarr P.I. Moseley S.L. Expression of putative virulence factors of Escherichia coli O157: H7 differs in bovine and human infections. Infect. Immun. 2006; 74 (7):4142-4148.
    10.
  10. Hopkins K.L. Hilton A.C. Restriction endonuclease analysis of RAPD-PCR amplicons derived from Shiga-like toxin-producing Escherichia coli O157 isolates. J. Med. Microbiol. 2001; 50 (1):90-95.
    11.
  11. Madico G. Akopyants N.S. Berg D.E. Arbitrarily primed PCR DNA fingerprinting of Escherichia coli O157: H7 strains by using templates from boiled cultures. J. Clin. Microbiol. 1995; 33 (6):1534-1536.
    12.
  12. Vidovic S. Korber D.R. Prevalence of Escherichia coli O157 in Saskatchewan cattle: characterization of isolates by using random amplified polymorphic DNA PCR, antibiotic resistance profiles and pathogenicity determinants. Appl. Environ. Microbiol. 2006; 72(6): 4347-4355.
    13.
  13. Bodendoerfer E. Marchesi M. Imkamp F. Courvalin P. Böttger E. Mancini S. Co-occurrence of aminoglycoside and β-lactam resistance mechanisms in aminoglycoside-non-susceptible Escherichia coli isolated in the Zurich area, Switzerland. Int. J.Antimicrob. Agent. 2020; 56(1): 106- 109.
    14.
  14. Weinstein MP. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute. 2021.
    15.
  15. Tayh G. Boubaker SM. Khedher RB. Jbeli M, Chehida FB. Mamlouk A. Daaloul-Jedidi M. Messadi L. Prevalence virulence genes and antimicrobial profiles of Escherichia coli O157: H7 isolated from healthy cattle in Tunisia. JIDC. 2022;16(08):1308-16.
    16.
  16. Mir R.A. Weppelmann T.A. Kang M. Bliss T.M. DiLorenzo N. Lamb G.C. Ahn S. Jeong K.C. Association between animal age and the prevalence of Shiga toxin-producing Escherichia coli in a cohort of beef cattle. Vet. Microbiol. 2015; 175(2-4): 325-331.
    17.
  17. Afshari A. Rad M. Seifi H.A. Ghazvini K. Genetic variation among Escherichia coli isolates from human and calves by using RAPD PCR. Iran. J. Vet. Res. 2016; 10(1): 33-40.
    18.
  18. Suardana I.W. Artama W.T. Widiasih D.A. Mahardika I. Genetic diversity of Escherichia coli O157: H7 strains using random amplified polymorphic DNA (RAPD). Int Res J Microbiol. 2013; 4 (72): 8
    19.
  19. Williams J.G.K. Kubelik A.R. Livak K.J. Rafalski J.A. Tingey S.V.'DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. Spec. Publ. 1990; 18 (22):6531-6535.
    20.
  20. Radu, S. Ling O.W. Rusul G. Karim M.I.A. Nishibuchi M. Detection of Escherichia coli O157: H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses. J. Microbiol. Methods. 2001; 46 (2):131-139.
    21.
  21. Saadatian Farivar A. Nowroozi J. Eslami G. Sabokbar A. RAPD PCR profile, antibiotic resistance, prevalence of armA Gene, and detection of KPC enzyme in Klebsiella pneumoniae isolates. Can. J. Infect. Dis. Med. Microbiol. 2018;2018.
    22.
  22. Chansiripornchai N. Ramasoota P. Sasipreeyajan J. Svenson S. Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplifed polymorphic DNA (RAPD) analysis. Vet. Microbiol. 2001; 80:75-83.
    23.
  23. Nielsen K. Dynesen P. Larsen P. Frimodt-Moller N. Faecal Escherichia coli from patients with E. coli urinary tract infection and healthy controls who have never had a urinary tract infection. J of Med Micro. 2014; 63:582–589.
    24.
  24. Zhang S. Huang Y. Chen M. Yang G. Zhang J. Wu Q. Wang J. Ding Y. Ye Q. Lei T. Su Y. Characterization of Escherichia coli O157: non-H7 isolated from retail food in China and first report of mcr-1/IncI2-carrying colistin-resistant E. coli O157: H26 and E. coli O157: H4. Int. J. Food Microbiol. 2022; 378:109805.
    25.
  25. Marialouis X.A. Santhanam A. Antibiotic resistance, RAPD-PCR typing of multiple drug resistant strains of Escherichia coli from urinary tract infection (UTI). JCDR. 2016; 10(3): pDC05.
    26.
  26. Cobbaut K. Houf K. Boyen F. Haesebrouck F.De Zutter L. Genotyping and antimicrobial resistance patterns of Escherichia coli O157 originating from cattle farms.  Foodborne Pathog. Dis. 2011; 8(6):719-724.
    27.
  27. Kim H.H. Samadpour M. Grimm L. Clausen C.R. Besser T.E. Baylor M. Kobayashi J.M. Neill M.A. Schoenknecht F.D. Tarr P.I. Characteristics of antibiotic-resistant Escherichia coli O157: H7 in Washington State, 1984–1991. J. Infect. Dis. 1994; 170(6):1606-1609.
    28.
  28. Fazel F. Jamshidi A. Khoramian B. Phenotypic and genotypic study on antimicrobial resistance patterns of E. coli isolates from bovine mastitis. Microb. Pathog. 2019; 132:355-361.
    29.

 

  1. Khan A and Yamasaki S. Prevalence and genetic profiling of virulence determinates of Non-O157 STEC isolate from cattle, beef and humans Calcutta India. Emerg Inf Dis. 2002; 8 (1): 54-62.
    2.
  2. Oluborode O.B. Smith S.I. Seriki T.A. Fowora M. Ajayi A. Coker A.O. Antibiotic susceptibility pattern and molecular typing by PCR-RAPD analysis of clinical and environmental isolates of Pseudomonas aeruginosa. Microbiol. Biotechnol. Lett. 2018; 46(4): 434-437.
    3.

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