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Confirmatory Detection of Escherichia coli O157:H7 in Diarrhoeic and Non-Diarrhoeic Calves by using Real Time PCR with Studying the Antimicrobial Susceptibility of These Bacteria


Affiliations
1 Department of Internal and Preventive Vet. Medicine, College of Vet. Med., University of Fallujah, Iraq
2 Department of Internal and Preventive Vet. Medicine, College of Vet. Med., University of Baghdad, Iraq
     

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Shiga toxigenic E. coli O157:H7 is one of the Shiga-toxin producing types of E. coli, which can produce disease in man and animals. The aims of this study were to detect O gene (rfbO157) and H gene( fliCH7) and determine the pattern of antimicrobial susceptibility of 32 E. coli O157:H7 isolates. The isolates isolated by conventional culture methods and latex test from faecal samples of 4 calves suffering from diarrhoea and 28 calves apparent normal. DNA was extracted from these isolates and the real time PCR was applied on the extracted DNA for confirming the diagnosis of rfbO157 and flic H7 genes. Seven antimicrobial discs were used in this study. The result presented that all the (32) isolates were possessed the rfbO157 and flics H7 genes. The susceptibility test of the isolates revealed that all isolates were highly resistant to Erythromycin (100%) and Cephalothin (96.87%). Less resistance to Tetracycline, Trimethoprim-Sulfamethoxazole and Chloramphenicol at different percentage (31.25%), (12.5%) and (9.37%) respectively and all isolates showed high sensitivity to Gentamicin (100%), Enrofloxacin (100%). In conclusion, the real time PCR can be used as a sensitive and reliable tool for diagnosis of E. coli O157:H7, and this bacteria showed high resistance to antibiotic.

Keywords

E. coli O157:H7, Real Time PCR, rfbO157, flic H7, Antibiotic Susceptibility.
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  • Abbasi P, Kargar M, Doosti A, Mardaneh J, Ghorbani-Dalini S and Dehyadegari M. Characterization of Shiga-toxin producing E. coli (STEC) and enteropathogenic E. coli (EPEC) using multiplex Real-Time PCR assays for stx1, stx2, eaeA. Iran. J. Microbiol., 6(3); 2014:169-174 .

  • Andrews JM (British society for antimicrobial chemotherapy). Methods for Antimicrobial Susceptibility Testing, 2012, Version 11.1.

  • Aranda K, Fabbricotti S, Fagundes-Neto U and Scaletsky IC. Single multiplex assay to identify simultaneously enterophatogenic, entero-aggregative, enterotoxigenic, enteroinvasive and Shiga toxin-producing Escherichia coli strains. FEMS Microbiology Letters, 267; 2007: 145-150.

  • Bauer AW, Kirby WM, Sherris JC and Turck M. Antibiotic susceptibility testing by a standardized single disk method. American journal of clinical pathology, 45(4); 1966: 493.

  • Cho S, Diez-Gonzalez F, Fossler CP, Wells SJ, Hedberg CW, Kaneene JB, Ruegg PL, Warnick LD and Bender JB "Prevalence of shiga toxin-encoding bacteria and shiga toxin-producing Escherichia coli isolates from dairy farms and county fairs." Vet.Microbiol. 118; (2006): 289–298.

  • Chui L, Lee M, Allen R, Bryks A, Haines L and Boras V. Comparison between ImmunoCard STAT!(®) and real-time PCR as screening tools for both O157:H7 and non-O157 Shiga toxinproducing Escherichia coli in Southern Alberta, Canada. Diagn. Microbiol. Infect. Dis. , 77(1); 2013:8-13.

  • Clinical and Laboratory Standards Institute antimicrobial susceptibility testing standards (CLSI). Performance Standards for Antimicrobial Susceptibility Testing, Wayne, PA,USA, Tenth Edition ,Vol. 30 No. 1 and Vol. 30 No. 15 M02-A10 and M07-A8, 2011.

  • Cornick NA, Pitzer J, Helgerson AF, Madsen ML, Kurth KT, Xiao Q and Minion F. Use of signature-tagged mutagenesis to identify genes associated with colonization of sheep by E. coli O157: H7. Veterinary microbiology, 201; 2017: 177-182.

  • Dunn JR. The epidemiology of shiga-toxigenic Escherichia coli O157:H7 in Louisiana cattle, and white-tailed deer. LSU Doctoral Dissertations; 2003.

  • El-Seedy FR, Abed AH, Yanni HA and El-Rahman SA. Prevalence of Salmonella and E. coli in neonatal diarrheic calves. Beni-Suef University Journal of Basic and Applied Sciences, 5(1); 2016: 45-51.

  • Galland JC, Hyatt DR, Crupper SS and Acheson DW. Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots. Appl. Environ. Microbiol., 67(4); 2001:1619-27.

  • Hasan MS, Yousif AA and Alwan MJ. Detection of virulent genes in E. coli O157: H7 isolated from puppies and adult dogs by polymerase chain reaction. Res. J. Vet. Pract, 4(1); 2016: 1-6.

  • Hussein MA and Yousif AA. Detection of stx1 and stx2 virulence genes from Escherichia coliO157:H7 isolated from calves by PCR assay.Int.J.Adv.Res.Biol.Sci.2(11); 2015: 324–329.

  • Jenkins C, Lawson A, Cheasty T and Willshaw G. Assessment of a real-time PCR for the detection and characterization of verocytotoxigenic Escherichia coli. Journal of Medical Microbiology, 61; 2012:1082–1085.

  • Jorgensen J and Ferraro M. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Infect Dis. 1;49(11); 2009:1749-55.

  • Lupindu AM. Epidemiology of Shiga toxin-producing Escherichia coli O157: H7 in Africa in review. Southern African Journal of Infectious Diseases, 33(1); 2018: 24-30.

  • Markey B, Leonard F, Archambault M, Cullinane A and Maguire D. Clinical Veterinary Microbiology. Second edition.; 2013: 246249.

  • Martorelli, L., Garbaccio, S., Vilte, D. A., Albanese, A. A., Mejías, M. P., Palermo, M. S., and Cataldi, A. A. (2017). Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157: H7. PloS one, 12(1), e0169422.

  • Mora, A.; Blanco, J.E.; Blanco, M.; Alonso, M.P.; Dhabi, G.; Echeita, A. and Gonazalez. (2005). Antimicrobial resistance of Shiga toxin (verotoxin) procucing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in spain. Res Microbiol., 156(7):793-806.

  • National Committee for Clinical Laboratory Standards Guidelines (NCCLS), (1990). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals,Wayne, PA,USA: NCCLS, M2-A4.

  • National Committee for Clinical Laboratory Standards (NCCLS),(1997). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals,Wayne, PA,USA: NCCLS, 6th ed, M2-A7

  • Osaili, T. , Akram, R. and Alaboudi, M. (2013). Prevalence and antimicrobial susceptibility of Escherichia coli O157:H7 on beef cattle slaughtered in Amman abattoir. Meat Science 93(3):463-8 ·

  • Osek, J. (2004). Phenotypic and genotypic characterization of Escherichia coli O157 strains isolated from humans, cattle and pigs. Vet. Med. – Czech, 49 (9): 317–326.

  • Osman, K. M., Mustafa, A. M., Elhariri, M., and Abdelhamed, G. S. (2013). The distribution of Escherichia coli serovars, virulence genes, gene association and combinations and virulence genes encoding serotypes in pathogenic E. coli recovered from diarrhoeic calves, sheep and goat. Transboundary and emerging diseases, 60(1), 69-78.

  • Qin, X. ; Klein, E. ; Galanakis, E. ; Thomas, A. ; Stapp, J. ; Rich, S. ; Buccat, A. and Tarr, P. (2015). Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples. J. Clin. Microbiol., 53(7):2148-53.

  • Rebekka, R.; Artz, L.; Davey, L. and Ken, K. (2006). Potential pitfalls in the quantitative molecular detection of Escherichia coli O157:H7 in environmental matrices. Can. J. Microbiol., 52: 482– 488.

  • Schaut RG, Loving CL, Sharma VK. Escherichia coli O157:H7 virulence factors differentially impact cattle and bison macrophage killing capacity. Microb Pathog., S0882-4010(17); 2018:31250-0.

  • Schildgen O. CDC Yellow Book 2018-Health information for International Travel. Chapter 3: Infectious Diseases Related to Travel. CDC; 2018. ISBN 978-0-19-937915-6.

  • Schroeder CM, Zhao C, DebRoy C, Torcolini J, Zhao S, White DG and Meng J. Antimicrobial resistance of Escherichia coli O157 isolated from humans, cattle, swine, and food. Applied and Environmental Microbiology, 68(2); 2002: 576-581.

  • Stanford K, Agopsowicz CA, McAllister TA. Genetic diversity and antimicrobial resistance among isolates of Escherichia coli O157:H7 from faeces and hides of super-shedders and lowshedding pen-mates in two commercial beef feedlots. BMC Vet. Res. 8; 2012:178.

  • Stefan A, Scaramagli S, Bergami R, Mazzini C, Barbanera M, Perelle S and Fach P. Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producing Escherichia coli in mincemeat samples. Canadian Journal of Microbiology, 53; 2007: 337-342.

  • Stein RA and Katz DE. Escherichia coli, cattle and the propagation of disease. FEMS microbiology letters, 364(6); 2017.

  • Tahamtan Y, Pourbakhsh A, Hayati SA, Namdar M, Shams N and Namvari Z. Prevalence and molecular characterization of verotoxin-producing Escherichia coli O157:H7 in cattle and sheep in Shiraz-Iran. Archives of Razi Institute. 66 (1); 2011: 29-36.

  • Talaro K and Chess B. Foundations in Microbiology. Ninth Edition. McGraw-Hill Education, New York.; 2013: 617.

  • Tarr GA, Shringi S, Phipps A, Besser T, Mayer J, Oltean H and Rabinowitz P. Geogenomic Segregation and Temporal Trends of Human Pathogenic Escherichia coli O157: H7, Washington, USA, 2005–2014. Emerging infectious diseases, 24(1); 2018: 32.

  • Thévenot J, Cordonnier C, Rougeron A, Le Goff O, Nguyen H, Denis Sand Blanquet-Diot. Enterohemorrhagic Escherichia coli infection has donor-dependent effect on human gut microbiota and may be antagonized by probiotic yeast during interaction with Peyer’s patches. Applied microbiology and biotechnology, 99(21); 2015: 9097-9110.

  • Vidovic, S. and Korber, D. (2006). 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.;72(6):4347-55.

  • Wong C, Jelacic S, Habeeb R, Watkins S and Tarr P .The risk of hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N. Engl. J. Med. 342; 2000:1930-1936.

  • Yousif AA, Hasan MS and Alwan MJ. Clinical and molecular study of E. coli O157:H7 isolated from Diarrheic and non diarrheic dogs. MRVSA 5(2); 2016: 1-10.

  • Yousif A, Hasan M, Najim T and Hussein M. Detection of virulence and antimicrobial susceptibility testing of Escherichia coli O157:H7 isolated from pets and stray dogs. Journal of Entomology and Zoology Studies, 5(4); 2017: 573-578.

  • Yousif A and Hussein M. Prevalence and molecular detection of intimin (eaeA) virulence gene in E. coli O157:H7 in calves. Res. J. Vet. Pract. 3(3) 2015: 47-52.

  • Zhang X, McDaniel A, Wolf L, Keusch G, Waldor M and Acheson D. Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J. Infect. Dis. 181; 2000:664-670.


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  • Confirmatory Detection of Escherichia coli O157:H7 in Diarrhoeic and Non-Diarrhoeic Calves by using Real Time PCR with Studying the Antimicrobial Susceptibility of These Bacteria

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Authors

Mustafa Salah Hasan
Department of Internal and Preventive Vet. Medicine, College of Vet. Med., University of Fallujah, Iraq
Mohammed Ali Hussein
Department of Internal and Preventive Vet. Medicine, College of Vet. Med., University of Fallujah, Iraq
Afaf Abdulrahman Yousif
Department of Internal and Preventive Vet. Medicine, College of Vet. Med., University of Baghdad, Iraq

Abstract


Shiga toxigenic E. coli O157:H7 is one of the Shiga-toxin producing types of E. coli, which can produce disease in man and animals. The aims of this study were to detect O gene (rfbO157) and H gene( fliCH7) and determine the pattern of antimicrobial susceptibility of 32 E. coli O157:H7 isolates. The isolates isolated by conventional culture methods and latex test from faecal samples of 4 calves suffering from diarrhoea and 28 calves apparent normal. DNA was extracted from these isolates and the real time PCR was applied on the extracted DNA for confirming the diagnosis of rfbO157 and flic H7 genes. Seven antimicrobial discs were used in this study. The result presented that all the (32) isolates were possessed the rfbO157 and flics H7 genes. The susceptibility test of the isolates revealed that all isolates were highly resistant to Erythromycin (100%) and Cephalothin (96.87%). Less resistance to Tetracycline, Trimethoprim-Sulfamethoxazole and Chloramphenicol at different percentage (31.25%), (12.5%) and (9.37%) respectively and all isolates showed high sensitivity to Gentamicin (100%), Enrofloxacin (100%). In conclusion, the real time PCR can be used as a sensitive and reliable tool for diagnosis of E. coli O157:H7, and this bacteria showed high resistance to antibiotic.

Keywords


E. coli O157:H7, Real Time PCR, rfbO157, flic H7, Antibiotic Susceptibility.

References