Abstract
Background
Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes.
Materials and methods
A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously.
Results
Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1, stx2, eae and ehlyA genes simultaneously.
Conclusion
It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.
Similar content being viewed by others
References
Chileshe J, Ateba CN (2013) Molecular identification of Escherichia coli O145: H28 from beef in the North West Province, South Africa. Life Sci J 10(4):1171–1176
Karmali MA, Gannon V, Sargeant JM (2010) Verocytotoxin-producing Escherichia coli (VTEC). Vet Microbiol 140(3–4):360–370
Sekhar MS, Sharif NM, Rao TS (2017) Serotypes of sorbitol-positive shiga toxigenic Escherichia coli (SP-STEC) isolated from freshwater fish. Int J Fish Aquatic Sci 5:503–505
Oporto B, Ocejo M, Alkorta M, Marimón JM, Montes M, Hurtado A (2019) Zoonotic approach to Shiga toxin-producing Escherichia coli: integrated analysis of virulence and antimicrobial resistance in ruminants and humans. Epidemiol Infect. https://doi.org/10.1017/S0950268819000566
Hunt JM (2010) Shiga toxin–producing Escherichia coli (STEC). Clin Lab Med 30(1):21–45
Fernandez TF (2008) E. coli O157: H7. Vet World 1(3):83
Pintara A, Jennison A, Rathnayake IU, Mellor G, Huygens F (2020) Core and accessory genome comparison of Australian and international strains of O157 Shiga toxin-producing Escherichia coli. Front Microbiol 11:2162
Mian AH, Fatima T, Qayyum S, Ali K, Shah R, Ali NM (2020) A study of bacterial profile and antibiotic susceptibility pattern found in drinking water at district Mansehra, Pakistan. Appl Nanosci 10:5435–5439. https://doi.org/10.1007/s13204-020-01411-0
Xia X, Meng J, McDermott PF, Ayers S, Blickenstaff K, Tran TT, Zhao S (2010) Presence and characterization of Shiga toxin-producing Escherichia coli and other potentially diarrheagenic E. coli strains in retail meats. Appl Environ Microbiol 76(6):1709–1717
Elson R, Grace K, Vivancos R, Jenkins C, Adak GK, O’Brien SJ, Lake IR (2018) A spatial and temporal analysis of risk factors associated with sporadic Shiga toxin-producing Escherichia coli O157 infection in England between 2009 and 2015. Epidemiol Infect 146(15):1928–1939
Persad AK, Lejeune JT (2015) Animal reservoirs of Shiga toxin-producing Escherichia coli. Enterohemorrhagic Escherichia coli and other shiga toxin-producing E. coli. ASM Press, Washington, DC
Rigobelo EC, Santo E, Marin JM (2008) Beef carcass contamination by Shiga toxin-producing Escherichia coli strains in an abattoir in Brazil: characterization and resistance to antimicrobial drugs. Foodborne Pathog Dis 5:811–817
Joris A, Vanrompay D, Verstraete K, De Reu K, De Zutter L (2012) Enterohemorrhagic Escherichia coli with particular attention to the German outbreak strain O104: H4. VDT 81(1):3–10
Kiranmayi C, Krishnaiah N, Mallika EN (2010) Escherichia coli O157: H7-an emerging pathogen in foods of animal origin. Vet World 3(8):382
Mughini-Gras L, Van Pelt W, Van der Voort M, Heck M, Friesema I, Franz E (2018) Attribution of human infections with Shiga toxin-producing Escherichia coli (STEC) to livestock sources and identification of source-specific risk factors, The Netherlands (2010–2014). Zoonoses Public Health 65(1):e8–e22
Rahimi E, Momtaz H, Anari MMH, Alimoradi M, Momen M, Riahi M (2012) Isolation and genomic characterization of Escherichia coli O157: NM and Escherichia coli O157: H7 in minced meat and some traditional dairy products in Iran. Afr J Biotech 11(9):2328–2332
Kim JS, Lee MS, Kim JH (2020) Recent updates on outbreaks of Shiga toxin-producing Escherichia coli and its potential reservoirs. Front Cell Infect Microbiol 10:273
Anonymous (2012) Summary of notifiable diseases-the United States, 2010. MMWR Morb Mortal Wkly Rep 59:1–111
Ecdc E (2013) The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA J 11:3129
On S, Lim E, Lopez L, Cressey P, Pirie R (2011) Annual report concerning foodborne disease in New Zealand. Enviromental Science and Research Limited (ESR), Christchurch, New Zealand, p 130
Carbonari CC, Fittipaldi N, Teatero S, Athey TB, Pianciola L, Masana M, Melano RG, Rivas M, Chinen I (2016) Whole-genome sequencing applied to the molecular epidemiology of shiga toxin-producing Escherichia coli O157:H7 in Argentina. Genome Announc 4:10. https://doi.org/10.1128/genomeA.01341-16
Qayyum S, Basharat S, Mian AH, Qayum S, Ali M, Changsheng P, Shahzad M (2020) Isolation, identification and antibacterial study of pigmented bacteria. Appl Nanosci 10:4495–4503. https://doi.org/10.1007/s13204-020-01363-5
Qayyum S, Nasir A, Mian AH, Rehman S, Qayum S, Siddiqui MF, Kalsoom U (2020) Extraction of peroxidase enzyme from different vegetables for biodetoxification of vat dyes. Appl Nanosci 10:5191–5199. https://doi.org/10.1007/s13204-020-01348-4
Group OW (2012) Monitoring the incidence and causes of diseases potentially transmitted by food in Australia: annual report of the OzFoodNet network, 2010. Commun Dis Intell Q Rep 36:E213
Ali NH, Farooqui A, Khan A, Khan AY, Kazmi SU (2010) Microbial contamination of raw meat and its environment in retail shops in Karachi, Pakistan. J Infect Dev Ctries 4:382–388
Fatima T, Mian AH, Khan Z, Khan AM, Anwar F, Tariq A, Sardar M (2020) Citrus sinensis a potential solution against superbugs. Appl Nanosci 10:5077–5083. https://doi.org/10.1007/s13204-020-01408-9
Irshad H, Binyamin I, Ahsan A, Riaz A, Shahzad MA, Qayyum M, Yousaf A (2020) Occurrence and molecular characterization of Shiga Toxin-producing Escherichia coli isolates recovered from cattle and goat meat obtained from retail meat shops in Rawalpindi and Islamabad, Pakistan. Pak Vet J 40(3):10. https://doi.org/10.29261/pakvetj/2020.045
Mohsin M, Haque A, Ali A, Sarwar Y, Bashir S, Tariq A, Afzal A, Iftikhar T, Saeed MA (2010) Effects of ampicillin, gentamicin, and cefotaxime on the release of Shiga Toxins from Shiga Toxin-producing Escherichia coli isolated during a diarrhea episode in Faisalabad, Pakistan. Foodborne Pathog Dis 7:85–90
Razzaq A, Shamsi S, Nawaz A, Nawaz A, Ali A, Malik K (2016) The occurrence of Shiga toxin producing E. coli from raw milk. Pure Appl Biol 5(2):270–276
Shahzad K, Muhammad K, Sheikh A, Yaqub T, Rabbani M, Hussain T, Anjum A, Anees M (2013) Isolation and molecular characterization of Shiga toxin producing E. coli O157. J Anim Plant Sci 23:1618–1621
Jeshveen SS, Chai LC, Pui CF, Son R (2012) Optimization of multiplex PCR conditions for rapid detection of Escherichia coli O157: H7 virulence genes. Int Food Res J 19(2)
Radu S, Ling OW, Rusul G, Karim MIA, Nishibuchi M (2001) Detection of Escherichia coli O157: H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses. J Microbiol Methods 46:131–139
Irshad H, Cookson A, Hotter G, Besser T, On S, French N (2012) Epidemiology of Shiga toxin-producing Escherichia coli O157 in very young calves in the North Island of New Zealand. N Z Vet J 60:21–26
Fortin NY, Mulchandani A, Chen W (2001) Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157: H7. Anal Biochem 289:281–288
Perelle S, Dilasser F, Grout J, Fach P (2004) Detection by 5′-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157: H7, associated with the world’s most frequent clinical cases. Mol Cell Probes 18:185–192
Stromberg ZR, Redweik GA, Mellata M (2018) Detection, prevalence, and pathogenicity of non-O157 Shiga toxin-producing Escherichia coli from cattle hides and carcasses. Foodborne Pathog Dis 15(3):119–131
Sharma VK, Dean-Nystrom EA (2003) Detection of enterohemorrhagic Escherichia coli O157: H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins. Vet Microbiol 93:247–260
Mori L, Perales R, Rodríguez J, Shiva C, Koga Y, Choquehuanca G, Palacios C (2014) Molecular identification of Shiga-toxin producing and enteropathogenic Escherichia coli (STEC and EPEC) in diarrheic and healthy young alpacas. Adv Microbiol 4:360
Bonardi S, Alpigiani I, Tozzoli R, Vismarra A, Zecca V, Greppi C, Brindani F (2015) Shiga toxin-producing Escherichia coli O157, O26 and O111 in cattle faeces and hides in Italy. Vet Record Open. https://doi.org/10.1136/vetreco-2014-000061
Govaris A, Angelidis AS, Katsoulis K, Pournaras S (2011) Occurrence, virulence genes and antimicrobial resistance of Escherichia coli O157 in bovine, caprine, ovine and porcine carcasses in Greece. J Food Saf 31(2):242–249
Pinaka O, Pournaras S, Mouchtouri V, Plakokefalos E, Katsiaflaka A, Kolokythopoulou F, Hadjichristodoulou C (2013) Shiga toxin-producing Escherichia coli in Central Greece: prevalence and virulence genes of O157: H7 and non-O157 in animal feces, vegetables, and humans. Eur J Clin Microbiol Infect Dis 32(11):1401–1408
Akanbi BO, Mbah IP, Kerry PC (2011) Prevalence of Escherichia coli O157: H7 on hides and faeces of ruminants at slaughter in two major abattoirs in Nigeria. Lett Appl Microbiol 53(3):336–340
Zarei M, Basiri N, Jamnejad A, Eskandari MH (2013) Prevalence of Escherichia coli O157: H7, Listeria monocytogenes and Salmonella spp. in beef, buffalo and lamb using multiplex PCR. Jundishapur J Microbiol 6(8)
Al-Ajmi D, Rahman S, Banu S (2020) Occurrence, virulence genes, and antimicrobial profiles of Escherichia coli O157 isolated from ruminants slaughtered in Al Ain, United Arab Emirates. BMC Microbiol 20(1):1–10
McPherson AS, Dhungyel OP, Ward MP (2015) Comparison of recto-anal mucosal swab and faecal culture for the detection of Escherichia coli O157 and identification of super-shedding in a mob of Merino sheep. Epidemiol Infect 143(13):2733–2742
Rice DH, Sheng HQ, Wynia SA, Hovde CJ (2003) Recto anal mucosal swab culture is more sensitive than fecal culture and distinguishes Escherichia coli O157: H7-colonized cattle and those transiently shedding the same organism. J Clin Microbiol 41(11):4924
Williams KJ, Ward MP, Dhungyel OP (2015) Longitudinal study of Escherichia coli O157 shedding and super shedding in dairy heifers. J Food Prot 78(4):636–642
De Boer E, Heuvelink AE (2000) Methods for the detection and isolation of Shiga toxin-producing Escherichia coli. J Appl Microbiol 88(S1):133S-143S
Vimont A, Vernozy-Rozand C, Delignette-Muller ML (2006) Isolation of E. coli O157: H7 and non-O157 STEC in different matrices: review of the most commonly used enrichment protocols. Lett Appl Microbiol 42(2):102–108
Conrad CC, Stanford K, McAllister TA, Thomas J, Reuter T (2016) Competition during enrichment of pathogenic Escherichia coli may result in culture bias. Facets 1(1):114–126
Wang F, Yang Q, Kase JA, Meng J, Clotilde LM, Lin A, Ge B (2013) Current trends in detecting non-O157 Shiga toxin–producing Escherichia coli in food. Foodborne Pathog Dis 10(8):665–677
EFSA Biohaz Panel, Koutsoumanis K, Allende A, Alvarez-Ordóñez A, Bover-Cid S, Chemaly M, Davies R, Cesare AD, Herman L, Hilbert F, Skandamis P, Suffredini E, Jenkins C, Pires SM, Morabito S, Niskanen T, Scheutz F, Felicio MTDS, Messens W, Bolton D (2020) Pathogenicity assessment of Shiga toxin-producing Escherichia coli (STEC) and the public health risk posed by contamination of food with STEC. EFSA J 18(1):e05967. https://doi.org/10.2903/j.efsa.2020.5967
Momtaz H, Farzan R, Rahimi E, Safarpoor Dehkordi F, Souod N (2012) Molecular characterization of Shiga toxin-producing Escherichia coli isolated from ruminant and donkey raw milk samples and traditional dairy products in Iran. Sci World J 2012(2):20. https://doi.org/10.1100/2012/231342
Brusa V, Piñeyro PE, Galli L, Linares LH, Ortega EE, Padola NL, Leotta GA (2016) Isolation of Shiga toxin-producing Escherichia coli from ground beef using multiple combinations of enrichment broths and selective agars. Foodborne Pathog Dis 13(3):163–170
Gill A, Huszczynski G, Gauthier M, Blais B (2014) Evaluation of eight agar media for the isolation of shiga toxin—producing Escherichia coli. J Microbiol Methods 96:6–11
Lupindu AM (2018) Epidemiology of Shiga toxin-producing Escherichia coli O157: H7 in Africa in review. South Afr J Infect Dis 33(1):24–30
Shridhar PB, Noll LW, Shi X, An B, Cernicchiaro N, Renter DG, Bai J (2016) Multiplex quantitative PCR assays for the detection and quantification of the six major non-O157 Escherichia coli serogroups in cattle feces. J Food Prot 79(1):66–74
Verhaegen B, De Reu K, De Zutter L, Verstraete K, Heyndrickx M, Van Coillie E (2016) Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces. Toxins 8(5):157
Macori G, McCarthy SC, Burgess CM, Fanning S, Duffy G (2019) A quantitative real time PCR assay to detect and enumerate Escherichia coli O157 and O26 serogroups in sheep recto-anal swabs. J Microbiol Methods 165:105703
Sethulekshmi C, Latha C, Anu CJ (2018) Occurrence and quantification of Shiga toxin-producing Escherichia coli from food matrices. Vet World 11(2):104
Macori G, McCarthy SC, Burgess CM, Fanning S, Duffy G (2020) Investigation of the causes of shigatoxigenic Escherichia coli PCR positive and culture negative samples. Microorganisms 8(4):587
Perera A, Clarke CM, Dykes GA, Fegan N (2015) Characterization of Shiga toxigenic Escherichia coli O157 and non-O157 isolates from Ruminant Feces in Malaysia. Biomed Res Int 2015:382403
Murphy BP, McCabe E, Murphy M, Buckley JF, Crowley D, Fanning S, Duffy G (2016) Longitudinal study of two Irish dairy herds: low numbers of Shiga toxin-producing Escherichia coli O157 and O26 Super-Shedders Identified. Front Microbiol 7:1850
Thomas KM, McCann MS, Collery MM, Logan A, Whyte P, McDowell DA, Duffy G (2012) Tracking verocytotoxigenic Escherichia coli O157, O26, O111, O103 and O145 in Irish cattle. Int J Food Microbiol 153(3):288–296
Bibbal D, Loukiadis E, Kérourédan M, Ferré F, Dilasser F, de Garam CP, Brugère H (2015) Prevalence of carriage of Shiga toxin-producing Escherichia coli serotypes O157: H7, O26: H11, O103: H2, O111: H8, and O145: H28 among slaughtered adult cattle in France. Appl Environ Microbiol 81(4):1397
McCabe E, Burgess CM, Lawal D, Whyte P, Duffy G (2019) An investigation of shedding and super-shedding of Shiga toxigenic Escherichia coli O157 and E. coli O26 in cattle presented for slaughter in the Republic of Ireland. Zoonoses Public Health 66(1):83–91
Fox J, Corrigan M, Drouillard J, Shi X, Oberst R, Nagaraja T (2007) Effects of concentrate level of diet and pen configuration on the prevalence of Escherichia coli O157 in finishing goats. Small Rumin Res 72:45–50
Mersha G, Asrat D, Zewde B, Kyule M (2010) The occurrence of Escherichia coli O157: H7 in feces, skin and carcasses from sheep and goats in Ethiopia. Lett Appl Microbiol 50:71–76
Menrath A, Wieler LH, Heidemanns K, Semmler T, Fruth A, Kemper N (2010) Shiga toxin producing Escherichia coli: identification of non-O157: H7-super-shedding cows and related risk factors. Gut Pathog 2(1):1–9
Mir RA, Weppelmann TA, Elzo M, Ahn S, Driver JD, Jeong KC (2016) Colonization of beef cattle by Shiga toxin-producing Escherichia coli during the first year of life: a cohort study. PLoS ONE 11(2):e0148518
Kilonzo C, Atwill ER, Mandrell R, Garrick M, Villanueva V, Hoar BR (2011) Prevalence and molecular characterization of Escherichia coli O157: H7 by multiple-locus variable-number tandem repeat analysis and pulsed-field gel electrophoresis in three sheep farming operations in California. J Food Prot 74(9):1413–1421
Moriarty EM, McEwan N, Mackenzie M, Karki N, Sinton LW, Wood DR (2011) Incidence and prevalence of microbial indicators and pathogens in ovine faeces in New Zealand. N Z J Agric Res 54(2):71–81
Gunn GJ, McKendrick IJ, Ternent HE, Thomson-Carter F, Foster G, Synge BA (2007) An investigation of factors associated with the prevalence of verocytotoxin producing Escherichia coli O157 shedding in Scottish beef cattle. Vet J 174(3):554–564
Dewell GA, Ransom JR, Dewell RD, McCurdy K, Gardner IA, Hill E, Salman MD (2005) Prevalence of and risk factors for Escherichia coli O157 in market-ready beef cattle from 12 US feedlots. Foodborne Pathog Dis 2(1):70–76
Acknowledgements
We would like to thank Matiullah (Department of Microbiology, Hazara University Mansehra, Pakistan) for his generous suggestion and help in revision of the final draft manuscript.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Shahzad, A., Ullah, F., Irshad, H. et al. Molecular detection of Shiga toxin-producing Escherichia coli (STEC) O157 in sheep, goats, cows and buffaloes. Mol Biol Rep 48, 6113–6121 (2021). https://doi.org/10.1007/s11033-021-06631-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-021-06631-3