Investigation of antibiotic resistance, virulence genes, and biofilm formation of Escherichia coli isolated from sheep feces in Shiraz industrial slaughterhouse, South of Iran

Document Type : Full paper (Original article)


1 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

2 MSc Student in Bacteriology, Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran


Background: With the increase in human population, the consumption of livestock products such as sheep meat has also increased. Sheep are the reservoir and shedder of Escherichia coli that can be transmitted to humans. Aims: Characterization of fecal E. coli isolated from sheep in slaughterhouse. Methods: Stool specimens were collected from 30 apparently healthy sheep from different flocks in Shiraz industrial slaughterhouse. The resistance of E. coli isolates against 10 antibiotics was determined by disk diffusion method. The presence of three major extended spectrum beta-lactamase (ESBL) genes and five tetracycline resistance genes as well as seven virulence genes were investigated by polymerase chain reaction (PCR) technique. Using the microtiter plate method, the biofilm formation ability of E. coli isolates was investigated. Results: The highest frequency of resistance was to amoxicillin (100%) followed by tetracycline (25%). All E. coli isolates were susceptible to gentamicin and nitrofurantoin, and only one isolate was resistant to the tested third-generation cephalosporins. Multidrug resistance phenotype was observed in 16.7% of the isolates. blaTEM (25%) was the most prevalent ESBL gene and tetA (62.5%) was the most prevalent tetracycline resistance gene in the isolates. crl, csgA, fimH, and bcsA genes were present in all isolates, and the prevalence of papC and afa genes was 95.8% and 83.3%, respectively. In total, 62.5% of the isolates were biofilm producers. Conclusion: According to the concept of One Health, the presence of virulent antibiotic-resistant biofilm producing strains of E. coli in sheep is a risk to public health.


Main Subjects

Aliasadi, S and Saei, HD (2015). Fecal carriage of Escherichia coli harboring extended-spectrum beta-lactamase (ESBL) genes by sheep and broilers in Urmia region, Iran. Iran. J. Vet. Med., 9: 93-101.
Brinas, L; Zarazaga, M; Sáenz, Y; Ruiz-Larrea, F and Torres, C (2002). Beta-lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrob. Agents Chemother., 46: 3156-3163.
CDC (2023). Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID). basics/index.html. Last Reviewed: August 17, 2023.
Cheney, TE; Smith, RP; Hutchinson, JP; Brunton, LA; Pritchard, G and Teale, CJ (2015). Cross-sectional survey of antibiotic resistance in Escherichia coli isolated from diseased farm livestock in England and Wales. Epidemiol. Infect., 143: 2653-2659.
CLSI (2018). Performance standards for antimicrobial susceptibility testing. CLSI Supplement M100. 28th Edn., Wayne, PA: Clinical and Laboratory Standards Institute.
Derakhshandeh, A; Firouzi, R and Naziri, Z (2014). Phylogenetic group determination of faecal Escherichia coli and comparative analysis among different hosts, Iran. J. Vet. Res., 15: 13-17.
Eltai, N; Al-Thani, A; Alhadidi, S; Abdfarag, A; Romaiha, H; Mahmoud, M; Alawad, O and Yassine, H (2020). Antibiotic resistance profile of commensal Escherichia coli isolated from healthy sheep in Qatar. J. Infect. Dev. Ctries., 14: 138-145.
Emett, J; David, R; McDaniel, J; McDaniel, S and Kingsley, K (2020). Comparison of DNA extracted from pediatric saliva, gingival crevicular fluid and site-specific biofilm samples. Methods Protoc., 3: 48.
Gemeda, BA; Wieland, B; Alemayehu, G; Knight-Jones, TJ; Wodajo, HD; Tefera, M; Kumbe, A; Olani, A; Abera, S and Amenu, K (2023). Antimicrobial resistance of Escherichia coli isolates from livestock and the environment in extensive smallholder livestock production systems in Ethiopia. Antibiotics. 12: 941.
Ghanbarpour, R; Askari, N; Ghorbanpour, M; Tahamtan, Y; Mashayekhi, K; Afsharipour, N and Darijani, N (2017). Genotypic analysis of virulence genes and antimicrobial profile of diarrheagenic Escherichia coli isolated from diseased lambs in Iran. Trop. Anim. Health Prod., 49: 591-597.
Gozi, KS; Froes, JR; Deus Ajude, LP; Da Silva, CR; Baptista, RS; Peiró, JR; Marinho, M; Mendes, LC; Nogueira, MC and Casella, T (2019). Dissemination of multidrug-resistant commensal Escherichia coli in feedlot lambs in Southeastern Brazil. Front. Microbiol., 10: 1394.
Gu, X; Ma, X; Wu, Q; Tao, Q; Chai, Y; Zhou, X; Han, M; Li, J; Huang, X; Wu, T and Zhang, X (2023). Isolation, identification, molecular typing, and drug resistance of Escherichia coli from infected cattle and sheep in Xinjiang, China. Vet. Med. Sci., 9: 1359-1368.
Hafez, AA (2020). Virulence and antimicrobial resistance genes of E. coli isolated from diarrheic sheep in the North-West Coast of Egypt. Sys. Rev. Pharm., 11: 609-617.
Hassan, A; Usman, J; Kaleem, F; Omair, M; Khalid, A and Iqbal, M (2011). Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz. J. Infect. Dis., 15: 305-311.
Johnson, JR and Stell, AL (2000). Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J. Infect. Dis., 181: 261-272.
Lanumtiang, Y; Jiemtaweeboon, S; Sungpradit, S; Leesombun, A and Boonmasawai, S (2022). The surveillance of antimicrobial susceptibility pattern and blaCTX-M gene encoding in Escherichia coli isolated from healthy goat farms in Sai Yok District, Kanchanaburi Province, Thailand. J. Appl. Anim. Sci., 15: 9-24.
Le Bouguenec, C; Archambaud, M and Labigne, A (1992). Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction. J. Clin. Microbiol., 30: 1189-1193.
Markey, B; Leonard, F; Archambault, M; Cullinane, A and Maguire, D (2013). Clinical veterinary microbiology. 2nd Edn., St. Louis, MO: Mosby Ltd., PP: .
Maurer, JJ; Brown, TP; Steffens, WL and Thayer, SG (1998). The occurrence of ambient temperature-regulated adhesions, curli, and the temperature-sensitive hemagglutinin tsh among avian Escherichia coli. Avian Dis., 42: 106-118.
Mohammed, YJ; Mustafa, JY and Abdullah, AR (2020). Isolation and molecular study of some bacterial urinary tract infections of sheep in Basrah province. Iraqi J. Agric. Sci., 51: 885-893.
Naziri, Z; Kilegolan, JA; Moezzi, MS and Derakhshandeh, A (2021). Biofilm formation by uropathogenic Escherichia coli: a complicating factor for treatment and recurrence of urinary tract infections. J. Hosp. Infect., 117: 9-16.
Nielsen, SS; Bicout, DJ; Calistri, P; Canali, E; Drewe, JA; Garin-Bastuji, B; Gonzales Rojas, JL; Gortázar, C; Herskin, M; Michel, V and Miranda Chueca, MÁ (2022). Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): antimicrobial-resistant Escherichia coli in dogs and cats, horses, swine, poultry, cattle, sheep and goats. EFSA J., 20: 1-93.
Safavi, EA and Shahbazi, Y (2017). Antimicrobial resistance in Escherichia coli isolated from different parts of the digestive tract of sheep. Bulg. J. Vet. Med., 20: 271-275.
Shabana, II and Al-Enazi, AT (2020). Investigation of plasmid-mediated resistance in E. coli isolated from healthy and diarrheic sheep and goats. Saudi J. Biol. Sci., 27: 788-796.
Singh, F; Sonawane, GG; Kumar, J; Dixit, SK; Meena, RK and Tripathi, BN (2019). Antimicrobial resistance and phenotypic and molecular detection of extended-spectrum ß-lactamases among extraintestinal Escherichia coli isolated from pneumonic and septicemic sheep and goats in Rajasthan, India. Turkish J. Vet. Anim. Sci., 43: 754-760.
Speer, BS; Shoemaker, NB and Salyers, AA (1992). Bacterial resistance to tetracycline: mechanisms, transfer, and clinical significance. Clin. Microbiol. Rev., 5: 387-399.
Srinivasan, V; Gillespie, BE; Lewis, MJ; Nguyen, LT; Headrick, SI; Schukken, YH and Oliver, SP (2007). Phenotypic and genotypic antimicrobial resistance patterns
of Escherichia coli isolated from dairy cows with mastitis. Vet. Microbiol., 124: 319-328.
Stepanović, S; Vuković, D; Hola, V; Di Bonaventura, G; Djukić, S; Cirković, I and Ruzicka, F (2007). Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS. 115: 891-899.
Tabar, MM; Mirkalantari, S and Amoli, RI (2016). Detection of ctx-M gene in ESBL-producing E. coli strains isolated from urinary tract infection in Semnan, Iran. Electron. Physician., 8: 2686-2690.
Tahamtan, Y; Pourbakhsh, S; Hayati, M; Namdar, N; Shams, Z and Namavari, M (2011). Prevalence and molecular characterization of verotoxin-producing Escherichia coli O157:H7 in cattle and sheep in Shiraz-Iran. Arch. Razi Inst., 66: 29-36.
The European Committee on Antimicrobial Susceptibility Testing (2019). Redefining susceptibility testing categories S, I and R. Accessed 1 January 2022.
Van Houdt, R and Michiels, CW (2005). Role of bacterial cell surface structures in Escherichia coli biofilm formation. Res. Microbiol., 156: 626-633.
Velhner, M and Milanov, D (2015). Resistance to tetracycline in Escherichia coli and Staphylococcus aureus: brief overview on mechanisms of resistance and epidemiology. Arch. Vet. Med., 8: 27-36.
Warsa, UC; Nonoyama, M; Ida, T; Okamoto, R; Okubo, T; Shimauchi, C; Kuga, A and Inoue, M (1996). Detection of tet(K) and tet(M) in Staphylococcus aureus of Asian countries by the polymerase chain reaction. J. Antibiot. (Tokyo), 49: 1127-1132.
Wu, Z; Chi, H; Han, T; Li, G; Wang, J and Chen, W (2024). Differences of Escherichia coli isolated from different organs of the individual sheep: molecular typing, antibiotics resistance, and biofilm formation. Folia Microbiol., 69: 567-578.
Zhao, X; Lv, Y; Adam, FE; Xie, Q; Wang, B; Bai, X; Wang, X; Shan, H; Wang, X; Liu, H and Dang, R (2021). Comparison of antimicrobial resistance, virulence genes, phylogroups, and biofilm formation of Escherichia coli isolated from intensive farming and free-range sheep. Front. Microbiol., 12: 699927.