Prevalence and Molecular Characterization of TEM and CTX-M Beta-Lactamase Genes of Escherichia coli Isolated From Cow Milk

Authors

  • Amr M. Aly Ministry of Agriculture, Agricultural Research Center, Egypt; Department of Botany and Microbiology, Faculty of Science, Kafrelsheikh University, 33511, Egypt.
  • Lina Ahmed Bahamdain Department of Biological Science, Faculty of Science, King Abdulaziz University, Saudi Arabia.
  • Aziza S. El-kholy Department of Botany and Microbiology, Faculty of Science, Kafrelsheikh University, 33511, Egypt.
  • Ehab F. Alsebaey Ministry of Agriculture, Agricultural Research Center, Animal Health Research Institute, Egypt.
  • Magda M. Aly Department of Botany and Microbiology, Faculty of Science, Kafrelsheikh University, 33511, Egypt; Department of Biological Science, Faculty of Science, King Abdulaziz University, Saudi Arabia.
  • Yasser H. El-Halmouch Department of Botany and Microbiology, Faculty of Science, Kafrelsheikh University, 33511, Egypt.

DOI:

https://doi.org/10.22317/jcms.v10i3.1578

Keywords:

E. coli, raw milk, antibiotic resistance, blaCTX-M gene, blaTEM gene

Abstract

Objective: The study objective was to investigate the presence and antibiotic resistance of toxigenic E. coli strains in the raw milk of dairy
cattle and to determine the prevalence of blaTEM and blaCTX-M genes in the selected isolates.

Methods: Two hundred raw milk samples were collected from 20 dairy farms located in Kafr El Sheikh City.

Results: Among the samples, 60 were positive for E. coli. The sensitivity of these isolates was detected against different antibiotics. Using
the disk diffusion test, all the isolates were resistant to at least two beta-lactam antibiotics. The resistance to beta-lactam antibiotics by
the selected E. coli was varied to be highly significant. Five percent of the tested E. coli was highly resistant with a multi-antibiotic resistant (MAR) index, of 1.0, while 8% had the lowest MAR (0.14). Both blaTEM and blaCTX-M resistant genes were detected in isolate No. 52, while, blaCTX-M was detected in isolate No. 34 and blaTEM was detected in isolates No. 9, 21, 28, and 40. The PCR products of blaTEM and blaCTX-M genes were sequenced and deposited in the GenBank of the NCBI database with the accession numbers OR450046 and OR879117, respectively.

Conclusion: The macrolides group of antibiotics especially erythromycin should not be a suitable treatment of dairy herds for mastitis
by E. coli in Egypt. The majority of E. coli was multiple-antibiotic resistant and co-carried many virulence genes, and it may pose a great
potential risk to public health.

References

Aidar-Ugrinovich, L., Blanco, J., Blanco, M., Blanco, J.E., Leomil, L., Dhabi, G., Mora, A.; Onuma, D.L., Silveira, W.D., and Pestana de Castro, A.F. )2007(. Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli isolated from calves in São Paulo, Brazil. Int. J. Food Microbiol., 115: 297-306.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipmanl, D.J. (1990). Basic Local Alignment Search Tool. J. Mol. Biol., 215: 403-410.

Archambault, M., Petrov, P., Hendriksen, R.S., Asseva, G., Bangtrakulnonth, A., Hasman, H., and Aarestrup, F.M. (2006). Molecular characterization and occurrence of extended-spectrum beta-lactamase resistance genes among Salmonella enterica serovar Corvallis from Thailand, Bulgaria, and Denmark. Microb. Drug Resist., 12(3): 192-8.

Awadallah, M.A., Ahmed, H.A., Merwad, A.M., and Selim, M.A. (2016). Occurrence, genotyping, Shiga toxin genes and associated risk factors of E. coli isolated from dairy farms, handlers and milk consumers. Vet. J., 217: 83-88.

Ayandele, A.A., Oladipo, E.K., Oyebisi, O., and Kaka, M.O. (2020). Prevalence of Multi-Antibiotic Resistant Escherichia coli and Klebsiella species obtained from a Tertiary Medical Institution in Oyo State, Nigeria. Qatar Med. J., 3(1): 9. doi: 10.5339/qmj.2020.9.

Bai, J., Shi, X. and Nagaraja, T.G. (2010). A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7. J. Microbiol. Methods, 82: 8589.

Bajpai, T., Pandey, M., Varma, M., and Bhatambare, G.S. (2017). Prevalence of TEM, SHV, and CTX-M Beta-Lactamase genes in the urinary isolates of a tertiary care hospital. Avicenna J. Med., 7(1): 12-16.

Bhoomika, Sanjay, S., Anil, P., and Eknath, G.N. (2016). Occurrence and characteristics of extended-spectrum β-lactamases producing Escherichia coli in foods of animal origin and human clinical samples in Chhattisgarh, India. Vet. World, 9: 996-1000.

Bolton, D.J. (2011). Verocytotoxigenic (shiga toxin producing) Escherichia coli: virulence factors and pathogenicity in the farm to fork paradigm. Foodborne Pathog. Dis., 8(3): 357-365.

Bradley, A.J. (2002). Bovine mastitis: an evolving disease. Vet. J., 164: 116-128.

Bradley, A.J., Leach, K.A., Breen, J.E., Green, L.E., and Green, M.J. (2007). Survey of the incidence and aetiology of mastitis in dairy farms in England and Wales. Vet. Rec., 160: 253-258.

CLSI (2020). Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute.

Colom, K., Pèrez, J., Alonso, R., Fernández-Aranguiz, A., Lariňo, E., and Cisterna, R. (2003). Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae. FEMS Microbiol. Lett., 223 (2): 147-151.

Cruickshank, R., Duguid, J.P., Marmion, B.P., and Swain, R.H.A. (1975). Medical Microbiology. Vol. 2, the practice of medical microbiology. 12th Ed., Churchill Livingstone, Edinburgh, London.

Daoud, N., Hamdoun, M., Hannachi, H., Gharsallah, C., Mallekh, W., and Bahri, O. (2020). Antimicrobial Susceptibility Patterns of Escherichia coli among Tunisian Outpatients with Community-Acquired Urinary Tract Infection (2012-2018). Curr. Urol., 14(4): 200-205. doi: 10.1159/000499238.

EFSA (2015). Scientific opinion on the public health risks related to the consumption of raw drinking milk. EFSA J., 13: 3940.

EFSA-ECDC (2012). Scientific report of EFSA and ECDC: the European Union summary report on trents and sources of zoonoses, agents and food-borne outbreaks in 2010. EFSA J., 10: 2597.

Gasanov, U., Hughes, D., and Hansbro, P.M. (2005). Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: A review. FEMS Microbiol. Rev., 29(5): 851-875. 10.1016/j.femsre.2004.12.002.

Gonggrijp, M.A., Santman-Berends, I., Heuvelink, A.E., Buter, G.J., and Lam, T. (2016). Prevalence and risk factors for extended-spectrum β-lactamase- and ampc-producing Escherichia coli in dairy farms. J. Dairy Sci., 99: 9001-9013.

Hagel, S., Makarewicz, O., Hartung, A., et al. (2019). ESBL colonization and acquisition in a hospital population: The molecular epidemiology and transmission of resistance genes. PloS one, 14(1).

Hinthong, W., Pumipuntu, N., Santajit, S., Kulpeanprasit, S., Buranasinsup, S., and Sookrung, N. (2017). Detection and drug resistance profile of Escherichia coli from subclinical mastitis cows and water supply in dairy farms in Saraburi Province, Thailand. PeerJ , 5: e3431.

Huang, S., Tian, P., Kou, X., An, N., Wu, Y., Dong, J., Cai, H., Li, B., Xue, Y., Liu, Y., and Ji, H. (2022). The prevalence and characteristics of extended-spectrum β-lactamase Escherichia coli in raw milk and dairy farms in Northern Xinjiang, China. Int. J. Food Microbiol., 381(16): 109908.

Idland, L., Bø-Granquist, E.G., Aspholm, M., and Lindbäck, T. (2022). The Ability of Shiga Toxin-Producing Escherichia coli to Grow in Raw Cow's Milk Stored at Low Temperatures. Foods, 11(21): 3411. doi: 10.3390/foods11213411.

Islam, M.A., Kabir, S.M.L., and Seel, S.K. (2016). Molecular detection and characterization of Escherichia coli isolated from raw milk sold in different markets of Bangladesh. Bangladesh J. Vet. Med., 14: 271-275.

Jayarao, B.M. and Henning, D.R. (2001). Prevalence of foodborne pathogens in bulk tank milk. J. Dairy Sci., 84: 2157-2162.

Jung, D., Park, S., Ruffini, J., Dussault, F., Dufour, S., and Ronholm, J. (2021). Comparative genomic analysis of Escherichia coli isolates from cases of bovine clinical mastitis identifies nine specific pathotype marker genes. Microb. Genom., 7(7):000597. doi: 10.1099/mgen.0.000597.

Kaipainen, T., Pohjanvirta, T., Shpigel, N.Y., Shwimmer, A., Pyörälä, S., and Pelkonen, S. (2002). Virulence factors of Escherichia coli isolated from bovine clinical mastitis. Vet. Microbiol., 85: 37-46. doi: 10.1038/nrmicro818.

Lan, X.Y., Zhao, S.G., Zheng, N., Li, S.L., Zhang, Y.D., Liu, H.M., et al. (2017). Short communication: microbiological quality of raw milk of raw cow milk and its association with herd management practices in Northern China. J. Dairy Sci., 100: 4294-4299.

Lang, N.L. and Smith, S.R. (2007). Influence of soil type, moisture content and biosolids application on the fate of Escherichia coli in agricultural soil Under controlled laboratory conditions. J. Apple. Microbiol., 103: 2122- 213.

Lange, M.E., Uwiera, R.R.E., and Inglis, G.D. (2022). Enteric Escherichia coli O157:H7 in Cattle, and the Use of Mice as a Model to Elucidate Key Aspects of the Host-Pathogen-Microbiota Interaction: A Review. Front. Vet. Sci., 9: 937866. doi: 10.3389/fvets.2022.937866.

Lippolis, J.D., Holman, D.B., Brunelle, B.W., Thacker, T.C., Bearson, B.L., and Reinhardt, T.A. (2017). Genomic and transcriptomic analysis of Escherichia coli strains associated with persistent and transient bovine mastitis and the role of colanic acid. Infect. Immun., 86: e00566-17.

Liu, H., Meng, L., Dong, L., Zhang, Y., Wang, J., and Zheng, N. (2021). Prevalence, Antimicrobial Susceptibility, and Molecular Characterization of Escherichia coli Isolated From Raw Milk in Dairy Herds in Northern China. Front Microbiol., 12: 730656. doi: 10.3389/fmicb.2021.730656.

Naseer, U. and Sundsford, A. (2011). The CTX-M conundrum: dissemination of plasmids and Escherichia coli clones. Microb. Drug Resist., 17(1): 83-97.

Navajas-Benito, E.V., Alonso, C.A., Sanz, S., Olarte, C., Martínez-Olarte, R., and Hidalgo-Sanz, S. (2016). Molecular characterization of antibiotic resistance in Escherichia coli strains from a dairy cattle farm and its surroundings. J. Sci. Food Agric., 97: 363-365. doi: 10.1002/jsfa.7709.

Ntuli, V., Njage, P.M.K., and Buys, E.M. (2016). Characterization of Escherichia coli and other Enterobacteriaceae in producer-distributor bulk milk. J. Dairy Sci., 99: 9534-9549.

Ombarak, R.A., Hinenoya, A., Awasthi, S.P., Iguchi, A., Shima, A., Elbagory, A.R.M., et al. (2016). Prevalence and pathogenic potential of Escherichia coli isolates from raw milk and raw milk cheese in Egypt. Int. J. Food Microbial., 221: 69-76.

Osman, K.M., Mustafa, A.M., Aly, M.A., and Abd Elhamed, G.S. (2012). Serotypes, virulence genes, and intimin types of shiga toxin producing Escherichia coli and enter pathogenic Escherichia coli isolated from mastitic milk relevant to human health in Egypt. Vector Borne Zoonotic Dis., 12(4): 297-305.

Oxoid (1987). Agents and main distributors, the manual. Sixth Edition.

Pishtiwan, A.H. and Khadija, K.M. (2019). Prevalence of blaTEM, blaSHV, and blaCTX-M Genes among ESBL-Producing Klebsiella pneumoniae and Escherichia coli Isolated from Thalassemia Patients in Erbil, Iraq. Mediterr. J. Hematol. Infect. Dis., 11(1): e2019041. doi: 10.4084/MJHID.2019.041.

Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J.C., Leonard, F.C., and Maguire, D. (2002). Veterinary Microbiology and Microbial Disease. Published by Blackwell. p. 113-116.

Radostits, O.M., Gay, C.C., Hinchcliff, K.W., and Constable, P.D. (2007). Veterinary Medicine, 10. Edition. Saunders Elsevier, Philadelphia, USA. p. 673-748.

Rugeles, L.C., Bai, J., Martinez, A.J., Vanegas, M.C., and Gomez-Duarte, O.G. (2010). Molecular characterization of diarrheagenic Escherichia coli strains from stools samples and food products in Colombia. Int. J. Food Microbiol., 138: 282-286.

Santman-Berends, I., Gonggrijp, M.A., Hage, J.J., Heuvelink, A.E., Velthuis, A., and Lam, T. (2016). Prevalence and risk factors for extended-spectrum beta-lactamase or AMPC-producing Escherichia coli in organic dairy herds in the Netherlands. J. Dairy Sci., 562: 120-128. doi: 10.3168/jds.2016-11839.

Sharma, S., Aarif, K., Dahiya, D.K., Jain, J., and Sharma, V. (2015). Prevalence, identification and drug resistance pattern of Staphylococcus aureus and Escherichia coli isolated from raw milk samples of Jaipur city of Rajasthan. J. Pure Appl. Microbiol., 9: 341-348.

Sharun, K., Dhama, K., Tiwari, R., Gugjoo, M.B., Iqbal Yatoo, M., Patel, S.K., Pathak, M., Karthik, K., Khurana, S.K., Singh, R., Puvvala, B., Amarpal, Singh, R., Singh, K.P., and Chaicumpa, W. (2021). Advances in therapeutic and managemental approaches of bovine mastitis: a comprehensive review. Vet. Quart., 41(1): 107-136. doi: 10.1080/01652176.2021.1882713.

Soomro, A.H., Arain, M.A., Khaskheli, M., and Bhutto, B. (2002). Isolation of E. coli from raw milk and milk products in relation to public health sold under market conditions at Tandojam, Pakistan. Pak. J. Nutr., 1: 151-152.

Su, Y.C., Yu, C.Y., Tsai, Y.L., Wang, S.H., Lee, C., and Chu, C. (2016). Fluoroquinolone-resistant and extended-spectrum β-lactamase-producing Escherichia coli from the milk of cows with clinical mastitis in Southern Taiwan. J. Microbiol. Immunol. Infect., 49: 892-901. doi: 10.1016/j. jmii.2014.10.003.

Tadesse, H.A., Gidey, N.B., Workelule, K., Hailu, A.B., Gidey, S., Bsrat, A., and Taddele, H. (2018). Antimicrobial resistance profile of E. coli isolated from raw cow milk and fresh fruit juice in Mekelle, Tigray, Ethiopia. Vet. Med. Int., 2018: 8903142.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol., 30: 2725-2729.

Tark, D.S., Moon, D.C., Kang, H.Y., Kim, S.R., Nam, H.M., Lee, H.S., et al. (2016). Antimicrobial susceptibility and characterization of extended-spectrum β-lactamases in Escherichia coli isolated from bovine mastitic milk in South Korea from 2012 to 2015. J. Dairy Sci., 100: 3463-3469. doi: 10.3168/jds.2016-12276.

Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22(22): 4673-4680.

Wang, X., Yu, D., Chui, L., Zhou, T., Feng, Y., Cao, Y., and Zhi, S. (2024). A Comprehensive Review on Shiga Toxin Subtypes and Their Niche-Related Distribution Characteristics in Shiga-Toxin-Producing E. coli and Other Bacterial Hosts. Microorganisms, 12(4): 687. doi.org/10.3390/microorganisms12040687.

Wenz, J.R., Barrington, G.M., Garry, F.B., Ellis, R.P., and Magnuson, R.J. (2006). Escherichia coli isolates serotypes, genotypes, and virulence genes and clinical coliform mastitis severity. J. Dairy Sci., 89: 3408-3412.

Downloads

Published

2024-07-02

How to Cite

Aly, A. M., Bahamdain, L. A., El-kholy, A. S., Alsebaey, E. F., M. Aly, M., & El-Halmouch, Y. H. (2024). Prevalence and Molecular Characterization of TEM and CTX-M Beta-Lactamase Genes of Escherichia coli Isolated From Cow Milk. Journal of Contemporary Medical Sciences, 10(3). https://doi.org/10.22317/jcms.v10i3.1578