Detection and Molecular Characterization of Some Virulence Genes of Escherichia Coli Isolated from Milk in Dairy Cow Farms

Document Type : Original Article

Authors

1 Giza Lab., Animal Health Research Institute, Giza, Agricultural Research Center (ARC), Egypt

2 Udder Health and Neonatal Diseases, Animal Reproduction Research Institute, Giza, Agricultural Research Center (ARC), Egypt

3 Serology Unit, Animal Health Research Institute, Giza, Agricultural Research Center (ARC), Egypt

4 Buffalo Diseases Research Department, Animal Health Research Institute, Giza, Agricultural Research Center (ARC), Egypt

Abstract

Coliform pathogens, primarily E. coli, were discovered throughout the farm, causing environmental mastitis and can be shed from the udder into the milk; they are concerned about severe gastrointestinal disruption and potential enteropathogenic and/or toxic strains, posing a risk to public health. The objectives of this study were to identify the incidence and harmful serotypes of pathogenic Escherichiacoliand some of their virulent genes, which were isolated from the collected milk of some dairy farms in the Delta region and Cairo-Alexandria desert road farms, in Egypt during one year using the polymerase chain reaction (PCR) technique after bacteriological and serological identification as well as determine the antimicrobial susceptibility of the isolated strains. 150 milk samples in total were gathered (100 milk samples from bulk milk tanks and the other 50 samples from clinically mastitic dairy cows). According to our finding, the mean values of somatic cell count (SCC), standard plate count (SPC), and coliform count (CC) in the hundred bulk tank milk samples were 3.67  1.08×104/ml, 7.08×104± 6.25×104 cfu/ml and 3.04×102±1.43×102/ml, respectively. The bacteriological investigation exhibited that, the Escherichiacoli incidences from bulk tank milk (BTM) and mastitic milk samples (MMS) were 12% and 18%, respectively. The detected   E.coli serotypes including, O26, O44, O55:K99, O111, O119 and O157:H7 from MMS, while O1, O55, O78, O86, O124 and O158:H10 from BTM. Molecular virulence characterization of E.coli strains showed that, Shiga toxins 2 (stx2) gene is present in O157:H7, while the stx1 gene present in O26.  The Intimin gene (eaeA) is involved in four strains, O44, O111, O119, and O157:H7. Positive amplification of a biofilm gene (adrA) appeared in all E.coli strains. The outcome of the antimicrobial susceptibility revealed high resistance to amoxicillin (85.71%), streptomycin (80.95%), ampicillin (71.43%), and flucloxacillin (61.90%). Meanwhile, the highest susceptibility was to ciprofloxacin (95.24%) followed by enrofloxacin (90.48%), neomycin (80.95%), and gentamycin (76.19%). Effective hygienic measurements are required to avoid toxigenic and pathogenic E.coli and more future studies should be performed to increase awareness in dairy farms.

Keywords

Main Subjects

References

ABDEL-TAWAB, A.A., NABIH, A. M., AGAG, M.A., and AL-ABBOU, M. A., 2017. Bacteriological and molecular studies on Shiga-Toxin producing Escherichia coli causing cattle clinical mastitis. Benha Vet. Med. J., 33(2):17–26.  https://doi.org/10.21608/bvmj.2017.29984
AHMADI, E., MARDANI, K., and AMIRI, A., 2020. Molecular detection and antimicrobial resistance patterns of shiga toxigenic Escherichia coli isolated from bovine subclinical mastitis milk samples in Kurdistan, Iran. Archives of Razi Institute, 75(2): 169–177. https://doi.org/10.22092/ari.2019.124238.1278
AHMED, A. S., DIAB, H.M.,  ALKAHTANI, M.A., ALSHEHRI, M.A., SABER, H., BADR, H. DANDRAWY, M.K., EL-MANSI, A.A., SHATI, A.A., and AHMED, A.E., 2020. Molecular epidemiology of virulent Escherichia coli among rural small scale dairy herds and shops: Efficacy of selected marine algal extracts and disinfectants. Int. J. of Environ. Health Res. https://doi.org/10.1080/09603123.2020.1727422
AMERICAN PUBLIC HEALTH ASSOCIATION (APHA), 1992. Standard methods for examination of dairy products. INC., 16th ed. New York. at: https://www.scirp.org/(S(i43dyn45teexjx455qlt3d2q
AZOOZ M. F., EL-WAKEEL S. A., and YOUSEF, H. M., 2020. Risk Factor Analysis of Salmonella typhimurium, Staphylococcus aureus, Standard Plate Count and Somatic Cell Count in Bulk Tank Milk in Cattle Dairies. World Vet. J., 10 (3): 338-361.  https://dx.doi.org/10.36380/scil.2020.wvj44
BELOIN, C., ROUX, A., and GHIGO, J., 2008. Escherichia coli biofilms. Samenvatting, pp. 249–289. https://doi.org/10.1007%2F978-3-540-75418-3_12
BHOWMICK, P.P., DEVEGOWDA, D., RUWANDEEPIKA, H.A.D., FUCHS, T.M., SRIKUMAR, S., KARUNASAGAR, I., and KARUNASAGAR, I., 2011.  gcpA (stm1987) is critical for cellulose production and biofilm formation on polystyrene surface by S. enterica serovar Weltevreden in both high and low nutrient medium. Micro. Path., 50:114–122.  https://doi.org/10.1016/j.micpath.2010.12.002
BISI-JOHNSON, M.A., OBI, C.L., VASAIKAR, S.D., BABA, K.A., and HATTORI, T., 2011. Molecular basis of virulence in clinical isolates of Escherichia coli and Salmonella species from a tertiary hospital in the Eastern Cape. S. Afr. Gut Path., 3: 9. https://doi.org/10.1186%2F1757-4749-3-9
 
CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI), 2015. Performance Standards for Antimicrobial Susceptibility Testing. 25.  https://clsi.org/standards/products/microbiology/documents/m100/
DIPINETO, L., SANTANIELLO, A., FONTANELLA, M., LAGOS, K., FIORETTI, A., and MENNA, L. F., 2006. Presence of Shiga toxin-producing Escherichia coli O157:H7 in living layer hens. Appl. Microbiol., 43:293–295.
EGYPTIAN STANDARDS (ES), 2010. ES: 7123/2010, Essential Requirements for Milk and Dairy products. Egyptian Organization for Standardization and Quality, Egypt.
EL-KHOLY, A., HASSAN,G., EL-SHATER, M., and BAKR, A., 2018. Indicator microorganisms as indices of quality for milk and some dairy products. Assiut vet. Med. J., 64 (158): 40-48. https://dx.doi.org/10.21608/avmj.2018.168949
EL MAHMOUDY, S., ADEL EL-GOHARY., MOHAMED, A., RAMADAN, H., and GWIDA, M., 2021. Characterization of Shiga toxin-producing Escherichia coli isolated from cattle and their contacts. Mansoura Vet. Med. J., 22 (1):13-19.  https://mvmj.journals.ekb.eg/article_151606_681de98e11a1c6ce0c60f96122673e75.pdf
FARROKH, C., JORDAN, K., AUVRAY, F., GLASS, K., OPPEGAARD, H., and RAYNAUD, S., 2013. Review of Shiga toxin– producing Escherichia coli (STEC) and their significance in dairy production. Int. J. Food Microbiol., 162:190–212. DOI: https://doi.org/10.1016/j.ijfoodmicro.2012.08.008
FILHO, E. G. F., FERREIRA, M. R. A., and PINTO, J. F. N., 2014. ‘Enterohemorrhagic Escherichia coli O157 : H7 from healthy dairy cattle in Mid-West Brazil : occurrence and molecular’, Pesq. Vet. Bras. 34(1):24-28 . https://doi.org/10.1590/S0100-736X2014000100004
GODINHO , F. M. S., KRUG, M., FIGUEIREDO, R. P.,  MÜLLER, A.,  JANK , L., TOMASZEWSKI, C.A.,  HILLESHEIM, D. R.,  KINAST, É. J.,  FRAZZON, A.P. G., and  MOTTA, A. S., 2020.  Microbiological and physicochemical characteristics of buffalo milk used for dairy products in southern Brazil. J. Dairy Res., 87(4):463-468. https://doi.org/10.1017/s002202992000093x
HARJANTI, D. W., and SAMBODHO, P., 2020. Effects of mastitis on milk production and composition in dairy cows. IOP Conference Series: Earth and Environ. Sci., 518(1): 6–10. doi:10.1088/1755-1315/518/1/012032
INTERNATIONAL COMMITTEE ON MICROBIOLOGICAL SPECIFICATIONS FOR FOODS (ICMSF), 1986. Microorganisms in food, their significance and methods of enumeration 2nd ed.Univ. torontopress, Toronto, Buffalo and London.
ISMAIL, Z.B., and ABUTARBUSH S.M., 2020. Molecular characterization of antimicrobial resistance and virulence genes of Escherichia coli isolates from bovine mastitis. Vet. World., 13(8):1588–1593. . DOI: https://www.doi.org/10.14202/vetworld.2020.1588-1593
KOK, T., WORSWICH, D., and GOWANS, E., 1996. Some serological techniques for microbial and viral infections. In Practical Medical Microbiology (Collee, J.; Fraser, A.; Marmion, B. and Simmons, A., eds), 14th ed., Edinburgh, Churchill Livingstone, UK.
LEE, M. S., and TESH, V. L., 2019. Roles of Shiga toxins in immunopathology. Toxins (Basel), 11:212:1-26. DOI: 10.3390/toxins11040212
MARRI, N., LOSITO, F., LE BOFFE, L., GIANGOLINI, G., AMATISTE, S., MURGIA, L., ARIENZO, A., and ANTONINI, G., 2020.  Rapid microbiological assessment in raw milk: Validation of a rapid alternative method for the assessment of microbiological quality in raw milk. Foods, 9:1186. https://doi.org/10.3390%2Ffoods9091186
METZ, M., SHEEHAN, J., and FENG, P. C. H., 2020. Use of indicator bacteria for monitoring sanitary quality of raw milk cheeses – A literature review. Food Microbiology, 85(June 2019), p. 103283. https://doi.org/10.1016/j.fm.2019.103283
MOMTAZ, H., DEHKORDI, F.S., TAKTAZ, T., REZVANI, A., and YARALI, S., 2012. Shiga toxin-producing Escherichia coli isolated from bovine mastitic milk: Serogroups, virulence factors, and antibiotic resistance properties. The Scientific World Journal,. Article ID 618709, 9 pages. DOI: 10.1100/2012/618709
MOTLAGH, A. M. and  YANG, Z., 2019. Detection and occurrence of indicator organisms and pathogens. Water Environ. Res.,  91(10): 1402–1408. https://doi.org/10.1002%2Fwer.1238
NATIONAL MASTITIS COUNCIL (NMC), 2001. National Mastitis Council recommended mastitis control program.  Available at: http://www.nmconline.org/docs/
NDAHETUYE, J. B. 2019.  Mastitis in dairy cows in Rwanda: Prevalence, aetiology, antimicrobial resistance, molecular epidemiology and effects on milk quality. Available at: http://epsilon.slu.se/eindex.html
OPORTO, B., ESTEBAN, J. I,, ADURIZ, G., JUSTE, R.A., and HURTADO, A., 2008. Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E.coli in healthy cattle, sheep and swine herds in Northern Spain. Zoonoses Public Health., 55(2):73-81. https://doi.org/10.1111/j.1863-2378.2007.01080.x
QUINN, P. J., MARKEY, B.K.,  LEONARD, F.C., HARTIGAN, P.,   FANNING, S., and FITZPATRICK, E.S., 2011. Veterinary Microbiology and Microbial Disease, 2nd Edition | Wiley.Wiley-Blackwell, p. 980.
RYSANEK, D., BABAK, V., and ZOUHAROVA, M., 2007. Bulk tank milk somatic cell count and sources of raw milk contamination with mastitis pathogens. Veterinary Research Institute, Brno, Czech Republic. Veterinarni Medicina, 52 (6): 223-230. https://www.agriculturejournals.cz/publicFiles/00217
SAMBROOK, J.; FRITSCGH, E. F.  and MENTIATE. 1989. Molecular coloning. A laboratory manual.Vol !., Cold spring Harbor Laboratotry press, New York.’,
UNITED STATES DEPARTMENT OF AGRICULTURE (USDA), 2013. National Agricultural Statistics Service Agricultural Statistics. at:https://www.nass.usda.gov/Publications/Ag_Statistics/2013
URSELER, N., BACHETTI, R., MORGANTE, V., AGOSTINI, E. and MORGANTE, C., 2022. Groundwater quality and vulnerability in farms from agricultural dairy basin of the Argentine Pampas. Environ. Sci. and Poll. Res., https://doi.org/10.1007/s11356-022-20073-9
WORLD HEALTH ORGANIZATION AND FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (WHO), 2018. Shiga toxin-producing Escherichia coli (STEC) and food: attribution, characterization, and monitoring: report. World Health Organization. https://apps.who.int/iris /handle/10665/272871
YOUNIS, G.; AWAD, A. and GHABOUR, R., 2018. Prevalence and virulence determinants of Escherichia coli isolated from raw cow's milk. Afr. J. Microbiol. Res., 12 (9): 225-229. https://doi.org/10.5897/AJMR2018.8798

Files

History

  • Receive Date: 13 December 2022
  • Revise Date: 23 January 2023
  • Accept Date: 23 February 2023

Share

How to cite

Statistics