Microbial species diversity and antibiotic-resistant Enterobacteriaceae spread on dairy farms

Introduction. Bacterial communities significantly affect the overall productivity of agricultural establishments, as animal health, milk production, and food quality and safety depend on them. Zoonotic bacteria not only have a negative impact on animal health, but also pose a risk to public health, so monitoring of the microbial species diversity on dairy farms to determine the predominant pathogen species and antibiotic resistance profiles is essential.

Objective. Study of bacterial species diversity on a dairy farm and monitoring of antibiotic resistance spread in Escherichia coli and Proteus mirabilis isolates in order to enable timely development of measures containing the spread of antibiotic-resistant microorganisms.

Materials and methods. To achieve this goal, microorganisms were identified by MALDI-ToF mass spectrometry and antibiotic susceptibility of the isolated cultures was determined using the disc diffusion test.

Results. The species diversity of microorganisms isolated from samples of cattle limb wound exudates, feces, and feed was established. Opportunistic and pathogenic Escherichia coli and Proteus mirabilis turned out to be the predominant microorganisms, and their antibiotic resistance profiles were determined. One of the Escherichia coli isolates was found to be multi-resistant; only a combination of amoxicillin and clavulanic acid proved effective in inhibiting the growth of this culture. A large proportion of Proteus mirabilis isolates were resistant to drugs included in the group of fluoroquinolones and sensitive to all other tested antibacterial agents.

Conclusion. The factors influencing the microbial species diversity in wound exudate, feces and feed were reported. Determination of Enterobacteriaceae antibiotic resistance profiles will allow for the rotation of antibacterial drugs on the studied livestock farms.

1. Perdomo A., Calle A. Assessment of microbial communities in a dairy farm from a food safety perspective. International Journal of Food Microbiology. 2024; 423:110827. https://doi.org/10.1016/j.ijfoodmicro.2024.110827

2. Tyrrell C., Burgess C. M., Brennan F. P., Münzenmaier D., Drissner D., Leigh R. J., Walsh F. Genomic analysis of antimicrobial resistant Escherichiacoli isolated from manure and manured agricultural grasslands. NPJ Antimicrobials and Resistance. 2025; 3:8. https://doi.org/10.1038/s44259-025-00081-8

3. Mahmud B., Vargas R. C., Sukhum K. V., Patel S., Liao J., Hall L. R., et al. Longitudinal dynamics of farmer and livestock nasal and faecal microbiomes and resistomes. Nature Microbiology. 2024; 9: 1007–1020. https://doi.org/10.1038/s41564-024-01639-4

4. Veloo Y., Rajendiran S., Zakaria Z., Ismail R., Rahman S. A., Mansor R., Thahir S. S. A. Prevalence and antimicrobial resistance patterns of Escherichia coli in the environment, cow dung, and milk of Selangor dairy farms. Antibiotics. 2025; 14 (2):137. https://doi.org/10.3390/antibiotics14020137

5. Liu L., Dong Z., Ai S., Chen S., Dong M., Li Q., et al. Virulence-related factors and antimicrobial resistance in Proteus mirabilis isolated from domestic and stray dogs. Frontiers in Microbiology. 2023; 14:1141418. https://doi.org/10.3389/fmicb.2023.1141418

6. Al-Qurashi E., Elbnna K., Ahmad I., Abulreesh H. H. Antibiotic resistance in Proteus mirabilis: mechanism, status, and public health significance. Journal of Pure and Applied Microbiology. 2022; 16 (3): 1550–1561. https://doi.org/10.22207/JPAM.16.3.59

7. Liegenfeld S. C., Stenzel S., Rembe J.-D., Dittmer M., Ramos P., Stuermer E. K. Pathogenic and non-pathogenic microbes in the wound microbiome – how to flip the switch. Microbiology Research. 2025; 16 (2):39. https://doi.org/10.3390/microbiolres16020039

8. Chalmers G., Anderson R. E. V., Murray R., Topp E., Boerlin P. Characterization of Proteus mirabilis and associated plasmids isolated from anaerobic dairy cattle manure digesters. PloS ONE. 2023; 18 (8):e0289703. https://doi.org/10.1371/journal.pone.0289703

9. Rzewuska M., Kwiecień E., Chrobak-Chmiel D., Kizerwetter-Świda M., Stefańska I., Gieryńska M. Pathogenicity and virulence of Trueperella pyogenes: a review. International Journal of Molecular Sciences. 2019; 20 (11):2737. https://doi.org/10.3390/ijms20112737

10. Lee H. K., Walls G., Anderson G., Sullivan C., Wong C. A. Prolonged Bacteroides pyogenes infection in a patient with multiple lung abscesses. Respirology Case Reports. 2024; 12 (3):e01314. https://doi.org/10.1002/rcr2.1314

11. Cunha F., Jeon S. J., Jeong K. C., Galvão K. N. Draft genome sequences of Bacteroides pyogenes strains isolated from the uterus of Holstein dairy cows with metritis. Microbiology Resource Announcements. 2019; 8 (41):e01043-19. https://doi.org/10.1128/MRA.01043-19

12. Pyakurel S., Caddey B. J., Dias A. P., De Buck J., Morck D. W., Orsel K. Profiling bacterial communities in feedlot cattle affected with bovine foot rot and bovine digital dermatitis lesions using 16S rRNA gene sequencing and quantitative real-time PCR. BMC Microbiology. 2025; 25:158. https://doi.org/10.1186/s12866-025-03869-w

13. Awoyomi O. J., Oyewusi J. A., Talabi A. O., Oyewusi I. K., Biobaku K. T., Mustapha O. A., Agbaje M. Isolation of Aeromonas hydrophila in a case of wound infection in cattle in Nigeria. Nigerian Journal of Animal Production. 2014; 41 (1): 213–219. https://doi.org/10.51791/njap.v41i1.2726

14. Kabelitz T., Aubry E., van Vorst K., Amon T., Fulde M. The role of Streptococcus spp. in bovine mastitis. Microorganisms. 2021; 9 (7):1497. https://doi.org/10.3390/microorganisms9071497

15. Corrêa P. S., Jimenez C. R., Mendes L. W., Rymer C., Ray P., Gerdes L., et al. Taxonomy and functional diversity in the fecal microbiome of beef cattle reared in Brazilian traditional and semi-intensive production systems. Frontiers in Microbiology. 2021; 12:768480. https://doi.org/10.3389/fmicb.2021.768480

16. Auffret M. D., Dewhurst R. J., Duthie C. A., Rooke J. A., Wallace R. J., Freeman T. C., et al. The rumen microbiome as a reservoir of antimicrobial resistance and pathogenicity genes is directly affected by diet in beef cattle. Microbiome. 2017; 5:159. https://doi.org/10.1186/s40168-017-0378-z

17. Abegewi U. A., Esemu S. N., Ndip R. N., Ndip L. M. Prevalence and risk factors of coliform-associated mastitis and antibiotic resistance of coliforms from lactating dairy cows in North West Cameroon. PloS ОNE. 2022; 17 (7):e0268247. https://doi.org/10.1371/journal.pone.0268247

18. Yamamura F., Sugiura T., Munby M., Shiokura Y., Murata R., Nakamura T., et al. Relationship between Escherichia coli virulence factors, notably kpsMTII, and symptoms of clinical metritis and endometritis in dairy cows. Journal of Veterinary Medical Science. 2022; 84 (3): 420–428. https://doi.org/10.1292/jvms.21-0586

19. Driehuis F., Wilkinson J. M., Jiang Y., Ogunade I., Adesogan A. T. Silage review: Animal and human health risks from silage. Journal of Dairy Science. 2018; 101 (5): 4093–4110. https://doi.org/10.3168/jds.2017-13836

20. Elad D., Shpigel N. Y., Winkler M., Klinger I., Fuchs V., Saran A., Faingold D. Feed contamination with Candida krusei as a probable source of mycotic mastitis in dairy cows. Journal of the American Veterinary Medical Association. 1995; 207 (5): 620–622. https://pubmed.ncbi.nlm.nih.gov/7649779

21. Zhai J., Wang B., Sun Y., Yang J., Zhou J., Wang T., et al. Effects of Aspergillus niger on cyanogenic glycosides removal and fermentation qualities of ratooning sorghum. Frontiers in Microbiology. 2023; 14:1128057. https://doi.org/10.3389/fmicb.2023.1128057

22. Seyedmousavi S., Guillot J., Arné P., de Hoog G. S., Mouton J. W., Melchers W. J. G., Verweij P. E. Aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease. Medical Mycology. 2015; 53 (8): 765–797. https://doi.org/10.1093/mmy/myv067

23. Manishimwe R., Moncada P. M., Bugarel M., Scott H. M., Loneragan G. H. Antibiotic resistance among Escherichia coli and Salmonella isolated from dairy cattle feces in Texas. PloS ONE. 2021; 16 (5):e0242390. https://doi.org/10.1371/journal.pone.0242390

24. Alharbi N. S., Khaled J. M., Kadaikunnan S., Alobaidi A. S., Sharafaddin A. H., Alyahya S. A., et al. Prevalence of Escherichia coli strains resistance to antibiotics in wound infections and raw milk. Saudi Journal of Biological Sciences. 2019; 26 (7): 1557–1562. https://doi.org/10.1016/j.sjbs.2018.11.016

25. Sarwar A., Aslam B., Mahmood S., Muzammil S., Siddique A. B., Sarwar F., et al. Distribution of multidrug-resistant Proteusmirabilis in poultry, live-stock, fish, and the related environment: One Health heed. Veterinary World. 2025; 18 (2): 446–454. https://doi.org/10.14202/vetworld.2025.446-454

26. Krivonogova A. S., Donnik I. M., Isaeva A. G., Loginov E. A., Petropavlovskiy M. V., Bespamyatnykh E. N. Antibiotic resistance of Enterobacteriaceae in microbiomes associated with poultry farming. Food Processing: Techniques and Technology. 2023; 53 (4): 710–717. https://doi.org/10.21603/2074-9414-2023-4-2472 (in Russ.)