PREVALENCE, ANTIMICROBIAL RESISTANCE PATTERNS, AND ASSOCIATED RISK FACTORS OF STAPHYLOCOCCUS AUREUS IN RAW MILK FROM DAIRY CATTLE IN TILOTTAMA MUNICIPALITY, NEPAL

Ishwari Tiwari; Ishwor Dhakal; Rubina Shah; Akash Adhikari; Sirjan Bastola

doi:10.56093/ijvasr.v55i6.185432

Authors

Ishwari Tiwari Veterinary Officer. Livestock Development and Veterinary Division, Governement of Nepal Narayanhiti, Kathmandu - 44600, Nepal
Ishwor Dhakal Veterinary Officer, Canine Breeding Centre, Government of Nepal, Bhaktapur - 44800. Nepal
Rubina Shah Veterinary Officer, Central Veterinary Laboratory, Tripureshwor, Kathmandu - 45104, Nepal
Akash Adhikari Veterinary Student (Graduated), Paklihawa Campus, Institute of Agriculture and Animal Sciences, Tribhuvan University, Siddharthanagar-01, Rupandehi-32900. Nepal
Sirjan Bastola Assistant Professor, Veterinary Microbiology, Paklihawa Campus, Institute of Agriculture and Animal Sciences, Tribhuvan University, Siddharthanagar - 01, Rupandehi - 32900, Nepal

https://doi.org/10.56093/ijvasr.v55i6.185432

Keywords:

S. aureus prevalence, antimicrobial resistance, cefoxitin non-susceptibility, dairy cattle

Abstract

Staphylococcus aureus (S. aureus)-associated bovine mastitis is a serious economic and animal health concern in Nepalese dairy production. Yet, antimicrobial resistance (AMR) data from commercial dairy herds remain scarce. A cross-sectional study was conducted from January to March 2020 in Tilottama Municipality, Rupandehi District, Nepal. A total of 207 lactating cows from 69 commercial dairy farms were enrolled by two-stage cluster sampling. Composite milk samples were pre-enriched in Buffered Peptone Water and cultured on Mannitol Salt Agar. S. aureus was considered positive by a five-step biochemical protocol with the tube coagulase test as the definitive criterion. Antimicrobial susceptibility to six agents was determined by the Kirby-Bauer disk diffusion method, and phenotypic methicillin-resistant S. aureus (MRSA) was identified by cefoxitin (30 µg) disk diffusion. Animal-level S. aureus prevalence was 33.3% (69/207) and herd-level prevalence was 53.6% (37/69). Cefoxitin non-susceptibility was detected in 52.2% of isolates; multidrug resistance (MDR) in 26.1% and a multiple antibiotic resistance (MAR) index ≥0.25 in 71.0% of isolates. Chloramphenicol showed the highest susceptibility rate (92.8%). After Bonferroni correction, animal age ≥5 years (OR=3.88) and early lactation stage ≤6 months post-calving (OR=3.30) were significantly associated with S. aureus positivity. Cefoxitin non-susceptibility and MDR were common among S. aureus isolates from commercial dairy farms of Tilottama Municipality. Older cows as well as early-lactating ones carried the highest infection risk. Culture-guided antimicrobial therapy, improved antibiotic stewardship, and sustained antimicrobial resistance (AMR) surveillance with molecular confirmation are recommended to prevent antimicrobial resistance.

Downloads

Download data is not yet available.

References

Abebe, R., Hatiya, H., Abera, M., Megersa, B. and Asmare, K. (2016). Bovine mastitis: prevalence, risk factors and isolation of Staphylococcus aureus in dairy herds at Hawassa milk shed, South Ethiopia. BMC Veterinary Research, 12: 270. https://doi. org/10.1186/s12917-016-0905-3

Acharya, K.P. and Wilson, R.T. (2019). Antimicrobial resistance in Nepal. Frontiers in Medicine, 6: 105. https:// doi.org/10.3389/fmed. 2019.00105

Anand, K.B., Agrawal, P., Kumar, S. and Kapila, K. (2009). Comparison of cefoxitin disc diffusion test, oxacillin screen agar, and PCR for mecA gene for detection of MRSA. Indian Journal of Medical Microbiology, 27: 27–29.

Brahma, U., Suresh, A., Murthy, S., Bhandari, V. and Sharma, P. (2022). Antibiotic resistance and molecular profiling of the clinical isolates of Staphylococcus aureus causing bovine mastitis from India. Microorganisms, 10: 833.

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

CLSI (2020b). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. 5th edn. Wayne, PA: Clinical and Laboratory Standards Institute.

Das, C., Rathore, R. and Singh, V.K. (2025). Prevalence, characterization and antibiogram of Staphylococcus aureus isolates from bovine and swine population in Bareilly, Uttar Pradesh, India. Scientific Reports, 15: 28171. https://doi.org/10.1038/s41598-025-13369-6

Dego, O.K. and Vidlund, J. (2024). ‘Staphylococcal mastitis in dairy cows’, Frontiers in Veterinary Science, 11: 1356259. https://doi. org/10.3389/fvets.2024.1356259.

Islam, M.M., Hossain, M.I., Islam, M.S., Azam, M.G. and Sultana, S. (2025). Prevalence, antibiotic resistance patterns, and virulence factors of Staphylococcus aureus isolates associated with bovine mastitis in northern Bangladesh. Heliyon, 11: e42107. https://doi.org/10.1016/j. heliyon.2025.e42107

Joshi, L.R., Tiwari, A., Devkota, S.P., Khatiwada, S., Paudyal, S. and Pande, K.R. (2014). Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in dairy farms of Pokhara, Nepal. International Journal of Veterinary Science, 3(2): 87- 90.

Khanal, S., Boonyayatra, S. and Awaiwanont, N. (2022). Prevalence of methicillin-resistant Staphylococcus aureus in dairy farms: a systematic review and meta-analysis. Frontiers in Veterinary Science, 9: 947154. https:// doi.org/10.3389/fvets.2022.947154

Krumperman, P.H. (1983). Multiple antibiotic resistance indexing of Escherichia coli to identify high- risk sources of fecal contamination of foods. Applied and Environmental Microbiology, 46: 165–170. https:// doi.or g/10.1 128/aem.46.1.165- 170.1983

Magiorakos, A.P., Srinivasan, A., Carey, R.B., Carmeli, Y., Falagas, M.E., Giske, C.G., Harbarth, S., Hindler, J.F., Kahlmeter, G., Olsson-Liljequist, B., Paterson, D.L., Rice, L.B., Stelling, J., Struelens, M.J., Vatopoulos, A., Weber, J.T. and Monnet, D.L. (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection, 18: 268–281. https://doi.org/10.1111/j.1469-0691.2011.03570.x

MoALD (2025). Livestock and Fisheries Statistics of Nepal. https://dls.gov. np/downloadfiles/Book-Livestock- website-1751864806.pdf.

MoHFS (2024). National Action Plan on Antimicrobial Resistance (2024–2028). https://mohp.gov.np/ content/254/national-action-plan-on- antimicrobial-resistance--2024-2028/.

Morales-Ubaldo, A.L., Rivero-Perez, N., Valladares-Carranza, B., Velazquez- Ordonez, V., Delgadillo-Ruiz, L. and Zaragoza-Bastida, A. (2023). Bovine mastitis, a worldwide impact disease: prevalence, antimicrobial resistance, and viable alternative approaches. Veterinary and Animal Science, 21: 100306. https://doi.org/10.1016/j.vas.2023.100306

Newcombe, R.G. (1998). Two-sided confidence intervals for the single proportion: comparison of seven methods. Statistics in Medicine, 17: 857–872.

Peduzzi, P., Concato, J., Kemper, E., Holford, T.R. and Feinstein, A.R. (1996). A simulation study of the number of events per variable in logistic regression analysis. Journal of Clinical Epidemiology, 49: 1373– 1379. https://doi.org/10.1016/S0895-4356(96)00236-3.

Peeler, E.J., Green, M.J., Fitzpatrick, J.L., Morgan, K.L. and Green, L.E. (2000). Risk factors associated with clinical mastitis in low somatic cell count British dairy herds. Journal of Dairy Science, 83: 2464–2472. https://doi.org/10.3168/jds.S0022-0302(00)75138-1

Rijal, K.R., Banjara, M.R., Dhungel, B., Kafle, S., Gautam, K., Ghimire, B., Ghimire,, P., Dhungel, S., Adhikari, N., Shrestha, U.T., Sunuwar, D.R., Adhikari, B. and Ghimire, P. (2021). 'Use of antimicrobials and antimicrobial resistance in Nepal: a nationwide survey,' Scientific Reports, 11(1): 11554. https://doi.org/10.1038/s41598-021-90812-4.

Sargeant, J.M., Leslie, K.E., Shirley, J.E., Pulkrabek, B.J. and Lim, G.H. (2001). Sensitivity and specificity of somatic cell count and California mastitis test for identifying intramammary infection in early lactation. Journal of Dairy Science, 84: 2018–2024. https://doi.org/10.3168/jds.S0022-0302(01)74645-0

Shahzad, M.A., Yousaf, A., Ahsan, A., Irshad, H., Riaz, A., Khan, A., Ullah, I., Sattar, S., Bostan, N. and Javed, S. (2024). Virulence and resistance profiling of Staphylococcus aureus isolated from subclinical bovine mastitis in the Pakistani Pothohar region. Scientific Reports, 14: 14569.

Shrestha, A., Bhattarai, R.K., Luitel, H., Karki, S. and Basnet, H.B. (2021). Prevalence of methicillin-resistant Staphylococcus aureus and pattern of antimicrobial resistance in mastitis milk of cattle in Chitwan, Nepal. BMC Veterinary Research, 17: 239. https:// doi.org/10.1186/s12917-021-02942-6

Sordillo, L.M. and Streicher, K.L. (2002). Mammary gland immunity and mastitis susceptibility. Journal of Mammary Gland Biology and Neoplasia, 7(2): 135–146.

Tiwari, B.B., Subedi, D., Bhandari, S., Shrestha, P., Pathak, C.R., Chandran, D., Pandey, G., Mohankumar, P. and Dhama, K. (2022). Prevalence and risk factors of staphylococcal subclinical mastitis in dairy animals of Chitwan, Nepal. Journal of Pure and Applied Microbiology, 16(2): 1392–1403.

Touaitia, R., Ibrahim, N.A., Touati, A. and Idres, T. (2025). Staphylococcus aureus in bovine mastitis: a narrative review of prevalence, antimicrobial resistance, and advances in detection strategies. Antibiotics, 14: 810. https:// doi.org/10.3390/antibiotics14080810

VHLSEC Rupandehi. (2019). Annual Report.

von Elm, E., Altman, D.G., Egger, M., Pocock, S.J., Gøtzsche, P.C. and Vandenbroucke, J.P. (2007). Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. BMJ, 335(7624): 806-808. doi:10.1136/bmj.39335.541782.AD