Assessment of antimicrobial resistance in bovine mastitis pathogens across smallholder farms in Southeast Asia

Thesis Abstract

Bovine mastitis remains a critical constraint to dairy production in Southeast Asia, where widespread antimicrobial use and limited veterinary oversight contribute to the emergence and dissemination of antimicrobial-resistant pathogens on smallholder farms. This study aims to quantify the prevalence and patterns of antimicrobial resistance (AMR) among bacterial pathogens isolated from bovine mastitis cases, identify farm-level and management factors associated with AMR, and evaluate the potential impact of AMR on treatment outcomes. Specific objectives are (1) to determine the prevalence of bacterial isolates from milk samples of clinical and subclinical mastitis across 60 smallholder farms in three Southeast Asian countries; (2) to characterize antimicrobial susceptibility profiles for isolates against a panel of 14 antibiotics representing veterinary- and human-use classes using standard disk diffusion and minimum inhibitory concentration (MIC) methods in accordance with CLSI guidelines; (3) to identify risk factors for AMR at herd and management levels through structured farmer interviews and on-farm observations; (4) to assess associations between AMR patterns and treatment outcomes, including cure rates and recurrence, using multivariable regression analyses; (5) to develop a predictive model incorporating Phylogenetic groupings, farm practices, and antimicrobial usage patterns to forecast AMR risk; and (6) to contextualize findings within the One Health framework and propose evidence-based stewardship interventions. A mixed-methods approach will be employed. The quantitative component involves a cross-sectional survey of 600 milk samples collected from 60 smallholder herds, with sampling stratified by mastitis status (clinical vs. subclinical). Isolates will be identified to species level (Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Klebsiella spp., and coagulase-negative staphylococci) using MALDI-TOF mass spectrometry and phenotypic confirmatory tests. Antimicrobial susceptibility will be assessed against antibiotics including penicillin, amoxicillin-clavulanate, cephalosporins, macrolides, tetracyclines, fluoroquinolones, sulfonamides, and/ or associated combinations. MIC determination will be conducted for isolates with inconclusive disk diffusion results. Data on antimicrobial usage, farm hygiene, housing system, milking practices, and veterinarian access will be gathered through structured questionnaires administered to farm owners/managers. Statistical analyses will include descriptive statistics, bivariate tests, and multilevel logistic regression to identify factors associated with AMR carriage, while generalized linear models will evaluate the relationship between AMR profiles and clinical outcomes. A multivariate analysis of variance (MANOVA) will compare susceptibility patterns across bacterial species and country contexts. The study will apply the Theory of Planned Behavior and the Health Belief Model to interpret farmers’ antibiotic use patterns and to identify leverage points for behavior change. Qualitative components will comprise semi-structured interviews with 40 veterinarians and farm workers to explore perceptions of AMR, antibiotic access, and stewardship barriers, followed by thematic analysis using a constant comparative method. The integration of quantitative and qualitative findings will be achieved through a convergent design, with triangulation to validate AMR risk factors and governance gaps. Expected findings include a higher prevalence of resistance to commonly used antibiotics such as penicillin and tetracyclines among S. aureus and E. coli isolates, with notable regional variation across the three countries. It is anticipated that AMR will be associated with inadequate veterinary oversight, non-prescription antibiotic access, poor mastitis management practices, and higher frequency of antibiotic usage without culture-guided therapy. The study will contribute to knowledge by delineating gene-level resistance indicators (if feasible within resources) and mapping AMR transmission risk within and between farms under smallholder conditions, thereby informing targeted stewardship interventions and policy recommendations. The main conclusion is that AMR in bovine mastitis pathogens on Southeast Asian smallholder farms is driven by a combination of antimicrobial access, farm management, and limited veterinary governance. Recommendations include promoting culture-based therapy, implementing farm-level antibiotic stewardship programs, improving record-keeping and surveillance for AMR, strengthening veterinarian engagement, and developing region-specific guidelines for prudent antimicrobial use in dairy cattle.