Microbiological quality, molecular characterization, and antimicrobial resistance profiling of foodborne bacterial pathogens in donkey milk and associated environmental sources in Maharashtra, India

Introduction: Donkey milk has attracted increasing attention because of its compositional similarity to human milk and its nutritional and therapeutic potential for infants, adults, and elderly individuals. However, the occurrence of foodborne pathogens and antimicrobial-resistant bacteria in donkey milk poses a potential public health concern. The present study evaluated the microbiological quality, occurrence of selected bacterial pathogens, and antimicrobial resistance patterns in donkey milk and its associated environment in Maharashtra, India.

Methods: A total of 50 raw donkey milk samples and 100 environmental samples, including udder swabs, utensils, farmers’ hand swabs, and drinking water, were analyzed using standard microbiological methods for the detection of Escherichia coli, Salmonella spp., Listeria spp., and Staphylococcus spp. Total bacterial counts were determined by standard plate count methods. Isolates were characterized by biochemical assays, PCR, and MALDI-TOF MS/VITEK® MS. Antimicrobial susceptibility testing was performed to assess multidrug resistance (MDR) patterns and multiple antibiotic resistance (MAR) indices.

Results: The total bacterial count in donkey milk ranged from 3.95 to 5.02 log CFU/mL, with a mean count of 4.39 ± 0.02 log CFU/mL. Escherichia coli was detected in 6% of milk samples, whereas Salmonella spp. and Listeria spp. were absent in all samples analyzed. Overall occurrence of Staphylococcus species was 5.33%, including Staphylococcus sciuri and Staphylococcus aureus. Molecular and MALDI-TOF MS identification showed strong concordance with microbiological findings. All bacterial isolates exhibited notable MDR patterns, with resistance to multiple commonly used antibiotics and MAR index values ranging from 0.32 to 0.56.

Conclusions: The detection of multidrug-resistant E. coli and Staphylococcus species in donkey milk and related environmental sources highlights potential public health risks associated with raw milk consumption. Implementation of strict hygiene measures, pasteurization, Good Dairy Farming Practices, and prudent antimicrobial use is essential to ensure the microbiological safety of donkey milk and limit the spread of antimicrobial resistance.