Raw milk as a reservoir of multidrug resistant bacteria in Khyber Pakhtunkhwa.

Antimicrobial resistance (AMR) in dairy production systems poses a major public health threat, particularly under the One Health framework. Raw milk can act as a reservoir for multidrug-resistant (MDR) pathogens, especially in regions like Khyber Pakhtunkhwa (KP), Pakistan, where unregulated antibiotic use and insufficient surveillance have promoted high-risk resistance hotspots. This study analyzed 172 bacterial isolates from raw milk, focusing on key pathogens, Staphylococcus aureus and Escherichia coli, to assess their prevalence, resistance profiles, and epidemiological distribution. A total of 172 bacterial isolates were analyzed using: (1) conventional microbiological isolation and identification, (2) antimicrobial susceptibility testing across twelve antibiotic classes, (3) molecular detection of resistance determinants (mecA, vanA, blaTEM, blaCTX-M-15, qnrS, aac(6')-Ib-cr) by PCR, and (4) geospatial modeling (GeoDa, R, ArcGIS) to identify AMR hotspots. E. coli was detected in 70.0% of samples, followed by S. aureus in 41.0%. Among E. coli isolates, 32.5% were MDR, with high β-lactam resistance (ampicillin 31.0%, amoxicillin 22.7%). S. aureus exhibited extensive MDR, with 55.2% resistant to three or more antibiotic classes; the Oxacillin-Penicillin-Tetracycline phenotype was most prevalent (28.6%), and 18.4% displayed complex hexa-resistant profiles. Vancomycin resistance was observed in 16.3% of S. aureus isolates, with 8.2% carrying vanA. Molecular screening confirmed mecA in 89.8% of S. aureus, blaTEM in 67.9% of β-lactam-resistant isolates, blaCTX-M-15 in 21.4% of E. coli, and plasmid-mediated quinolone/aminoglycoside determinants (qnrS 8.2%, aac(6')-Ib-cr 10.9%). Geospatial analysis identified three resistance hotspots across KP, with 68% of vancomycin-resistant S. aureus concentrated in northern districts and a strong correlation between β-lactam and tetracycline resistance ([Formula: see text], [Formula: see text]). Raw milk in KP harbors pathogens with multidrug resistance exceeding previous regional estimates by 2-3 fold, including resistance to last-resort antibiotics such as vancomycin. These findings emphasize the urgent need for enhanced veterinary antibiotic stewardship, targeted surveillance of resistance hotspots, improved dairy hygiene practices, and community education regarding raw milk consumption. Integrated One Health strategies are critical to mitigate the amplification and spread of AMR in dairy production systems.