Resistome and Mobilome Profiling of Raw Cow and Buffalo Milk from the Brazilian Amazon via Shotgun Metagenomics

Background/Objectives: Antimicrobial resistance (AMR) is a global health threat, with raw milk serving as a potential reservoir for antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). This study characterized the resistome and mobilome of raw milk from cows (Bos taurus) and water buffalo (Bubalus bubalis) in the Brazilian Amazon, a region where unpasteurized dairy consumption is culturally ingrained. Methods: Using shotgun metagenomic sequencing, we analyzed 32 pooled milk samples from extensive and semi-intensive farms in the Manaus Metropolitan Region. Results: Sequencing yielded over 3.1 million contigs. While cow milk showed a higher prevalence of positive samples (80%), buffalo milk exhibited a significantly higher abundance and diversity of ARG-associated contigs (301 contigs vs. 85 in cows). Clinically relevant genes were identified, including AbaQ, ArnT, and KpnF, alongside complex multi-AMR cassettes co-occurring with plasmids and widespread viral sequences (dominated by Caudoviricetes). Integrons were ubiquitous in cattle and highly prevalent in buffalo samples. Conclusions: These findings indicate that raw milk in the Amazon harbors a rich reservoir of resistance determinants and MGEs, likely driven by farm-level antibiotic usage. This underscores a critical food safety risk and highlights the need for One Health-based surveillance in the region.