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

<b>Background/Objectives:</b> 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 (<i>Bos taurus</i>) and water buffalo (<i>Bubalus bubalis</i>) in the Brazilian Amazon, a region where unpasteurized dairy consumption is culturally ingrained. <b>Methods:</b> Using shotgun metagenomic sequencing, we analyzed 32 pooled milk samples from extensive and semi-intensive farms in the Manaus Metropolitan Region. <b>Results:</b> 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 <i>AbaQ</i>, <i>ArnT</i>, and <i>KpnF</i>, alongside complex multi-AMR cassettes co-occurring with plasmids and widespread viral sequences (dominated by <i>Caudoviricetes</i>). Integrons were ubiquitous in cattle and highly prevalent in buffalo samples. <b>Conclusions:</b> 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.