Shotgun Metagenomics Reveals Microbial Diversity, Resistome, and Plasmidome in Dairy Cattle Feces

Fecal microbiota are shaped by upstream digestive processes and reflect the outcome of host-microbe interactions, including the resistant microbial fraction that survives to be excreted. This is particularly crucial for assessing zoonotic risks and environmental contamination, as feces are the primary source of dissemination, which is considered an emerging One Health threat. Therefore, we conducted a pilot study to obtain the exploratory findings regarding the cattle GIT microbial composition, potential resistome, and their transmission drivers, such as plasmids, using metagenomic analysis from different districts in Khyber Pakhtunkhwa (KP) province, Pakistan. For this purpose, a total of 150 fecal samples (50 from each district) of healthy cattle were collected from various farms in Mardan (FC1), Peshawar (FC2), and Dera Ismail Khan (FC3) districts. Total DNA from each sample was extracted, pooled (FC1, FC2, and FC3), and sequenced via the Illumina platform. Bacteria were the highly abundant kingdom, while Pseudomonadota and Bacillota were dominant phyla in all samples. <i>Caryophanon latum</i> and <i>Escherichia coli</i> were highly abundant at the species level. A large resistome (40-49 genes), including critical genes, such as <i>tet</i>(X), <i>bla</i>_OXA-427, and plasmidomes (16-22), such as IncF, was detected in the samples. The prominence of certain commensal or opportunistic pathogens in the fecal microbiota may indicate the presence of sub-clinical gastrointestinal disruptions or disease that may affect cattle herds. The fecal resistome is extensive, identifying dairy cattle in these regions as important reservoirs for AMR genes capable of spreading via HGT. This pilot study establishes that the fecal microbiota of dairy cattle in this region are not merely a waste product but a complex ecosystem, rich in microbiota of One Health significance.