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

## TL;DR

This study explores the microbial diversity and antibiotic resistance in dairy cattle feces from Pakistan, highlighting their role in One Health.

## Contribution

The study provides the first metagenomic analysis of cattle fecal microbiota, resistome, and plasmidome in Khyber Pakhtunkhwa, Pakistan.

## Key findings

- Bacteria dominated the fecal microbiota, with Escherichia coli and other potential pathogens being highly abundant.
- A large resistome and plasmidome were detected, indicating active horizontal gene transfer and antibiotic resistance.
- Fecal microbiota reflect host-microbe interactions and serve as a hotspot for zoonotic and environmental risks.

## Abstract

Gastrointestinal tract (GIT) microbiota play a crucial role in maintaining health and improving the production performance of livestock. While fecal samples do not directly represent these foregut communities, they provide a highly relevant and practical snapshot of the terminal gastrointestinal ecosystem. Upstream digestive processes shape the fecal microbiota 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. However, metagenomic data on livestock fecal microbiota in Pakistan remain scarce. This study explored the microbial composition, resistome, and plasmidome in cattle feces from three districts in Khyber Pakhtunkhwa, Pakistan, via a metagenomics approach. Bacteria were highly abundant (84.00–91.00%) at the kingdom level, followed by viruses (2.00–4.00%), archaea (0.20–1.00%), and Eukaryota (0.02–0.06%). Escherichia coli, Providencia stuartii, and Aliarcobacter skirrowii were highly abundant at the species level in feces, suggesting a GIT environment that may be conducive to the colonization of commensal and opportunistic pathogens of human and animal health concern. We observed that the highest number of species was in Peshawar (FC2; n = 9147), followed by Mardan (FC1; n = 8481) and Dera Ismail Khan (FC3; n = 8462), which may reflect underlying variations in farm management practices, local antibiotic usage, dietary compositions, or environmental exposures. A large resistome (40–49 genes) and plasmidome (16–22) were detected from all regions, indicating an active hotspot for horizontal gene transfer. In conclusion, 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.

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. Caryophanon latum and Escherichia coli were highly abundant at the species level. A large resistome (40–49 genes), including critical genes, such as tet(X), blaOXA-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.