Hybrid assembly of an agricultural slurry virome reveals a diverse and stable community with the potential to alter the metabolism and virulence of veterinary pathogens

Background
Viruses are the most abundant biological entities on Earth, known to be crucial components of microbial ecosystems. However, there is little information on the viral community within agricultural waste. There are currently ~2.7 million dairy cattle in the UK which produce 7-8% of their own bodyweight in manure daily, and 28 million tonnes annually. To avoid pollution of UK freshwaters, manure must be stored and spread in accordance with guidelines set by DEFRA. Manures are used as fertiliser, and widely spread over crop fields, yet very little is known about their microbial composition.

We analysed the virome of agricultural slurry over a five-month period using short and long-read sequencing.

Results
Hybrid sequencing uncovered more high-quality viral genomes than long or short-reads alone; yielding 7,682 vOTUs, 174 of which were complete viral genomes. The slurry virome was highly diverse and dominated by lytic bacteriophage, the majority of which represent novel genera (~98%). Despite constant influx and efflux of slurry, the composition and diversity of the slurry virome was extremely stable over time, with 55% of vOTUs detected in all samples over a five-month period.

Functional annotation revealed a diverse and abundant range of auxiliary metabolic genes and novel features to the community. Including the agriculturally relevant virulence factor VapE, which was widely distributed across different phage genera that were predicted to infect a wide range of hosts. Furthermore, we identified and an abundance of phage-encoded diversity-generating retroelements, which were previously thought to be rare on lytic viral genomes. Additionally, we identified a group of crAssphages, including lineages that were previously thought only to be found in the human gut.

Conclusions
The slurry virome is complex, diverse and dominated by novel genera, many of which are not recovered using long or short-reads alone. These phages carry a wide range of AMGs that are not constrained to particular groups or predicted hosts, including virulence determinants and putative ARGs. Application of agricultural slurry may therefore be a driver of bacterial virulence and antimicrobial resistance in the environment.