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

Abstract

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 producing 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 little is known about their microbial composition. We analysed the virome of agricultural slurry over a 5-month period using short and long-read sequencing. Results Hybrid sequencing uncovered more high-quality viral genomes than long or short-reads alone; yielding 7682 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 5-month period. Functional annotation revealed a diverse and abundant range of auxiliary metabolic genes and novel features present in the community, including the agriculturally relevant virulence factor VapE, which was widely distributed across different phage genera that were predicted to infect several hosts. Furthermore, we identified 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 cattle slurry virome is complex, diverse and dominated by novel genera, many of which are not recovered using long or short-reads alone. Phages were found to encode a wide range of AMGs that are not constrained to particular groups or predicted hosts, including virulence determinants and putative ARGs. The application of agricultural slurry to land may therefore be a driver of bacterial virulence and antimicrobial resistance in the environment.

Global Composition of the Bacteriophage Community in Honey Bees

“…Sequencing methods influence viral genome assembly and community characterization ( 36 , 37 ). As a proof of concept, we sequenced whole-genome-amplified viral DNA extracted from Texas honey bees using Illumina MiSeq short-read technology.…”

“…In all three honey bee viromes, virulent phages dominate the phage community in abundance and diversity. Virulent phages are often abundant in dense, productive microbial communities such as in animal (including human) digestive tracts ( 22 , 54 ), moist soils ( 55 ), and animal waste slurry ( 36 ), etc. ( 30 ).…”

Insights into the Human Gut Virome by Sampling a Population from the Indian Subcontinent

“…They have been classified into 4 subfamilies and 10 genera. 23,27 Using similar methods, we were able to classify only 30 % of our crAss-like phages, with genera VI, VII and VIII being the most represented. In the Western population, the most common genus is I and in the Malawian cohorts, they are genera VIII and IX.…”

“…The database comprised of prototypical crAssphage genome and proteome (p-crAssphage; NC_024711.1), 249 crAssphage genome and 2684 crAssphage family, which were reported by Guerin et al, and Yutin et al, respectively. 23,27 From the search result, putative crAss-like phages were selected using the following criteria (i) a BLAST hit against databases with an E-value less than 1E-05, (ii) a BLAST query alignment length 350bp, and (iii) a minimum contig length of 70kb (representing nearcomplete crAss-like phage contigs).…”

Complete Genome Sequence of Pseudomonas aeruginosa Bacteriophage vB_PaeP_PaCe

“…Annotations predict 73 coding features, including four tRNAs (Pro, Tyr, Asp, and Asn). The genome lacks known lysogeny genes using a set of HMMs, suggesting that PaCe has a strictly lytic lifestyle ( 11 ). Furthermore, no virulence factor or antibiotic resistance genes were identified using Abricate with ResFinder and the Virulence Factor Database (VFDB) ( 12 – 14 ).…”