In Silico Isolation of a Novel Phage for Food Safety Applications against Pathogenic E. coli

With the advent of next-generation sequencing (NGS) on metagenomes, the elucidation of all genetic material from microbiomes has prompted a renewed interest towards uncultivated members of the virosphere. We describe the discovery of a novel phage from a metagenomic dataset on the West African fermented dairy product, nunu, with a custom bioinformatics workflow to potentially serve as a biocontrol agent against pathogenic E. coli. Initial dataset of ERR2014814 from NCBI was first subjected to Kraken2 to extract novel sequencing reads for further de novo assembly into contigs by MetaVelvet. Resultant contigs served as potential partial phage genomes, then searched against databases to find closest characterized homologous sequences for potential functional determinations and any previously published phages for phylogenetic relatedness. References further assembled several contigs for the genome of this novel phage with both SPAdes and Bowtie2.  Gaps were then closed with AlignGraph.  A full assembled genome at 41,601bp in length was generated using Escherichia phage vB_EcoM_ECOO78 as a reference genome. The average GC content was 55%, similar to vB_EcoM_ECOO78, but lower by 4%. Local similarity on the 5’ end to Enterobacter bacteriophage Arya was also observed. Several similar open reading frames (ORFs) were found to share similarity with ones from vB_EcoM_ECOO78. For example, an ORF from this novel phage shares 75% identity on the amino acid level to the tail protein of vB_EcoM_ECOO78. Its overall demonstrable sequence similarity towards vB_EcoM_ECOO78 also implies the potential to infect E. coli. Our discovery of a novel phage will provide a potentially new biocontrol agent as a natural enemy of E. coli with targeted and timely actions to improve the overall food safety in various products. Furthermore, our customized bioinformatics workflow could also serve as an enhanced protocol towards the detection of novel phages from other microbiomes.