Enrichment Culture but Not Metagenomic Sequencing Identified a Highly Prevalent Phage Infecting Lactiplantibacillus plantarum in Human Feces

As Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human gut-related diseases, its bacteriophages may pose a certain threat to their further application and should be identified and characterized more often from the human intestine. Here, we isolated and identified the first gut L. plantarum phage that is prevalent in a Chinese population. ABSTRACT Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human diseases, but its phages in the human gut remain unexplored. Here, we report its first gut phage, Gut-P1, which we systematically screened using metagenomic sequencing, virus-like particle (VLP) sequencing, and enrichment culture from 35 fecal samples. Gut-P1 is virulent, belongs to the Douglaswolinvirus genus, and is highly prevalent in the gut (~11% prevalence); it has a genome of 79,928 bp consisting of 125 protein coding genes and displaying low sequence similarities to public L. plantarum phages. Physiochemical characterization shows that it has a short latent period and adapts to broad ranges of temperatures and pHs. Furthermore, Gut-P1 strongly inhibits the growth of L. plantarum strains at a multiplicity of infection (MOI) of 1e−6. Together, these results indicate that Gut-P1 can greatly impede the application of L. plantarum in humans. Strikingly, Gut-P1 was identified only in the enrichment culture, not in our metagenomic or VLP sequencing data nor in any public human phage databases, indicating the inefficiency of bulk sequencing in recovering low-abundance but highly prevalent phages and pointing to the unexplored hidden diversity of the human gut virome despite recent large-scale sequencing and bioinformatics efforts. IMPORTANCE As Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human gut-related diseases, its bacteriophages may pose a certain threat to their further application and should be identified and characterized more often from the human intestine. Here, we isolated and identified the first gut L. plantarum phage that is prevalent in a Chinese population. This phage, Gut-P1, is virulent and can strongly inhibit the growth of multiple L. plantarum strains at low MOIs. Our results also show that bulk sequencing is inefficient at recovering low-abundance but highly prevalent phages such as Gut-P1, suggesting that the hidden diversity of human enteroviruses has not yet been explored. Our results call for innovative approaches to isolate and identify intestinal phages from the human gut and to rethink our current understanding of the enterovirus, particularly its underestimated diversity and overestimated individual specificity.