What Lies Beneath? Taking the Plunge into the Murky Waters of Phage Biology

The sequence revolution revealed that bacteria-infecting viruses, known as phages, are Earth's most abundant biological entities. Phages have far-reaching impacts on the form and function of microbial communities and play a fundamental role in ecological processes. However, even well into the sequencing revolution, we have only just begun to explore the murky waters around the phage biology iceberg. Many viral reads cannot be assigned to a culturable isolate, and reference databases are biased toward more easily collectible samples, which likely distorts our conclusions. This minireview points out alternatives to mapping reads to reference databases and highlights innovative bioinformatic and experimental approaches that can help us overcome some of the challenges in phage research and better decipher the impact of phages on microbial communities. Moving beyond the identification of novel phages, we highlight phage metabolomics as an important influencer of bacterial host cell physiology and hope to inspire the reader to consider the effects of phages on host metabolism and ecosystems at large. We encourage researchers to report unassigned/unknown sequencing reads and contigs and to continue developing alternative methods to investigate phages within sequence data

Longitudinal Analysis of the Microbiome and Metabolome in the 5xfAD Mouse Model of Alzheimer's Disease.

Recent reports implicate gut microbiome dysbiosis in the onset and progression of Alzheimer's disease (AD), yet studies involving model animals overwhelmingly omit the microbial perspective. Here, we evaluate longitudinal microbiomes and metabolomes from a popular transgenic mouse model for familial AD (5xfAD). Cecal and fecal samples from 5xfAD and wild-type B6J (WT) mice from 4 to 18 months of age were subjected to shotgun Illumina sequencing. Metabolomics was performed on plasma and feces from a subset of the same animals. Significant genotype, sex, age, and cage-specific differences were observed in the microbiome, with the variance explained by genotype at 4 and 18 months of age rising from 0.9 to 9% and 0.3 to 8% for the cecal and fecal samples, respectively. Bacteria at significantly higher abundances in AD mice include multiple Alistipes spp., two Ligilactobacillus spp., and Lactobacillus sp. P38, while multiple species of Turicibacter, Lactobacillus johnsonii, and Romboutsia ilealis were less abundant. Turicibacter is similarly depleted in people with AD, and members of this genus both consume and induce the production of gut-derived serotonin. Contradicting previous findings in humans, serotonin is significantly more concentrated in the blood of older 5xfAD animals compared to their WT littermates. 5xfAD animals exhibited significantly lower plasma concentrations of carnosine and the lysophospholipid lysoPC a C18:1. Correlations between the microbiome and metabolome were also explored. Taken together, these findings strengthen the link between Turicibacter abundance and AD, provide a basis for further microbiome studies of murine models for AD, and suggest that greater control over animal model microbiomes is needed in AD research. IMPORTANCE Microorganisms residing within the gastrointestinal tract are implicated in the onset and progression of Alzheimer's disease (AD) through the mediation of inflammation, exchange of small-molecules across the blood-brain barrier, and stimulation of the vagus nerve. Unfortunately, most animal models for AD are housed under conditions that do not reflect real-world human microbial exposure and do not sufficiently account for (or meaningfully consider) variations in the microbiome. An improved understanding of AD model animal microbiomes will increase model efficacy and the translatability of research findings into humans. Here, we present the characterization of the microbiome and metabolome of the 5xfAD mouse model, which is one of the most common animal models for familial AD. The manuscript highlights the importance of considering the microbiome in study design and aims to lay the groundwork for future studies involving mouse models for AD

Phage Cocktails Constrain the Growth of Enterococcus.

Phages that infect pathogenic bacteria present a valuable resource for treating antibiotic-resistant infections. We isolated and developed a collection of 19 Enterococcus phages, including myoviruses, siphoviruses, and a podovirus, that can infect both Enterococcus faecalis and Enterococcus faecium. Several of the Myoviridae phages that we found in southern California wastewater were from the Brockvirinae subfamily (formerly Spounavirinae) and had a broad host range across both E. faecium and E. faecalis. By searching the NCBI Sequence Read Archive, we showed that these phages are prevalent globally in human and animal microbiomes. Enterococcus is a regular member of healthy human gut microbial communities; however, it is also an opportunistic pathogen responsible for an increasing number of antibiotic-resistant infections. We tested the ability of each phage to clear Enterococcus host cultures and delay the emergence of phage-resistant Enterococcus. We found that some phages were ineffective at clearing Enterococcus cultures individually but were effective when combined into cocktails. Quantitative PCR was used to track phage abundance in cocultures and revealed dynamics ranging from one dominant phage to an even distribution of phage growth. Genomic characterization showed that mutations in Enterococcus exopolysaccharide synthesis genes were consistently found in the presence of phage infection. This work will help to inform cocktail design for Enterococcus, which is an important target for phage therapy applications. IMPORTANCE Due to the rise in antibiotic resistance, Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy offers an alternative to antibiotics and has shown promise in both in vitro and early clinical studies. Here, we established a collection of 19 Enterococcus phages and tested whether combining phages into cocktails could delay growth and the emergence of resistant mutants in comparison with individual phages. We showed that cocktails of two or three phages often prevented the growth of phage-resistant mutants, and we identified which phages were replicating the most in each cocktail. When resistant mutants emerged to single phages, they showed consistent accumulation of mutations in exopolysaccharide synthesis genes. These data serve to demonstrate that a cocktail approach can inform efforts to improve efficacy against Enterococcus isolates and reduce the emergence of resistance