Loss of expression of the glutamate dehydrogenase (gdh) of Streptococcus suis serotype 2 compromises growth and pathogenicity.

Streptococcus suis serotype 2 is a zoonotic agent that causes substantial economic losses to the swine industry and threatens human public health. Factors that contribute to its ability to cause disease are not yet fully understood. Glutamate dehydrogenase (GDH) is an enzyme found in living cells and plays vital roles in cellular metabolism. It has also been shown to affect pathogenic potential of certain bacteria. In this study, we constructed a S. suis serotype 2 GDH mutant (Δgdh) by insertional inactivation mediated by a homologous recombination event and confirmed loss of expression of GDH in the mutant by immunoblot and enzyme activity staining assays. Compared with the wild type (WT) strain, Δgdh displayed a different phenotype. It exhibited impaired growth in all conditions evaluated (solid and broth media, increased temperature, varying pH, and salinity) and formed cells of reduced size. Using a swine infection model, pigs inoculated with the WT strain exhibited fever, specific signs of disease, and lesions, and the strain could be re-isolated from the brain, lung, joint fluid, and blood samples collected from the infected pigs. Pigs inoculated with the Δgdh strain did not exhibit any clinical signs of disease nor histologic lesions, and the strain could not be re-isolated from any of the tissues nor body fluid sampled. The Δgdh also showed a decreased level of survival in pig blood. Taken together, these results suggest that the gdh is important in S. suis physiology and its ability to colonize, disseminate, and cause disease.

Genomic differentiation among wild cyanophages despite widespread horizontal gene transfer.

BACKGROUND: Genetic recombination is a driving force in genome evolution. Among viruses it has a dual role. For genomes with higher fitness, it maintains genome integrity in the face of high mutation rates. Conversely, for genomes with lower fitness, it provides immediate access to sequence space that cannot be reached by mutation alone. Understanding how recombination impacts the cohesion and dissolution of individual whole genomes within viral sequence space is poorly understood across double-stranded DNA bacteriophages (a.k.a phages) due to the challenges of obtaining appropriately scaled genomic datasets. RESULTS: Here we explore the role of recombination in both maintaining and differentiating whole genomes of 142 wild double-stranded DNA marine cyanophages. Phylogenomic analysis across the 51 core genes revealed ten lineages, six of which were well represented. These phylogenomic lineages represent discrete genotypic populations based on comparisons of intra- and inter- lineage shared gene content, genome-wide average nucleotide identity, as well as detected gaps in the distribution of pairwise differences between genomes. McDonald-Kreitman selection tests identified putative niche-differentiating genes under positive selection that differed across the six well-represented genotypic populations and that may have driven initial divergence. Concurrent with patterns of recombination of discrete populations, recombination analyses of both genic and intergenic regions largely revealed decreased genetic exchange across individual genomes between relative to within populations. CONCLUSIONS: These findings suggest that discrete double-stranded DNA marine cyanophage populations occur in nature and are maintained by patterns of recombination akin to those observed in bacteria, archaea and in sexual eukaryotes.

Long-read powered viral metagenomics in the oligotrophic Sargasso Sea.

Dominant microorganisms of the Sargasso Sea are key drivers of the global carbon cycle. However, associated viruses that shape microbial community structure and function are not well characterised. Here, we combined short and long read sequencing to survey Sargasso Sea phage communities in virus- and cellular fractions at viral maximum (80 m) and mesopelagic (200 m) depths. We identified 2,301 Sargasso Sea phage populations from 186 genera. Over half of the phage populations identified here lacked representation in global ocean viral metagenomes, whilst 177 of the 186 identified genera lacked representation in genomic databases of phage isolates. Viral fraction and cell-associated viral communities were decoupled, indicating viral turnover occurred across periods longer than the sampling period of three days. Inclusion of long-read data was critical for capturing the breadth of viral diversity. Phage isolates that infect the dominant bacterial taxa Prochlorococcus and Pelagibacter, usually regarded as cosmopolitan and abundant, were poorly represented.

Viral tag and grow: a scalable approach to capture and characterize infectious virus-host pairs.

Viral metagenomics (viromics) has reshaped our understanding of DNA viral diversity, ecology, and evolution across Earth's ecosystems. However, viromics now needs approaches to link newly discovered viruses to their host cells and characterize them at scale. This study adapts one such method, sequencing-enabled viral tagging (VT), to establish "Viral Tag and Grow" (VT + Grow) to rapidly capture and characterize viruses that infect a cultivated target bacterium, Pseudoalteromonas. First, baseline cytometric and microscopy data improved understanding of how infection conditions and host physiology impact populations in VT flow cytograms. Next, we extensively evaluated "and grow" capability to assess where VT signals reflect adsorption alone or wholly successful infections that lead to lysis. Third, we applied VT + Grow to a clonal virus stock, which, coupled to traditional plaque assays, revealed significant variability in burst size-findings that hint at a viral "individuality" parallel to the microbial phenotypic heterogeneity literature. Finally, we established a live protocol for public comment and improvement via protocols.io to maximally empower the research community. Together these efforts provide a robust foundation for VT researchers, and establish VT + Grow as a promising scalable technology to capture and characterize viruses from mixed community source samples that infect cultivable bacteria.

Long-read viral metagenomics captures abundant and microdiverse viral populations and their niche-defining genomic islands.

Marine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in host culturing and a lack of reference genomes and 'universal' gene markers to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution, but their assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Such features also challenge assembly across genomic islands containing niche-defining genes that drive ecological speciation. These populations and features may be successfully captured by single-virus genomics and fosmid-based approaches, at least in abundant taxa, but at considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative sequencing and informatics workflow to improve viral metagenomic assemblies using short-read and long-read technology. The 'VirION' (Viral, long-read metagenomics via MinION sequencing) approach was first validated using mock communities where it was found to be as relatively quantitative as short-read methods and provided significant improvements in recovery of viral genomes. We then then applied VirION to the first metagenome from a natural viral community from the Western English Channel. In comparison to a short-read only approach, VirION: (i) increased number and completeness of assembled viral genomes; (ii) captured abundant, highly microdiverse virus populations, and (iii) captured more and longer genomic islands. Together, these findings suggest that VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

Fighting Fire with Fire: Phage Potential for the Treatment of E. coli O157 Infection.

Hemolytic⁻uremic syndrome is a life-threating disease most often associated with Shiga toxin-producing microorganisms like Escherichia coli (STEC), including E. coli O157:H7. Shiga toxin is encoded by resident prophages present within this bacterium, and both its production and release depend on the induction of Shiga toxin-encoding prophages. Consequently, treatment of STEC infections tend to be largely supportive rather than antibacterial, in part due to concerns about exacerbating such prophage induction. Here we explore STEC O157:H7 prophage induction in vitro as it pertains to phage therapy-the application of bacteriophages as antibacterial agents to treat bacterial infections-to curtail prophage induction events, while also reducing STEC O157:H7 presence. We observed that cultures treated with strictly lytic phages, despite being lysed, produce substantially fewer Shiga toxin-encoding temperate-phage virions than untreated STEC controls. We therefore suggest that phage therapy could have utility as a prophylactic treatment of individuals suspected of having been recently exposed to STEC, especially if prophage induction and by extension Shiga toxin production is not exacerbated.

Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study.

BACKGROUND: Autism spectrum disorders (ASD) are complex neurobiological disorders that impair social interactions and communication and lead to restricted, repetitive, and stereotyped patterns of behavior, interests, and activities. The causes of these disorders remain poorly understood, but gut microbiota, the 1013 bacteria in the human intestines, have been implicated because children with ASD often suffer gastrointestinal (GI) problems that correlate with ASD severity. Several previous studies have reported abnormal gut bacteria in children with ASD. The gut microbiome-ASD connection has been tested in a mouse model of ASD, where the microbiome was mechanistically linked to abnormal metabolites and behavior. Similarly, a study of children with ASD found that oral non-absorbable antibiotic treatment improved GI and ASD symptoms, albeit temporarily. Here, a small open-label clinical trial evaluated the impact of Microbiota Transfer Therapy (MTT) on gut microbiota composition and GI and ASD symptoms of 18 ASD-diagnosed children. RESULTS: MTT involved a 2-week antibiotic treatment, a bowel cleanse, and then an extended fecal microbiota transplant (FMT) using a high initial dose followed by daily and lower maintenance doses for 7-8 weeks. The Gastrointestinal Symptom Rating Scale revealed an approximately 80% reduction of GI symptoms at the end of treatment, including significant improvements in symptoms of constipation, diarrhea, indigestion, and abdominal pain. Improvements persisted 8 weeks after treatment. Similarly, clinical assessments showed that behavioral ASD symptoms improved significantly and remained improved 8 weeks after treatment ended. Bacterial and phagedeep sequencing analyses revealed successful partial engraftment of donor microbiota and beneficial changes in the gut environment. Specifically, overall bacterial diversity and the abundance of Bifidobacterium, Prevotella, and Desulfovibrio among other taxa increased following MTT, and these changes persisted after treatment stopped (followed for 8 weeks). CONCLUSIONS: This exploratory, extended-duration treatment protocol thus appears to be a promising approach to alter the gut microbiome and virome and improve GI and behavioral symptoms of ASD. Improvements in GI symptoms, ASD symptoms, and the microbiome all persisted for at least 8 weeks after treatment ended, suggesting a long-term impact. TRIAL REGISTRATION: This trial was registered on the ClinicalTrials.gov, with the registration number  NCT02504554.

Optimization of viral resuspension methods for carbon-rich soils along a permafrost thaw gradient.

Permafrost stores approximately 50% of global soil carbon (C) in a frozen form; it is thawing rapidly under climate change, and little is known about viral communities in these soils or their roles in C cycling. In permafrost soils, microorganisms contribute significantly to C cycling, and characterizing them has recently been shown to improve prediction of ecosystem function. In other ecosystems, viruses have broad ecosystem and community impacts ranging from host cell mortality and organic matter cycling to horizontal gene transfer and reprogramming of core microbial metabolisms. Here we developed an optimized protocol to extract viruses from three types of high organic-matter peatland soils across a permafrost thaw gradient (palsa, moss-dominated bog, and sedge-dominated fen). Three separate experiments were used to evaluate the impact of chemical buffers, physical dispersion, storage conditions, and concentration and purification methods on viral yields. The most successful protocol, amended potassium citrate buffer with bead-beating or vortexing and BSA, yielded on average as much as 2-fold more virus-like particles (VLPs) g(-1) of soil than other methods tested. All method combinations yielded VLPs g(-1) of soil on the 10(8) order of magnitude across all three soil types. The different storage and concentration methods did not yield significantly more VLPs g(-1) of soil among the soil types. This research provides much-needed guidelines for resuspending viruses from soils, specifically carbon-rich soils, paving the way for incorporating viruses into soil ecology studies.