Meeting report of the third annual Tri-Service Microbiome Consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations and to facilitate resource, material and information sharing among consortium members. The 2019 annual symposium was held 22-24 October 2019 at Wright-Patterson Air Force Base in Dayton, OH. Presentations and discussions centered on microbiome-related topics within five broad thematic areas: 1) human microbiomes; 2) transitioning products into Warfighter solutions; 3) environmental microbiomes; 4) engineering microbiomes; and 5) microbiome simulation and characterization. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the presentations and outcomes of the 3rd annual TSMC symposium.

Phylosymbiosis Impacts Adaptive Traits in Nasonia Wasps.

Phylosymbiosis is defined as microbial community relationships that recapitulate the phylogeny of hosts. As evidence for phylosymbiosis rapidly accumulates in different vertebrate and invertebrate holobionts, a central question is what evolutionary forces cause this pattern. We use intra- and interspecific gut microbiota transplants to test for evidence of selective pressures that contribute to phylosymbiosis. We leverage three closely related species of the parasitoid wasp model Nasonia that recently diverged between 0.4 and 1 million years ago: N. vitripennis, N. giraulti, and N. longicornis Upon exposure of germfree larvae to heat-inactivated microbiota from intra- or interspecific larvae, we measure larval growth, pupation rate, and adult reproductive capacity. We report three key findings: (i) larval growth significantly slows when hosts receive an interspecific versus intraspecific gut microbiota, (ii) marked decreases in pupation and resulting adult survival occur from interspecific gut microbiota exposure, and (iii) adult reproductive capacities including male fertility and longevity are unaffected by early life exposure to an interspecific microbiota. Overall, these findings reveal developmental and survival costs to Nasonia upon larval exposures to interspecific microbiota and provide evidence that selective pressures on phenotypes produced by host-microbiota interactions may underpin phylosymbiosis.IMPORTANCE Phylosymbiosis is an ecoevolutionary hypothesis and emerging pattern in animal-microbiota studies whereby the host phylogenetic relationships parallel the community relationships of the host-associated microbiota. A central prediction of phylosymbiosis is that closely related hosts exhibit a lower microbiota beta diversity than distantly related hosts. While phylosymbiosis has emerged as a widespread trend in a field often challenged to find trends across systems, two critical and understudied questions are whether or not phylosymbiosis is consequential to host biology and if adaptive evolutionary forces underpin the pattern. Here, using germfree rearing in the phylosymbiosis model Nasonia, we demonstrate that early life exposure to heat-inactivated microbiota from more distantly related species poses more severe developmental and survival costs than microbiota from closely related or the same species. This study advances a functional understanding of the consequences and potential selective pressures underpinning phylosymbiosis.

The Maternal Effect Gene Wds Controls Wolbachia Titer in Nasonia.

Maternal transmission of intracellular microbes is pivotal in establishing long-term, intimate symbioses. For germline microbes that exert negative reproductive effects on their hosts, selection can theoretically favor the spread of host genes that counteract the microbe's harmful effects. Here, we leverage a major difference in bacterial (Wolbachia pipientis) titers between closely related wasp species with forward genetic, transcriptomic, and cytological approaches to map two quantitative trait loci that suppress bacterial titers via a maternal effect. Fine mapping and knockdown experiments identify the gene Wolbachia density suppressor (Wds), which dominantly suppresses bacterial transmission from mother to embryo. Wds evolved by lineage-specific non-synonymous changes driven by positive selection. Collectively, our findings demonstrate that a genetically simple change arose by positive Darwinian selection in less than a million years to regulate maternally transmitted bacteria via a dominant, maternal effect gene.

Correction: Phylosymbiosis: Relationships and Functional Effects of Microbial Communities across Host Evolutionary History.

[This corrects the article DOI: 10.1371/journal.pbio.2000225.].

Phylosymbiosis: Relationships and Functional Effects of Microbial Communities across Host Evolutionary History.

Phylosymbiosis was recently proposed to describe the eco-evolutionary pattern, whereby the ecological relatedness of host-associated microbial communities parallels the phylogeny of related host species. Here, we test the prevalence of phylosymbiosis and its functional significance under highly controlled conditions by characterizing the microbiota of 24 animal species from four different groups (Peromyscus deer mice, Drosophila flies, mosquitoes, and Nasonia wasps), and we reevaluate the phylosymbiotic relationships of seven species of wild hominids. We demonstrate three key findings. First, intraspecific microbiota variation is consistently less than interspecific microbiota variation, and microbiota-based models predict host species origin with high accuracy across the dataset. Interestingly, the age of host clade divergence positively associates with the degree of microbial community distinguishability between species within the host clades, spanning recent host speciation events (~1 million y ago) to more distantly related host genera (~108 million y ago). Second, topological congruence analyses of each group's complete phylogeny and microbiota dendrogram reveal significant degrees of phylosymbiosis, irrespective of host clade age or taxonomy. Third, consistent with selection on host-microbiota interactions driving phylosymbiosis, there are survival and performance reductions when interspecific microbiota transplants are conducted between closely related and divergent host species pairs. Overall, these findings indicate that the composition and functional effects of an animal's microbial community can be closely allied with host evolution, even across wide-ranging timescales and diverse animal systems reared under controlled conditions.

Disentangling a Holobiont - Recent Advances and Perspectives in Nasonia Wasps.

The parasitoid wasp genus Nasonia (Hymenoptera: Chalcidoidea) is a well-established model organism for insect development, evolutionary genetics, speciation, and symbiosis. The host-microbiota assemblage which constitutes the Nasonia holobiont (a host together with all of its associated microbes) consists of viruses, two heritable bacterial symbionts and a bacterial community dominated in abundance by a few taxa in the gut. In the wild, all four Nasonia species are systematically infected with the obligate intracellular bacterium Wolbachia and can additionally be co-infected with Arsenophonus nasoniae. These two reproductive parasites have different transmission modes and host manipulations (cytoplasmic incompatibility vs. male-killing, respectively). Pioneering studies on Wolbachia in Nasonia demonstrated that closely related Nasonia species harbor multiple and mutually incompatible Wolbachia strains, resulting in strong symbiont-mediated reproductive barriers that evolved early in the speciation process. Moreover, research on host-symbiont interactions and speciation has recently broadened from its historical focus on heritable symbionts to the entire microbial community. In this context, each Nasonia species hosts a distinguishable community of gut bacteria that experiences a temporal succession during host development and members of this bacterial community cause strong hybrid lethality during larval development. In this review, we present the Nasonia species complex as a model system to experimentally investigate questions regarding: (i) the impact of different microbes, including (but not limited to) heritable endosymbionts, on the extended phenotype of the holobiont, (ii) the establishment and regulation of a species-specific microbiota, (iii) the role of the microbiota in speciation, and (iv) the resilience and adaptability of the microbiota in wild populations subjected to different environmental pressures. We discuss the potential for easy microbiota manipulations in Nasonia as a promising experimental approach to address these fundamental aspects.

An optimized approach to germ-free rearing in the jewel wasp Nasonia.

Development of a Nasonia in vitrogerm-free rearing system in 2012 enabled investigation of Nasonia-microbiota interactions and real-time visualization of parasitoid metamorphosis. However, the use of antibiotics, bleach, and fetal bovine serum introduced artifacts relative to conventional rearing of Nasonia. Here, we optimize the germ-free rearing procedure by using filter sterilization in lieu of antibiotics and by removing residual bleach and fetal bovine serum. Comparison of these methods reveals no influence on larval survival or growth, and a 52% improvement in adult production. Additionally, adult males produced in the new germ-free system are similar in size to conventionally reared males. Experimental implications of these changes are discussed.

Vancomycin Treatment Alters Humoral Immunity and Intestinal Microbiota in an Aged Mouse Model of Clostridium difficile Infection.

BACKGROUND: The elderly host is highly susceptible to severe disease and treatment failure in Clostridium difficile infection (CDI). We investigated how treatment with vancomycin in the aged host influences systemic and intestinal humoral responses and select intestinal microbiota. METHODS: Young (age, 2 months) and aged (age, 18 months) C57BL/6 mice were infected with VPI 10463 after exposure to broad-spectrum antibiotics. Vancomycin was given 24 hours after infection, and treatment was continued for 5 days. At select time points, specimens of serum and intestinal tissue and contents were collected for histopathologic analysis, to measure antibody levels and the pathogen burden, and to determine the presence and levels of select intestinal microbiota and C. difficile toxin. RESULTS: Levels of disease severity, relapse, and mortality were increased, and recovery from infection was slower in aged mice compared to young mice. Serum levels of immunoglobulin M, immunoglobulin A, and immunoglobulin G against C. difficile toxin A were depressed in aged mice, and vancomycin treatment reduced antibody responses in both age groups. While baseline levels of total bacterial load, Bacteroidetes, Firmicutes, and Enterobacteriaceae were mostly similar, aged mice had a significant change in the Firmicutes to Bacteroidetes ratio with vancomycin treatment. CONCLUSIONS: Vancomycin treatment decreases the systemic humoral response to CDI. Increased mortality from and recurrence of CDI in the aged host are associated with an impaired humoral response and a greater susceptibility to vancomycin-induced alteration of intestinal microbiota.

Treatment of Clostridium difficile infection using SQ641, a capuramycin analogue, increases post-treatment survival and improves clinical measures of disease in a murine model.

OBJECTIVES: Clostridium difficile infection (CDI) is a primary cause of antibiotic-associated diarrhoeal illness. Current therapies are insufficient as relapse rates following antibiotic treatment range from 25% for initial treatment to 60% for treatment of recurrence. In this study, we looked at the efficacy of SQ641 in a murine model of CDI. SQ641 is an analogue of capuramycin, a naturally occurring nucleoside-based compound produced by Streptomyces griseus. METHODS: In a series of experiments, C57BL/6 mice were treated with a cocktail of antibiotics and inoculated with C. difficile strain VPI10463. Animals were treated orally with SQ641 for 5 days at a dose range of 0.1-300 mg/kg/day, 20 mg/kg/day vancomycin or drug vehicle. Animals were monitored for disease severity, clostridial shedding and faecal toxin levels for 14 days post-infection. RESULTS: Five day treatment of CDI with SQ641 resulted in higher 14 day survival rates in mice compared with either vancomycin or vehicle alone. CDI survival rates were 100% (13 of 13) and 94% (32 of 34), respectively, in the 1 and 10 mg/kg/day SQ641 treatment groups, 37% (7 of 19) with vancomycin treatment at 20 mg/kg/day and 32% (14 of 44) in the vehicle-only control group. Secondary measures of efficacy, such as prevention of weight loss, decreased disease severity, decreased C. difficile shedding and decreased toxin in faeces, were observed with SQ641 and vancomycin treatment. CONCLUSIONS: SQ641 is effective for CDI treatment with prevention of relapse in the murine model of CDI.

Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model.

BACKGROUND: Clostridium difficile is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of C. difficile infection in a murine model. METHODS: C57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with C. difficile strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and C. difficile growth and toxin production were quantified ex vivo in intestinal contents from untreated or antibiotic-treated mice fed with the different diets. RESULTS: C. difficile infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. C. difficile shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. Ex vivo inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and C. difficile growth in both defined diets compared with a traditional diet. CONCLUSIONS: Low protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.

High temporal resolution of glucosyltransferase dependent and independent effects of Clostridium difficile toxins across multiple cell types.

BACKGROUND: Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins' glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated. RESULTS: Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB's rapid effects. gdTcdB did not clearly delay TcdA or TcdB's toxin-induced effects on macrophages. CONCLUSIONS: Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.

Vancomycin treatment's association with delayed intestinal tissue injury, clostridial overgrowth, and recurrence of Clostridium difficile infection in mice.

Antibiotic treatment, including vancomycin, for Clostridium difficile infection (CDI) has been associated with recurrence of disease in up to 25% of infected persons. This study investigated the effects of vancomycin on the clinical outcomes, intestinal histopathology, and anaerobic community during and after treatment in a murine model of CDI. C57BL/6 mice were challenged with C. difficile strain VPI 10463 after pretreatment with an antibiotic cocktail. Twenty-four hours after infection, mice were treated daily with vancomycin, nitazoxanide, fidaxomicin, or metronidazaole for 5 days. Mice were monitored for either 6 or 12 days postinfection. Clinical, diarrhea, and histopathology scores were measured. Cecal contents or stool samples were assayed for clostridial or Bacteroides DNA and C. difficile toxins A and B. Vancomycin treatment of infected mice was associated with improved clinical, diarrhea, and histopathology scores and survival during treatment. However, after discontinuation of the drug, clinical scores and histopathology were worse in treated mice than in untreated infected controls. At the end of the study, 62% of the vancomycin-treated mice succumbed to recurrence, with an overall mortality rate equivalent to that of the untreated infected control group. Fidaxomicin-treated mice had outcomes similar to those of vancomycin-treated mice. C. difficile predominated over Bacteroides in cecal contents of vancomycin-treated mice, similar to findings for untreated infected mice. Decreasing the duration of vancomycin treatment from 5 days to 1 day decreased recurrence and deaths. In conclusion, vancomycin improved clinical scores and histopathology acutely but was associated with poor outcome posttreatment in C. difficile-infected mice. Decreasing vancomycin exposure may decrease relapse and improve survival in CDI.

Contribution of adenosine A(2B) receptors in Clostridium difficile intoxication and infection.

Clostridium difficile toxins A (TcdA) and B (TcdB) induce a pronounced systemic and intestinal inflammatory response. A(2B) adenosine receptors (A(2B)ARs) are the predominant adenosine receptors in the intestinal epithelium. We investigated whether A(2B)ARs are upregulated in human intestinal cells by TcdA or TcdB and whether blockade of A(2B)ARs can ameliorate C. difficile TcdA-induced enteritis and alter the outcome of C. difficile infection (CDI). Adenosine receptor subtype (A(1), A(2A), A(2B), and A(3)) mRNAs were assayed in HCT-8 cells. Ileal loops from wild-type rabbits and mice and A(2B)AR(-/-) mice were treated with TcdA, with or without the selective A(2B)AR antagonist ATL692 or PSB1115. A murine model of CDI was used to determine the effect of A(2B)AR deletion or blockade with the orally available agent ATL801, on clinical outcome, histopathology and intestinal interleukin-6 (IL-6) expression from infection. TcdA and TcdB upregulated A(2B)AR gene expression in HCT-8 cells. ATL692 decreased TcdA-induced secretion and epithelial injury in rabbit ileum. Deletion of A(2B)ARs reduced secretion and histopathology in TcdA-challenged mouse ileum. Deletion or blockade of A(2B)ARs reduced histopathology, IL-6 expression, weight loss, diarrhea, and mortality in C. difficile-infected mice. A(2B)ARs mediate C. difficile toxin-induced enteritis and disease. Inhibition of A(2B)AR activation may be a potential strategy to limit morbidity and mortality from CDI.

Amixicile, a novel inhibitor of pyruvate: ferredoxin oxidoreductase, shows efficacy against Clostridium difficile in a mouse infection model.

Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-µg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.