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C. difficile infection (CDI) is a worldwide healthcare problem with ~30% of cases failing primary therapy, placing a burden on healthcare systems and increasing patient morbidity. We have little understanding of why these therapies fail. Here, we use a clinically validated in vitro gut model to assess the contribution of biofilms towards recurrent disease and to investigate biofilm microbiota-C. difficile interactions. Initial experiments show that C. difficile cells became associated with the colonic biofilm microbiota and are not depleted by vancomycin or faecal microbiota transplant therapies. We observe that transferring biofilm encased C. difficile cells into a C. difficile naïve but CDI susceptible model induces CDI. Members of the biofilm community can impact C. difficile biofilm formation by acting either antagonistically or synergistically. We highlight the importance of biofilms as a reservoir for C. difficile, which can be a cause for recurrent infections.

Immunotherapies have drastically improved clinical outcomes in a wide range of malignancies. Nevertheless, patient responses remain highly variable, and reliable biomarkers that predict responses accurately are not yet fully understood. Compelling evidence from preclinical studies and observational data from clinical cohorts have shown that commensal microorganisms that reside in the human gastrointestinal tract, collectively termed the ‘microbiome', can actively modify responses to chemotherapeutic agents and immunotherapies by influencing host immunosurveillance. Notably, microbial correlates are largely context specific, and response signatures may vary by patient population, geographic location and type of anticancer treatment. Therefore, the incongruence of beneficial microbiome signatures across studies, along with an emerging understanding of the mechanisms underlying the interactions between the microbiome, metabolome and host immune system, highlight a critical need for additional comprehensive and standardized multi-omics studies. Future research should consider key host factors, such as diet and use of medication, in both preclinical animal models and large-scale, multicenter clinical trials. In addition, there is a strong rationale to evaluate the microbiome as a tumor-extrinsic biomarker of clinical outcomes and to test the therapeutic potential of derived microbial products (e.g. defined microbial consortia), with the eventual goal of improving the efficacy of existing anticancer treatments. This review discusses the importance of the microbiome from the perspective of cancer immunotherapies, and outlines future steps that may contribute to wide-ranging clinical and translational benefits that may improve the health and quality of life of patients with cancer.

Background Recurrent Clostridioides difficile infection (rCDI) is associated with loss of microbial diversity and microbe-derived secondary bile acids, which inhibit C. difficile germination and growth. SER-109, an investigational microbiome drug of donor-derived, purified spores, reduced recurrence in a dose-ranging, phase (P) 1 study in subjects with multiple rCDIs. Methods In a P2 double-blind trial, subjects with clinical resolution on standard-of-care antibiotics were stratified by age (< or ≥65 years) and randomized 2:1 to single-dose SER-109 or placebo. Subjects were diagnosed at study entry by PCR or toxin testing. Safety, C. difficile–positive diarrhea through week 8, SER-109 engraftment, and bile acid changes were assessed. Results 89 subjects enrolled (67% female; 80.9% diagnosed by PCR). rCDI rates were lower in the SER-109 arm than placebo (44.1% vs 53.3%) but did not meet statistical significance. In a preplanned analysis, rates were reduced among subjects ≥65 years (45.2% vs 80%, respectively; RR, 1.77; 95% CI, 1.11–2.81), while the, In a phase 2 trial, SER-109, an investigational microbiome drug, did not reduce rates of recurrent CDI, despite a previously successful open-label study. Key contributing factors, which led to a redesign of the currently enrolling phase 3 trial, are highlighted.

e15062 Background: Gastrointestinal (GI) mucositis is a frequent complication of chemotherapy for cancer treatment and conditioning regimens for hematopoietic stem cell transplant (HSCT). Clinical and preclinical data suggest that disruption of the gut microbiome may be implicated in GI mucositis pathophysiology by affecting GI barrier integrity, immune homeostasis, and colonization resistance to pathogens. Here, we evaluated the ability of a rationally-designed microbial consortium, DE486, to reduce systemic inflammation and toxicity in a chemotherapy-induced murine mucositis model. Methods: Germ-free mice were used to evaluate the impact of the microbiome in a 5-fluorouracil-induced (5-FU) mucositis model. Mice were colonized with either DE486 or an inflammatory composition (INFL2). DE486 was rationally designed as a consortium of strains for experimental investigation to restore colonization resistance, improve GI barrier integrity, and reduce inflammatory responses. After 4-5 weeks of colonization, mice were intraperitoneally injected with 5-FU or vehicle control on three consecutive days (days 0-2) to initiate mucositis. The degree of 5-FU-induced toxicity was assessed by daily body weight measurements from the start of 5-FU treatment (day 0) until study end (day 8) and expressed as the mean body weight loss ± SD. Serum levels of pro-inflammatory cytokines were assessed on days 5 and 8 by mouse cytokine/chemokine immunoassays (Luminex xMAP). Data reported are the group mean pg/mL and the statistical significance of group mean differences was analyzed using one-way ANOVA. Results: In the 5-FU-induced mucositis model, mice colonized with DE486 or INFL2 exhibited a mean body weight loss of 10% ± 2% by day 4. However, on day 5, mice colonized with DE486 began to recover body weight and by day 8 returned to their body weight before 5-FU treatment. In contrast, mice colonized with INFL2 continued to lose body weight and by day 8 had a mean body weight loss of 18% ± 4%. Serum analyses showed that on day 5 cytokine levels in mice colonized with DE486 or INFL2 remained low and did not differ significantly between the two groups. However, on day 8, mice colonized with INFL2 developed significantly higher proinflammatory cytokine levels than mice colonized with DE486 (TNFα: 21.68 vs. 2.26, P = 0.018, IL-6: 1860.55 vs. 2.15, P = 0.007, IFN-γ: 177.37 vs. 0.37, P < 0.001). Conclusions: Preclinical assessments in a murine model of chemotherapy-induced mucositis showed that colonization with DE486 promoted body weight recovery and protected against increased levels of key pro-inflammatory cytokines, as compared to colonization with an inflammatory microbiome. These data support the ability of a rationally-designed microbial consortium to ameliorate systemic inflammation following chemotherapy as a strategy to prevent or manage GI mucositis.

Background There is intense interest about whether modulating gut microbiota can impact systemic metabolism. We investigated the safety of weekly oral fecal microbiota transplantation (FMT) capsules from healthy lean donors and their ability to alter gut microbiota and improve metabolic outcomes in patients with obesity. Methods and findings FMT-TRIM was a 12-week double-blind randomized placebo-controlled pilot trial of oral FMT capsules performed at a single US academic medical center. Between August 2016 and April 2018, we randomized 24 adults with obesity and mild–moderate insulin resistance (homeostatic model assessment of insulin resistance [HOMA-IR] between 2.0 and 8.0) to weekly healthy lean donor FMT versus placebo capsules for 6 weeks. The primary outcome, assessed by intention to treat, was change in insulin sensitivity between 0 and 6 weeks as measured by hyperinsulinemic euglycemic clamps. Additional metabolic parameters were evaluated at 0, 6, and 12 weeks, including HbA1c, body weight, body composition by dual-energy X-ray absorptiometry, and resting energy expenditure by indirect calorimetry. Fecal samples were serially collected and evaluated via 16S V4 rRNA sequencing. Our study population was 71% female, with an average baseline BMI of 38.8 ± 6.7 kg/m2 and 41.3 ± 5.1 kg/m2 in the FMT and placebo groups, respectively. There were no statistically significant improvements in insulin sensitivity in the FMT group compared to the placebo group (+5% ± 12% in FMT group versus −3% ± 32% in placebo group, mean difference 9%, 95% CI −5% to 28%, p = 0.16). There were no statistically significant differences between groups for most of the other secondary metabolic outcomes, including HOMA-IR (mean difference 0.2, 95% CI −0.9 to 0.9, p = 0.96) and body composition (lean mass mean difference −0.1 kg, 95% CI −1.9 to 1.6 kg, p = 0.87; fat mass mean difference 1.2 kg, 95% CI −0.6 to 3.0 kg, p = 0.18), over the 12-week study. We observed variable engraftment of donor bacterial groups among FMT recipients, which persisted throughout the 12-week study. There were no significant differences in adverse events (AEs) (10 versus 5, p = 0.09), and no serious AEs related to FMT. Limitations of this pilot study are the small sample size, inclusion of participants with relatively mild insulin resistance, and lack of concurrent dietary intervention. Conclusions Weekly administration of FMT capsules in adults with obesity results in gut microbiota engraftment in most recipients for at least 12 weeks. Despite engraftment, we did not observe clinically significant metabolic effects during the study. Trial registration ClinicalTrials.gov NCT02530385., Elain W Yu et al investigate the potential of fecal microbiota transplantation to improve metabolism in obese individuals., Author summary Why was this study done? Animal studies show that body weight and glycemic regulation can be markedly altered by manipulation of the intestinal microbiota. Two prior studies in men with obesity and metabolic syndrome showed that a single nasoduodenal fecal microbiota transplantation (FMT) from lean donors led to small and transient improvements in glycemic outcomes. We hypothesized that repeated dosing with oral FMT capsules from lean donors could lead to lasting improvements in metabolic outcomes. What did the researchers do and find? We randomized adults with obesity who were at high risk for development of type 2 diabetes to receive either weekly oral FMT capsules from healthy lean donors or placebo capsules for 6 weeks. Oral FMT was safe and tolerable, and we observed durable microbial shifts in most participants receiving FMT. We found no significant differences between groups in most glycemic outcomes, weight, or body composition over a 12-week period. There was a minor improvement in HbA1c after FMT as compared to placebo. Exploratory analyses suggest possible improvement in metabolism after FMT among study participants with low baseline microbiome diversity, similar to what was observed in a prior study. What do these findings mean? Our results suggest that intestinal microbial manipulation by FMT capsules does not meaningfully alter human metabolism and weight in adults with obesity. Regional differences between study populations, differences in route of FMT administration, and differences in engrafting microbial species all might explain the overall negative findings and discordance with prior trials. Future studies should evaluate pre-selection of donors and recipients, and consider microbiome and lifestyle modifications concurrently.

Background & Aims Firmicutes bacteria produce metabolites that maintain the intestinal barrier and mucosal immunity. Firmicutes are reduced in the intestinal microbiota of patients with ulcerative colitis (UC). In a phase 1b trial of patients with UC, we evaluated the safety and efficacy of SER-287, an oral formulation of Firmicutes spores, and the effects of vancomycin preconditioning on expansion (engraftment) of SER-287 species in the colon. Methods We conducted a double-blind trial of SER-287 in 58 adults with active mild-to-moderate UC (modified Mayo scores 4–10, endoscopic subscores ≥1). Participants received 6 days of preconditioning with oral vancomycin (125 mg, 4 times daily) or placebo followed by 8 weeks of oral SER-287 or placebo. Patients were randomly assigned (2:3:3:3) to groups that received placebo followed by either placebo or SER-287 once weekly, or vancomycin followed by SER-287 once weekly or SER-287 once daily. Clinical endpoints included safety and clinical remission (modified Mayo score ≤ 2; endoscopic subscores 0 or 1). Microbiome endpoints included SER-287 engraftment (dose species detected in stool after, but not before, SER-287 administration). Engraftment of SER-287 and changes in microbiome composition and associated metabolites were measured by analyses of stool specimens collected at baseline, after preconditioning, and during and 4 weeks after administration of SER-287 or placebo. Results Proportions of patients with adverse events did not differ significantly among groups. A higher proportion of patients in the vancomycin/SER-287 daily group (40%) achieved clinical remission at week 8 than patients in the placebo/placebo group (0), placebo/SER-287 weekly group (13.3%), or vancomycin/SER-287 weekly group (17.7%) (P=.024 for vancomycin/SER-287 daily vs placebo/placebo). By day 7, higher numbers of SER-287 dose species were detected in stool samples from all SER-287 groups compared with the placebo group (P

Background: There is intense interest about the therapeutic potential of altering gut microbiota to improve metabolism, based primarily on intriguing animal studies. One prior trial of fecal microbiota transplantation (FMT) in obese men found that improved metabolic response after FMT was predicted by low baseline microbiome diversity. In the current trial, we investigated the safety and efficacy of weekly oral FMT capsules to improve glycemic outcomes in obese adults, and also explored determinants of successful microbiome engraftment and metabolic improvement after FMT. Methods: FMT-TRIM was a double-blind randomized placebo-controlled pilot trial of weekly oral FMT vs placebo capsules for 6 weeks in 24 obese adults with mild-moderate insulin resistance. Each participant in the FMT arm received capsules derived from one of 4 metabolically healthy lean donors (BMI 18.5-23 kg/m2). The primary outcome was change in insulin sensitivity assessed by hyperinsulinemic euglycemic clamp at 0 and 6 weeks. Secondary outcomes included body weight, metabolic labs, and body composition assessed by DXA over 12 weeks. 16SV4 rRNA sequencing was performed to assess microbiome composition and engraftment. Post-hoc exploratory analyses investigated metabolic outcomes after stratification by baseline microbiome diversity. Results: FMT and placebo groups were well balanced in terms of age (mean±SD 40±9 yrs), BMI (40±6 kg/m2), sex (72% female), and baseline metabolic measures. During the study, there were no statistically significant differences in insulin sensitivity between the FMT and placebo groups (+5 ± 12% FMT vs -3 ± 32% placebo, mean percent difference 9%, 95% CI -5% to 28%; p=0.16). There was a minor improvement in HbA1c at 12 weeks after FMT as compared to placebo (mean difference -0.1, 95% CI -0.3-0.01), but no significant differences in other metabolic labs, body weight, or body composition. Microbial engraftment varied by donor but was present in most FMT recipients, with persistence of engrafting strains throughout the 12-week study. Subgroup analyses of subjects with low microbiome diversity at baseline (FMT n=4, placebo n=7) showed a relative benefit of FMT over placebo at 12 weeks for HbA1c (mean difference -0.2, 95% CI -0.4 to -0.01), total cholesterol (-22 mg/dL, 95% CI -40 to -4 mg/dL), and fasting glucose (-10 mg/dL, 95% CI -19 to -1 mg/dL). There were no significant differences in adverse events between FMT and placebo groups. Conclusion: Weekly administration of FMT capsules results in gut microbiota engraftment for at least 12 weeks but does not meaningfully alter human metabolism in an unselected population of obese adults. Future studies are needed to elucidate the role of baseline recipient microbial diversity and other factors on the impact of FMT.

Background In a Phase 1b study of mild-to-moderate ulcerative colitis (UC) (Henn et al. Gastro 2020), oral daily dosing of SER-287, a purified consortium of Firmicutes spores administered after vancomycin pre-conditioning, led to significantly higher rates of clinical remission compared to placebo (40% versus 0%, respectively; p=0.024). Engraftment, defined as SER-287 spore germination and growth of metabolically-active bacteria, was associated with remission and shifts in disease relevant microbe-associated metabolites. To better understand the mechanism underlying these favorable clinical observations, we evaluated metabolite production by SER-287 and their impact on specific inflammatory pathways and barrier epithelial integrity in vitro. Methods Three SER-287 lots and a negative control pro-inflammatory consortium including three strains isolated from UC subjects (Escherichia coli, Bifidobacterium dentium, and Streptococcus parasanguinis) were grown in vitro in a complex medium designed to mimic the nutrient composition found in the human colon. Culture supernatants were assessed for the presence of anti-inflammatory and remission associated metabolites by GC-MS or LC-MS. Anti- and pro-inflammatory activities were determined by measuring IL-8 secretion, an inflammatory cytokine whose expression is elevated in UC patients, by HT29 epithelial cells after incubation with bacterial supernatants in the presence or absence of TNF-α. Epithelial integrity was assayed by measuring FITC-dextran leakage in a trans-well culture of differentiated primary human colonic epithelial cells challenged with IFN-γ in the presence or absence of culture supernatants. Results When cultured in vitro, all SER-287 lots tested produced the anti-inflammatory short-chain fatty acids butyrate and propionate and other metabolites associated with remission in the Phase 1b trial, including tryptophan metabolites, bile acids and medium-chain fatty acids. SER-287 culture supernatants significantly reduced IL-8 secretion in TNF-α stimulated HT29 cells while the pro-inflammatory consortium increased IL-8 secretion compared to TNF-α alone (Figure 1A). SER-287 lots induced minimal IL-8 secretion in the absence of TNF-α while the pro-inflammatory consortium strongly induced IL-8 (Figure 1B). SER-287 supernatants significantly decreased IFN-γ-mediated barrier disruption, while the pro-inflammatory consortium was not protective (Figure 2). Conclusions Production of remission-associated metabolites, reduction of epithelial inflammation, and protection from epithelial barrier damage are shown to be pharmacological properties of SER-287 which may underlie the mechanism of action of this microbiome therapeutic for mild-to-moderate UC.

Background: Antibiotics targeted against Clostridioides difficile bacteria are necessary, but insufficient, to achieve a durable clinical response because they have no effect on C. difficile spores that germinate within a disrupted microbiome. ECOSPOR-III evaluated SER-109, an investigational, biologically derived microbiome therapeutic of purified Firmicute spores for treatment of rCDI. Herein, we present the interim analysis in the ITT population at 8 and 12 weeks. Methods: Adults ≥18 years with rCDI (≥3 episodes in 12 months) were screened at 75 US and CAN sites. CDI was defined as ≥3 unformed stools per day for Results: Overall, 287 participants were screened and 182 were randomized (59.9% female; mean age, 65.5 years). The most common reason for screen failure was a negative C. difficile toxin assay. A significantly lower proportion of SER-109 participants had rCDI after dosing compared to placebo at week 8 (11.1% vs 41.3%, respectively; relative risk [RR], 0.27; 95% confidence interval [CI], 0.15–0.51; p-value Conclusions: SER-109, an oral live microbiome therapeutic, achieved high rates of sustained clinical response with a favorable safety profile. By enriching for Firmicute spores, SER-109 achieves high efficacy while mitigating risk of transmitting infectious agents, beyond donor screening alone. SER-109 represents a major paradigm shift in the clinical management of patients with recurrent CDI. Clinicaltrials.gov Identifier NCT03183128. These data were previously presented as a late breaker at American College of Gastroenterology 2020.Funding: Seres TherapeuticsDisclosures: None

7005 Background: Studies suggest that decreased microbial diversity due to chemotherapeutic and antibiotic exposure may be associated with acute graft vs host disease (aGvHD) and mortality in patients undergoing allogeneic HSCT. In addition, disruption of the microbiome by antibiotics may lead to intestinal domination by pro-inflammatory bacteria, resulting in increased risk of aGvHD. This relationship has been described in settings with prophylactic antibiotic use, a standard of care in most transplant centers. Here we assessed how the microbiome and GvHD outcomes differ when prophylactic use of antibiotics is avoided. Methods: We collaborated on an observational study (COLLECT) to evaluate changes in microbial diversity over time in subjects undergoing allogeneic HSCT. According to protocol at the University Hospital of Cologne, antibiotics were administered only as empiric treatment for febrile neutropenia or as targeted treatment. Stool was collected weekly from 65 subjects at baseline (pre-HSCT) to day 28 with additional time points taken at day 56, day 90, day 365, and upon diagnosis of intestinal GvHD (GvHD-day 0 and GvHD-day 7). Patients were monitored for incidence of GvHD, including acute GvHD of the liver, intestine, and skin. Microbiome 16SV4 profiles were generated from 381 stool samples. Linear effects models were developed to evaluate the association between Shannon diversity, intestinal domination, and the incidence of intestinal GvHD and mortality. Results: Of the 65 subjects, 28 subjects (42%) went on to develop intestinal GvHD, and 16 subjects (25%) did not survive to day 365. A decline in Shannon diversity was observed during the neutropenic period following HSCT. Subjects who went on to develop intestinal GvHD had significantly lower Shannon diversity at the time of stem cell engraftment (p < 0.0468). Furthermore, lower diversity was observed throughout the study period in subjects experiencing intestinal GvHD. We developed a linear model evaluating the association between mortality and Shannon diversity and found a significant relationship at days 28 and 90 post HSCT (p < 0.0001 and 0.0121, resp). Intestinal domination by Enterobacteriaceae or Enterococcus was significantly associated with the incidence of intestinal GvHD (p < 0.0082) or mortality (p < 0.001), respectively. Conclusions: Data from this observational study (COLLECT) suggests decreases in microbial diversity over time occur in subjects undergoing allogeneic HSCT despite the lack of prophylactic antibiotics. Investigation of whether administration of microbiome therapeutic drugs prior to transplant and/or at the time of engraftment can reduce morbidity and mortality in this high-risk patient population is warranted. Clinical trial information: NCT03148197.

2574 Background: Clinical and preclinical experiments suggest that the gut microbiome can affect outcome in cancer patients treated with immune checkpoint inhibitors (ICI). Most data to date has been in melanoma, so the relationship of the gut microbiome with treatment outcome in other cancers is poorly understood. Here, we evaluated the microbiome composition in correlation to ICI response in patients with metastatic lung, urothelial, or renal cancer, as well as metastatic melanoma. Methods: Fecal microbiome samples were obtained from patients with metastatic melanoma, lung, urothelial, or renal cancer immediately before ICI therapy was initiated. Bacterial genomic DNA was isolated and profiled by whole metagenome sequencing. Sequence data were analyzed using a custom implementation of MetaPhlAn2. Response to ICI was defined as partial or complete response or remaining on therapy for more than 6 months. Results: Samples were prospectively collected from 94 patients, including metastatic melanoma (n = 17), lung (n = 44), urothelial (n = 23), or renal cancer (n = 10). Treatment included anti-PD(L)1 monotherapy (n = 51), anti-PD1 + anti-CTLA4 combination therapy (n = 17), or a combination of anti-PD1 and chemotherapy (n = 26). Clinical response was observed in 58% of patients, including partial or complete response (45%) and on treatment for more than 6 months (55%, with 31% on treatment for more than 1 year). Although the variance in the composition of pretreatment microbiome samples did not explain response alone (R vs NR, PERMANOVA, p = 0.273), a significant portion of the variance in microbiome composition was explained by the interaction of cancer type and outcome (PERMANOVA, p = 0.014), suggesting a cancer-specific microbiome relationship. Notably, there was some similarity in the signature of NR across three cancer types (lung, urothelial and melanoma). One sample in this NR cluster was from a patient whose metastatic NSCLC was nonresponsive to pembrolizumab and carboplatin/pemetrexed. This microbiome sample was evaluated in vivo using subcutaneous MC38 and CT26 tumor models in germ-free mice. In contrast to mice colonized with stool from a healthy donor, mice colonized with stool from this patient yielded a nonresponsive result upon treatment with anti-PD1 or anti-PD-L1 in combination with anti-CTLA4. Conclusions: Analysis of the fecal microbiome composition from patients with metastatic lung, urothelial, renal cancer, and melanoma identified a cancer-specific signature of R and NR to ICI. Across three cancer types, a consistent signature of NR was identified and corroborated experimentally in preclinical models.

Background C. difficile recurs when dormant spores germinate in the dysbiotic gut, facilitated by an increase of 1° vs. 2° bile acids. SER-109, an ecology of bacterial spores purified from stool of healthy donors, is an investigational first-in-class microbiome therapeutic intended to facilitate microbiome restoration and reduce risk of recurrent C. difficile (rCDI). Rapid engraftment of spore-forming species is associated with (i) higher doses of SER-109 in our dose-ranging Phase 1b study (Ph1b) and (ii) reduced rCDI in our Phase 2 trial (Ph2). We explored whether higher doses of SER-109 were associated with an increase in 2° bile acids. Methods Whole metagenomic shotgun (WMS) data were generated from stool, and species were identified using a proprietary build of MetaPhlAn. Evaluation of spore-forming species richness and bile acid concentrations identified effects of SER-109 treatment. A triple stage bioreactor model of the human gut and rCDI was used to evaluate the impact of microbial therapeutics. Results Ph1b subjects who received a higher dose (>1.5 × 108 SporQ) had significantly higher spore-forming species richness than subjects who received a low dose (

Background A healthy gut microbiome is associated with colonization resistance against C. difficile and other pathogens, including bacteria carrying antibiotic resistance genes (AbRG). Designed to facilitate microbiome restoration and reduce the risk of recurrent C. difficile infection (rCDI), SER-109, an investigational microbiome therapeutic, is an ecology of bacterial spores purified from stool from healthy screened donors. We evaluated the impact of engraftment of SER-109 dose species on the abundance of AbRG in rCDI subjects. Methods We generated whole metagenomic shotgun (WMS) data for a subset of study subjects with available stool samples receiving SER-109 (n = 66) or placebo (n = 25) from 2 clinical trials (a dose-ranging Phase 1b study and a fixed-dose Phase 2 trial). WMS data from stool was analyzed to (1) quantify the abundance of AbRG (Comprehensive Antibiotic Resistance Database CARD v.1.1.8) and (2) define subjects with significant engraftment of SER-109 dose species. For each subject and antibiotic drug class, we calculated the change in abundance of AbRG between samples collected at baseline (after antibiotic therapy for an episode of C. difficile infection) and following treatment with SER-109 or placebo. We evaluated the effect of SER-109 engraftment on AbRG abundance, independent of dose. Results In subjects with significant high-confidence engraftment of SER-109 organisms (n = 30) we observed significantly greater reduction in AbRG relative to placebo at week 1 post treatment. These AbRG were associated with multiple classes of antibiotics including, but not limited to, cephalosporins (P = 0.035), and fluoroquinolones (P = 0.035) (Figure 1). Furthermore, the reduction of AbRG was correlated with the increased abundance of SER-109 dose species, and with a reduction in Proteobacteria (e.g., Enterobactericeae) (Figure 2). Conclusion Restoration of the gut microbiome with SER-109 in subjects with a history of rCDI is associated with a reduction in abundance of antibiotic resistance genes. These observations suggest that microbiome therapeutics could play a role in more rapidly decolonizing drug-resistant bacteria. Disclosures C. Ford, Seres Therapeutics, Inc: Employee and Shareholder, Salary. M. Henn, Seres Therapeutics, Inc: Employee and Shareholder, Salary. J. Bryant, Seres Therapeutics, Inc: Employee and Shareholder, Salary. L. Diao, Seres Therapeutics, Inc: Employee and Shareholder, Salary. J. Wortman, Seres Therapeutics, Inc: Employee and Shareholder, Salary. A. Tomlinson, Seres Therapeutics, Inc: Employee and Shareholder, Salary. K. Litcofsky, Seres Therapeutics, Inc: Employee and Shareholder, Salary. P. Bernardo, Seres Therapeutics, Inc: Employee and Shareholder, Salary. B. McGovern, Seres Therapeutics, Inc: Employee and Shareholder, Salary. J. G. Aunins, Seres Therapeutics, Inc: Employee and Shareholder, Salary. D. N. Cook, Seres Therapeutics, Inc: Employee and Shareholder, Salary. M. Trucksis, Seres Therapeutics, Inc: Employee and Shareholder, Salary.

The human gut microbiota forms a diverse, dynamic and complex ecosystem that modulates numerous host processes including metabolism, inflammation and cellular and humoral immune responses. Emerging data suggest that the gut microbiota of cancer patients may predict tumor response to immune checkpoint inhibitors (ICI). To better understand how the microbiome may impact response to ICI, we have developed and validated robust tumor models using either conventional mice treated with antibiotics or germ-free (GF) mice. We have confirmed in both models, that mice lacking a diverse microbiome fail to mount an efficient antitumor immune response after treatment with anti-PD-1, primarily due to T-cell dysfunction. This response to anti-PD-1 can be restored by introduction of a microbiome using fecal microbial transplant (FMT) prepared from stool from healthy donors in GF mice, and is driven by increased tumor-infiltrating lymphocytes (TILs), specifically CD8+ T-cells. Importantly, for the first time, we show that the bacterial spore fraction from healthy donor stool can also restore response to anti-PD-1 and increase CD8+ TILs in both conventional mice treated with antibiotics and GF mice. We have also tested cancer patient-derived microbiome samples that are efficacious or non-efficacious in GF mouse models. We have sequenced the input material as well as the engrafted gut microbiota in these mice via 16S rDNA sequencing. Comparison of these sequences has enabled us to develop a “microbiome signature” of response that is being used in the development of a microbiome based therapy for combination with ICI. Based on these encouraging animal model data we plan to initiate a randomized, placebo-controlled clinical study at MD Anderson Cancer Center in 2018, sponsored by the Parker Institute for Cancer Immunotherapy, in patients with advanced metastatic melanoma. The clinical trial will evaluate the impact of an anti-PD-1 checkpoint inhibitor with adjunctive microbiome therapy on patient outcomes. Seres is developing SER-401, a preclinical stage oral microbiome therapy to improve the efficacy and safety of immunotherapy. Our drug discovery strategy combines computational analyses and empirical in vitro, in vivo and ex-vivo screening of strains and consortia to inform selection and drive microbiome drug design. Data from such a comprehensive approach are invaluable for designing compositions of bacteria that form “functional ecological networks” that can impact response to ICI therapy. We believe these data will provide insight into how microbiome drugs can be developed in the setting of immunotherapy to augment the efficacy of ICIs by altering the cancer-immune set point. Citation Format: Lata Jayaraman, Jaclyn Sceneay, Srimathi Srinivasan, Keith Halley, Meghna Bist, Kathleen Cieciuch, George Marnellos, Christopher Desjardins, Jennifer Wortman, Matthew Henn, David Cook. Leveraging gut microbiota networks to impact tumor immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B063.

Lien vers le texte intégral : http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=22677620; International audience; The Dengue National Control Program was established in Cambodia in 2000 and has reported between 10,000 and 40,000 dengue cases per year with a case fatality rate ranging from 0.7 to 1.7. In this study 39 DENV-2 and 57 DENV-3 viruses isolated from patients between 2000 and were fully sequenced. Five DENV2 and four DENV3 distinct lineages with different dynamics were identified. Each lineage was characterized by the presence of specific mutations with no evidence of recombination. In both DENV-2 and DENV-3 the lineages present prior to 2003 were replaced after that date by unrelated lineages. After 2003, DENV-2 lineages D2-3 and D2-4 cocirculated until 2007 when they were almost completely replaced by a lineage D2-5 which emerged from Conversely, all DENV-3 lineages remained, diversified and cocirculated with novel lineages emerging. Years 2006 and 2007 were marked by a high prevalence of DENV-3 and 2007 with a large dengue outbreak and a high proportion of patients with severe disease. Selective sweeps in DENV-1 and DENV-2 were linked to immunological escape to a predominately DENV-3-driven immunological response. The complex dynamic of dengue in Cambodia in the last ten years has been associated with a combination of stochastic climatic events, cocirculation, coevolution, adaptation to different vector populations, and with the human population immunological landscape.

National Science Foundation (U.S.). Center for Microbial Oceanography: Research and Education (Grant OCE-042560)

Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples ( n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia.

Dengue virus (DENV) causes dengue fever and dengue haemorrhagic fever/dengue shock syndrome, both considered major public-health problems worldwide. We generated a lethal DENV-2 strain (D220) by 10 additional cycles of subcutaneous inoculation of mice with supernatant from mosquito cells infected with the previously characterized strain D2S10, followed by harvesting of serum. D220 induces mortality at ten-fold lower doses than D2S10 in mice lacking only the alpha/beta interferon (IFN-α/β) receptor in C57BL/6 or 129 backgrounds under both non-enhanced and antibody-enhanced conditions. Sequence analysis of the complete viral genome revealed five amino acid changes between D220 and D2S10, of which two (K122I in envelope and V115A in NS4B) appear to account for the observed phenotypic differences between the viruses. By causing mortality at lower doses in C57BL/6 mice lacking only the IFN-α/β receptor, D220 constitutes an improved tool for study of DENV-induced pathogenesis, as well as for testing potential vaccines and antiviral drugs against DENV.

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ∼75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.

Background. The dengue virus serotype 3 (DENV-3) Indian subcontinent strain emerged in Puerto Rico in 1998 after a 21-year absence. The rapid expansion of DENV-3 on the island correlated with the withdrawal of the other serotypes for 7 years. The DENV-3 prevalence declined in 2008 and remains undetected. Methods. We sequenced complete genomes of 92 DENV-3 clinical isolates to characterize the molecular evolution and phylogeography throughout 10 years of continued sampling (1998–2007). Results. We documented 8 distinct lineages that emerged simultaneously and evolved independently. Two of the 8 lineages were highly associated with transient introductions of foreign viruses, and 2 of the 3 endemic lineages covered the entire study period. We found evidence of temporal-geographical clustering only within the 3 endemic lineages. The phylogeography analysis combined with serotype-specific incidence data showed that transmission of a DENV serotype in a given location and time is usually correlated with the absence of the other serotype. Conclusions. Our study shows the cotransmission of DENV-3 lineages through a complex dissemination pattern dissimilar to the evolutionary dynamics of the other serotypes in the island. High virus genetic diversity and a large naive population were underlying factors in the expansion and collapse of DENV-3 in Puerto Rico.

Background Recurrence of CDI occurs within a few weeks after treatment due to antibiotic-induced dysbiosis. SER-109, an investigational, first-in-class microbiome drug, was designed to sustain a clinical response through microbiome restoration with a purified ecology of spores. In an open-label Phase 1b (Ph1b) trial of SER-109 for prevention of recurrent CDI, 26 of 30 subjects did not recur following treatment. In a Phase 2 (Ph2) double-blind controlled trial of SER-109 (n = 59) vs. placebo (n = 30), no significant difference was observed in the proportions of subjects with recurrence (44.1% vs. 53.3%, respectively). Here we contrast gut microbiome changes among subjects in both trials to understand differences in clinical outcomes observed 8-weeks after dosing. Methods We used 16S v4 and high-resolution whole metagenomic shotgun (WMS) sequencing to characterize microbiome changes from stool samples collected at baseline and 1, 4, and 8 weeks post-treatment. Microbiome analyses focused on subjects diagnosed with recurrence via EIA toxin testing (high confidence recurrence; HCR). Results Significantly greater richness of commensal spore-former species was observed in Ph2 subjects treated with SER-109 compared with PBO at weeks 1 and 4 post-treatment (Mann–Whitney P = 0.008, P = 0.044 respectively) consistent with drug engraftment. In addition, the number of spore-forming species at 1 week post-treatment was significantly greater in non-recurrent subjects vs. HCR subjects (Mann–Whitney P = 0.011). Furthermore, we identified 10 spore-former species that were significantly more prevalent in both SER-109 and non-recurrent subjects (Fig 1). In comparison to Ph1 subjects, SER-109 engraftment was significantly reduced and delayed among Ph2 subjects at all time points (Fig 2). Moreover, Ph1b subjects who received higher doses of SER-109 than that used in Ph2 had increased levels of engraftment. Conclusion In patients with recurrent CDI and dysbiosis, a focused spore-based therapeutic approach leads to engraftment of SER-109 strains. In addition, microbiome signatures of engraftment were associated with a favorable clinical outcome. Although SER-109 was biologically active, a higher dose may improve the rate and degree of microbiome repair. Disclosures M. Henn, Seres Therapeutics: Employee and Shareholder, Salary and Stock Options; C. Ford, Seres Therapeutics: Employee and Shareholder, Salary and Stock Options; E. O’Brien, Seres Therapeutics: Employee and Shareholder, Salary and Stock Options; J. Wortman, Seres Therapeutics: Employee and Shareholder, Salary and Stock Options; S. Simmons, Seres Therapeutics: Employee and Shareholder, Salary and Stock options; L. Diao, Seres Therapeutics: Employee and Shareholder, Salary and Stock; K. Litcofsky, Seres Therapeutics: Employee and Shareholder, Salary and stock options; P. Bernardo, Seres Therapeutics: Employee and Shareholder, Salary and Stock options; J. Aunins, Seres Therapeutics, Inc.: Employee and Shareholder, Salary and Stock options; D. Cook, Seres Therapeutics: Employee and Shareholder, Salary and Stock Options; M. Trucksis, Seres Therapeutics Inc.: Employee and Shareholder, Salary and stock options

Human leukocyte antigen B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant, HLA-B27-restricted, HCV-specific, cluster of differentiation (CD)8+ T-cell epitope, nonstructural protein (NS)5B2841-2849 (ARMILMTHF), has been shown to be limited by viral fitness costs as well as broad T-cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27-restricted epitope, NS5B2936-2944 (GRAAICGKY), to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a–infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T-cell receptor contact site, neither of which impaired viral replication capacity, as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27+ subjects, rare escape mutations arose at the HLA-B27 anchor residue, R2937, which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Coexpression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from

Epidemic dengue fever (DF) and the emergence of dengue hemorrhagic fever (DHF) in the Americas are associated with increased endemicity and cocirculation of the 4 dengue virus (DENV) serotypes, 1–4 (1). These increases have been particularly evident in Puerto Rico, where transmission increased during the past 25 years (2–4). The first DHF epidemics in the Americas occurred in the 1980s and were caused by the Asian/American genotype of DENV-2, then new to the region, which rapidly replaced the American genotype (5–7). This replacement has been linked to a potential to cause higher viremia and severe illness (8–10). Introduction of DENV-3 in the mid 1990s and increased human population and travel further fostered larger and more frequent DF and DHF epidemics in the region (11–13). Although all 4 DENV serotypes circulate on the island, DENV-2 circulated continuously for 25 years. Previously, a partial sequence analysis from 74 DENV-2 isolates collected in Puerto Rico during 7 years throughout a 14-year period (1987–2001) showed a DENV-2 lineage evolving through a series of turnover events (14). A lineage replacement in 1994 appeared to be associated with a foreign virus but only 3 other reintroductions were found, all linked to the 1998 epidemic, the largest in Puerto Rico history (14). This was a turning point in the epidemiology of dengue, with DENV-2 (and DENV-1 and -4) rapidly declining during the expansion of DENV-3. However, transmission of DENV-2 persisted at low levels during 1999–2003 and increased thereafter. This serotype turnover offers new opportunities to study the evolution of DENV-2. Our analysis illustrates the genetic composition and population diversity of DENV-2 throughout 22 consecutive years of sampling in Puerto Rico and may explain the evolutionary resilience and long-term establishment of this virus.

T4-like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4-like genomes, including 10 from non-cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non-cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl-CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage-encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2-oxoglutarate. Patterns among non-core genes that may drive niche diversification revealed that phosphorus-related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome-wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross-infection. Finally, genome-wide comparisons of both diverse and closely related, co-isolated genomes provide a locus-to-locus variability metric that will prove valuable for interpreting metagenomic data sets.

Background Dengue is the most prevalent mosquito-borne viral disease in humans and a major urban public health problem worldwide.

Resistance mutations to hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease inhibitors in1% of the viral quasispecies may still allow1000-fold viral load reductions upon treatment, consistent with their reported reduced replicative fitness in vitro. Recently, however, an R155K protease mutation was reported as the dominant quasispecies in a treatment-naïve individual, raising concerns about possible full drug resistance. To investigate the prevalence of dominant resistance mutations against specifically targeted antiviral therapy for HCV (STAT-C) in the population, we analyzed HCV genome sequences from 507 treatment-naïve patients infected with HCV genotype 1 from the United States, Germany, and Switzerland. Phylogenetic sequence analysis and viral load data were used to identify the possible spread of replication-competent, drug-resistant viral strains in the population and to infer the consequences of these mutations upon viral replication in vivo. Mutations described to confer resistance to the protease inhibitors Telaprevir, BILN2061, ITMN-191, SCH6 and Boceprevir; the NS5B polymerase inhibitor AG-021541; and to the NS4A antagonist ACH-806 were observed mostly as sporadic, unrelated cases, at frequencies between 0.3% and 2.8% in the population, including two patients with possible multidrug resistance. Collectively, however, 8.6% of the patients infected with genotype 1a and 1.4% of those infected with genotype 1b carried at least one dominant resistance mutation. Viral loads were high in the majority of these patients, suggesting that drug-resistant viral strains might achieve replication levels comparable to nonresistant viruses in vivo.Naturally occurring dominant STAT-C resistance mutations are common in treatment-naïve patients infected with HCV genotype 1. Their influence on treatment outcome should further be characterized to evaluate possible benefits of drug resistance testing for individual tailoring of drug combinations when treatment options are limited due to previous nonresponse to peginterferon and ribavirin.

Summary Acidobacteria constitute a still poorly studied phylum that is well represented in soils. Recent studies suggest that members of this phylum may be also abundant in deep-sea plankton, but their relative abundance and ecological role in this ecosystem are completely unknown. A recent screening of three metagenomic deep-sea libraries of bathypelagic plankton from the South Atlantic (1000 m depth), the Adriatic (1000 m depth) and the Ionian (3000 m depth) seas in the Mediterranean revealed an unexpected relative proportion of acidobacterial fosmids, which affiliated to the Solibacterales (Group 3), to the Group 11 and, most frequently, to the Group 6 of this diverse phylum. Here, we present the comparative analysis of 11 acidobacterial genome fragments containing the rrn operon from these Mediterranean libraries. A highly conserved syntenic region spanning up to 30 kb and containing up to 25 open reading frames was shared by Group 6 Acidobacteria. Synteny was also partially conserved in distantly related acidobacterial genome fragments derived from a metagenomic soil library, indicating a remarkable conservation of this genomic region within these Acidobacteria. A search for Acidobacteria-specific hits in directly comparable, available fosmid-end sequences from soil and marine metagenomic libraries showed a significant increase of their relative proportion in plankton libraries as a function of increasing depth reaching, at high depth, levels nearly comparable to those of soil. Thus, our results suggest that Acidobacteria are abundant and represent a significant proportion of the microbial community in the deep-sea ecosystem.

Marine planktonic archaea are widespread and abundant in deep oceanic waters but, despite their obvious ecological importance, little is known about them. Metagenomic analyses of large genome fragments allow access to both gene content and genome structure from single individuals of these cultivation-reluctant organisms. We present the comparative analysis of 22 archaeal genomic clones containing 16S rRNA genes that were selected from four metagenomic libraries constructed from meso- and bathypelagic plankton of different oceanic regions (South Atlantic, Antarctic Polar Front, Adriatic and Ionian Sea; depths from 500 to 3000 m). We sequenced clones of the divergent archaeal lineages Group 1A (Crenarchaeota) and Group III (Euryarchaeota) as well as clones from the more frequent Group I Crenarchaeota and Group II Euryarchaeota. Whenever possible, we analysed clones that had identical or nearly identical 16S rRNA genes and that were retrieved from distant geographical locations, that is, that defined pan-oceanic operational taxonomic units (OTUs). We detected genes involved in nitrogen fixation in Group 1A Crenarchaeota, and genes involved in carbon fixation pathways and oligopeptide importers in Group I Crenarchaeota, which could confirm the idea that these are mixotrophic. A two-component system resembling that found in ammonia-oxidizing bacteria was found in Group III Euryarchaeota, while genes for anaerobic respiratory chains were detected in Group II Euryarchaeota. Whereas gene sequence conservation was high, and recombination and gene shuffling extensive within and between OTUs in Group I Crenarchaeota, gene sequence conservation was low and global synteny maintained in Group II Euryarchaeota. This implies remarkable differences in genome dynamics in Group I Crenarchaeota and Group II Euryarchaeota with recombination and mutation being, respectively, the dominant genome-shaping forces. These observations, along with variations in GC content, led us to hypothesize that the two groups of organisms have fundamentally different lifestyles.

Summary • 15N natural abundance was examined to determine the fractionation associated with ammonium assimilation into fungi. The importance of fungi to the movement of nitrogen (N) within soils and into plants highlights the need to consider such fractionations in the interpretation of ecosystem 15N distributions. • To separate the influence of physiological fractionations from N source preferences on fungal δ15N, both ectomycorrhizal (EM) and saprotrophic (SAP) fungi were grown in liquid culture containing (NH4)2HPO4 of known δ15N and harvested after 35 d. Time series experiments were employed for two taxa to determine how growth stage affects fractionation. • Mycelium δ15N was positively associated with several measures of growth and no distinction was observed between EM and SAP fungi. Kinetic isotope exchange models explain the decrease in discrimination against 15N at larger biomasses in terms of the amount of N utilized. The models indicate a fractionation of approx. −20‰, and suggest that assimilated N is excreted back into the medium during growth. • The observed fractionations at high and low N concentrations provide insight into the mechanisms that could drive fungal, plant and soil δ15N. We suggest that detailed information on fungal life histories, territorial behaviours, and sink–source relationships in the fungus–plant–soil continuum could aid accurate ecological interpretations of δ15N.

We grew 11 basidiomycetes in axenic culture to characterize their physiological capacities to fractionate stable C isotopes. Generally, δ 13 C values of the fungal biomass were (i) enriched in 13 C relative to the growth medium, (ii) variable among the isolates, and (iii) dependent on the growth rate and growth stage of the fungi. We found a multiphasic dynamic of fractionation for Cryptoporus volvatus and Marasmius androsaceus during various growth stages. The first phase, P1, corresponded to the exponential growth stage and was characterized by an increasing enrichment in 13 C content of the fungal biomass relative to the growth medium ranging between 4.6 and 6.9‰. The second phase, P2, exhibited a continual depletion in 13 C of the fungal biomass, with the δ 13 C values of the fungal biomass asymptotically returning to the δ 13 C value of the growth medium at inoculation. The expression of the various fractionation phases was dependent on the amount of low-concentration micronutrients and growth factors added to the growth medium. The onset of P2 occurred at reduced concentrations of these elements. All of the sugars in the growth medium (sucrose, maltose, and glucose) were utilized for growth, indicating that the observed fractionation was not an artifact derived from the preferential use of 13 C-rich maltose, which was found at low concentrations in the growth medium. In this study, we establish a framework with which to explore the impact of physiological fractionations by fungal interfaces on natural distributions of stable C isotopes.

High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium’s minimal information (MIxS) and NCBI’s BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.

Exotic pine plantations are promoted for their presumed capacity to provide a net sink of atmospheric C. Millions of hectares worldwide will be subjected to conversion into plantations during the next decades. However, pine introductions are known to result in a marked depletion of soil C, a phenomenon which has remained unexplained. We studied plantations in paramo grasslands of Ecuador, where the effect of the exotic introduction of radiata pines ( Pinus radiata ) and their accompanying ectomycorrhizal fungi can be studied in isolation from other ecosystem disturbances. We suggest that ectomycorrhizal fungi can extract C previously accumulated by paramo grasslands based on (a) a drastic simplification of the ectomycorrhizal community shown by direct DNA identification, (b) a loss of up to 30% soil C within 2 levels.

To quantify and characterize N and C isotopic fractionation effects due to fungal transformation of organic substrates in forest ecosystems, we performed a field study in California and a meta-analysis of three additional studies conducted by others across the Northern Hemisphere. Basidiomycete fungal biomass was consistently enriched for the heavier isotope for C relative to substrate and either enriched or depleted for N relative to atmospheric N. Extent and pattern of fractionation was very variable, but the distinction between ectomycorrhizal and saprotrophic basidiomycetes was strongly supported, particularly when dual isotope analyses were performed. This differentiation, which we call the "EM-SAP Divide" holds for studies within a restricted ecosystem, but becomes less distinct over larger geographical regions, removing the rationale for using direct isotopic values from single specimens as diagnostic of ecophysiological role. For C, the EM-SAP Divide seems to reflect substrate effects, potentially due to differential access to recently synthesized versus recycled organic compounds, rather than distinct physiological pathways. Once substrate and ecophysiological role effects are removed, our meta-analysis suggests the existence of more than one mechanism causing C fractionations in fungi which is found equally in ectomycorrhizal and saprotrophic fungi. Similarly, a multimodal distribution of δ15N values suggests that physiological effects may play a much stronger influence on N natural isotopic distributions in fungi. Our meta-analysis provides a firm statistical base to evaluate fungal ecological statements based on natural isotopic distributions of C and N. We call into question the current practice of using direct isotopic measurements to make statements about trophic relationships of fungi in the absence of other supporting evidence.