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Background: Neratinib is a potent small molecule tyrosine kinase inhibitor (TKI) of human epidermal growth factor receptors (HER1,2,4). One of the major side effects of neratinib is diarrhea. The human gut contains a dense microbiome ecosystem that is essential in maintaining a healthy host physiology, and its disruption may lead to increased risk of toxicities from cancer therapy. In this study, we aimed to develop a machine learning model based on analysis of gut microbiota data to predict neratinib-induced diarrhea. Methods: Patients were enrolled in a phase II trial evaluating safety and tolerability of neratinib in older adults with HER2+ breast cancer (NCT02673398). Neratinib was administered as single agent, 240 mg oral daily in a 28-day cycle. Stool samples were collected at baseline and during treatment for 16S rRNA gene sequencing. Using microbial relative abundance data, we developed gradient-boosted tree models with two nested loops of cross validations to classify whether diarrhea would occur or not after treatment onset. For the inner validation loop, we used ten-fold cross validation to determine the optimal model from hyper-parameters including regularization. For the outer validation loop, we utilized a leave-one-patient-out cross validation to test this model on the hold-out patient’s baseline data and the predictions were used for model assessment. Results: A total of 11 patients and 50 longitudinal stool samples were collected. The median age was 66 years. 73% developed grade ≥ 1 diarrhea attributed to neratinib. Shannon diversity index of gut microbiome was not associated with diarrhea. For predictive modeling, the outer validation loop Area Under the Receiver Operating Characteristic Curve (AUROC) and Area Under the Precision Recall Curve (AUPRC) were 0.92 and 0.97, respectively. The two most important taxa predictive of protection from diarrhea were Ruminiclostridium 9, and Bacteroides sp. HPS0048. We found that patients with a larger relative abundance of Ruminiclostridium 9 and Bacteroides sp. HPS0048 have reduced risk of neratinib-related diarrhea. Conclusions: The machine learning model can identify breast cancer patients at risk of diarrhea prior to neratinib use. Future studies are required to validate this finding. Citation Format: Chi Wah Wong, Susan E. Yost, Jin Sun Lee, John D. Gillece, Megan Folkerts, Lauren Reining, Sarah K. Highlander, Zahra Eftekhari, Joanne Mortimer, Yuan Yuan. Machine learning model of gut microbiota predicts neratinib induced diarrhea in patients with breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-41.

Studies suggest a link between the gut microbiome and metastatic renal cell carcinoma (mRCC) outcomes, including evidence that mRCC patients possess a lower abundance of Bifidobacterium spp. compared to healthy adults. We sought to assess if a Bifidobacterium‐containing yogurt product could modulate the gut microbiome and clinical outcome from vascular endothelial growth factor‐tyrosine kinase inhibitors (VEGF‐TKIs). mRCC patients initiating VEGF‐TKIs, regardless of the line of therapy, were randomized to probiotic‐supplemented (two 4 oz. servings of the probiotic yogurt product daily) or probiotic‐restricted arms. Stool samples were collected prior to therapy and at weeks 2, 3, 4, and 12. Microbiome composition was assessed using whole‐metagenome sequencing. A total of 20 patients were randomized. Bifidobacterium animalis, the active ingredient of the probiotic supplement, reached detectable levels in all patients in the probiotic‐supplemented arm versus two patients in the probiotic‐restricted arm. Clinical benefit rate was similar in probiotic‐supplemented versus probiotic‐restricted arms (70% vs. 80%, p = 0.606). Linear discriminant analysis (LDA) effect size analysis of MetaPhIAn2 abundance data predicted 25 enriched species demonstrating an LDA score >3 in either clinical benefit or no clinical benefit. In patients with clinical benefit (vs. no clinical benefit), Barnesiella intestinihominis and Akkermansia muciniphila were significantly more abundant (p = 7.4 × 10−6 and p = 5.6 × 10−3, respectively). This is the first prospective randomized study demonstrating modulation of the gut microbiome with a probiotic in mRCC. Probiotic supplementation successfully increased the Bifidobacterium spp. levels. Analysis of longitudinal stool specimens identified an association between B. intestinihominis, A. muciniphila, and clinical benefit with therapy. Trial Registration: NCT02944617, Our study provides novel evidence regarding gut microbiome modulation with a Bifidobacterium‐containing yogurt in metastatic renal cell carcinoma patients receiving targeted therapies and specifies the microbiota members (Barnesiella intestinihominis, Akkermansia muciniphila) associated with clinical benefit from targeted therapies in mRCC.

Preclinical models and early clinical data suggest an interplay between the gut microbiome and response to immunotherapy in solid tumors including metastatic renal cell carcinoma (mRCC). We sought to characterize the stool microbiome of mRCC patients receiving a checkpoint inhibitor (CPI) and to assess treatment-related changes in microbiome composition over the course of CPI therapy. Stool was collected from 31 patients before initiation of nivolumab (77%) or nivolumab plus ipilimumab (23%) therapy, of whom 58% experienced clinical benefit. Greater microbial diversity was associated with clinical benefit from CPI therapy (p = 0.001), and multiple species were associated with clinical benefit or lack thereof. Temporal profiling of the microbiome indicated that the relative abundance of Akkermansia muciniphila increased in patients deriving clinical benefit from CPIs. This study substantiates results from previous CPI-related microbiome profiling studies in mRCC. Temporal changes in microbiome composition suggest potential utility in modulating the microbiome for more successful CPI outcomes. Patient summary We compared the composition and diversity of the gut microbiome in patients receiving immunotherapy for renal cell carcinoma. We found that higher microbial diversity is associated with better treatment outcomes. Treatment response is characterized by changes in microbial species over the course of treatment.

Previous studies have suggested that the gut microbiome influences the response to checkpoint inhibitors (CPIs) in patients with cancer. CBM588 is a bifidogenic live bacterial product that we postulated could augment CPI response through modulation of the gut microbiome. In this open-label, single-center study (NCT03829111), 30 treatment-naive patients with metastatic renal cell carcinoma with clear cell and/or sarcomatoid histology and intermediate- or poor-risk disease were randomized 2:1 to receive nivolumab and ipilimumab with or without daily oral CBM588, respectively. Stool metagenomic sequencing was performed at multiple timepoints. The primary endpoint to compare the relative abundance of Bifidobacterium spp. at baseline and at 12 weeks was not met, and no significant differences in Bifidobacterium spp. or Shannon index associated with the addition of CBM588 to nivolumab–ipilimumab were detected. Secondary endpoints included response rate, progression-free survival (PFS) and toxicity. PFS was significantly longer in patients receiving nivolumab–ipilimumab with CBM588 than without (12.7 months versus 2.5 months, hazard ratio 0.15, 95% confidence interval 0.05–0.47, P = 0.001). Although not statistically significant, the response rate was also higher in patients receiving CBM588 (58% versus 20%, P = 0.06). No significant difference in toxicity was observed between the study arms. The data suggest that CBM588 appears to enhance the clinical outcome in patients with metastatic renal cell carcinoma treated with nivolumab–ipilimumab. Larger studies are warranted to confirm this clinical observation and elucidate the mechanism of action and the effects on microbiome and immune compartments.

Background: While early life exposures such as mode of birth, breastfeeding, and antibiotic use are established regulators of microbiome composition in early childhood, recent research suggests that the social environment may also exert influence. Two recent studies in adults demonstrated associations between socioeconomic factors and microbiome composition. This study expands on this prior work by examining the association between family socioeconomic status (SES) and host genetics with microbiome composition in infants and children. Methods: Family SES was used to predict a latent variable representing six genera abundances generated from whole-genome shotgun sequencing. A polygenic score derived from a microbiome genome-wide association study was included to control for potential genetic associations. Associations between family SES and microbiome diversity were assessed. Results: Anaerostipes, Bacteroides, Eubacterium, Faecalibacterium, and Lachnospiraceae spp. significantly loaded onto a latent factor, which was significantly predicted by SES (p &lt, 0.05) but not the polygenic score (p &gt, 0.05). Our results indicate that SES did not predict alpha diversity but did predict beta diversity (p &lt, 0.001). Conclusions: Our results demonstrate that modifiable environmental factors influence gut microbiome composition at an early age. These results are important as our understanding of gut microbiome influences on health continue to expand.

Simple Summary Given their high degree of identity and even greater similarity at the amino acid level, Kat3 coactivators, CBP (Kat3A) and p300 (Kat3B), have long been considered redundant. We describe the generation of novel p300 S89A knock-in mice carrying a single site directed amino acid mutation in p300, changing the highly evolutionarily conserved serine 89 to alanine, thus enhancing Wnt/CBP/catenin signaling (at the expense of Wnt/p300/catenin signaling). p300 S89A knock-in mice exhibit multiple organ system, immunologic and metabolic differences, compared with their wild type counterparts. In particular, these p300 S89A knock-in mice are highly sensitive to intestinal injury resulting in colitis which is known to significantly predispose to colorectal cancer. Our results highlight the critical role of this region in p300 as a signaling nexus and provide further evidence that p300 and CBP are non-redundant, playing definite and distinctive roles in development and disease. Abstract Differential usage of Kat3 coactivators, CBP and p300, by β-catenin is a fundamental regulatory mechanism in stem cell maintenance and initiation of differentiation and repair. Based upon our earlier pharmacologic studies, p300 serine 89 (S89) is critical for controlling differential coactivator usage by β-catenin via post-translational phosphorylation in stem/progenitor populations, and appears to be a target for a number of kinase cascades. To further investigate mechanisms of signal integration effected by this domain, we generated p300 S89A knock-in mice. We show that S89A mice are extremely sensitive to intestinal insult resulting in colitis, which is known to significantly increase the risk of developing colorectal cancer. We demonstrate cell intrinsic differences, and microbiome compositional differences and differential immune responses, in intestine of S89A versus wild type mice. Genomic and proteomic analyses reveal pathway differences, including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. The diverse effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the critical role of this region in p300 as a signaling nexus and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution.

4510 Background: The negative association between ICI response and abx therapy is well defined (Derosa et al Cancer Discov 2021). Paradoxically, a retrospective assessment of the live bacterial product (LBP) CBM588 in patients (pts) with advanced lung cancer showed improved outcome with ICIs when the combination of CBM588 and abx (as compared to CBM588 alone) was employed (Tomita et al Cancer Immunol Res 2020). We postulated that the microbial resistome (genes encoding antimicrobial resistance) could shift in a manner with CBM588 therapy that facilitated ICI response. Methods: Pts with newly diagnosed mRCC with clear cell and/or sarcomatoid histology and intermediate/high risk disease per IMDC criteria were randomized to nivolumab/ipilimumab (nivo/ipi) or nivo/ipi/CBM588 in a 1:2 ratio. Stool samples were collected at baseline and week 12. Whole-metagenome sequencing was performed to analyze stool microbiome composition. Abx resistance genes (RGs) were inferred using publicly available database (McArthur et al. Antimicrob Agents Chemother 2013), and groups of abx RGs for various classes of abx were characterized. Wilcoxon signed-rank test was used for comparison of abx RG abundance between baseline and week 12 in each treatment arm and in responders (R) and non-responders (NR). Results: The study enrolled 30 pts, with the final analysis including 29 eligible pts (median age: 66 years, M:F 21:8, nivo/ipi: 19 pts, nivo/ipi/CBM588:10 pts). Objective response was 20% and 58% in nivo/ipi and nivo/ipi/CBM588 arms, respectively. The overall abundance of abx RGs remained unchanged between baseline and week 12 in pts receiving nivo/ipi alone. In contrast, a decrease in abx RGs was observed in pts receiving nivo/ipi with CBM588 arm from baseline to week 12 (p = 0.042 in Rs; p = 0.078 in NRs). More specifically, nivo/ipi/CBM588 treatment led to a significant reduction in fosfomycin RGs and nitroimidazole (e.g., metronidazole) RGs in both pts with R (p = 0.019 and 0.042, respectively) and NR (p = 0.031 and p = 0.031, respectively). A multitude of other clinically relevant abx RGs were downregulated in pts receiving CBM588, including those mediating resistance to glycopeptide (e.g., vancomycin) and lincosamide (e.g., clindamycin) abx. Conclusions: In the first interrogation of the resistome in mRCC, we demonstrate that CBM588 decreases abx RGs associated with multiple commonly used classes of abx. Abx clear commensals and increase pathogenic (abx resistant) bacteria in the gut. Based on our data, we formulate the hypothesis that combining abx with CBM588 may decrease potentially pathobionts and favor butyrogenic species, thereby improving CPI response. Clinical studies using CBM588 with abx priming may be warranted. Clinical trial information: NCT03829111.

Neratinib has great efficacy in treating HER2+ breast cancer but is associated with significant gastrointestinal toxicity. The objective of this pilot study was to understand the association of gut microbiome and neratinib-induced diarrhea. Twenty-five patients (age ≥ 60) were enrolled in a phase II trial evaluating safety and tolerability of neratinib in older adults with HER2+ breast cancer (NCT02673398). Fifty stool samples were collected from 11 patients at baseline and during treatment. 16S rRNA analysis was performed and relative abundance data were generated. Shannon’s diversity was calculated to examine gut microbiome dysbiosis. An explainable tree-based approach was utilized to classify patients who might experience neratinib-related diarrhea (grade ≥ 1) based on pre-treatment baseline microbial relative abundance data. The hold-out Area Under Receiver Operating Characteristic and Area Under Precision-Recall Curves of the model were 0.88 and 0.95, respectively. Model explanations showed that patients with a larger relative abundance of Ruminiclostridium 9 and Bacteroides sp. HPS0048 may have reduced risk of neratinib-related diarrhea and was confirmed by Kruskal-Wallis test (p ≤ 0.05, uncorrected). Our machine learning model identified microbiota associated with reduced risk of neratinib-induced diarrhea and the result from this pilot study will be further verified in a larger study.Clinical Trial RegistrationClinicalTrials.gov, identifier NCT02673398.

371 Background: The role of gut microbial composition as a determinant of clinical outcomes has been well established in several cancers, including metastatic renal cell carcinoma (mRCC) (Routy et al Science 2018). A growing body of evidence suggests that examining the metabolic function of microbial communities may provide a more insightful understanding of these associations (Helmink et al Nature Medicine 2019). Herein, we aimed to examine the effect of nivolumab/ipilimumab with or without CBM588 on clinical outcomes and gut microbiome functionality. Methods: Treatment naïve mRCC pts with clear cell and/or sarcomatoid histology and IMDC intermediate/high risk disease were enrolled and randomized into receiving nivolumab/ipilimumab or nivolumab/ipilimumab with CBM588 in 1:2 fashion. Whole metagenome sequencing was performed on stool samples collected at baseline and week 12. Generated MetaPhlan 3.0 data was run through HUMAnN 3.0 to identify differentially expressed metabolic pathways between two timepoints in each arm and with respect to treatment response. Results: A total of 30 pts were enrolled and randomized, and 29 pts were eligible for analysis as one patient was excluded as tumor tissue next-generation sequencing revealed genomic alterations pathognomonic for sarcoma after initiation of protocol-based therapy. Median age of the participants was 66 years, 21 pts (72%) were male, 10 pts (34%) had sarcomatoid features, and 29 pts (45%) had prior nephrectomy. Objective response was achieved in 58% and 20% of the pts in nivolumab/ipilimumab/CBM-588 and nivolumab/ipilimumab arm, respectively. Significant changes in 40 metabolic pathways (37 with upregulation and 3 with downregulation) in nivolumab/ipilimumab arm and 52 metabolic pathways (49 with downregulation and 3 with upregulation) in nivolumab/ipilimumab with CBM588 arm were identified. In detail, dTDP-β-L-rhamnose biosynthesis, L-lysine biosynthesis II and superpathway of pyrimidine ribonucleosides degradation pathways were found upregulated while O-antigen building blocks biosynthesis (E. coli) pathway was found downregulated after treatment with nivolumab/ipilimumab and CBM588 (p = 0.001, p = 0.007, p = 0.037, p = 0.005 respectively). Heatmaps detailing the dynamics of metabolic pathway expressions in regard to response in each arm will be presented. Conclusions: We observed an increase in the activity of the pathways associated with butyrate consumption and a resultant decrease in glycolytic dependence. Further, suppression of the pathogenic E. coli function was observed, suggesting a role for CBM588 in protection from pathogenic species. Our findings provide mechanistic evidence for the effect of the addition of CBM588 to nivolumab/ipilimumab on gut microbiome function and resultant improvement in clinical outcomes in mRCC. Clinical trial information: NCT03829111.

372 Background: Multiple studies have suggested that the gut microbiome plays a modulatory role in ICI activity and that specific bacteria and/or cumulative microbial diversity may drive response in patients (pts) with mRCC (Routy et al Science 2018; Salgia et al Eur Urol 2020). Even though the tumor microenvironment has substantial bacterial proliferation (Heymann et al Cancer L. 2021), there is a paucity of data assessing the impact of intra-tumoral microbiota in response to ICI therapy. In this study, we sought to explore this association in pts with mRCC. Methods: Pts diagnosed with mRCC who had available RNA sequencing (RNA-seq) data collected in the course of routine clinical care and who were treated with ICIs were retrospectively identified.Intra-tumoral microbiome analysis was performed on formalin-fixed paraffin-embedded samples. Following quality and adapter trimming, RNA-seq reads were mapped to a human genome to filter host reads using the Burrows-Wheeler alignment (BWA) tool. Taxonomic classification was performed using Kraken2 and the absolute abundances of species were estimated using Bracken. The relative abundances among all non-human species were calculated. Statistical testing with Student’s t-test was performed to compare the relative abundance for all species seen within pts who responded to ICIs and those who did not. Results: Among the 28 pts (22:6, M:F) included in this analysis, 24 (86%) had clear cell histology and 20 (71%) were IMDC intermediate/poor risk. All of the samples were collected prior to starting treatment with ICIs and the majority of these (57%) were collected from the primary site. 11 pts (39%) received ICIs as first line treatment and 17 (61%) as second line. Clinical response was seen in 50% of pts included in the study and the most common rendered treatment was nivolumab (17 pts). In the overall cohort, Cutibacterium acne, Moraxella osloensis, and Pasteurella multocida had the highest relative abundances. Additionally, significant differences in relative abundances of specific bacteria were found between ICI responders and non-responders. Among these, Stenotrophomonas maltophilia (p = 0.037) and Corynebacterium sp. zg-917 (p = 0.035) had significantly higher relative abundances in pts who responded to ICIs. Conclusions: This is the first study evaluating the association between intra-tumoral microbiome and response to ICIs in pts with mRCC. Among bacteria associated with response, several have particular relevance – for instance, Corynebacterium spp. have been studied for decades as a possible adjunct to immunotherapeutic agents such as BCG. Efforts are ongoing to validate these findings in a larger cohort.

374 Background: Treatment with N/I with the addition of CBM588, a live bacterial product comprised primarily of Clostridium butyricum, improved PFS and RR versus N/I alone when used as first line treatment for patients with mRCC (Meza et al ASCO 2021). Increased abundance of certain bacterial species in the gut microbiome have been associated with the development of treatment related adverse events (TREAs) in lung cancer patients receiving immunotherapy (Chau et al BMC Cancer 2021). However, this association has not yet been delineated in the setting of mRCC. Here, we present results of an exploratory analysis assessing the differences in stool microbiome composition between patients who experienced grade (G) 3/4 TREAs and those who did not. Methods: Patients were randomized 2:1 to receive N/I with or without CBM588. Stool collection for bacteriomic profiling was planned at baseline and after 12 weeks of therapy for all randomized patients. Whole metagenome sequencing was performed using previously published methods (Dizman et al Cancer Med 2021) and differences in microbiome composition were measured based on the occurrence of G 3/4 TREAs. Results: 30 patients were enrolled and 29 included in the study. At the time of data cutoff (April 15, 2021) the median follow up was 12.2 months (95% confidence intervals [CI], 10.6-13.8). Grade 3/4 TRAEs were experienced in 52, 50, and 53% of patients in the overall cohort, control, and intervention arms, respectively (p = NS). Among the most common G 3/4 TRAEs, subjects experienced fatigue, diarrhea, and hyperglycemia. Patients with a complete set of stool samples were included for the microbiome analysis (n = 26). In patients who experienced G 3/4 TRAEs, a significantly greater baseline abundance of Escherichia coli, Klebsiella spp. and Blautia spp. (p = 0.02, 0.03, 0.05) were seen when compared to those not experiencing G 3/4 TRAEs. In contrast, Bacteroides intestinalis and B. thetaiotamicron, were observed in significantly higher abundances in baseline stool specimens of patients who did not experience G 3/4 TREAs (p = 0.03 for both). No significant differences were seen for any of these species at the 12-week timepoint. Conclusions: We are among the first to investigate the differences in baseline stool microbiome in mRCC patients experiencing G 3/4 TRAEs while receiving immunotherapy. Our results suggest that certain taxa of bacteria are predictors of the development of serious TRAEs. Larger cohorts are needed to corroborate these findings. Clinical trial information: NCT03829111.

336 Background: The high incidence of esophageal adenocarcinoma (EAC) remains a major clinical challenge as majority of these cases are diagnosed at advanced stages, with poor survival outcomes. Emerging evidence indicate intriguing associations between unique microbial profiles and cancer pathogenesis. More recent data also indicates the potential clinical significance of specific microbial signatures as diagnostic and prognostic biomarkers. However, to date, no studies have systematically interrogated circulating metagenome profiling in EAC patients, particularly as non-invasive, early detection, surveillance and prognostic classifiers that may improve the current management paradigms. Methods: Metagenome sequencing was performed on 81 serum specimens collected from 51 EAC, 10 high grade dysplasia (HGD), 10 Barretts’s esophagus (BE), and 10 gastro-esophageal reflux disease (GERD) patients, respectively. Sequencing reads were classified using Bracken and MetaPhlAn3 to determine relative abundance between various classes. The Linear Discriminant Analysis effect size (LEfSe) method was performed to identify potential conserved and discrete microbial profiles between groups. Logistic regression and Kaplan-Meier analyses were used to build a model according to clinical and metagenomic classifiers to examine the diagnostic and prognostic potential of the identified metagenomic signature. Results: A significant loss of alpha and beta diversity was identified in serum specimens from EAC patients. We observed a shift in microbial taxa between each group – at the phylum, genus, and species level – with Lactobacillus sakei as the most prominent species in GERD vs. other patient groups. Interestingly, LEfSe analysis identified a complete loss of Lactobacillus ( L.Sakei and L. Curvatus), Collinsella stercoris and Bacteroides stercoris, but conversely a significant increase in Escherichia coli in patients with EAC. Finally, we developed a metagenome panel that discriminated EAC from GERD patients with an AUC value of 0.89 (95% CI, 0.78 – 0.95), and this panel in conjunction with the TNM stage was a robust predictor of overall survival (≥24 months; AUC = 0.84 [95% CI 0.66 – 0.92]) and an accompanying hazard ratio (HR) of 6.23 (95% CI, 2.65 – 14.46, P

The gut microbiota plays an important role in maintaining intestinal homeostasis by regulating the maturation of the mucosal immune system, which constitutes an immune barrier for the integrity of the intestinal tract. In recent years, the role of the human GI microbiota in graft-versus-host disease (GVHD) and other outcomes after allogeneic hematopoietic cell transplantation (HCT) has been increasingly evaluated in observational studies. However, there have been limited interventional trials specifically designed to alter the microbiota of HCT recipients. CBM588 (clostridium butyricum MIYAIRI 588) is a novel Live Biotherapeutic Product (LBP) that produces short chain organic acids, mainly butyric acid, which plays a key role in the maintenance of colonic homeostasis by regulating fluid and electrolyte uptake, epithelial cell growth, and inflammatory responses. In this pilot trial (NCT03922035) we sought to determine the safety, feasibility, biologic activities, and preliminary efficacy of CBM588 in HCT recipients. Patients age ≥18 years, scheduled to undergo HCT from an 8/8 or 7/8 matched related/unrelated donor with reduced intensity conditioning (RIC) were eligible. Following the patient safety lead-in (SLI; n=6), 30 patients were randomized (1:1 ratio) to receive either standard peri-transplant supportive care alone (control arm) or with CBM588 (treatment arm, open label) at the fixed dose of 160 mg orally (2x/day) from day -8 or hospital admission until day +28 or discharge (figure 1). Patients received prophylactic antibiotics per intuitional SOPs. Study objectives were to evaluate the safety/feasibility of CBM588 (Primary), and to compare the incidence and severity of adverse events (AE), HCT outcomes including GVHD, and gut microbiome diversity between the Treatment and Control arms. Feasibility was defined as the ability to consume CDM588 for 14 days during the SLI phase. For microbiome analysis, we isolated DNA from weekly collected stool samples, and amplified the V4 region of the bacterial 16S rRNA gene from each total DNA sample. Between April, 2018 and January, 2020, we enrolled 36 patients (20 were female) at the median age of 66 years (range: 34-77). The indication for HCT was Leukemias (n=22), MDS (n=5), lymphoma (n=3), myeloma (n=3), or other (n=3). All but one patient received fludarabine/melphalan-based RIC and tacrolimus/sirolimus-based GVHD prophylaxis. Graft source was peripheral blood stem cell from a matched related (n=13) or unrelated (n=23) donor (Table 1). One patient assigned to the Treatment arm declined to receive CBM588 before the first dose; but remained on the study with clinical data/biospecimen collections and safety/feasibility/biologic endpoints were analyzed as treated for this patient. All the other patients who were assigned to the treatment arm (n=21, including the patients in SLI segment) were able to take the prescribed study drug; with the median 52 doses (range: 0-55), and 19 of 21 subjects (90.5%) consumed at least 14 days of the study drug. There were no serious adverse events (SAE) related to CBM588. The overall AEs and infection- or GI-specific AEs were similar between the Treatment and Control arms. All but one patient (who died of sepsis in the Control arm - on day 8) engrafted with a median of 15 days for neutrophils. The 100-day non-relapse mortality (NRM) was 0% in the Treatment and 6.7% in the Control arm. According to the intent-to-treat principle, acute GVHD (grade 2-4) was observed in 4 of 15 patients in the Treatment arm and 5 of 15 in the Control arm. The lower GI GVHD was seen in 2 patients in the Treatment and 4 in the control arm. As treated analyses showed the overall grade 2-4 GVHD in 3 of 14 (21.4%) with the use of CBM588 and 6 of 16 (37.5%) without CBM588 (one case of lower GI GVHD with CBM, 5 cases without; (Table 2). The Shannon Diversity Index was similar between the two groups at each time point tested. (Figure 1). However, had favorable microbial profile was detected as the pathogens Enterobacteriaceae, Clostridium baratii, and Clostridiodes difficile were reduced in the treatment group. (Figure 2) In summary, our data demonstrate the feasibility and safety of CBM588 administration during the peri-transplant period, which was associated with an intended biologic impact on the gut microbiome, and an early favorable sign of GI-GVHD incidence and HCT outcomes in this older population who underwent RIC HCT. Figure 1 Figure 1. Disclosures Dadwal: Astellas: Speakers Bureau; Aseptiscope: Consultancy; Shire/Takeda: Research Funding; AlloVir: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Other: Investigator; Karius: Other: Investigator. Pullarkat: AbbVie, Amgen, Genentech, Jazz Pharmaceuticals, Novartis, Pfizer, and Servier: Membership on an entity's Board of Directors or advisory committees; Amgen, Dova, and Novartis: Consultancy, Honoraria. Al Malki: CareDx: Consultancy; Neximmune: Consultancy; Hansa Biopharma: Consultancy; Rigel Pharma: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy. Ali: Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees. Artz: Radiology Partners: Other: Spouse has equity interest in Radiology Partners, a private radiology physician practice. Stein: Amgen: Consultancy, Speakers Bureau; Celgene: Speakers Bureau; Stemline: Speakers Bureau. Budde: Roche: Consultancy; BeiGene: Consultancy; IGM Biosciences: Research Funding; Merck, Inc: Research Funding; Gilead: Consultancy; AstraZeneca: Research Funding; Mustang Bio, Inc: Research Funding; Novartis: Consultancy; Amgen: Research Funding. Popplewell: Hoffman La Roche: Other: Food; Pfizer: Other: Travel; Novartis: Other: Travel. Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Forman: Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy.

Background: Collection of fecal samples is non-invasive, and DNA sequencing of microorganisms can provide a profile of the gut microbiome which may be an indicator of general health and disease, including inflammation, digestive inefficiencies, and the presence of pathogens. The gut microbiome has been associated with response to immune checkpoint inhibitors (ICI) in patients with solid tumors. We aim to study the association of gut microbiome and response to therapy in patients with metastatic breast cancer (BC) undergoing ICI therapies. Methods: 50 fecal samples from two ICI clinical trials (NCT02971761, N=22; NCT02778685, N=28) were collected at baseline, on treatment and at the end of treatment. DNA was extracted, and 16S rRNA gene libraries were generated targeting the variable region 4 (V4) region of the bacterial and archaeal rRNA gene. Libraries were sequenced on the Illumina MiSeq and sequence reads were analyzed using QIIME2. Results: A total of 22 dominant taxa (present ≥5% in any one sample) were identified in samples from 12 triple negative BC patients treated with pembrolizumab + GTx-024 (Enobosarm) and 17 dominant taxa were identified taxa 10 ER+ BC patients treated with pembrolizumab, letrozole, and palbociclib. As is true for most microbiome studies, inter-subject variation was much greater than intra-subject variation. However, in some patients there was a significant change in the composition of the microbial communities over time. In the first study, two patients had partial response (PR) to treatment and both had relatively “healthy” gut microbiota dominated by Bacteroides, Faecalibacterium prausnitzii, and other short chain fatty acid producers like Ruminococcus bromii or Roseburia faecis. Most of the patients who progressed had dysbiotic gut microbiomes and four had very high (≥20%) relative abundance of Prevotella copri, an organism associated with inflammation. In the second study, four patients had PR, and as before, these patients had “healthy” gut signatures dominated by Bacteroides and short chain fatty acid-producing Firmicutes. In one of these patients, a high level of Prevotella stercorea (57%)at baseline was replaced by Bacteroides and Lachnospira as treatment proceeded. Three additional patients had very high levels of P. copri (≥34%); one resolved during treatment, the others did not. Shannon’s diversity index was used to measure abundance and evenness in the fecal communities. Several samples in both studies had very low diversity at baseline. In most cases, the diversity increased following treatment. Conclusion: Most patients who progressed on ICI had dysbiotic gut microbiomes present at baseline and later time points, such as a high relative abundance of Prevotella (implicated in inflammatory processes); this often resolved during therapy. Our observations provide promise for future use of the gut microbiome as a predictor of response to immunotherapy for BC and the potential for modulating the gut microbiome to improve responses in patients with dysbiosis. Citation Format: Yuan Yuan, Sarah Highlander, Susan E Yost, Kim Robinson, Simran Padam, Aileen Tang, Norma Martinez, John Gillece, Lauren Reining, Mina Sedrak, Joanne Mortimer, James Waisman. Gut microbiome profiling of patients with metastatic breast cancer undergoing immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-26.

4513 Background: Recent evidence suggests that the gut microbiome is a potent mediator of immune checkpoint inhibitor (ICI) activity in metastatic renal cell carcinoma (mRCC), with both specific bacterial species and cumulative microbial diversity driving response (Routy et al Science 2018; Salgia et al Eur Urol 2020). We examined whether the butyrate-producing bacterium Clostridium butyricum, the key constituent of CBM-588, could modulate the gut microbiome in patients (pts) with mRCC receiving nivolumab/ipilimumab (N/I) and secondarily improve clinical outcome. Methods: An open-label, randomized study was conducted, with key eligibility criteria including confirmed clear cell and/or sarcomatoid mRCC, intermediate/poor risk by IMDC criteria and no systemic therapy for metastatic disease. Patients were randomized 2:1 to receive either N/I+CBM-588 or N/I alone. N/I was dosed at 3 mg/kg and 1 mg/kg IV every 3 weeks for 12 weeks, followed by N at 480 mg IV every 4 weeks. CBM-588 was dosed orally at 80 mg bid. Stool was collected for bacteriomic profiling at baseline and 12 weeks. Metagenomic sequencing was employed using previously published methods (Dizman et al Cancer Med 2020). The primary endpoint of the study was change in Bifidobacterium spp. from baseline to week 12. Secondary endpoints included change in microbial diversity and clinical outcomes including response rate (RR) and progression-free survival (PFS). Results: 30 pts were randomized between April 2019 and Nov 2020; 1 pt was excluded after genomic sequencing clarified a diagnosis of sarcoma. Among 29 evaluable patients (21:8 M:F), median age was 66, 10 pts (34%) had sarcomatoid features and 24 pts (83%) were intermediate risk. Metagenomic sequencing of paired stool specimens showed an 8-fold increase in B. bifidum and a 6-fold increase in B. adolescentis in pts receiving N/I+CBM-588 from baseline to week 12. C. butyricum was detected only in pts receiving CBM-588. Pathogenic species (e.g., Escherichia. coli and Klebsiella spp.) were more prevalent in pts not receiving CBM-588. RR was significantly higher among pts receiving N/I+CBM-588 vs N/I alone (59% vs 11%; P = 0.024). Median PFS was also prolonged with the addition of CBM-588 to N/I (NR vs 11 weeks; P < 0.001). No significant difference in grade 3/4 toxicities were observed between study arms. Conclusions: This is the first randomized, prospective study to suggest enhancement of ICI response with a live bacterial product. The observed clinical impact is corroborated by biologic findings supporting gut modulation by CBM-588. Clinical trial information: NCT03829111.

351 Background: Plasma cytokines and the gut microbiome have been shown separately to influence the response to systemic therapy in mRCC. We sought associations between serum cytokines and gut microbial composition in patients (pts) with mRCC. Methods: Eligibility requirements included histologically proven mRCC and an intent to receive either vascular endothelial growth factor-tyrosine kinase inhibitor (VEGF-TKI) or immune checkpoint inhibitor (ICI). Blood samples were collected prior to treatment initiation and immunologic profiles were evaluated using a Human Cytokine 30-plex protein assay (Invitrogen). Stool was collected at baseline and shotgun metagenomic sequencing was performed to quantify gut microbial populations using previously published methods (Salgia et al Eur Urol 2020). Results: A total of 50 pts were studied (36:14 M:F) with a median age of 67 (range, 32-85). Twenty pts and 30 pts had subsequent initiation of VEGF-TKI and ICI therapy, respectively. Levels of Akkermansia spp were significantly higher in pts who were IL-6 low (P = 0.023). In contrast, pts who were IL-6 high had higher levels of enteric pathogens, including Salmonella spp and Enterococcus spp. Both Akkermansia spp and Bacteroides spp levels were higher in pts who were IL-8 low. Associations between cytokine levels, microbiome composition, and treatment response will be presented. Conclusions: Given studies suggesting the role of Akkermansia spp in enhancing ICI response (Routy et al Science 2018), our data provide a critical link between the gut microbiome and systemic immunomodulation.

337 Background: Previous studies have associated specific stool bacterial species with response to both targeted therapy and immunotherapy (Routy et al Science 2018; Dizman et al Cancer Med 2020). Abundant fungal elements also constitute the human microbiome (the so-called “mycobiome”); we explore whether these could be related to clinical benefit (CB) from TT. Methods: Pts from 2 simultaneously conducted studies were included in the analysis. Both studies enrolled patients with histologically confirmed RCC with metastatic disease; in one study, pts received standard of care (SOC) TT, while in the other pts received SOC IO. In both studies, stool was collected at baseline and at multiple timepoints thereafter. Whole metagenome sequencing was performed for fungal microbiome composition (TGen North, AZ). Linear discriminant analysis (LDA) effect size (LEfSe) was used for comparison of the gut mycobiome in patients who obtained clinical benefit (CB; complete response, partial response or stable disease > 6mos) versus no clinical benefit (NCB; progressive disease or stable disease ≤6mos) from TT or IO. Results: A total of 50 samples from 24 pts (19:5 M:F) were included in the analysis. The majority of pts (19; 79%) had clear cell histology. 15 pts received TT while 9 pts received IO. The fungal genera demonstrating the highest abundance was Saccharomyces with a median relative abundance of 86.9% (range, 11%-99%). LEfSe performed in different taxonomic levels revealed Malassezia globosa (LDA = 4.93; P = 0.038) and Alternaria infectoria (LDA 4.94; P = 0.018) as gut mycobiome components associated with NCB, and order Russulales associated with CB from TT (LDA = 4.93; P = 0.018). In contrast, no association was identified between mycobiome profile and CB in the IO-treated group. The presence of several pathogenic fungi such as Candida albicans and Aspergillus fumigatus was noted in a minority of pts and did not have any bearing on clinical outcome. In pts with serial samples, a trend towards decreasing Saccharomyces spp. (the most abundant species) and increasing fungal diversity was noted. Conclusions: Our study is the first to highlight potential associations between the mycobiome and CB with TT. Our finding of Malassezia spp resulting in lack of CB with TT bolsters findings from Aykut et al (Nature 2019), implicating the same genera in progression of pancreatic cancer. Confirmation of these findings in larger series is underway.

The presence of advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is the most important predictor of liver mortality. There are limited data on the diagnostic accuracy of gut microbiota-derived signature for predicting the presence of advanced fibrosis. In this prospective study, we characterizedthe gut microbiome compositions using whole-genome shotgun sequencing of DNA extracted from stool samples. This study included 86 uniquely well-characterized patients with biopsy-proven NAFLD, of which 72 had mild/moderate (stage 0-2 fibrosis) NAFLD, and 14 had advanced fibrosis (stage 3 or4fibrosis). We identified a set of 40 features (p< 0.006), which included 37 bacterial species that were used to construct a Random Forest classifier model to distinguish mild/moderate NAFLD from advanced fibrosis. The model had a robust diagnosticaccuracy (AUC 0.936) for detecting advanced fibrosis. This study provides preliminary evidence for a fecal-microbiome-derived metagenomic signature to detect advanced fibrosis in NAFLD.

Alterations in the gut microbiota are correlated with ailments such as obesity, inflammatory bowel disease, and diarrhea. Up to 60% of individuals traveling from industrialized to developing countries acquire a form of secretory diarrhea known as travelers' diarrhea (TD), and enterotoxigenic Escherichia coli (ETEC) and norovirus (NoV) are the leading causative pathogens. Presumably, TD alters the gut microbiome, however the effect of TD on gut communities has not been studied. We report the first analysis of bacterial gut populations associated with TD. We examined and compared the gut microbiomes of individuals who developed TD associated with ETEC, NoV, or mixed pathogens, and TD with no pathogen identified, to healthy travelers. We observed a signature dysbiotic gut microbiome profile of high Firmicutes:Bacteroidetes ratios in the travelers who developed diarrhea, regardless of etiologic agent or presence of a pathogen. There was no significant difference in α-diversity among travelers. The bacterial composition of the microbiota of the healthy travelers was similar to the diarrheal groups, however the β-diversity of the healthy travelers was significantly different than any pathogen-associated TD group. Further comparison of the healthy traveler microbiota to those from healthy subjects who were part of the Human Microbiome Project also revealed a significantly higher Firmicutes:Bacteriodetes ratio in the healthy travelers and significantly different β-diversity. Thus, the composition of the gut microbiome in healthy, diarrhea-free travelers has characteristics of a dysbiotic gut, suggesting that these alterations could be associated with factors such as travel.

Host-associated microbial communities are influenced by both host genetics and environmental factors. However, factors controlling the human oral microbiome and their impact on disease remain to be investigated. To determine the combined and relative effects of host genotype and environment on oral microbiome composition and caries phenotypes, we profiled the supragingival plaque microbiome of 485 dizygotic and monozygotic twins aged five to eleven. Oral microbiome similarity always increased with shared host genotype, regardless of caries state. Additionally, although most of the variation in the oral microbiome was determined by environmental factors, highly heritable oral taxa were identified. The most heritable oral bacteria were not associated with caries state, did not tend to co-occur with other taxa, and decreased in abundance with age and sugar consumption frequency. Thus, while the human oral microbiome composition is influenced by host genetic background, potentially cariogenic taxa are likely not controlled by genetic factors.

Background: Pancreatic cancer remains a deadly disease, and immunotherapy is effective in only 1-2% of microsatellite instability-high pancreatic cancers. The response to immunotherapy is very heterogeneous across tumor types, but recently there is evidence that the gastrointestinal microbiome may influence the response to immunotherapy. The SU2C Catalyst Trial is a randomized phase II maintenance trial to compare outcomes using a combination regimen of the vitamin D agonist, paricalcitol, plus the PD-1 inhibitor pembrolizumab vs. pembrolizumab alone. As a part of this trial, we are examining the composition of the gut microbiome. Methods: Patients with metastatic pancreatic cancer receiving standard first-line chemotherapy are eligible after achieving best response of at least stable disease and are then randomized to pembrolizumab +/- paricalcitol groups. Patients remain on study treatment until disease progression or treatment-ending toxicities. Patients collect stool samples within 72 hours prior to day one of each cycle, 24-72 later, and ca. 30 days after treatment discontinuation. DNA is extracted from fecal samples and 16S rRNA gene libraries are generated targeting the V4 region of bacterial and archaeal rRNA gene. Libraries are sequenced on the Illumina MiSeq and sequence reads are analyzed using QIIME2. Results: To date, we have analyzed the gut microbiota of six patients who have completed between one to four cycles of study treatment. We identified 28 taxa that occurred at ≥5% relative abundance in any given patient. As is true for most microbiome studies, intersubject variation was much greater than intrasubject variation. However, in three patients, there was significant change in the composition of the microbial communities over time. One patient had a “healthy” microbiome across the one treatment cycle with a good balance of Bacteroidetes and Firmicutes and the presence of Faecalibacterium prausnitzii. This patient discontinued the study due to treatment-related hypophysitis Grade 2, which was resolved by prednisone treatment and remains in survival follow-up. One patient had a relatively healthy microbiome during cycle one and two, but the composition of the gut microbiome became severely dysbiotic at the time the patient discontinued study treatment due to disease progression; this patient died five months later. The remaining four patients were dysbiotic across all treatment times, with some having very low alpha diversity. Conclusions: The gut microbiota of pancreatic cancer patients in the SU2C trial was very heterogeneous and usually dysbiotic. In one patient, who survives progression free, a “healthy” microbiome was observed. This provides promise for future use of the gut microbiome as a predictor of response to immunotherapy for pancreatic cancer and the potential for modulating the gut microbiome to improve responses in patients with dysbiosis. (ClinicalTrials.gov Identifier: NCT03331562. Support: Stand Up To Cancer (SU2C) Catalyst Merck Grant.) Citation Format: Sarah K. Highlander, Vincent Chung, Angela T. Alistar, Erkut Borazanci, Gayle Jameson, Talima Pearson, Nina A. Cantafio, Daniel D. Von Hoff. Gastrointestinal microbiome changes in stage IV pancreatic cancer patients treated with pembrolizumab with or without paricalcitol on the Stand Up to Cancer (SU2C) Pancreas Catalyst Trial [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B24.

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.

Mannheimia haemolytica, one of the agents associated with bovine respiratory disease complex, can cause severe lung pathology including the leakage of vascular products into the airways and alveoli. Previous work by this laboratory has demonstrated that bovine lung endothelial and epithelial cells undergo dramatic permeability increases when exposed to adenosine-5'-triphosphate (ATP). Therefore, we wanted to determine if ATP levels were elevated in bronchoalveolar lavage (BAL) samples from calves experimentally infected with M. haemolytica. In addition, cultured bovine pulmonary epithelial (BPE) cells were stimulated with heat-killed and live M. haemolytica bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), interleukin-1 (IL-1), and zymosan activated plasma (ZAP) to determine whether they might release extracellular ATP during in vitro infection. Calves experimentally exposed to M. haemolytica had an approximately 2-fold higher level of ATP in their BAL samples compared to control. BPE cells exposed to increasing numbers of heat-killed or live M. haemolytica had significantly increased levels of ATP release as compared to time-matched controls. Finally, BPE cells treated with several concentrations of LPS and IL-1 had increases in ATP release as compared to time-matched controls. This increase appeared to be a result of active ATP secretion by the cells, as cell viability was similar between treated and non-treated cells. Neither ZAP nor LTA induced any ATP release by the cells. In conclusion, ATP levels are elevated in lung secretions from calves infected with M. haemolytica. In addition, lung epithelial cells can actively release ATP when exposed to heat-killed or live M. haemolytica, LPS or IL-1.

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies.

Analysis of microbial communities using high throughput sequencing methods began in the mid 2000s permitting the production of 1000s to 10,000s of sequence reads per sample and megabases of data per sequence run. This then unprecedented depth of sequencing allowed, for the first time, the discovery of the ‘rare biosphere’ in environmental samples. The technology was quickly applied to studies in several human subjects. Perhaps these early studies served as a reminder that though the microbes that inhabit mammals are known to outnumber host cells by an order of magnitude or more, most of these are unknown members of our second genome, or microbiome (as coined by Joshua Lederberg), because of our inability to culture them. High throughput methods for microbial 16S ribosomal RNA gene and whole genome shotgun (WGS) sequencing have now begun to reveal the composition and identity of archaeal, bacterial and viral communities at many sites, in and on the human body. Surveys of the microbiota of food production animals have been published in the past few years and future studies should benefit from protocols and tools developed from large-scale human microbiome studies. Nevertheless, production animal-related resources, such as improved host genome assemblies and increased numbers and diversity of host-specific microbial reference genome sequences, will be needed to permit meaningful and robust analysis of 16S rDNA and WGS sequence data.

It is expected that Mannheimia hemolytica A1 expresses a particular collection of genes during infection in the host. The bacterial gene products are produced in the in vivo environment to facilitate growth and survival. Here, we examined gene expression by M. hemolytica A1 in the bovine host after 6 days of infection. Total RNA from M. hemolytica A1 recovered from pneumonic lungs of two animals was used to produce cDNA to screen a custom M. hemolytica A1 microarray. The expression profile was compared to a RNA sample from an in vitro grown culture. The data showed that 44 genes were differentially expressed by more than eightfold when compared with the in vitro sample. Seventeen genes were found to have higher expression in vivo and 27 genes had lower expression. Several virulence-associated genes including those encoding leukotoxin, a capsule biosynthetic enzyme and the serotype-specific antigen, Ssa, had reduced expression, suggesting that their products may not be important during the later stages of infection. Most of the genes up-regulated in vivo encoded hypothetical or conserved hypothetical proteins. Three Mu-like bacteriophage-related genes were up-regulated in the in vivo sample, suggesting that the prophage may be transcriptionally active. The results provide a glimpse of gene expression by the bacterium in the host after pulmonary infection has been established.

Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences--the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental packages' apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

Background: The Human Microbiome Project has ushered in a new era for human metagenomics and high-throughput next-generation sequencing strategies. Content: This review describes evolving strategies in metagenomics, with a special emphasis on the core technology of DNA pyrosequencing. The challenges of microbial identification in the context of microbial populations are discussed. The development of next-generation pyrosequencing strategies and the technical hurdles confronting these methodologies are addressed. Bioinformatics-related topics include taxonomic systems, sequence databases, sequence-alignment tools, and classifiers. DNA sequencing based on 16S rRNA genes or entire genomes is summarized with respect to potential pyrosequencing applications. Summary: Both the approach of 16S rDNA amplicon sequencing and the whole-genome sequencing approach may be useful for human metagenomics, and numerous bioinformatics tools are being deployed to tackle such vast amounts of microbiological sequence diversity. Metagenomics, or genetic studies of microbial communities, may ultimately contribute to a more comprehensive understanding of human health, disease susceptibilities, and the pathophysiology of infectious and immune-mediated diseases.

The ferric uptake regulator (Fur) is an iron-dependent transcriptional regulator that regulates genes related to iron acquisition, oxidative stress response, and various other functions. Transcription of fur is typically self-regulating and sensitive to iron and oxidative stress. Following the identification of a fur gene in the genome of the bovine pathogen Mannheimia haemolytica, an attempt was made to characterize the transcriptional control of M. haemolytica fur. Northern blotting, RT-PCR, and primer extension were done to determine that M. haemolytica fur is transcribed using three distinct promoters, two of which are located within the upstream fldA gene. The third promoter is located upstream of a conserved hypothetical protein and drives transcription of a tricistronic message. Quantitative real time PCR experiments indicated that unlike current models of Fur regulation, M. haemolytica fur transcription is unchanged by iron depletion at logarithmic phase and repressed by iron depletion at stationary phase.

Observations from human microbiome studies are often conflicting or inconclusive. Many factors likely contribute to these issues including small cohort sizes, sample collection, and handling and processing differences. The field of microbiome research is moving from 16S rDNA gene sequencing to a more comprehensive genomic and functional representation through whole-genome sequencing (WGS) of complete communities. Here we performed quantitative and qualitative analyses comparing WGS metagenomic data from human stool specimens using the Illumina Nextera XT and Illumina TruSeq DNA PCR-free kits, and the KAPA Biosystems Hyper Prep PCR and PCR-free systems. Significant differences in taxonomy are observed among the four different next-generation sequencing library preparations using a DNA mock community and a cell control of known concentration. We also revealed biases in error profiles, duplication rates, and loss of reads representing organisms that have a high %G+C content that can significantly impact results. As with all methods, the use of benchmarking controls has revealed critical differences among methods that impact sequencing results and later would impact study interpretation. We recommend that the community adopt PCR-free-based approaches to reduce PCR bias that affects calculations of abundance and to improve assemblies for accurate taxonomic assignment. Furthermore, the inclusion of a known-input cell spike-in control provides accurate quantitation of organisms in clinical samples.

Mannheimia haemolytica causes pneumonia in domestic and wild ruminants. Leukotoxin (Lkt) is the most important virulence factor of the bacterium. It is encoded within the four-gene lktCABD operon: lkt A encodes the structural protoxin, and lktC encodes a trans -acylase that adds fatty acid chains to internal lysine residues in the protoxin, which is then secreted from the cell by a type 1 secretion system apparatus encoded by lktB and lktD . It has been reported that LktC-mediated acylation is necessary for the biological effects of the toxin. However, an LktC mutant that we developed previously was only partially attenuated in its virulence for cattle. The objective of this study was to elucidate the role of LktC-mediated acylation in Lkt-induced cytotoxicity. We performed this study in bighorn sheep ( Ovis canadensis ) (BHS), since they are highly susceptible to M. haemolytica infection. The LktC mutant caused fatal pneumonia in 40% of inoculated BHS. On necropsy, a large number of necrotic polymorphonuclear leukocytes (PMNs) were observed in the lungs. Lkt from the mutant was cytotoxic to BHS PMNs in an in vitro cytotoxicity assay. Flow cytometric analysis of mutant Lkt-treated PMNs revealed the induction of necrosis. Scanning electron microscopic analysis revealed the presence of pores and blebs on mutant-Lkt-treated PMNs. Mass spectrometric analysis confirmed that the mutant secreted an unacylated Lkt. Taken together, these results suggest that acylation is not necessary for the cytotoxic activity of M. haemolytica Lkt but that it enhances the potency of the toxin.

The human microbiome includes bacteria, viruses, and small eukaryotes, such as fungi, and this chapter focuses on the bacterial members of the microbiome. The Human Microbiome Project (HMP) aims at developing tools and resources for characterization of the human microbiota and to relate this microbiota to human health and disease. The goals of the jumpstart phase have been to sequence 900 reference genomes to provide a catalog of genomes for metagenomic studies, to sample at least 300 healthy adults between 18 and 40 years of age at five body sites, and to develop sequencing and analysis protocols for the samples derived from human subjects. The second phase of the HMP includes human microbiome studies that target particular disease states. In a recent study, four phyla comprised 92.3% of bacterial DNA sequences analyzed from multiple human sources, including hair, oral cavity, skin, genitourinary, and gastrointestinal tract. A study by Pei et al. showed that the distal esophageal microbiomes of four adults had compositions similar to that of the oropharynx, with the exception that no spirochetes were found in the esophagus. The chapter concludes by highlighting that pathogen discovery efforts will be enhanced by new metagenomics strategies, and these studies may uncover single etiologic agents of infections as well as relative shifts in groups of bacterial pathogens that may contribute to human disease.

Tolypothrix sp. PCC 7601 is a freshwater filamentous cyanobacterium with complex responses to environmental conditions. Here, we present its 9.96-Mbp draft genome sequence, containing 10,065 putative protein-coding sequences, including 305 predicted two-component system proteins and 27 putative phytochrome-class photoreceptors, the most such proteins in any sequenced genome.

The γ-proteobacterium Francisella tularensis is one of the most infectious human pathogens, and the highly virulent organism F. tularensis subsp. tularensis (type A) and less virulent organism F. tularensis subsp. holarctica (type B) are most commonly associated with significant disease in humans and animals. Here we report the complete genome sequence and annotation for a low-passage type B strain (OSU18) isolated from a dead beaver found near Red Rock, Okla., in 1978. A comparison of the F. tularensis subsp. holarctica sequence with that of F. tularensis subsp. tularensis strain Schu4 (P. Larsson et al., Nat. Genet. 37: 153-159, 2005) highlighted genetic differences that may underlie different pathogenicity phenotypes and the evolutionary relationship between type A and type B strains. Despite extensive DNA sequence identity, the most significant difference between type A and type B isolates is the striking amount of genomic rearrangement that exists between the strains. All but two rearrangements can be attributed to homologous recombination occurring between two prominent insertion elements, IS Ftu1 and IS Ftu2 . Numerous pseudogenes have been found in the genomes and are likely contributors to the difference in virulence between the strains. In contrast, no rearrangements have been observed between the OSU18 genome and the genome of the type B live vaccine strain (LVS), and only 448 polymorphisms have been found within non-transposase-coding sequences whose homologs are intact in OSU18. Nonconservative differences between the two strains likely include the LVS attenuating mutation(s).

Using a bacteriophage lambda complementation system in Escherichia coli, we cloned genes encoding subunits of the heterodimeric DNA binding/bending protein, integration host factor, from the bovine pathogen, Pasteurella haemolytica. Complementation of ihfA and ihfB mutations in E. coli demonstrated that the P. haemolytica gene products form functional heterologous heterodimers. The ihfA and ihfB genes encode polypeptides predicted to be 99 and 93 amino acids long, respectively, and are very similar to integration host factor subunits from other Gram-negative bacteria, although phylogenetic analysis indicated that the P. haemolytica sequences are distantly related to those from other bacteria. Most significant amino acid differences were restricted to the amino-terminal domains of the predicted peptides.

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality, but short sequences. These platforms have allowed researchers to significantly improve the design of their experiments. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The synthetic mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 2.16% to 0.32%. Unfortunately, this error rate was still 16-times higher than the error rate that has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the longer reads frequently provided better classification, the wider adoption of this approach for 16S rRNA gene sequencing is likely limited by its high sequencing error and low yield of sequencing data relative to the other available platforms.

The vertebrate gut symbiont Lactobacillus reuteri has diversified into separate clades reflecting host origin. Strains show evidence of host adaptation, but how host–microbe coevolution influences microbial-derived effects on hosts is poorly understood. Emphasizing human-derived strains of L. reuteri, we combined comparative genomic analyses with functional assays to examine variations in host interaction among genetically distinct ecotypes. Within clade II or VI, the genomes of human-derived L. reuteri strains are highly conserved in gene content and at the nucleotide level. Nevertheless, they share only 70–90% of total gene content, indicating differences in functional capacity. Human-associated lineages are distinguished by genes related to bacteriophages, vitamin biosynthesis, antimicrobial production, and immunomodulation. Differential production of reuterin, histamine, and folate by 23 clade II and VI strains was demonstrated. These strains also differed with respect to their ability to modulate human cytokine production (tumor necrosis factor, monocyte chemoattractant protein-1, interleukin [IL]-1β, IL-5, IL-7, IL-12, and IL-13) by myeloid cells. Microarray analysis of representative clade II and clade VI strains revealed global regulation of genes within the reuterin, vitamin B12, folate, and arginine catabolism gene clusters by the AraC family transcriptional regulator, PocR. Thus, human-derived L. reuteri clade II and VI strains are genetically distinct and their differences affect their functional repertoires and probiotic features. These findings highlight the biological impact of microbe:host coevolution and illustrate the functional significance of subspecies differences in the human microbiome. Consideration of host origin and functional differences at the subspecies level may have major impacts on probiotic strain selection and considerations of microbial ecology in mammalian species.

The genus Streptococcus comprises important pathogens that have a severe impact on human health and are responsible for substantial economic losses to agriculture. Here, we utilize 46 Streptococcus genome sequences (44 species), including eight species sequenced here, to provide the first genomic level insight into the evolutionary history and genetic basis underlying the functional diversity of all major groups of this genus. Gene gain/loss analysis revealed a dynamic pattern of genome evolution characterized by an initial period of gene gain followed by a period of loss, as the major groups within the genus diversified. This was followed by a period of genome expansion associated with the origins of the present extant species. The pattern is concordant with an emerging view that genomes evolve through a dynamic process of expansion and streamlining. A large proportion of the pan-genome has experienced lateral gene transfer (LGT) with causative factors, such as relatedness and shared environment, operating over different evolutionary scales. Multiple gene ontology terms were significantly enriched for each group, and mapping terms onto the phylogeny showed that those corresponding to genes born on branches leading to the major groups represented approximately one-fifth of those enriched. Furthermore, despite the extensive LGT, several biochemical characteristics have been retained since group formation, suggesting genomic cohesiveness through time, and that these characteristics may be fundamental to each group. For example, proteolysis: mitis group; urea metabolism: salivarius group; carbohydrate metabolism: pyogenic group; and transcription regulation: bovis group.

Objective—To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT). Sample Population—Partially purified LKT from a wild type P haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant P haemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves. Procedure—Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation. Results—Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes. Conclusions and Clinical Relevance—Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host. (Am J Vet Res 2000;61:51–56)

It has been reported that Pasteurella haemolytica leukotoxin (LKT) induces morphologic changes in bovine leukocytes consistent with apoptosis in vitro, but DNA fragmentation was not observed. We investigated whether bovine lymphocytes undergo DNA fragmentation during LKT-induced apoptosis. Bovine peripheral blood lymphocytes were isolated by density gradient centrifugation and exposed to LKT or inactive pro-LKT protein from a lktC- mutant strain. After exposure, DNA fragmentation in lymphocytes was quantified colorimetrically by diphenylamine assay and visualized by agarose gel electrophoresis. At high LKT concentrations, bovine lymphocytes were lysed, but at low concentrations, LKT caused DNA fragmentation characteristic of apoptosis. Maximal DNA fragmentation in bovine lymphocytes was induced by 0.1 TU ml(-1) LKT following 3 h exposure, but only background level of DNA fragmentation was observed with the inactive pro-LKT. Equine lymphocytes that are resistant to LKT intoxication did not show DNA fragmentation following exposure to LKT. Preincubation of LKT with a neutralizing anti-LKT monoclonal antibody inhibited LKT-induced DNA fragmentation. Electrophoresis of DNA from bovine lymphocytes treated with 0.1 TU ml(-1) LKT demonstrated the typical 'ladder' pattern of internucleosomal DNA cleavage, the hallmark of apoptosis associated with activation of endonucleases. LKT-induced DNA fragmentation was inhibited by 0.5 mM ZnCl2, an endonuclease inhibitor. The results indicated that LKT at low concentrations induced apoptotic cell death of bovine lymphocytes, which may play a role in initiation and persistence of P. haemolytica infection.

Iron is essential for bacterial survival, being required for numerous biological processes. NEAr-iron Transporter (NEAT) domains have been studied in pathogenic Gram-positive bacteria to understand how their proteins obtain heme as an iron source during infection. While a 2002 study initially discovered and annotated the NEAT domain encoded by the genomes of several Gram-positive bacteria, there remains a scarcity of information regarding the conservation and distribution of NEAT domains throughout the bacterial kingdom, and whether these domains are restricted to pathogenic bacteria. This study aims to expand upon initial bioinformatics analysis of predicted NEAT domains, by exploring their evolution and conserved function. This information was used to identify new candidate domains in both pathogenic and nonpathogenic organisms. We also searched metagenomic datasets, specifically sequence from the Human Microbiome Project. Here, we report a comprehensive phylogenetic analysis of 343 NEAT domains, encoded by Gram-positive bacteria, mostly within the phylum Firmicutes, with the exception of Eggerthella sp. (Actinobacteria) and an unclassified Mollicutes bacterium (Tenericutes). No new NEAT sequences were identified in the HMP dataset. We detected specific groups of NEAT domains based on phylogeny of protein sequences, including a cluster of novel clostridial NEAT domains. We also identified environmental and soil organisms that encode putative NEAT proteins. Biochemical analysis of heme binding by a NEAT domain from a protein encoded by the soil-dwelling organism Paenibacillus polymyxa demonstrated that the domain is homologous in function to NEAT domains encoded by pathogenic bacteria. Together, this study provides the first global bioinformatics analysis and phylogenetic evidence that NEAT domains have a strong conservation of function, despite group-specific differences at the amino acid level. These findings will provide information useful for future projects concerning the structure and function of NEAT domains, particularly in pathogens where they have yet to be studied.

A Pasteurella haemolytica cosmid clone that activates leukotoxin transcription in Escherichia coli has been isolated. The activator locus, alxA, is part of a continuous open reading frame that includes the type I hsdM methylase gene. AlxA and HsdM peptides are processed from a precursor, and translation of the polyprotein can be modulated by slipped-strand mispairing across a pentanucleotide repeat, ACAGC, within the 5' end of alxA-hsdM. Extracts containing AlxA can bind to a leukotoxin promoter fragment.