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Background: Imbalance in tryptophan (TRP) metabolism and its neuroactive metabolites, serotonin and kynurenine (KYN), is a known pathogenic mechanism underlying neurocognitive impairment. Gut microbiota plays an important role in TRP metabolism, and the production of these neuroactive molecules affects neurocognitive function. Although both HIV infection and normal aging independently induce gut dysbiosis and influence TRP metabolism, their interactive effects on compositional/functional changes in gut microbiota and consequent alterations in TRP metabolites remain largely undetermined. Methods: Older people living with HIV infection (PLWH, aged 50–70 years, n = 22) were enrolled in this cross-sectional pilot study. Metagenomic analysis of fecal microbiome using 16S Ribosomal ribonucleic acid gene sequencing and metabolomics analysis of plasma using mass spectrometry with a reverse-phase iquid chromatography tandem mass spectrometry were performed. Statistical analyses included the univariate linear regression and Spearman correlation analyses. Results: Age-associated changes in plasma levels of key neuroactive TRP metabolites, serotonin and KYN, were seen in PLWH. Specifically, we observed age-dependent decreases in serotonin and increases in KYN and KYN-to-TRP ratio, indicative of dysfunctional TRP metabolism. Furthermore, the gut dysbiosis seen in older PLWH is characterized by a reduction of Firmicutes/Bacteroidetes ratio and butyrate-producing microbial families Lachnospiraceae and Lactobacillaceae. Of importance, correspondent with gut dysbiosis, increasing age was significantly associated with decreased plasma butyrate levels, which in turn correlated positively with serotonin and negatively with KYN/TRP ratio. Conclusions: Age-dependent gut microbial dysbiosis distinguished by a decrease in butyrogenic potential is a key pathogenic feature associated with the shift in TRP metabolism from serotonin to KYN in older PLWH.

Purpose Patients with non-infectious complications have worse clinical outcomes in common variable immunodeficiency (CVID) than those with infections-only. Non-infectious complications are associated with gut microbiome aberrations, but there are no reductionist animal models that emulate CVID. Our aim in this study was to uncover potential microbiome roles in the development of non-infectious complications in CVID. Methods We examined fecal whole genome shotgun sequencing from patients CVID, and non-infectious complications, infections-only, and their household controls. We also performed Fecal Microbiota transplant from CVID patients to Germ-Free Mice. Results We found potentially pathogenic microbes Streptococcus parasanguinis and Erysipelatoclostridium ramosum were enriched in gut microbiomes of CVID patients with non-infectious complications. In contrast, Fusicatenibacter saccharivorans and Anaerostipes hadrus, known to suppress inflammation and promote healthy metabolism, were enriched in gut microbiomes of infections-only CVID patients. Fecal microbiota transplant from non-infectious complications, infections-only, and their household controls into germ-free mice revealed gut dysbiosis patterns in recipients from CVID patients with non-infectious complications, but not infections-only CVID, or household controls recipients. Conclusion Our findings provide a proof of concept that fecal microbiota transplant from CVID patients with non-infectious complications to Germ-Free mice recapitulates microbiome alterations observed in the donors.

Introduction: Vascular cognitive impairment (VCI) is the second most common cause of clinical dementia after Alzheimer’s disease. VCI results from injury to the cerebral blood vessels. Cerebral perfusion is diminished in elderly individuals and additional reduction of cerebral blood flow increases the risk of developing VCI. These findings have been successfully modeled in mice with bilateral common carotid artery stenosis (BCAS). Age is associated with gut dysbiosis and transplantation of aged microbiome leads to cognitive decline in young animals. However, there are large gaps in our understanding of the molecular mechanisms induced by chronic hypoperfusion contributes to impaired cognitive function. Methods: C57Bl6 aged (~18m) male mice were subjected to a sham or a BCAS surgery using 0.18mm titanium coils placed on both common carotid arteries. Mice were followed for 90d after surgery to assess both gut microbial content and behavioral changes. 16S ribosomal RNA (rRNA) sequencing analysis was performed on fecal samples collected from aged baseline, sham and BCAS animals at 7 and 28 days. Cognition was assessed using Y-maze. Tissues were collected at the time of euthanasia for metabolomics and histological analysis. Results: BCAS resulted in significant reduction of cerebral blood flow, measured using laser speckle (p Conclusions: We found that chronic cerebral hypoperfusion is associated with significant changes in gut microbiome and cognitive impairment in aged mice. Importantly, shifts in the microbiome preceded cognitive decline. These findings suggest that targeting these detrimental changes in the gut microbiome might be a novel therapeutic strategy to delay or prevent progression of VCI.

The border city of El Paso, Texas, and its water utility, El Paso Water, initiated a SARS-CoV-2 wastewater monitoring program to assess virus trends and the appropriateness of a wastewater monitoring program for the community. Nearly weekly sample collection at four wastewater treatment facilities (WWTFs), serving distinct regions of the city, was analyzed for SARS-CoV-2 genes using the CDC 2019-Novel coronavirus Real-Time RT-PCR diagnostic panel. Virus concentrations ranged from 86.7 to 268,000 gc/L, varying across time and at each WWTF. The lag time between virus concentrations in wastewater and reported COVID-19 case rates (per 100,00 population) ranged from 4-24 days for the four WWTFs, with the strongest trend occurring from November 2021 - June 2022. This study is an assessment of the utility of a geographically refined SARS-CoV-2 wastewater monitoring program to supplement public health efforts that will manage the virus as it becomes endemic in El Paso.

Introduction Gut microbiota plays a critical role in the regulation of immune homeostasis. Accordingly, several autoimmune disorders have been associated with dysbiosis in the gut microbiota. Notably, the dysbiosis associated with central nervous system (CNS) autoimmunity involves a substantial reduction of bacteria belonging toClostridiaclusters IV and XIVa, which constitute major producers of short-chain fatty acids (SCFA). Here we addressed the role of the surface receptor-mediated effects of SCFA on mucosal T-cells in the development of CNS autoimmunity. Methods To induce CNS autoimmunity we used the mouse model of experimental autoimmune encephalomyelitis (EAE) induced by immunization with the myelin oligodendrocyte glycoprotein (MOG)-derived peptide (MOG35 − 55peptide). To address the effects of GPR43 stimulation on colonic TCRαβ+T-cells upon CNS autoimmunity, mucosal lymphocytes were isolated and stimulated with a selective GPR43 agonistex vivoand then transferred into congenic mice undergoing EAE. Several subsets of lymphocytes infiltrating the CNS or those present in the gut epithelium and gut lamina propria were analysed by flow cytometry.In vitromigration assays were conducted with mucosal T-cells using transwells. Results Our results show a sharp and selective reduction of intestinal propionate at the peak of EAE development, accompanied by increased IFN-γ and decreased IL-22 in the colonic mucosa. Further analyses indicated that GPR43 was the primary SCFA receptor expressed on T-cells, which was downregulated on colonic TCRαβ+T-cells upon CNS autoimmunity. The pharmacologic stimulation of GPR43 increased the anti-inflammatory function and reduced the pro-inflammatory features in several TCRαβ+T-cell subsets in the colonic mucosa upon EAE development. Furthermore, GPR43 stimulation induced the arrest of CNS-autoreactive T-cells in the colonic lamina propria, thus avoiding their infiltration into the CNS and dampening the disease development. Mechanistic analyses revealed that GPR43-stimulation on mucosal TCRαβ+T-cells inhibits their CXCR3-mediated migration towards CXCL11, which is released from the CNS upon neuroinflammation. Conclusions These findings provide a novel mechanism involved in the gut-brain axis by which bacterial-derived products secreted in the gut mucosa might control the CNS tropism of autoreactive T-cells. Moreover, this study shows GPR43 expressed on T-cells as a promising therapeutic target for CNS autoimmunity.

BACKGROUND: Comorbid anxiety and depression are common and are associated with greater disease burden than either alone. Our recent efforts have identified an association between gut microbiota dysfunction and severity of anxiety and depression. In this follow-up, we applied Differential Co-Expression Analysis (DiffCoEx) to identify potential gut microbiota biomarker(s) candidates of treatment resistance among psychiatric inpatients. METHODS: In a sample of convenience, 100 psychiatric inpatients provided clinical data at admission and discharge; fecal samples were collected early during the hospitalization. Whole genome shotgun sequencing methods were used to process samples. DiffCoEx was used to identify clusters of microbial features significantly different based on treatment resistance status. Once overlapping features were identified, a knowledge-mining tool was used to review the literature using a list of microbial species/pathways and a select number of medical subject headlines (MeSH) terms relevant for depression, anxiety, and brain-gut-axis dysregulation. Network analysis used overlapping features to identify microbial interactions that could impact treatment resistance. RESULTS: DiffCoEx analyzed 10,403 bacterial features: 43/44 microbial features associated with depression treatment resistance overlapped with 43/114 microbial features associated with anxiety treatment resistance. Network analysis resulted in 8 biological interactions between 16 bacterial species. Clostridium perfringens evidenced the highest connection strength (0.95). Erysipelotrichaceae bacterium 6_1_45 has been most widely examined, is associated with inflammation and dysbiosis, but has not been associated with depression or anxiety. CONCLUSION: DiffCoEx potentially identified gut bacteria biomarker candidates of depression and anxiety treatment-resistance. Future efforts in psychiatric microbiology should examine the mechanistic relationship of identified pro-inflammatory species, potentially contributing to a biomarker-based algorithm for treatment resistance.

Although micronutrients modulate immunity and inflammation, it remains elusive whether they are implicated in the development and progression of chronic pancreatitis (CP). This study aimed to investigate differences in the circulating levels of selected carotenoids and vitamins between CP and controls and trends in the levels of these micronutrients across controls, early CP, and definite CP.Demographic and lifestyle data were extracted from medical records for 53 patients with CP (13 early and 38 definite) and obtained using a questionnaire for 52 controls. Plasma β-carotene, lycopene, cryptoxanthin, zeaxanthin, and α-tocopherol and serum 25(OH)D, folate, IL-6, TNF-α, and MCP-1 were measured with state-of-the-art methods.The levels of all micronutrients (except folate) were significantly lower in CP than in controls. There was a progressive decrease in the levels of these micronutrients across controls, early CP, and definite CP (all p values for trend: ≤0.0012); e.g., plasma lycopene was 36.6, 21.5, and 14.5 μg/dL for controls, early CP, and definite CP, respectively. After adjustment for confounders, there were strong, inverse associations between the levels of all micronutrients (except folate) and CP (e.g., OR (95% CI) for ≥ median vs.median: 0.10 (0.04, 0.27) for lycopene, 0.15 (0.05, 0.38) for α-tocopherol, and 0.24 (0.09, 0.64) for 25(OH)D). These associations became weaker after additional adjustment for inflammation markers (IL-6, TNF-α, and MCP-1).The circulating levels of some carotenoids, α-tocopherol, and vitamin D were reduced in CP patients compared with controls and this reduction was more pronounced in definite CP than in early CP.

Mounting evidence supports synergistic roles for the gut microbiome in cancer progression. Yet, the interplay between the gut microbiome and immune responses in cancer is still poorly understood. We recently showed that gut microbiome changes are closely associated with development of Kras-mutant lung adenocarcinoma (KM-LUAD) in a human-relevant, tobacco-associated mouse model (Gprc5a-/-; G). Knockout of the antimicrobial protein Lcn2 in these mice (Gprc5a-/-/Lcn2-/-; GL) further reduced microbial diversity while enhancing inflammation and tumor development. We thus hypothesized that microbial dysbiosis in the gut, such as that incurred by loss of Lcn2, may exacerbate LUAD development. Here, we investigated the effects of gut microbiome modulation on LUAD pathogenesis using fecal microbiota transfer (FMT) in both syngeneic and tobacco carcinogenesis models. Syngeneic G mice (transplant of G LUAD cells) that received FMT from GL donors (G < GL) exhibited significantly increased tumor growth relative to littermates with FMT from G mice (G < G). These effects were recapitulated in an independent syngeneic model (KrasG12D LKR13 cells in wild type mice). Tobacco carcinogen-exposed G < GL mice also exhibited increased lung tumor development compared with similarly exposed G < G littermates. 16S rDNA-Seq analysis of fecal pellets revealed significant differences in gut beta diversity between syngeneic G < G and G < GL mice. G < GL mice additionally displayed elevated relative abundance of tumor-promoting Alistipes, while Ruminoccocus and Akkermansia, taxa associated with favorable response to immunotherapy, were reduced. We next performed single-cell RNA-sequencing to comprehensively probe the tumor immune microenvironment (TIME) and the immune milieu near the gut of tumors and mesenteric lymph nodes (MLNs), respectively. The TIME in G < GL mice displayed an overall enhanced immunosuppressive phenotype evidenced by prominently increased fractions of T regulatory and Cd4+ Izumo1r+ exhausted T cells and, conversely, reduced levels of activated Isg15+ Cd8a+ T cells. MLNs from G < GL mice showed markedly increased fractions of memory B cells expressing the immunosuppressor Bank1 and reduced levels of follicular B cells and Cd8a+ Clec9a+ class 1 dendritic cells (cDC1). Flow cytometry further showed enhanced immunosuppression in G < GL relative to G < G mice, including increased fractions of myeloid-derived suppressor cells in the TIME of the former group. Our findings show that gut microbiome dysbiosis fosters lung cancer development by promoting immunosuppression, perhaps via a local and systemic gut microbiota-immune system crosstalk. Modulating the gut microbiome may be a promising strategy for interception or early treatment of lung cancer. Citation Format: Zahraa Rahal, Fuduan Peng, Yuejiang Liu, Matthew C. Ross, Ansam Sinjab, Ke Liang, Jiping Feng, Chidera O. Chukwuocha, Manvi Sharma, Elizabeth Tang, Camille Abaya, Joseph Petrosino, Junya Fujimoto, Seyed Javad Moghaddam, Linghua Wang, Kristi L. Hoffman, Humam Kadara. Gut microbiome dysbiosis promotes immune suppression and lung cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2883.

Introduction: There is a critical need for complex microbiome quality control standards representing population-based samples for microbial community profiling and analysis in large scale epidemiologic studies. Methods: We developed standard quality control samples from five volunteers with different phenotypes, comprising one obese female, one healthy male, one male on a low-carb diet, one infant, and one male with Crohn’s Disease, and evaluated their microbial metagenomic profiles within three laboratories at two different timepoints. To quantify the percentage of microbiome variability explained by donors, laboratory and sequencing run, a distance-based coefficient of determination R2 was estimated using a permutational multivariate analysis of variance. In addition, we calculated the intraclass correlation coefficients (ICC) for the relative abundance of the most abundant species, two alpha diversity metrics (i.e., observed number of species and Shannon index) and the first principal coordinates of three beta diversity matrices (i.e., Bray-Curtis, Jaccard and Aitchison) to estimate the accuracy of fecal microbial profiles between the three different laboratories as well as within the laboratories. Results: The variability introduced by the phenotype of the donors explained 82.7% to 95.3% of the overall variability, which was higher than the variability introduced by the laboratories (1.8% to 3.1%) and the sequencing runs (0.6% to 1.7%) - the residual percent variance explained varied between 2.2% and 12.4%. Observations based on principal coordinates analysis showed that samples clustered by donor and not by laboratory or sequencing runs. The five donor clusters were well separated and very distinct. Based on the comparison of species relative abundances, each donor displayed very different microbial profiles; and the microbial profiles of each donor were comparable between the three different laboratories and the two sequencing runs in each laboratory. The reproducibility within and between the laboratories was good to excellent for most diversity metrics (ICCs higher than 0.97) and species relative abundances (range, ICCs=0.70-0.99); however, the reproducibility of the observed number of species was moderate (ICC=0.64 for the first laboratory, ICC=0.78 for the second laboratory, ICC=0.81 for the third laboratory, and ICC=0.42 between the laboratories). Conclusions: These standard quality control samples can be used as a reference in future population-based epidemiologic studies to pool or meta-analyze microbiome data. Citation Format: Semi Zouiouich, Smriti Karwa, Yunhu Wan, Andrew Chan, Joseph Petrosino, Emma Allen-Vercoe, Rob Knight, Jianxin Shi, Mitchell Gail, Christian Abnet, Emily Vogtmann, Rashmi Sinha. Quality control samples for future population-based microbiome studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3050.

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here, we reported a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with one of four strains of RSV representing both major subgroups as well as historic and more contemporary genotypes (RSV/A/Tracy [GA1], RSV/A/Ontario [ON], RSV/B/18537 [GB1], and RSV/B/Buenos Aires [BA]) via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response, including transcriptional changes and levels of secreted cytokines and growth factors.

Background: Behavioral factors for cardiometabolic outcomes have been shown to influence the composition and function of the gut microbiota, which has been associated with cardiometabolic outcomes. Observational studies have documented associations between physical activity and gut microbial composition using 16S rRNA sequencing data, but there is a paucity of data on functional measures, which can be generated from whole-metagenomics sequencing (WMS). Methods: Data are from 480 CARDIA participants who attended the Year 30 follow-up exam (2015-2016, ages 48-60, 47:53 Black:White race, 47:53 male:female gender). Physical activity (exercise units) was estimated from reported frequency and duration of 13 activity types of at least moderate intensity during the previous year. Species, metabolic pathways, and enzyme families were assigned using standard reference databases. Multivariate measures of beta-diversity (between-person) was assessed with principal coordinates analysis (PCoA) and tested with PERMANOVA. Multivariable-adjusted linear regression was used to test associations between distinct microbial features and physical activity, adjusting for false discovery rate (FDR). Results: Sequence data mapped to 105 species 1,307 enzyme families, and 318 metabolic pathways. Beta-diversity of species, but not for enzyme families or metabolic pathways, was significantly differentiated according to physical activity. In analysis of distinct microbial features, 7 species, 15 enzyme families, and 3 metabolic pathways were significantly associated with physical activity at an FDR-adjusted p-value of 0.10 (Table). Conclusion: These findings support associations between gut microbial features and physical activity in a middle-aged, bi-race population-based cohort. Although temporality cannot be established from these cross-sectional analyses, our results are consistent with data suggesting that physical activity may influence composition and function of the gut microbiota.

691 Background: Pancreatic cancer (PC) is the third leading cause of cancer death in the United States. The high mortality associated with PC is attributed to multiple reasons: lack of effective therapies, aggressive biology and late diagnosis. Due to the absence of reliable early disease biomarkers, PC screening is largely dependent on imaging. Recent studies have highlighted the importance of the gut and tumor microbiome in PC. We present here the first report of oral and gut microbiome prospective analysis of PC high-risk individuals (PC-HRI) undergoing screening. Methods: We collected periodontal and stool samples at the University of Texas MD Anderson Cancer Center from 2017-2022. A total of 448 samples, consisting of 250 oral and 198 gut samples were obtained. Samples were collected from PC (n=73), PC-HRI (n=34), and healthy (n=143) individuals. 16s rRNA sequencing was used to characterize the oral and gut microbiome and statistical analysis and correlation with imaging and clinical characteristics were performed. Results: We identified three phyla, namely Proteobacteria, Actinobacteria, and Fusobacteria as significantly more abundant in the gut microbiome of PC patients. Conversely, Proteobacteria was decreased in the oral microbiome of PC patients. At the class level, Gammaproteobacteria (GP) oral/gut ratio was significantly decreased in PC patients compared with healthy individuals (p=0.02). Analysis of PC-HRI revealed also low GP oral/gut ratio in high-risk individuals who were diagnosed with worrisome pancreatic focal lesions. Interestingly, Gammaproteobacteria (GP) is one of the main classes of bacteria detected in pancreatic cancer tissue. GP shifts in oral and gut environments could be implicated in pancreatic early tumorigenesis and serve as biomarker of the disease. Additionally, gut bacteria with metabolic pathways related to lipid metabolism were more enriched in PC patients and PC-HRI with focal lesions compared to healthy controls. Conclusions: Gammaproteobacteria oral/gut ratio represents a potential novel biomarker which could predict presence of early high risk-pancreatic focal lesions in PC-HRI. Taken together, this report provides observational evidence about changes in oral and gut microbiome in patients with pancreatic cancer but even more importantly, the fact that those changes could be detected in PC-HRI with early high-risk lesions. Broader validation in other high-risk cohorts would be required. Detection of GP oral/gut ratio would represent an inexpensive, non-invasive method that could be useful for PC screening. Functional studies should be performed to determine how GP shifts can contribute with pancreatic tumorigenesis. Research supported by CPRIT (Grant Number: RP200173) and philanthropic funding through the MD Anderson Moonshot Program.

To conduct an exploratory examination of dietary patterns and quality during pregnancy in African-American women who were class I, II, or III obese, and those women with normal pre-pregnancy body mass index (pBMI), as well to identify dietary factors associated with GWG, and changes in the distal gut microbiome. African American women represent the largest group affected by pre-pregnancy obesity, a risk factor for several adverse birth outcomes.This prospective study investigated the association between diet, distal gut microbiome, and GWG among African-American women (n = 21) with obesity (n = 15) compared to women with a normal pre-pregnancy body mass index (pBMI) (n = 6) at two time points, 27-29 and 37-39 weeks gestation. Dietary patterns associated with obesity severity and GWG gain were assessed using Welch's T-test and Mann-Whitney U. The association between the gut microbiome and dietary patterns was assessed using a regression-based kernel association test and the adaptive microbiome-based sum of powered score test.In early pregnancy, dietary intake of Total Fruits and Greens and Beans was significantly different between pBMI and GWG groups; significance was 0.022 and 0.028 respectively. Women with Class II/III obesity and those with GWG above guidelines had Healthy Eating Index (HEI) scores below 50, meeting less than 75% of dietary guidelines, and did not meet recommendations for fruit and vegetable or fiber intake. We found no significant associations between the microbiome composition and diet (HEI Scores).Overall, the results indicate that women with pBMI obesity are not meeting minimum dietary guidelines for nutrient intakes during pregnancy, specifically fruits, vegetables, and fiber, regardless of GWG. Interventions for African-American women with pre-pregnancy obesity, with a focus on increasing consumption of fruits and vegetables, would be beneficial to control GWG and improve birth outcomes.

Microbial dysbiosis has emerged as a modulator of oncogenesis and response to therapy, particularly in lung cancer. Here, we investigate the evolution of the gut and lung microbiomes following exposure to a tobacco carcinogen. We performed 16S rRNA-Seq of fecal and lung samples collected prior to and at several timepoints following (nicotine-specific nitrosamine ketone/NNK) exposure in Gprc5a−/− mice that were previously shown to exhibit accelerated lung adenocarcinoma (LUAD) development following NNK exposure. We found significant progressive changes in human-relevant gut and lung microbiome members (e.g., Odoribacter, Alistipes, Akkermansia, and Ruminococus) that are closely associated with the phenotypic development of LUAD and immunotherapeutic response in human lung cancer patients. These changes were associated with decreased short-chain fatty acids (propionic acid and butyric acid) following exposure to NNK. We next sought to study the impact of Lcn2 expression, a bacterial growth inhibitor, given our previous findings on its protective role in LUAD development. Indeed, we found that the loss of Lcn2 was associated with widespread gut and lung microbiome changes at all timepoints, distinct from those observed in our Gprc5a−/− mouse model, including a decrease in abundance and diversity. Our overall findings apprise novel cues implicating microbial phenotypes in the development of tobacco-associated LUAD.

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but a high rate of immune-related adverse events (irAE). Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 advanced melanoma patients treated with CICB, with a high rate of any ≥Grade 3 irAEs (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1b in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Microbiota, both within the gut and the tumors, has emerged as a significant player influencing tumor growth and responses to therapies. Recent evidence links microbiota and pancreatic ductal adenocarcinoma (PDAC), an aggressive cancer surrounded by a highly immuno-suppressive tumor microenvironment which limits efficacy of most available therapies. The immunosuppressive tumor microenvironment of PDAC is partially facilitated by a proinflammatory cytokine Interleukin 17 (IL-17). IL-17 can be stimulated by intestinal commensal bacteria under normal physiological conditions. It is critical for microbial defense as well as immunopathology. Both IL-17 neutralization and antibiotics reduce murine PDAC growth. While the vital role of microbiota in affecting tumor immunity has been identified, there is still a gap of knowledge as to how microbes may regulate pro-tumorigenic IL-17 signaling. We wanted to investigate the systemic and local role of IL-17 in regulating the microbial-tumor immune axis. For this purpose, we genetically deleted the IL-17 receptor A (IL-17RA) in different compartments and evaluated tumor growth. We found that IL-17RA signaling was important for maintaining microbial homeostasis and its disruption resulted in differential tumor growth. Absence of IL-17RA signaling in the gut lead to local inflammation as well as systemic immune effects. Our data suggests that modulation of IL-17 signaling could serve as a therapeutic intervention to alter microbial mediated tumor effects. The significance of these findings may extend to other cancers as well. Citation Format: Vidhi Chandra, Yu Zhang, Olivereen Le Roux, Joseph Petrosino, Jay Kolls, Florencia McAllister. Microbial-Interleukin 17 receptor A (IL-17RA) signaling axis modulates tumor growth and microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3530.

Composition of the intestinal microbiome has been shown to impact generation of a tumor-specific immune response following chemotherapy and immune checkpoint blockade. We hypothesized that the local and intestinal microbiota may also impact immune activation and radiation response. We prospectively assessed the microbiota and T-cell repertoire of 30 cervical cancer patients throughout CRT. Greater diversity of the intestinal microbiome was associated with exceptional response and development of clonal T-cell expansion. Predominance of immunosuppressive Lactobacillus species in the cervix was associated with failure to expand clonal T-cells and decreased relapse-free survival. Patients with a robust response to CRT developed clonal expansion of T-cells, suggesting that highly radiation responsive cervical cancers generate an antigen specific immune response. Depletion of gut microbiome diversity and presence of vaginal Lactobacillus were associated with decreased presence of activated CD8 T-cell infiltration and decreased radiation response in an orthotopic model of HPV cancer, suggesting that microbial diversity is required for generation of effective antitumor immunity in radiation responsive HPV cancers.

Recent advances in sequencing technology have made it feasible to characterize human gut microbial communities (or microbiota) and examine their associations with disease outcomes, including cardiovascular disease (CVD) related traits. However, there is a paucity of research examining how the human gut microbiome may influence overall CVD risk in population based studies. The objective of the current study was to identify composite measures of microbiota diversity and specific microbial genera that associate with CVD risk among participants of the Bogalusa Heart Study (BHS). The current BHS cohort includes 1,257 participants who have been screened at least two times in childhood and two times in adulthood for CVD risk factors. For the microbiome study, we selected the 55 participants at highest CVD risk and 57 participants at lowest CVD risk using a z-score based data reduction technique, leveraging longitudinal mean measures of systolic BP, low-density lipoprotein cholesterol, and fasting plasma glucose levels. Next generation 16S rRNA sequencing of the V4 hypervariable region was conducted using genomic bacterial DNA extracted from stool samples of the 112 participants. The Wilcoxon rank sum test was used to examine differences in measures of alpha diversity, including observed operational taxonomic units (OTUs) and the Chao1 index, along with specific microbial taxa between CVD risk groups. Permutation multivariate analysis of variance (PERMANOVA) was used to test compositional differences based on unweighted Unifrac distance as a measure of beta diversity. We observed significantly less alpha diversity in those at high versus those at low CVD risk (P=0.04 and 0.02 for observed OTU and Chao1 index measures, respectively). The PERMANOVA test indicated marginal significance of compositional differences between the two groups (P=0.07). In addition, 10 microbial genera significantly differed between high and low CVD risk participants, including Faecalibacterium (P=0.0014), Subdoligranulum (P=0.01), Turicibacter (P=0.01), Rothia (P=0.01), Lachnospira (P=0.01), Haemophilus (P=0.03), Dialister (P=0.03), Bifidobacterium (P=0.03), Oscillibacter (P=0.04), and Megamonas (P=0.05). In summary, we provide empirical evidence of lower microbial enrichment among BHS participants at high versus those at low CVD risk, along with promising findings of differences in specific microbial genera between CVD risk groups.

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.

The collections of eukaryotic and prokaryotic viral genomes that are found in or on humans are referred to as the human virome. There are an estimated 1031 viral particles on Earth. Human feces, one of the richest human samples, contain at least 109 virus-like particles per gram. Viruses have an evident effect on human health either by shaping the structure and function of bacterial communities (prokaryotic viruses) or by directly infecting human cells resulting in acute, persistent, or latent infections. The use of next-generation sequencing methods to study viromes of all types has propelled research across the globe contributing to the discovery and our understanding of viruses, their impact in human health, and applications in biotechnology. Improved extraction methods and high-throughput platforms have emerged accompanied by a continuous decrease in sequencing costs. The immediate future of viral metagenomics includes rapid clinical diagnosis, modulation of clinical therapies, and genome mining with industrial applications.

The Human Microbiome Project used rigorous good clinical practice standards to complete comprehensive body site sampling in healthy 18- to 40-yr-old adults, creating an unparalleled reference set of microbiome specimens. To ensure that specimens represented minimally perturbed microbiomes, we first screened potential participants using exclusion criteria based on health history, including the presence of systemic diseases (e.g., hypertension, cancer, or immunodeficiency or autoimmune disorders), use of potential immunomodulators, and recent use of antibiotics or probiotics. Subsequent physical examinations excluded individuals based on body mass index (BMI), cutaneous lesions, and oral health. We screened 554 individuals to enroll 300 (149 men and 151 women, mean age 26 yr, mean BMI 24 kg/m2, 20.0% racial minority, and 10.7% Hispanic). We obtained specimens from the oral cavity, nares, skin, gastrointestinal tract, and vagina (15 specimens from men and 18 from women). The study evaluated longitudinal changes in an individual's microbiome by sampling 279 participants twice (mean 212 d after the first sampling; range 30-359 d) and 100 individuals 3 times (mean 72 d after the second sampling; range 30-224 d). This sampling strategy yielded 11,174 primary specimens, from which 12,479 DNA samples were submitted to 4 centers for metagenomic sequencing. Our clinical design and well-defined reference cohort has laid a foundation for microbiome research.—Aagaard, K., Petrosino, J., Keitel, W., Watson, M., Katancik, J., Garcia, N., Patel, S., Cutting, M., Madden, T., Hamilton, H., Harris, E., Gevers, D., Simone, G., McInnes, P., Versalovic, J. The Human Microbiome Project strategy for comprehensive sampling of the human microbiome and why it matters.

This project explores the nature of the human intestinal microbiome in healthy children and children with recurrent abdominal pain. The overall goal is to obtain a robust knowledge base of the intestinal microbiome in children without evidence of pain or gastrointestinal disease and in those with recurrent abdominal pain (functional abdominal pain (FAP) and FAP associated with changes in bowel habits, i.e., irritable bowel syndrome or IBS). Specific aims include: 1. Characterize the composition of the gut microbiome in healthy children by DNA sequencing. 2. Determine the presence of disease-specific organism signatures of variable gut microbiomes in children with recurrent abdominal pain. 3. Perform functional gut metagenomics by evaluation of whole community gene expression profiles and discovery of disease-specific pathway signatures. Multiple strategies have been deployed to navigate and understand the nature of the intestinal microbiome in childhood. These strategies included 454 pyrosequencing-based strategies to sequence 16S rRNA genes and understand the detailed composition of microbes in healthy and disease groups. Microarray-based hybridization with the PhyloChip and quantitative real-time PCR (qPCR) probes were applied as complementary strategies to gain an understanding of the intestinal microbiome from various perspectives. Data collected and analyzed during the HMP UH2 Demo project, from a set of healthy and IBS children (7-12 yo) may enable the identification of core microbiomes in children, in addition to variable components that may distinguish healthy from diseased pediatric states. Twenty-two children with IBS and twenty-two healthy children were enrolled and analyzed in the UH2 phase of this study. The planned enrollment targets for the UH2/3 phases include 50 healthy children, 50 children with FAP and 50 children with IBS (minimum of 3 time points per child). We are currently analyzing the dataset for the presence of disease-specific signatures in the human microbiome, and correlating these microbial signatures with pediatric health or IBS disease status in addition to IBS subtype (e.g., diarrhea-vs constipation-predominant). In the next phase, whole genome shotgun sequencing and metatranscriptomics will be performed with a subset of children in each group. This study explores the nature of core and variable human microbiome in pre-adolescent healthy children and children with IBS.

This project explores the nature of the human intestinal microbiome in healthy children and children with recurrent abdominal pain. The overall goal is to obtain a robust knowledge base of the intestinal microbiome in children without evidence of pain or gastrointestinal disease and in those with recurrent abdominal pain (functional abdominal pain (FAP) and FAP associated with changes in bowel habits, i.e., irritable bowel syndrome or IBS). Specific aims include: 1. Characterize the composition of the gut microbiome in healthy children by DNA sequencing. 2. Determine the presence of disease-specific organism signatures of variable gut microbiomes in children with recurrent abdominal pain. 3. Perform functional gut metagenomics by evaluation of whole community gene expression profiles and discovery of disease-specific pathway signatures. Multiple strategies have been deployed to navigate and understand the nature of the intestinal microbiome in childhood. These strategies included 454 pyrosequencing-based strategies to sequence 16S rRNA genes and understand the detailed composition of microbes in healthy and disease groups. Microarray-based hybridization with the PhyloChip and quantitative real-time PCR (qPCR) probes were applied as complementary strategies to gain an understanding of the intestinal microbiome from various perspectives. Data collected and analyzed during the HMP UH2 Demo project, from a set of healthy and IBS children (7-12 yo) may enable the identification of core microbiomes in children, in addition to variable components that may distinguish healthy from diseased pediatric states. Twenty-two children with IBS and twenty-two healthy children were enrolled and analyzed in the UH2 phase of this study. The planned enrollment targets for the UH2/3 phases include 50 healthy children, 50 children with FAP and 50 children with IBS (minimum of 3 time points per child). We are currently analyzing the dataset for the presence of disease-specific signatures in the human microbiome, and correlating these microbial signatures with pediatric health or IBS disease status in addition to IBS subtype (e.g., diarrhea-vs constipation-predominant). In the next phase, whole genome shotgun sequencing and metatranscriptomics will be performed with a subset of children in each group. This study explores the nature of core and variable human microbiome in pre-adolescent healthy children and children with IBS.

We present the Genomic Standards Consortium’s (GSC) “Minimum Information about an ENvironmental Sequence” (MIENS) standard for describing marker genes. Adoption of MIENS will enhance our ability to analyze natural genetic diversity across the Tree of Life as it is currently being documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

TEM-1 beta-lactamase catalyzes the hydrolysis of beta-lactam antibiotics such as the penicillins and cephalosporins, thus providing for bacterial resistance to these compounds. To determine the amino acid residues critical for the structure and function of TEM-1 beta-lactamase, the codons for each of the 263 amino acid residues that constitute the mature form of the enzyme were randomized using a site-directed mutagenesis procedure. Functional random mutants were selected based on their ability to confer ampicillin resistance to Escherichia coli. The DNA sequence of several functional mutants was determined for each set of random mutants. It was found that 43 out of the 263 amino acid residues do not tolerate substitutions and therefore are critical for the structure and activity of the enzyme. In addition, a comparison of conserved residue positions among functional beta-lactamase mutants with conserved residues in the beta-lactamase gene family identified many positions which did not tolerate substitutions in the mutagenesis studies but are freely substituted among members of the gene family. This observation may be due to the accumulation of compensating mutations among members of the gene family. Finally, the sequence variability at residue positions among functional mutants was quantitated by calculating the effective number of substitutions at each position using information-theoretical entropy. These values were used to obtain a quantitative estimate of the correlation between the sequence variability at a position and the fractional accessible surface area of the residue. The correlation is found to be statistically significant in that buried residues tend to exhibit low variability and invariant residues tend to exhibit low solvent exposure. However, the correlation is weak because most residues are neither completely buried nor invariant.

This report details the outcome of the 1st Skin Microbiota Workshop, Boulder, CO, held on October 15th-16th 2012. The workshop was arranged to bring Department of Defense personnel together with experts in microbial ecology, human skin physiology and anatomy, and computational techniques for interrogating the microbiome to define research frontiers at the intersection of these important areas. The workshop outlined a series of questions and created several working groups to address those questions, specifically to promote interdisciplinary activity and potential future collaboration. The US Army provided generous grant support and the meeting was organized and hosted by the University of Colorado at Boulder. A primary forward vision of the meeting was the importance of understanding skin microbial communities to improve the health and stealth of US Army warfighters., U.S. Army Research Laboratory, United States. Army Research Office (grant number W911NF-12-1-0513)