Increased Alpha Diversity Research Articles

Transplantation of an obesity-associated human gut microbiota to mice induces vascular dysfunction and glucose intolerance

ABSTRACT Recent preclinical data suggest that alterations in the gut microbiota may be an important factor linking obesity to vascular dysfunction, an early sign of cardiovascular disease. The purpose of this study was to begin translation of these preclinical data by examining whether vascular phenotypes in humans are transmissible through the gut microbiota. We hypothesized that germ-free mice colonized with gut microbiota from obese individuals would display diminished vascular function compared to germ-free mice receiving microbiota from lean individuals. We transplanted fecal material from obese and lean age-and sex-matched participants with disparate vascular function to germ-free mice. Using Principle Component Analysis, the microbiota of colonized mice separated by donor group along the first principle component, accounting for between 70–93% of the total variability in the dataset. The microbiota of mice receiving transplants from lean individuals was also characterized by increased alpha diversity, as well as increased relative abundance of potentially beneficial bacteria, including Bifidobacterium, Lactobacillus, and Bacteroides ovatis. Endothelium-dependent dilation, aortic pulse wave velocity and glucose tolerance were significantly altered in mice receiving microbiota from the obese donor relative to those receiving microbiota from the lean donor or those remaining germ-free. These data indicate that the obesity-associated human gut microbiota is sufficient to alter the vascular phenotype in germ-free mice in the absence of differences in body weight or dietary manipulation, and provide justification for future clinical trials to test the efficacy of microbiota-targeted therapies in the prevention or treatment of cardiovascular disease.

Reduced alpha diversity of the oral microbiome correlates with short progression-free survival in patients with relapsed/refractory multiple myeloma treated with ixazomib-based therapy (AGMT MM 1, phase II trial).

Alterations in the human microbiome have been linked to several malignant diseases. Here, we investigated the oral microbiome of 79 patients with relapsed/refractory multiple myeloma (MM) treated with ixazomib‐thalidomide‐dexamethasone. Increased alpha diversity (Shannon index) at the phylum level was associated with longer progression‐free survival (PFS) (10.2 vs 8.5 months, P = .04), particularly in patients with very long (>75% quartile) PFS . Additionally, alpha diversity was lower in patients with progressive disease (P < .05). These findings suggest an interrelationship between the oral microbiome and outcome in patients with MM and encourage a novel direction for diagnostic and/or therapeutic strategies.

Dynamic of Composition and Diversity of Gut Microbiota in Triatoma rubrofasciata in Different Developmental Stages and Environmental Conditions.

Triatoma rubrofasciata (T. rubrofasciata), one kind of triatomine insects, is the vector of Trypanosoma cruzi (T. cruzi), which lead to American trypanosomiasis. Although the gut microbiome may play an essential role in the development and susceptibility of triatomine, there is limited research on the gut microbiota of T. rubrofasciata. To elucidate the effect of the vector’s developmental stages and environmental conditions on the gut microbiome, we employed 16S rRNA gene sequencing to profile the gut bacterial community diversity and composition of T. rubrofasciata. Significant shifts were observed in the overall gut microbe diversity and composition across the development of T. rubrofasciata and specific bacteria were detected in different stages. Serratia and Burkholderia-Caballeronia-Paraburkholderia were dominant in the 1st nymphal stage, while the abundance of Staphylococcus was low in the 1st nymphal stage. Oceanicaulis were undetectable in the adult stage and Odoribacter peaked in the 2nd nymphal stage. Moreover, Staphylococcus was correlated negatively with Serratia. Likewise, the total gut microbiota diversity and composition of T. rubrofasciata differentiated significantly by environmental conditions. The ingestion of a bloodmeal increased alpha diversity of gut bacterial communities, and Staphylococcus was more abundant in laboratory-reared bugs whereas Enterococcus enriched in wild-caught bugs. Furthermore, Pantoea was negatively correlated with Staphylococcus, and positively related to Bacillus only. The phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) algorithm showed obvious metagenomic functional differences by environmental conditions, and Chagas disease relevant pathway was enriched in wild-caught T. rubrofasciata.

Fifteen-year no tillage of a Mollisol with residue retention indirectly affects topsoil bacterial community by altering soil properties

Soil microbes play an important role in nutrient cycling in agricultural soils and can be influenced by tillage. Conservation tillage aims to reduce energy inputs and conserve soil and water by decreasing disturbance of soil and returning a portion of crop residue. The response of the soil microbial community to conservation tillage is complex and quantitative analysis is largely absent regarding how tillage and depth, combined with soil properties, affect soil microbial diversity and community composition. Here, the diversity and composition of the soil bacterial community and its relationship with soil properties were explored by high-throughput sequencing technology and Structural Equation Modeling (SEM) at two soil depths (0–5 cm and 15–20 cm) under conventional tillage and no tillage with residue retention. No tillage significantly increased alpha diversity (Chao 1 and Shannon) at 0-5 cm and altered the composition of the bacterial community, coinciding with changes in physical-chemical properties. Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, and Gemmatimonadetes were the most abundant phyla across all samples. Alpha diversity was significantly correlated with soil bulk density (BD) and pH. The SEM showed that tillage and depth explained 86% of the bacterial diversity and 84% of the composition. In addition, tillage and depth had indirect effects on bacterial diversity and composition by affecting soil BD, pH and soil organic carbon. Results indicate that the soil bacterial community is altered by conservation tillage, especially in the topsoil, and highlight the importance of soil physical-chemical properties in shaping the diversity and composition of the soil bacterial community. Our findings contribute to a broad understanding of tillage disturbance and differentiated effects in the soil profile for the bacterial community.

Delayed Establishment of Gut Microbiota in Infants Delivered by Cesarean Section.

The maternal vaginal microbiome is an important source for infant gut microbiome development. However, infants delivered by Cesarean section (CS) do not contact the maternal vaginal microbiome and this delivery method may perturb the early establishment and development of the gut microbiome. The aim of this study was to investigate the early gut microbiota of Korean newborns receiving the same postpartum care services for two weeks after birth by delivery mode using fecal samples collected at days 3, 7, and 14. Early gut microbiota development patterns were examined using 16S rRNA gene-based sequencing from 132 infants either born vaginally (VD, n = 64) or via Cesarean section (CS, n = 68). VD-born neonates showed increased alpha diversity in infant fecal samples collated at days 7 and 14 compared to those from day 3, while those of CS infants did not differ (p < 0.015). Bacterial structures of infants from both groups separated at day 7 (p < 0.001) and day 14 (p < 0.01). The bacterial structure of VD infants gradually changed over time (day 3 vs. day 7, p < 0.012; day 3 vs. day 14, p < 0.001). Day 14 samples of CS infants differed from day 3 and 7 samples (day 3 vs. day 14, p < 0.001). VD infant relative abundance of Bifidobacterium (days 7, 14), Bacteroides (days 7, 14), and Lachnospiraceae (day 7) significantly increased compared to CS infants, with a lower abundance of Enterobacteriaceae (found in all periods of the CS group) (LDA > 3.0). Relative abundances of Bifidobacterium, Lactobacillus, and Staphylococcus were significantly increased in both VD and CS groups at day 14 (LDA > 3.0). Predicted functional analysis showed that VD infants had overrepresented starch/sucrose, amino acid and nucleotide metabolism in gut microbiota with depleted lipopolysaccharide biosynthesis until day 14 compared to CS infants. This study confirmed that delivery mode is the major determinant of neonatal intestinal microbiome establishment and provides a profile of microbiota perturbations in CS infants. Our findings provide preliminary insight for establishing recovery methods to supply the specific microbes missing in CS infants.

Abstract 4638: Associations of fecal microbial profiles with non-malignant breast disease and breast cancer in the Ghana Breast Health Study

Abstract Background and objective: Breast cancer burden is rising in Africa; however, breast cancer risk factors remain largely understudied in this population. The gut microbiome may play a role in the etiology of breast cancer through the regulation of estrogen homeostasis and other metabolic or immunologic pathways. We investigated the association of fecal bacteria with breast neoplasms in a case-control study conducted in Accra and Kumasi, Ghana, comprising 102 non-malignant breast disease cases, 379 invasive breast cancer cases, and 414 population-based controls nested within the Ghana Breast Health Study. Methods: We used the MO-BIO PowerMag kit for DNA extraction and sequenced the V4 region of 16S rRNA gene to characterize bacteria in fecal samples. We used DADA2 to process sequencing data. We calculated estimates of alpha diversity (observed sequence variants, Shannon index, and Faith's phylogenetic diversity), beta diversity (Bray Curtis and unweighted/weighted UniFrac), and presence/absence and relative abundance of select taxa. We then estimated the associations of the microbial metrics with non-malignant breast disease and malignant breast cancer using multivariable unconditional logistic regression. Results: Odds of malignant breast cancer were statistically significantly lower with increasing alpha diversity (all Ptrends &amp;lt;0.0001). For example, comparing those in the highest vs. lowest tertile of observed sequence variants, the odds ratio and 95% confidence interval was 0.22 (0.13-0.36). Alpha diversity estimates were also lower for non-malignant breast disease cases compared to controls but were similar to malignant cases. Likewise, based on the multivariable MiRKAT test, which compares kernel similarity matrices based on beta diversity metrics, overall microbiota composition was similar between non-malignant and malignant cases (all p's &amp;gt; 0.1) but was statistically significantly different for both case groups compared controls (all p's &amp;lt;0.001). After correction for multiple testing, 49 taxa were significantly differentially prevalent among non-malignant and malignant cases compared to controls. Cases tended to have a lower prevalence of the Families Lachnospiraceae, Ruminococcaceae, and Prevotellaceae, and the genera Bifidobacterium, Collinsella, Coprococcus, and Mitsuokella. Notably, previous evidence suggests that some of these differentially prevalent taxa encode the enzymes ß-glucuronidase and/or ß-galactosidase, which are involved in estrogen metabolism. Conclusions: Our findings suggest lower fecal alpha diversity, differences in overall microbial composition, and prevalence of certain taxa involved in estrogen metabolism may be associated with both non-malignant breast disease and invasive breast cancer. Given that the gut bacteria are potentially modifiable and targetable, our findings warrant further investigation into the role of the gut microbiota in hormone regulation and etiology of breast cancer in integrative, prospective studies. Citation Format: Doratha Byrd, Emily Vogtmann, Zeni Wu, Autumn Hullings, Xing Hua, Thomas Ahearn, James J. Goedert, Jianxin Shi, Jonine Figueroa, Louise Brinton, Montserrat Garcia-Closas, Rashmi Sinha. Associations of fecal microbial profiles with non-malignant breast disease and breast cancer in the Ghana Breast Health Study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4638.

Blood Bacterial Profiles Associated With Human Immunodeficiency Virus Infection and Immune Recovery.

Human immunodeficiency virus (HIV) infection impairs mucosal immunity and leads to bacterial translocation, fueling chronic inflammation and disease progression. While this is well established, questions remain about the compositional profile of the translocated bacteria, and to what extent it is influenced by antiretroviral therapy (ART). Using 16S ribosomal DNA targeted sequencing and shotgun proteomics, we showed that HIV increases bacterial translocation from the gut to the blood. HIV increased alpha diversity in the blood, which was dominated by aerobic bacteria belonging to Micrococcaceae (Actinobacteria) and Pseudomonadaceae (Proteobacteria) families, and the number of circulating bacterial proteins was also increased. Forty-eight weeks of ART attenuated this phenomenon. We found that enrichment with Lactobacillales order, and depletion of Actinobacteria class and Moraxellaceae and Corynebacteriacae families, were significantly associated with greater immune recovery and correlated with several inflammatory markers. Our findings suggest that the molecular cross talk between the host and the translocated bacterial products could influence ART-mediated immune recovery.

1867-P: White and Brown Rice Supplementation Changes High-Fat Diet–Induced Dysbiosis of Gut Microbiome and Improves Energy and Glucose Metabolism

Increased white rice (WR) with parallel decreased brown rice (BR) consumption has been associated with increased prevalence of type 2 diabetes (T2DM). To test whether WR and BR have differential effects on energy and glucose metabolism, C57BL/6 mice were fed 6 different diets: control diet (CD, 10% Fat), high-fat diet (HFD, 46% Fat) with either no rice, WR or BR. BR and, surprisingly, WR activated PGC1α and UCP-1 expressions in brown adipose tissue (BAT) and increased energy expenditure as measured by oxygen consumption, CO2 release and heat production, resulting in reduced body weight (36%, 23%) and improvements of glucose tolerance and insulin sensitivity while on HFD after 18 weeks. To investigate the underlying mechanism, gut microbial analysis was performed. Gut microbiota composition showed clear differences between HFD vs. CD or vs. the addition of BR or WR. Ratio of Firmicutes to Bacteroidetes was 3.7-fold increased in HFD vs.CD, but decreased by the WR (46%) or BR (59%). Increased alpha diversity was observed by WR and BR on HFD. Heat map binary showed that several genera are different in HFD+BR vs. HFD or HFD+WR vs. HFD. Analysis of family abundance revealed that Lachnospiraceae, Ruminococcaceae and Streptococcaceae are 2.5, 1.2 and 2.6-fold increased in HFD, which were reduced by WR or BR. Analysis of genus abundance showed that Lactococcus, Dorea, Peptococcus, Clostridium XIVb, Johnsonella, Anaerovorax are 2.6, 3.7, 2.9, 2.7, 4.7 and 1.7-fold respectively, increased in HFD, which were reduced by WR or BR on HFD. In addition, mice on BR+HFD showed greater reductions than mice on WR+HFD in family and genus levels. Our results demonstrated that supplementation with BR or WR, to a lesser degree, changed HFD-induced gut microbial composition at family and genus levels, in parallel with improved systemic energy and glucose metabolism via activation of BAT and increased energy expenditure, resulting in reduced body weight and improved insulin sensitivity. Disclosure H. Yokomizo: None. A. Ishikado: Employee; Self; Sunstar Group. Employee; Spouse/Partner; Sunstar Group. T. Shinjo: None. K. Park: None. Y. Maeda: None. Q. Li: None. I. Wu: None. M. Matsumoto: Employee; Self; Sunstar Group. S. Devkota: None. A. Kostic: None. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc.

Airway microbial diversity is decreased in young children with cystic fibrosis compared to healthy controls but improved with CFTR modulation

BackgroundCulture-independent next generation sequencing has identified diverse microbial communities within the cystic fibrosis (CF) airway. The study objective was to test for differences in the upper airway microbiome of children with CF and healthy controls and age-related differences in children with CF. MethodsOropharyngeal swabs and clinical data were obtained from 25 children with CF and 50 healthy controls aged ≤6 years. Bacterial DNA was amplified and sequenced for the V4 region of 16S rRNA marker-gene. Alpha diversity was measured using operational taxonomic units (OTUs), Shannon diversity, and the inverse Simpson's index. Beta diversity was measured using Morisita-Horn and Bray-Curtis and Jaccard distances. General linear models were used for comparison of alpha diversity measures between groups to account for differences in demographics and exposures. Mixed effects general linear models were used for longitudinal comparisons 1) between children with CF of different ages and 2) between children with CF receiving CF transmembrane conductance regulator (CFTR) modulators, children with CF not receiving CFTR modulators, and healthy controls to adjust for repeated measures per subject. ResultsChildren with CF were more likely to have received antibiotics in the prior year than healthy controls (92% vs 24%, p < 0.001). Controlling age, race, ethnicity, length of breastfeeding, and having siblings, children with CF had a lower richness than healthy controls: OTUs 62.1 vs 83, p = 0.022; and trended toward lower diversity: Shannon 2.09 vs 2.35, p = 0.057; inverse Simpson 5.7 vs 6.92, p = 0.118. Staphylococcus, three Rothia OTUs, and two Streptococcus OTUs were more abundant in CF children versus healthy controls (all p < 0.05). Bray-Curtis and Jaccard distances, which reflect overall microbial community composition, were also significantly different (both p = 0.001). In longitudinally collected samples from children with CF, Morisita-Horn trended toward more similarity in those aged 0–2 years compared to those aged 3–6 years (p = 0.070). In children >2 years of age, there was a significant trend in increasing alpha diversity measures between children with CF not receiving CFTR modulators, children with CF receiving CFTR modulators, and healthy controls: OTUs 63.7 vs 74.7 vs 97.6, p < 0.001; Shannon 2.11 vs 2.34 vs 2.56, p < 0.001; inverse Simpson 5.78 vs 7.23 vs 7.96, p < 0.001. ConclusionsChildren with CF have lower bacterial diversity and different composition of organisms compared with healthy controls. This appears to start in early childhood, is possibly related to the use of antibiotics, and may be partially corrected with the use of CFTR modulators.

Cruciferous Vegetable Intake Results in Differentially Modified Gut Microbiome Composition of Mice Fed a High Fat Diet or a Western Diet

Abstract Objectives Cruciferous (CRU; rich in glucosinolates) and apiaceous (API; rich in furanocoumarins) vegetable intake decrease colon cancer risk markers, likely through different mechanisms. Previous reports suggest background diets influence efficacy of bioactives. Here, we determined the effects on the composition of the gut microbiome of CRU and API supplementation to different background diets, diet-induced obesity (DIO) and the total western diet (TWD). Methods C57BL/6J male mice were fed standard diet (AIN93G), DIO, DIO with 21% (w/w) CRU (DIO + CRU), DIO with 21% (w/w) API (DIO + API), TWD, TWD with CRU (TWD + CRU), or TWD with API (TWD + API). After 12 weeks, cecal contents were collected for 16S rRNA sequencing and data analyzed by mothur. Results There were no differences in body weight gain except mice fed DIO + CRU gained more than mice fed AIN-93G or TWD. Lachnospiraceae was increased by CRU supplementation to both DIO and TWD and by API supplementation to TWD. CRU increased alpha diversity [Shannon Index, number of observed Operational Taxonomic Unit (OTUs)] compared to DIO and TWD. Regarding beta diversity, DIO + CRU showed distinct cluster compared to DIO (Bray-Curtis, ANOSIM, R = 0.35, P &amp;lt; 0.001; Jaccard distance, R = 0.47, P &amp;lt; 0.001). TWD + CRU showed distinct cluster compared to TWD (Bray-Curtis, R = 0.59, P &amp;lt; 0.001; Jaccard distance, R = 0.62, P &amp;lt; 0.001). API did not change alpha diversity, but did affect beta diversities with distinct clusters between API groups and their basal diet groups (Jaccard distance, R = 0.36 and 0.31 for DIO and TWD, respectively, P &amp;lt; 0.05). Among top 25 discriminating features between DIO and TWD and their supplementation of API and CRU, there were 9 shared OTUs including Lachnospiraceae, Clostridium XlVa, Clostridiales, Eisenbergiella, and Clostridium IV. Akkermansia were decreased in DIO + CRU compared with DIO. In TWD panel, Bifidobacterium and Erysipelotrichaceae decreased in TWD + CRU, while Turicibacter were identified as TWD + CRU signature. Erysipelotrichaceae and Bifidobacterium differentiated AIN-93G, DIO, and TWD. Conclusions CRU supplementation of DIO and TWD altered gut microbiome composition with some differences based on background diet. API also altered composition, albeit to a lesser extent. Funding Sources University of Arkansas, Fulbright Nicaragua Fellow.

Changes of Human Gut Microbiome Correlated with Metabolomics After Cranberry Juice Consumption in a Double-Blinded, Placebo Controlled, Crossover Study

Abstract Objectives 1) To investigate the changes of gut microbiome after 3 days and 21 days of cranberry juice consumption; 2) to correlate changes of microbiome with metabolome. Methods A double blinded, placebo-controlled, cross-over intervention study were conducted in 17 healthy young women aged 18–29 with normal BMI. Fecal, urine, and plasma samples were collected at baseline, after 3 days, and after 21 days consumption of double strength cranberry juice or a placebo juice. DNA was extracted from fecal samples and 16s rRNA was sequenced. Discriminant metabolites in urine and plasma was analyzed by UHPLC-Q-Orbitrap-HRMS and 0 identified using OPLS-DA models. Results 21-days but not 3 days of cranberry juice consumption significantly increased alpha diversity of colon microbiome compared to baseline. Significant increase in the abundance of Firmicutes, Ruminococcaceae, Faecalibacterium, and F/B (Firmicutes to Bacteroidetes) ratios were found after 21-days of cranberry juice intake. This was accompanied by decreases in Bacteroidetes, Proteobacteria, Clostridia, Enterobacteriaceae, Veillonellaceae, Bacteroides, Parabacteroides, and Enterobacter. Gut microbial composition before and after 21-days of cranberry juice consumption was significantly per Bray-Curtis dissimilarity analysis. However, cranberry consumption did not alter fecal content of short chain fatty acids, ammonia, or mucin. Spearman's correlation analysis showed significant positive or negative correlations between selected strains of bacteria and discriminant metabolites. 4-O-methylgallic acid was a discriminant metabolite in urine after 21-days of cranberry juice consumption. Its content positively correlated with Parabacteroides but negatively correlated with Faecalibacterium (P &amp;lt; 0.05). 3-(Hydroxyphenyl) propionic acid was a discriminant metabolite in plasma after cranberry juice consumption. Its content negatively correlated with Enterobacteriaceae (P &amp;lt; 0.05). Conclusions 21-days but not 3 days of cranberry juice consumption significantly altered colon microbiome. Correlation between gut bacteria and plasma, urine metabolites suggested interaction between gut microbiome and serum and urine metabolome. Funding Sources This research is funded in part by Ocean Spray Cranberries, inc.

Sex Work Is Associated With Increased Vaginal Microbiome Diversity in Young Women From Mombasa, Kenya

Although nonoptimal vaginal bacteria and inflammation have been associated with increased HIV risk, the upstream drivers of these phenotypes are poorly defined in young African women. Mombasa, Kenya. We characterized vaginal microbiome and cytokine profiles of sexually active young women aged 14-24 years (n = 168) in 3 study groups: those engaging in formal sex work, in transactional sex, and nonsex workers. Vaginal secretions were collected using self-inserted SoftCup, and assayed for cytokines and vaginal microbiome through multiplex ELISA and 16S rRNA sequencing, respectively. Epidemiological data were captured using a validated questionnaire. The median age of participants was 20 years (interquartile range: 18-22 years). Approximately two-thirds of young women (105/168) had vaginal microbial communities characterized by Gardnerella and/or Prevotella spp. dominance; a further 29% (49/168) were predominantly Lactobacillus iners. Microbiome clustering explained a large proportion of cytokine variation (>50% by the first 2 principal components). Age was not associated with vaginal microbial profiles in bivariable or multivariable analyses. Women self-identifying as sex workers had increased alpha (intraindividual) diversity, independent of age, recent sexual activity, HIV, and other sexually transmitted infections (beta = 0.47, 95% confidence interval: 0.05 to 0.90, P = 0.03). Recent sex (number of partners or sex acts last week, time since last vaginal sex) correlated with increased alpha diversity, particularly in participants who were not involved in sex work. Nonoptimal vaginal microbiomes were common in young Kenyan women and associated with sex work and recent sexual activity, but independent of age. Restoring optimal vaginal microflora may represent a useful HIV prevention strategy.

HIV-associated gut dysbiosis is independent of sexual practice and correlates with noncommunicable diseases

Loss of gut mucosal integrity and an aberrant gut microbiota are proposed mechanisms contributing to chronic inflammation and increased morbidity and mortality during antiretroviral-treated HIV disease. Sexual practice has recently been uncovered as a major source of microbiota variation, potentially confounding prior observations of gut microbiota alterations among persons with HIV (PWH). To overcome this and other confounding factors, we examine a well-powered subset of AGEhIV Cohort participants comprising antiretroviral-treated PWH and seronegative controls matched for age, body-mass index, sex, and sexual practice. We report significant gut microbiota differences in PWH regardless of sex and sexual practice including Gammaproteobacteria enrichment, Lachnospiraceae and Ruminococcaceae depletion, and decreased alpha diversity. Men who have sex with men (MSM) exhibit a distinct microbiota signature characterized by Prevotella enrichment and increased alpha diversity, which is linked with receptive anal intercourse in both males and females. Finally, the HIV-associated microbiota signature correlates with inflammatory markers including suPAR, nadir CD4 count, and prevalence of age-associated noncommunicable comorbidities.

Potential role of gut microbiota, the proto-oncogene PIKE (Agap2) and cytochrome P450 CYP2W1 in promotion of liver cancer by alcoholic and nonalcoholic fatty liver disease and protection by dietary soy protein

We have previously demonstrated promotion of diethylnitrosamine (DEN) initiated liver tumorigenesis after feeding diets high in fat or ethanol (EtOH) to male mice. This was accompanied by hepatic induction of the proto-oncogene PIKE (Agap2). Switch of dietary protein from casein to soy protein isolate (SPI) significantly reduced tumor formation in these models. We have linked EtOH consumption in mice to microbial dysbiosis. Adoptive transfer studies demonstrate that microbiota from mice fed ethanol can induce hepatic steatosis in the absence of ethanol suggesting that microbiota or the microbial metabolome play key roles in development of fatty liver disease. Feeding SPI significantly changed gut bacteria in mice increasing alpha diversity (P < 0.05) and levels of Clostidiales spp. Feeding soy formula to piglets also resulted in significant changes in microbiota, the pattern of bile acid metabolites and in inhibition of the intestinal-hepatic FXR/FGF19-SHP pathway which has been linked to both steatosis and hepatocyte proliferation. Moreover, feeding SPI also resulted in induction of hepatic PPARα signaling and inhibition of PIKE mRNA expression coincident with inhibition of steatosis and cancer prevention. Feeding studies in the DEN model with differing dietary fats demonstrated tumor promotion specific to the saturated fat, cocoa butter relative to diets containing olive oil or corn oil associated with microbial dysbiosis including dramatic increases in Lachnospiraceae particularly from the genus Coprococcus. Immunohistochemical analysis demonstrated that tumors from EtOH-fed mice and patients with alcohol-associated HCC also expressed high levels of a novel cytochrome P450 enzyme CYP2W1. Additional adoptive transfer experiments and studies in knockout mice are required to determine the exact relationship between soy effects on the microbiota, expression of PIKE, CYP2W1, PPARα activation and prevention of tumorigenesis.

Bladder bacterial diversity differs in continent and incontinent women: a cross-sectional study

Since the discovery of the bladder microbiome (urobiome), interest has grown in learning whether urobiome characteristics have a role in clinical phenotyping and provide opportunities for novel therapeutic approaches for women with common forms of urinary incontinence. This study aimed to test the hypothesis that the bladder urobiome differs among women in the control cohort and women affected by urinary incontinence by assessing associations between urinary incontinence status and the cultured urobiome. With institutional review board oversight, urine specimens from 309 adult women were collected through transurethral catheterization. These women were categorized into 3 cohorts (continent control, stress urinary incontinence [SUI], and urgency urinary incontinence [UUI]) based on their responses to the validated Pelvic Floor Distress Inventory (PFDI) questionnaire. Among 309 women, 150 were in the continent control cohort, 50 were in the SUI cohort, and 109 were in the UUI cohort. Symptom severity was assessed by subscale scoring with the Urinary Distress Inventory (UDI), subscale of the Pelvic Floor Distress Inventory. Microbes were assessed by expanded quantitative urine culture protocol, which detects the most common bladder microbes (bacteria and yeast). Microbes were identified to the species level by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Alpha diversity indices were calculated for culture-positive samples and compared across the 3 cohorts. The correlations of UDI scores, alpha diversity indices, and species abundance were estimated. Participants had a mean age of 53 years (range 22-90); most were whites (65%). Women with urinary incontinence were slightly older (control, 47; SUI, 54; UUI, 61). By design, UDI symptom scores differed (control, 8.43 [10.1]; SUI, 97.95 [55.36]; UUI, 93.71 [49.12]; P<.001). Among 309 participants, 216 (70%) had expanded quantitative urine culture-detected bacteria; furthermore, the urinary incontinence cohorts had a higher detection frequency than the control cohort (control, 57%; SUI, 86%; UUI, 81%; P<.001). In addition, the most frequently detected species among the cohorts were as follows: continent control, Lactobacillus iners (12.7%), Streptococcus anginosus (12.7%), L crispatus (10.7%), and L gasseri (10%); SUI, S anginosus (26%), L iners (18%), Staphylococcus epidermidis (18%), and L jensenii (16%); and UUI, S anginosus (30.3%), L gasseri (22%), Aerococcus urinae (18.3%), and Gardnerella vaginalis (17.4%). However, only Actinotignum schaalii (formerly Actinobaculum schaalii), A urinae, A sanguinicola, and Corynebacterium lipophile group were found at significantly higher mean abundances in 1 of the urinary incontinence cohorts when compared with the control cohort (Wilcoxon rank sum test; P<.02), and no individual genus differed significantly between the 2 urinary incontinence cohorts. Both urinary incontinence cohorts had increased alpha diversity similar to continent control cohort with indices of species richness, but not evenness, strongly associated with urinary incontinence. In adult women, the composition of the culturable bladder urobiome is associated with urinary incontinence, regardless of common incontinence subtype. Detection of more unique living microbes was associated with worsening incontinence symptom severity. Culturable species richness was significantly greater in the urinary incontinence cohorts than in the continent control cohort.

Abstract A39: The human oral microbiota and risk of lung cancer: An analysis of three prospective cohort studies

Abstract Background: The oral microbiota may be associated with lung cancer risk through direct mechanisms, including infection, immune responses, and periodontal disease, and through indirect mechanisms such as the modification of the oral microbiota by tobacco. We conducted a case-cohort study nested within three US prospective cohort studies to evaluate the association between oral microbiota ascertained years before a cancer diagnosis and risk of lung cancer. Methods: Incident lung cancer cases within the Agricultural Health Study (AHS; N=244), NIH-AARP Diet and Health Study (N=376), and the Prostate, Lung, Colorectum, and Ovarian Cancer Screening Trial (PLCO; N=700) who provided an oral wash sample were identified. The median time between oral sample collection and diagnosis was approximately 6.6 years, 3.4 years, and 4.5 years for AHS, NIH-AARP, and PLCO, respectively. A referent subcohort was randomly selected by strata of age, sex, and cigarette smoking history. We extracted DNA using the DSP DNA Virus Pathogen kit, and the V4 region of the 16S rRNA gene was PCR amplified and sequenced using the MiSeq. The sequencing data were processed using QIIME2 with the DADA2 plugin and we generated alpha and beta diversity metrics. Cox proportional hazards models were used to evaluate the hazard ratios (HR) and 95% confidence intervals (CI) for the association between the oral microbial measures and the risk of lung cancer with adjustment for known lung cancer risk factors, and estimates from the three cohorts were meta-analyzed. Results: Increased alpha diversity was associated with decreased lung cancer risk, although only the association with the Shannon Index reached statistical significance (HR for 5th quintile versus 1st quintile 0.74; 95% CI 0.60, 0.92) with no evidence of between-study heterogeneity (p = 0.5968). Specific principal coordinate vectors from the beta diversity matrices were also significantly associated with lung cancer risk, suggesting differing bacterial communities between future lung cancer cases. When stratified by histologic subtypes, the inverse association with alpha diversity was restricted to squamous cell carcinoma, with all alpha diversity metrics reaching statistical significance (e.g., Faith’s phylogenetic diversity HR for 5th quintile versus 1st quintile 0.57; 95% CI: 0.37, 0.87). Similarly, when stratified by smoking history, the inverse association with alpha diversity was restricted to former smokers (e.g., observed species HR for 5th quintile versus 1st quintile 0.63; 95% CI: 0.44, 0.89). Conclusions: In oral wash samples collected years before diagnosis, we found significant associations between both alpha and beta diversity metrics of the oral microbial communities and risk of lung cancer. Additional work is required to understand the associations by histologic subtype and smoking history. Citation Format: Emily Vogtmann, Xing Hua, Guoqin Yu, Autumn Hullings, Yunhu Wan, Casey L Dagnall, Kristine Jones, Belynda D. Hicks, Amy Hutchinson, Shalabh Suman, Bin Zhu, Barry Graubard, Mitchell H. Gail, J. Gregory Caporaso, William Wheeler, Dale Sandler, Laura E. Beane Freeman, Linda Liao, Neal D. Freedman, Neil Caporaso, Rashmi Sinha, Jianxin Shi, Christian C Abnet. The human oral microbiota and risk of lung cancer: An analysis of three prospective cohort studies [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A39.

Abstract B09: Unravelling the gastric microbiome in health and disease: Gastric cancer beyond Helicobacter pylori in a Brazilian cohort

Abstract Although numerous studies have shown the relevance of the gut microbiome in several diseases, underlying questions remain concerning the stomach microbiome and the establishment of a causal link between the microbiota and the development of gastric diseases, much beyond Helicobacter pylori and Epstein Barr virus. In this study, we aimed to characterize the bacterial composition of the stomach of subjects undergoing upper endoscopy, including gastric cancer (GC) individuals, aiming to identify fluctuations in bacterial populations that might be associated with stomach health. During endoscopic examination at A.C. Camargo Cancer Center (Sao Paulo, Brazil), gastric fluids (GF) were recovered from either GC patients (113) or individuals with gastric-related complaints, such as superficial gastritis (SG; 79), atrophic gastritis (AG; 12), and intestinal metaplasia (IM; 33). For eubacteria identification, the V3-V4 region of the 16S rRNA gene was amplified and paired-end sequenced (Illumina MiSeq). Analyses were carried out using Qiime2 and phyloseq packages. On average, we identified between 14 and 104 OTUs per subject, evidencing the potential of GFs for determining the stomach microbial composition and the interindividual variation. Testing of sample richness between GC and controls showed significant differences between the number of OTUs observed in each group (an average of 44 and 52 OTUs, respectively—Mann-Whitney test, p&amp;lt;0.05) and SG patients had a significantly increased alpha diversity (Shannon, p&amp;lt;0.05) as compared to AG, IM, and GC patients (both intestinal and diffuse subtypes), indicating dysbiosis already in early carcinogenesis steps. Additionally, the prolonged use of proton pump inhibitors or the presence of H. pylori (except for SG) did not seem to interfere with bacterial diversity. Specific genera are enriched in the sample subsets, including a lower presence of Corynebacterium and increased Streptococcus (Linear discriminant analysis Effect Size, LDA score &amp;gt;2) in AG samples as compared to SG patients, which are also increased in the IM, and GC. These results indicate these bacteria to be potentially associated with the stomach dysbiosis that may lead to the carcinogenesis cascade. Besides the regular turnover of the gastric epithelial tissue and pouring of cells onto the gastric cavity, GF certainly contains a high proportion of transient oral-derived bacteria, while stomach-resident microbiota is expected to be in close contact with the gastric epithelia. Preliminary data analyzing the bacterial content of the saliva x GF in a subset of patients showed no significant beta-diversity differences, but both fluids differ significantly from their biopsies’ composition. We are currently performing culturomics and transcriptomics to identify bacteria that are alive in the stomach. As GC is a complex malignancy with limited treatment options, these results may contribute to developing new interventions to treat and to better understand this disease. Citation Format: Thais F. Bartelli, Gabriela E. Albuquerque, Alexandre Defelicibus, Marianna S. Serpa, Lais L.S. Abrantes, Rodrigo Borges, Felipe Coimbra, Adriane G. Pelosof, Israel T. Silva, Diana N. Nunes, Emmanuel Dias-Neto. Unravelling the gastric microbiome in health and disease: Gastric cancer beyond Helicobacter pylori in a Brazilian cohort [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B09.

Age and environmental exposures influence the fecal bacteriome of young children with cystic fibrosis.

Mechanisms that facilitate early infection and inflammation in cystic fibrosis (CF) are unclear. We previously showed that young CF children with secondhand smoke exposure (SHSe) have increased susceptibility to respiratory infections. We aimed to define the impact of SHSe and other external factors upon the fecal bacteriome in early CF. Twenty CF infants and children were enrolled, clinical data recorded, and hair nicotine measured as an objective surrogate of SHSe. Fecal samples were collected at clinic visits and bacteriome 16S rRNA gene sequencing performed. SHSe was associated with increased alpha diversity and increased relative abundance of Acinetobacter and Akkermansia, along with decreased Bifidobacterium and Lactobacillus. Recent antibiotic exposure predicted bacterial population structure in children less than 2 years of age and was associated with decreased Bacteroides relative abundance. Age was the strongest predictor of overall fecal bacterial composition and positively associated with Blautia and Parabacteroides. Weight for length was negatively associated with Staphylococcus relative abundance. SHSe and other external factors such as antibiotics appear to alter fecal bacterial composition in young CF children, but the strongest predictor of overall composition was age. These findings have implications for understanding the intestinal microbiome in young CF children.

Microbiome Composition in Pediatric Populations from Birth to Adolescence: Impact of Diet and Prebiotic and Probiotic Interventions.

Diet is a key regulator of microbiome structure and function across the lifespan. Microbial colonization in the first year of life has been actively researched; however, studies during childhood are sparse. Herein, the impact of dietary intake and pre- and probiotic interventions on microbiome composition of healthy infants and children from birth to adolescence is discussed. The microbiome of breastfed infants has lower microbial diversity and richness, higher Proteobacteria, and lower Bacteroidetes and Firmicutes than those formula-fed. As children consume more complex diets, associations between dietary patterns and the microbiota emerge. Like adults, the microbiota of children consuming a Western-style diet is associated with greater Bacteroidaceae and Ruminococcaceae and lower Prevotellaceae. Dietary fibers and pre- or/and probiotics have been tested to modulate the gut microbiota in early life. Human milk oligosaccharides and prebiotics added to infant formula are bifidogenic and decrease pathogens. In children, prebiotics, such as inulin, increase Bifidobacterium abundance and dietary fibers reduce fecal pH and increase alpha diversity and calcium absorption. Probiotics have been administered to the mother during pregnancy and breastfeeding or directly to the infant/child. Findings on maternal probiotic administration on bacterial taxa are inconsistent. When given directly to the infant/child, some changes in individual taxa are observed, but rarely is overall alpha or beta diversity affected. Cesarean-delivered infants appear to benefit to a greater degree than those born vaginally. Infancy and childhood represent an opportunity to beneficially manipulate the microbiome through dietary or prebiotic interventions, which has the potential to affect both short- and long-term health outcomes.

Cortex Phellodendri extract’s anti-diarrhea effect in mice related to its modification of gut microbiota

Cortex Phellodendri extract (CPE) has been used in China to treat diarrhea whereas the underlying mechanisms remain poorly understood. Given that dysbiosis of gut microbiota is a potential reason for diarrhea, and that oral CPE has a low absorption rate in intestine, we hypothesized that modification of gut microbiota is an important factor in CPE’s anti-diarrhea effect. To test this hypothesis, we established a diarrhea model by challenging post-weaning mice with oral Enterotoxigenic-Escherichia coli (ETEC), and then the mice were treated with two doses of CPE (80 mg/kg bodyweight and 160 mg/kg bodyweight) or the vehicle control (phosphate buffered saline). Diarrhea indices, inflammatory factors, morphology of jejunum, short-chain fatty acids (SCFAs), and serum endocrine were determined. Modification of gut microbiota was analyzed using 16S rDNA high-throughput sequencing. The changes in functional profiles of gut microbiota were predicted using software PICRUSt. We then explored the association between CPE-responding bacteria and the symptoms indices with the spearman’s rank correlation coefficient and significance test. Compared with diarrheal mice, CPE decreased Gut/Carcass ratio and water content of stool, increased goblet cell density and villus height/crypt depth of jejunum, as well as decreased inflammatory indices (Tumour Necrosis Factor-α, Myeloperoxidase and Interleukin-1α). CPE shifted the gut microbiota significantly by increasing alpha diversity (observed species, ace, Shannon, and Simpson) and restoring the gut microbiota. CPE increased Firmicutes and decreased Bacteroidetes. The reduced genus Prevotella, Acinetobacter, and Morganella were positively associated with the diarrhea indices, whereas increased genus Odoribacter, Rikenella, and Roseburia were negatively associated with the diarrhea indices. The abundance of carbohydrate metabolism-related gene and SCFAs-producing bacteria were increased, which was evidenced by increased butyric acid and total SCFAs concentration in the caecum. Consequently, endocrine peptides glucagon-like peptide-1, epidermal growth factor, and peptide tyrosine tyrosine in serum were elevated. ConclusionsCPE shows a shift function on the gut microbiota in alleviating the diarrhea of mice in a dose-dependent manner. In addition, the microbial metabolites SCFAs may mediate CPE’s anti-diarrhea effect by enhancing endocrine secretion in mice.