The Oral Microbiome and Lung Cancer Risk: An Analysis of 3 Prospective Cohort Studies.

BACKGROUND: Previous studies suggested associations between the oral microbiome and lung cancer, but studies were predominantly cross-sectional and underpowered. METHODS: Using a case-cohort design, 1306 incident lung cancer cases were identified in the Agricultural Health Study; National Institutes of Health-AARP Diet and Health Study; and Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Referent subcohorts were randomly selected by strata of age, sex, and smoking history. DNA was extracted from oral wash specimens using the DSP DNA Virus Pathogen kit, the 16S rRNA gene V4 region was amplified and sequenced, and bioinformatics were conducted using QIIME 2. Hazard ratios and 95% confidence intervals were calculated using weighted Cox proportional hazards models. RESULTS: Higher alpha diversity was associated with lower lung cancer risk (Shannon index hazard ratio = 0.90, 95% confidence interval = 0.84 to 0.96). Specific principal component vectors of the microbial communities were also statistically significantly associated with lung cancer risk. After multiple testing adjustment, greater relative abundance of 3 genera and presence of 1 genus were associated with greater lung cancer risk, whereas presence of 3 genera were associated with lower risk. For example, every SD increase in Streptococcus abundance was associated with 1.14 times the risk of lung cancer (95% confidence interval = 1.06 to 1.22). Associations were strongest among squamous cell carcinoma cases and former smokers. CONCLUSIONS: Multiple oral microbial measures were prospectively associated with lung cancer risk in 3 US cohort studies, with associations varying by smoking history and histologic subtype. The oral microbiome may offer new opportunities for lung cancer prevention.

MicrobiomeGWAS: A Tool for Identifying Host Genetic Variants Associated with Microbiome Composition.

The microbiome is the collection of all microbial genes and can be investigated by sequencing highly variable regions of 16S ribosomal RNA (rRNA) genes. Evidence suggests that environmental factors and host genetics may interact to impact human microbiome composition. Identifying host genetic variants associated with human microbiome composition not only provides clues for characterizing microbiome variation but also helps to elucidate biological mechanisms of genetic associations, prioritize genetic variants, and improve genetic risk prediction. Since a microbiota functions as a community, it is best characterized by ß diversity; that is, a pairwise distance matrix. We develop a statistical framework and a computationally efficient software package, microbiomeGWAS, for identifying host genetic variants associated with microbiome ß diversity with or without interacting with an environmental factor. We show that the score statistics have positive skewness and kurtosis due to the dependent nature of the pairwise data, which makes p-value approximations based on asymptotic distributions unacceptably liberal. By correcting for skewness and kurtosis, we develop accurate p-value approximations, whose accuracy was verified by extensive simulations. We exemplify our methods by analyzing a set of 147 genotyped subjects with 16S rRNA microbiome profiles from non-malignant lung tissues. Correcting for skewness and kurtosis eliminated the dramatic deviation in the quantile-quantile plots. We provided preliminary evidence that six established lung cancer risk SNPs were collectively associated with microbiome composition for both unweighted (p = 0.0032) and weighted (p = 0.011) UniFrac distance matrices. In summary, our methods will facilitate analyzing large-scale genome-wide association studies of the human microbiome.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Induced Parkinson's Disease in Mouse: Potential Association between Neurotransmitter Disturbance and Gut Microbiota Dysbiosis.

Recent studies have revealed significant roles of neurotransmitters and gut microbiota along the gut-brain axis in Parkinson's disease (PD); however, the potential mechanisms remain poorly understood. In the current study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced characteristic PD neurobehavior changes accompanied by increased α-synuclein, apoptotic protein Bim, and cleaved caspase-3 and decreased expression of tyrosine hydroxylase (TH). Meanwhile, the tryptophan (Trp) and tyrosine (Tyr) neurotransmitter metabolites involving kynurenine (KYN), serotonin (5-HT), and dopamine (DA) pathways were significantly changed in serum. Furthermore, the step-limited enzymes, which are responsible for the key metabolic pathways of these neurotransmitters, were obviously dysregulated. The 16S rRNA gene sequence results indicated that the abundance and diversity of the microbiota were obviously decreased in MPTP-treated mice, the presence of Ruminococcus, Parabacteroides and Parasutterella genera were obviously increased, while Coriobacteriaceae, Flavonifractor, Lachnospiraceae, Lactobacillaceae, and Rikenellaceae abundance was markedly decreased. The connectivity between the gut microbiota and neurotransmitter metabolism revealed that the gut microbiota dysbiosis was associated with disturbance of the DA, KYN, and 5-HT metabolic pathways. Therefore, our results provide evidence that gut-microbiota-brain axis disturbance may play an important role in PD development and targeting this axis might provide a promising therapeutic strategy for PD.

Whole-Exome Sequencing of Nasopharyngeal Carcinoma Families Reveals Novel Variants Potentially Involved in Nasopharyngeal Carcinoma.

Genetic susceptibility is likely involved in nasopharyngeal carcinoma (NPC), a cancer caused by Epstein-Barr virus (EBV) infection. Understanding of genetic factors involved in NPC and how they contribute to EBV-induced carcinogenesis is limited. We conducted whole-exome capture/sequencing among 251 individuals from 97 multiplex families from Taiwan (205 affected, 21 obligate carriers, and 25 unaffected) using SeqCap EZ Human Exome Library v3.0 and Illumina HiSeq. Aligned sequences were filtered to identify likely-to-be-functional deleterious variants that co-segregated with disease. Ingenuity Pathway analysis was performed. Circulating magnesium levels were measured in 13 individuals in 2 families with NIPAL1 mutations and in 197 sporadic NPC cases and 237 controls. We identified variants in 12 genes likely involved in cancer pathogenesis, viral infection or immune responses to infection. These included genes postulated to be involved in magnesium transport (NIPAL1), EBV cell entry (ITGB6), modulation of EBV infection (BCL2L12, NEDD4L), telomere biology (CLPTM1L, BRD2, HNRNPU), modulation of cAMP signaling (RAPGEF3), DNA repair (PRKDC, MLH1), and Notch signaling (NOTCH1, DLL3). Pathway based analysis demonstrated enrichment for Notch signaling genes (p-value = 0.0006). Evaluation of individuals within NIPAL1 families suggested lower serum magnesium in NPC compared to unaffected members. A significant reduction in serum magnesium levels was observed among sporadic NPC cases compared to controls (7.1% NPC/1.7% controls below normal range; OR = 4.5; 95% CI = 1.4,14) and is consistent with findings demonstrating a role for magnesium channeling in T-cell responses to EBV. We identified novel genes associated with NPC that point to new areas of inquiry to better understand genetic factors that determine the fate of viral infections and/or otherwise predisposes to NPC.

Evaluation of Rare and Common Variants from Suspected Familial or Sporadic Nasopharyngeal Carcinoma (NPC) Susceptibility Genes in Sporadic NPC.

BACKGROUND: Genetic susceptibility is associated with nasopharyngeal carcinoma (NPC). We previously identified rare variants potentially involved in familial NPC and common variants significantly associated with sporadic NPC. METHODS: We conducted targeted gene sequencing of 20 genes [16 identified from the study of multiplex families, three identified from a pooled analysis of NPC genome-wide association study (GWAS), and one identified from both studies] among 819 NPC cases and 938 controls from two case-control studies in Taiwan (independent from previous studies). A targeted, multiplex PCR primer panel was designed using the custom Ion AmpliSeq Designer v4.2 targeting the regions of the selected genes. Gene-based and single-variant tests were conducted. RESULTS: We found that NPC was associated with combined common and rare variants in CDKN2A/2B (P = 1.3 × 10-4), BRD2 (P = 1.6 × 10-3), TNFRSF19 (P = 4.0 × 10-3), and CLPTM1L/TERT (P = 5.4 × 10-3). Such associations were likely driven by common variants within these genes, based on gene-based analyses evaluating common variants and rare variants separately (e.g., for common variants of CDKN2A/2B, P = 4.6 × 10-4; for rare variants, P = 0.04). We also observed a suggestive association with rare variants in HNRNPU (P = 3.8 × 10-3) for NPC risk. In addition, we validated four previously reported NPC risk-associated SNPs. CONCLUSIONS: Our findings confirm previously reported associated variants and suggest that some common variants in genes previously linked to familial NPC are associated with the development of sporadic NPC. IMPACT: NPC-associated genes, including CLPTM1L/TERT, BRD2, and HNRNPU, suggest a role for telomere length maintenance in NPC etiology.

Mammographic breast density and its association with urinary estrogens and the fecal microbiota in postmenopausal women.

BACKGROUND: Breast density, as estimated by mammography, is a strong risk factor for breast cancer in pre- and postmenopausal women, but the determinants of breast density have not yet been established. The aim of this study was to assess if urinary estrogens or gut microbiota alterations are associated with mammographic density in postmenopausal women. METHODS: Among 54 cancer-free, postmenopausal controls in the Breast and Colon Health study, we classified low- versus high-density women with Breast Imaging Reporting and Data System (BI-RADS, 5th edition) mammographic screening data, then assessed associations with urinary estrogens and estrogen metabolites (determined by liquid chromatography/tandem mass spectrometry), and fecal microbiota alpha and beta diversity (using Illumina sequencing of 16S rRNA amplicons). RESULTS: Multiple logistic regression revealed no significant association between breast density and fecal microbiota metrics (PD_tree P-value = 0.82; un-weighted and weighted UniFrac P = 0.92 and 0.83, respectively, both by MiRKAT). In contrast, total urinary estrogens (and all 15 estrogens/estrogen metabolites) were strongly and inversely associated with breast density (P = 0.01) after adjustment for age and body mass index. CONCLUSION: Mammographic density was not associated with the gut microbiota, but it was inversely associated with urinary estrogen levels. IMPACT: The finding of an inverse association between urinary estrogens and breast density in cancer-free women adds to the growing breast cancer literature on understanding the relationship between endogenous estrogens and mammographic density.

MiR-142a-3p and miR-155-5p reduce methamphetamine-induced inflammation: Role of the target protein Peli1.

Methamphetamine (METH) is a highly addictive stimulant and METH exposure can induce a series of neuroinflammatory effects. Peli1 is a novel and important E3 ubiquitin-protein ligase contributing to neuroinflammation; targeting Peli1 may thus provide promising therapeutic strategies for neuroinflammation. In addition to the classic MyD88-dependent or MyD88-independent pathways, miRNAs may also be involved in Peli1 modulation. In the present study, two novel miRNAs, miR-142a-3p and miR-155-5p, that were predicted to target Peli1 using bioinformatics were chosen, and their unique roles in METH-induced neuroinflammation via regulating Peli1 expression were identified. Our results showed that miR-142a-3p was significantly reduced in METH-induced neuroinflammation and was negatively associated with Peli1 expression both in BV2 cells and in the brain of mouse. MiR-155-5p was significantly reduced by METH in vitro but increased in vivo. A luciferase reporter assay was performed to reveal that miR-142a-3p and miR-155-5p bound specifically to Peli1, an effect that was completely abolished by the Peli1 binding site mutation. Reciprocally, the overexpression of miR-142a-3p and miR-155-5p could directly suppress Peli1 expression and could protect against the inflammatory effects of METH treatment partially through activating p38 MAPK and NF-κB inflammatory pathways. In conclusion, the present study reveals a novel signaling pathway, the miR-142a-3p/miR-155-5p/Peli1 axis in METH-mediated neuroinflammation, and this pathway could be a potential therapeutic target for METH-mediated neurotoxicity.

Elevated antibodies against Epstein-Barr virus among individuals predicted to carry nasopharyngeal carcinoma susceptibility variants.

Epstein-Barr virus (EBV) is an obligatory factor in the development of nasopharyngeal carcinoma (NPC), and anti-EBV IgA antibodies are elevated many years prior to the development of NPC. Nearly all adults are infected with EBV, but only a few develop cancer, suggesting that additional co-factors, including genetic susceptibility, must be required for the disease to manifest. Individuals were selected from the Taiwan Family Study, a cohort of 3389 individuals from NPC multiplex families. Primary analyses were conducted among 671 individuals from 69 pedigrees with the strongest family history of disease (>3 NPC-affected family members). The likelihood that a given family member carried a NPC susceptibility variant was estimated using Mendelian segregation rules, assuming a dominant mode of inheritance. We compared anti-EBV IgA antibody seropositivity between family members predicted to be carriers of NPC-linked genetic variants and those with a lower likelihood of carrying such variants. Obligate carriers of NPC susceptibility variants (100 % predicted probability of harbouring the genetic mutation) were nine-fold more likely to be anti-EBV IgA positive compared to family members predicted not to carry disease-causing variants (OR=9.2; P-trend<0.001). This elevated risk was confirmed in analyses restricted to both unaffected individuals and pedigrees with EBV-related pathway variants identified through exome sequencing. Our data indicate that family members who are more likely to carry NPC susceptibility variants are also more likely to be anti-EBNA1 IgA seropositive. Genetic susceptibility associated with control over this common herpes virus is likely a co-factor in determining which EBV-infected adults develop NPC.

Quantification of Human Microbiome Stability Over 6 Months: Implications for Epidemiologic Studies.

Temporal variation in microbiome measurements can reduce statistical power in research studies. Quantification of this variation is essential for designing studies of chronic disease. We analyzed 16S ribosomal RNA profiles in paired biological specimens separated by 6 months from 3 studies conducted during 1985-2013 (a National Cancer Institute colorectal cancer study, a Costa Rica study, and the Human Microbiome Project). We evaluated temporal stability by calculating intraclass correlation coefficients (ICCs). Sample sizes needed in order to detect microbiome differences between equal numbers of cases and controls for a nested case-control design were calculated on the basis of estimated ICCs. Across body sites, 12 phylum-level ICCs were greater than 0.5. Similarly, 11 alpha-diversity ICCs were greater than 0.5. Fecal beta-diversity estimates had ICCs over 0.5. For a single collection with most microbiome metrics, detecting an odds ratio of 2.0 would require 300-500 cases when matching 1 case to 1 control at P = 0.05. Use of 2 or 3 sequential specimens reduces the number of required subjects by 40%-50% for low-ICC metrics. Relative abundances of major phyla and alpha-diversity metrics have low temporal stability. Thus, detecting associations of moderate effect size with these metrics will require large sample sizes. Because beta diversity for feces is reasonably stable over time, smaller sample sizes can detect associations with community composition. Sequential prediagnostic specimens from thousands of prospectively ascertained cases are required to detect modest disease associations with particular microbiome metrics.

Fast coarse-fine locating method for φ-OTDR.

We proposed and demonstrated a coarse-fine method to achieve fast locating of external vibration for the phase-sensitive optical time-domain reflectometer (φ-OTDR) sensing system. Firstly, the acquired backscattered traces from heterodyne coherent φ-OTDR systems are spatially divided into a few segments along a sensing fiber for coarse locating, and most of the acquired data can be excluded by comparing the phase difference between the endpoints in adjacent segments. Secondly, the amplitude-based locating is implemented within the target segments for fine locating. By using the proposed coarse-fine locating method, we have numerically and experimentally investigated a distributed vibration sensor based on the heterodyne coherent φ-OTDR system with a 50-km-long sensing fiber. We find that the computation cost of signal processing for locating is significantly reduced in the long-haul sensing fiber, showing a potential application in real-time locating of external vibration.

On the Search to Elucidate the Role of Microbiota in the Genesis of Cancer: The Cases of Gastrointestinal and Cervical Cancer.

The microbiota that inhabits the human body plays an important role in health and disease, by their fundamental role in food digestion, training of the immune system or protection against pathogen colonization. However, when the equilibrium with its host is altered, some diseases like cancer might be promoted. In this review we describe the information collected in recent studies between the microbiota and its association with cancer. We conducted the review of the relation of microbiome and cancer etiology focusing on the gastrointestinal and cervical cancer. The MEDLINE database was used for the search. The gastrointestinal tract harbours a diverse and site specific microbiota, and several studies have demonstrated that perturbation of these microbial communities might be associated with different types of cancer. In particular, alteration of the colorectal, gastric and oesophageal microbiota have been reported associated with cancer development. Likewise, cervical microbiome studies suggest that some members of the cervical microbiota are possible modifiers of the cytokine profile of the cervical microenvironment during the development of cervical lesions and cervical cancer. Larger prospective studies are needed to examine whether microbiome dysbiosis could cause cancer, and to evaluate the utility of microbiome profiles as biomarkers for prevention and early diagnosis. This is an important area of research if we consider that microbiota may be a modifiable factor by the use of pre- and probiotics, in order to prevent cancer evolution or even to potentiate cancer treatment.

Molecular Characterization of the Human Stomach Microbiota in Gastric Cancer Patients.

Helicobacter pylori (Hp) is the primary cause of gastric cancer but we know little of its relative abundance and other microbes in the stomach, especially at the time of gastric cancer diagnosis. Here we characterized the taxonomic and derived functional profiles of gastric microbiota in two different sets of gastric cancer patients, and compared them with microbial profiles in other body sites. Paired non-malignant and tumor tissues were sampled from 160 gastric cancer patients with 80 from China and 80 from Mexico. The 16S rRNA gene V3-V4 region was sequenced using MiSeq platform for taxonomic profiles. PICRUSt was used to predict functional profiles. Human Microbiome Project was used for comparison. We showed that Hp is the most abundant member of gastric microbiota in both Chinese and Mexican samples (51 and 24%, respectively), followed by oral-associated bacteria. Taxonomic (phylum-level) profiles of stomach microbiota resembled oral microbiota, especially when the Helicobacter reads were removed. The functional profiles of stomach microbiota, however, were distinct from those found in other body sites and had higher inter-subject dissimilarity. Gastric microbiota composition did not differ by Hp colonization status or stomach anatomic sites, but did differ between paired non-malignant and tumor tissues in either Chinese or Mexican samples. Our study showed that Hp is the dominant member of the non-malignant gastric tissue microbiota in many gastric cancer patients. Our results provide insights on the gastric microbiota composition and function in gastric cancer patients, which may have important clinical implications.

Gastric microbiota features associated with cancer risk factors and clinical outcomes: A pilot study in gastric cardia cancer patients from Shanxi, China.

Little is known about the link between gastric microbiota and the epidemiology of gastric cancer. In order to determine the epidemiologic and clinical relevance of gastric microbiota, we used 16 S ribosomal RNA gene sequencing analysis to characterize the composition and structure of the gastric microbial community of 80 paired samples (non-malignant and matched tumor tissues) from gastric cardia adenocarcinoma (GCA) patients in Shanxi, China. We also used PICRUSt to predict microbial functional profiles. Compared to patients without family history of upper gastrointestinal (UGI) cancer in the non-malignant gastric tissue microbiota, patients with family history of UGI cancer had higher Helicobacter pylori (Hp) relative abundance (median: 0.83 vs. 0.38, p = 0.01) and lower alpha diversity (median observed species: 51 vs. 85, p = 0.01). Patients with higher (vs. lower) tumor grade had higher Hp relative abundance (0.73 vs. 0.18, p = 0.03), lower alpha diversity (observed species, 66 vs. 89, p = 0.01), altered beta diversity (weighted UniFrac, p = 0.002) and significant alterations in relative abundance of five KEGG functional modules in non-malignant gastric tissue microbiota. Patients without metastases had higher relative abundance of Lactobacillales than patients with metastases (0.05 vs. 0.01, p = 0.04) in non-malignant gastric tissue microbiota. These associations were observed in non-malignant tissues but not in tumor tissues. In conclusion, this study showed a link of gastric microbiota to a major gastric cancer risk factor and clinical features in GCA patients from Shanxi, China. Studies with both healthy controls and gastric cardia and noncardia cancer cases across different populations are needed to further examine the association between gastric cancer and the microbiota.

The effect of cigarette smoking on the oral and nasal microbiota.

BACKGROUND: The goal of the study was to investigate whether cigarette smoking alters oral and nasal microbial diversity, composition, and structure. Twenty-three current smokers and 20 never smokers were recruited. From each subject, nine samples including supra and subgingiva plaque scrapes, saliva, swabs from five soft oral tissue sites, and one nasal swab from both the anterior nares were collected. 16S rRNA V3-V4 region was sequenced for microbial profiles. RESULTS: We found that alpha diversity was lower in smokers than in nonsmokers in the buccal mucosa, but in other sample sites, microbial diversity and composition were not significantly different by smoking status. Microbial profiles differed significantly among eight oral sites. CONCLUSIONS: This study investigates the effect of cigarette smoking on different sites of the oral cavity and shows a potential effect of cigarette smoking on the buccal mucosa microbiota. The marked heterogeneity of the oral microbial ecosystem that we found may contribute to the stability of the oral microbiota in most sites when facing environmental perturbations such as that caused by cigarette smoking.

Evaluation of Buccal Cell Samples for Studies of Oral Microbiota.

BACKGROUND: The human microbiota is postulated to affect cancer risk, but collecting microbiota specimens with prospective follow-up for diseases will take time. Buccal cell samples have been obtained from mouthwash for the study of human genomic DNA in many cohort studies. Here, we evaluate the feasibility of using buccal cell samples to examine associations of human microbiota and disease risk. METHODS: We obtained buccal cells from mouthwash in 41 healthy participants using a protocol that is widely employed to obtain buccal cells for the study of human DNA. We compared oral microbiota from buccal cells with that from eight other oral sample types collected by following the protocols of the Human Microbiome Project. Microbiota profiles were determined by sequencing 16S rRNA gene V3-V4 region. RESULTS: Compared with each of the eight other oral samples, the buccal cell samples had significantly more observed species (P < 0.002) and higher alpha diversity (Shannon index, P < 0.02). The microbial communities were more similar (smaller beta diversity) among buccal cells samples than in the other samples (P < 0.001 for 12 of 16 weighted and unweighted UniFrac distance comparisons). Buccal cell microbial profiles closely resembled saliva but were distinct from dental plaque and tongue dorsum. CONCLUSIONS: Stored buccal cell samples in prospective cohort studies are a promising resource to study associations of oral microbiota with disease. IMPACT: The feasibility of using existing buccal cell collections in large prospective cohorts allows investigations of the role of oral microbiota in chronic disease etiology in large population studies possible today. Cancer Epidemiol Biomarkers Prev; 26(2); 249-53. ©2016 AACR.

Characterizing human lung tissue microbiota and its relationship to epidemiological and clinical features.

BACKGROUND: The human lung tissue microbiota remains largely uncharacterized, although a number of studies based on airway samples suggest the existence of a viable human lung microbiota. Here we characterized the taxonomic and derived functional profiles of lung microbiota in 165 non-malignant lung tissue samples from cancer patients. RESULTS: We show that the lung microbiota is distinct from the microbial communities in oral, nasal, stool, skin, and vagina, with Proteobacteria as the dominant phylum (60 %). Microbiota taxonomic alpha diversity increases with environmental exposures, such as air particulates, residence in low to high population density areas, and pack-years of tobacco smoking and decreases in subjects with history of chronic bronchitis. Genus Thermus is more abundant in tissue from advanced stage (IIIB, IV) patients, while Legionella is higher in patients who develop metastases. Moreover, the non-malignant lung tissues have higher microbiota alpha diversity than the paired tumors. CONCLUSIONS: Our results provide insights into the human lung microbiota composition and function and their link to human lifestyle and clinical outcomes. Studies among subjects without lung cancer are needed to confirm our findings.

Fecal Microbiota, Fecal Metabolome, and Colorectal Cancer Interrelations.

BACKGROUND AND AIMS: Investigation of microbe-metabolite relationships in the gut is needed to understand and potentially reduce colorectal cancer (CRC) risk. METHODS: Microbiota and metabolomics profiling were performed on lyophilized feces from 42 CRC cases and 89 matched controls. Multivariable logistic regression was used to identify statistically independent associations with CRC. First principal coordinate-component pair (PCo1-PC1) and false discovery rate (0.05)-corrected P-values were calculated for 116,000 Pearson correlations between 530 metabolites and 220 microbes in a sex*case/control meta-analysis. RESULTS: Overall microbe-metabolite PCo1-PC1 was more strongly correlated in cases than in controls (Rho 0.606 vs 0.201, P = 0.01). CRC was independently associated with lower levels of Clostridia, Lachnospiraceae, p-aminobenzoate and conjugated linoleate, and with higher levels of Fusobacterium, Porphyromonas, p-hydroxy-benzaldehyde, and palmitoyl-sphingomyelin. Through postulated effects on cell shedding (palmitoyl-sphingomyelin), inflammation (conjugated linoleate), and innate immunity (p-aminobenzoate), metabolites mediated the CRC association with Fusobacterium and Porphyromonas by 29% and 34%, respectively. Overall, palmitoyl-sphingomyelin correlated directly with abundances of Enterobacteriaceae (Gammaproteobacteria), three Actinobacteria and five Firmicutes. Only Parabacteroides correlated inversely with palmitoyl-sphingomyelin. Other lipids correlated inversely with Alcaligenaceae (Betaproteobacteria). Six Bonferroni-significant correlations were found, including low indolepropionate and threnoylvaline with Actinobacteria and high erythronate and an uncharacterized metabolite with Enterobacteriaceae. CONCLUSIONS: Feces from CRC cases had very strong microbe-metabolite correlations that were predominated by Enterobacteriaceae and Actinobacteria. Metabolites mediated a direct CRC association with Fusobacterium and Porphyromonas, but not an inverse association with Clostridia and Lachnospiraceae. This study identifies complex microbe-metabolite networks that may provide insights on neoplasia and targets for intervention.

Allergy associations with the adult fecal microbiota: Analysis of the American Gut Project.

BACKGROUND: Alteration of the gut microbial population (dysbiosis) may increase the risk for allergies and other conditions. This study sought to clarify the relationship of dysbiosis with allergies in adults. METHODS: Publicly available American Gut Project questionnaire and fecal 16S rRNA sequence data were analyzed. Fecal microbiota richness (number of observed species) and composition (UniFrac) were used to compare adults with versus without allergy to foods (peanuts, tree nuts, shellfish, other) and non-foods (drug, bee sting, dander, asthma, seasonal, eczema). Logistic and Poisson regression models adjusted for potential confounders. Odds ratios and 95% confidence intervals (CI) were calculated for lowest vs highest richness tertile. Taxonomy associations considered 122 non-redundant taxa (of 2379 total taxa) with ≥ 0.1% mean abundance. RESULTS: Self-reported allergy prevalence among the 1879 participants (mean age, 45.5 years; 46.9% male) was 81.5%, ranging from 2.5% for peanuts to 40.5% for seasonal. Fecal microbiota richness was markedly lower with total allergies (P = 10(-9)) and five particular allergies (P ≤ 10(-4)). Richness odds ratios were 1.7 (CI 1.3-2.2) with seasonal, 1.8 (CI 1.3-2.5) with drug, and 7.8 (CI 2.3-26.5) with peanut allergy. These allergic participants also had markedly altered microbial community composition (unweighted UniFrac, P = 10(-4) to 10(-7)). Total food and non-food allergies were significantly associated with 7 and 9 altered taxa, respectively. The dysbiosis was most marked with nut and seasonal allergies, driven by higher Bacteroidales and reduced Clostridiales taxa. INTERPRETATION: American adults with allergies, especially to nuts and seasonal pollen, have low diversity, reduced Clostridiales, and increased Bacteroidales in their gut microbiota. This dysbiosis might be targeted to improve treatment or prevention of allergy.

Collection media and delayed freezing effects on microbial composition of human stool.

BACKGROUND: Different bacteria in stool have markedly varied growth and survival when stored at ambient temperature. It is paramount to develop optimal biostabilization of stool samples during collection and assess long-term storage for clinical specimens and epidemiological microbiome studies. We evaluated the effect of collection media and delayed freezing up to 7 days on microbial composition. Ten participants collected triplicate stool samples each into no media as well as RNAlater® with and without kanamycin or ciprofloxacin. For each set of conditions, triplicate samples were frozen on dry ice immediately (time = 0) or frozen at -80 °C after 3-days and 7-days incubation at 25 °C. Microbiota metrics were estimated from Illumina MiSeq sequences of 16S rRNA gene fragments (V3-V4 region). Intraclass correlation coefficients (ICC) across triplicates, collection media, and incubation time were estimated for taxonomy and alpha and beta diversity metrics. RESULTS: RNAlater® alone yielded the highest ICCs for diversity metrics at time = 0 [ICC median 0.935 (range 0.89-0.97)], but ICCs varied greatly (range 0.44-1.0) for taxa with relative abundances <1%. The 3- and 7-day freezing delays were generally associated with stable beta diversity for all three media conditions. Freezing delay caused increased variance for Shannon index (median ICC 0.77) and especially for observed species abundance (median ICC 0.47). Variance in observed species abundance and in phylogenetic distance whole tree was similarly increased with a 7-day delay. Antibiotics did not mitigate variance. No media had inferior ICCs at time 0 and differed markedly from any media in microbiome composition (e.g., P =0.01 for relative abundance of Bacteroidetes). CONCLUSION: Bacterial community composition was stable for 7 days at room temperature in RNAlater® alone. RNAlater® provides some stability for beta diversity analyses, but analyses of rare taxa will be inaccurate if specimens are not frozen immediately. RNAlater® could be used as collection media with minimal change in the microbiota composition.