In vitro gut microbiome response to carbohydrate supplementation is acutely affected by a sudden change in diet.

BACKGROUND: Interactions between diet, stress and the gut microbiome are of interest as a means to modulate health and performance. Here, in vitro fermentation was used to explore the effects of a sudden change in diet, 21 days sole sustenance on the Meal, Ready-to-Eat (MRE) U.S. military combat ration, on inter-species competition and functional potential of the human gut microbiota. Human fecal samples collected before and after MRE intervention or consuming a habitual diet (HAB) were introduced to nutrient-rich media supplemented with starch for in vitro fermentation under ascending colon conditions. 16S rRNA amplicon and Whole-metagenome sequencing (WMS) were used to measure community composition and functional potential. Specific statistical analyses were implemented to detect changes in relative abundance from taxa, genes and pathways. RESULTS: Differential changes in relative abundance of 11 taxa, Dorea, Lachnospira, Bacteroides fragilis, Akkermansia muciniphila, Bifidobacterium adolescentis, Betaproteobacteria, Enterobacteriaceae, Bacteroides egerthii, Ruminococcus bromii, Prevotella, and Slackia, and nine Carbohydrate-Active Enzymes, specifically GH13_14, over the 24 h fermentation were observed as a function of the diet intervention and correlated to specific taxa of interest. CONCLUSIONS: These findings suggest that consuming MRE for 21 days acutely effects changes in gut microbiota structure in response to carbohydrate but may induce alterations in metabolic capacity. Additionally, these findings demonstrate the potential of starch as a candidate supplemental strategy to functionally modulate specific gut commensals during stress-induced states.

Association Between Sulfur-Metabolizing Bacterial Communities in Stool and Risk of Distal Colorectal Cancer in Men.

BACKGROUND & AIMS: Sulfur-metabolizing microbes, which convert dietary sources of sulfur into genotoxic hydrogen sulfide (H2S), have been associated with development of colorectal cancer (CRC). We identified a dietary pattern associated with sulfur-metabolizing bacteria in stool and then investigated its association with risk of incident CRC using data from a large prospective study of men. METHODS: We collected data from 51,529 men enrolled in the Health Professionals Follow-up Study since 1986 to determine the association between sulfur-metabolizing bacteria in stool and risk of CRC over 26 years of follow-up. First, in a subcohort of 307 healthy men, we profiled serial stool metagenomes and metatranscriptomes and assessed diet using semiquantitative food frequency questionnaires to identify food groups associated with 43 bacterial species involved in sulfur metabolism. We used these data to develop a sulfur microbial dietary score. We then used Cox proportional hazards modeling to evaluate adherence to this pattern among eligible individuals (n = 48,246) from 1986 through 2012 with risk for incident CRC. RESULTS: Foods associated with higher sulfur microbial diet scores included increased consumption of processed meats and low-calorie drinks and lower consumption of vegetables and legumes. Increased sulfur microbial diet scores were associated with risk of distal colon and rectal cancers, after adjusting for other risk factors (multivariable relative risk, highest vs lowest quartile, 1.43; 95% confidence interval 1.14-1.81; P-trend = .002). In contrast, sulfur microbial diet scores were not associated with risk of proximal colon cancer (multivariable relative risk 0.86; 95% CI 0.65-1.14; P-trend = .31). CONCLUSIONS: In an analysis of participants in the Health Professionals Follow-up Study, we found that long-term adherence to a dietary pattern associated with sulfur-metabolizing bacteria in stool was associated with an increased risk of distal CRC. Further studies are needed to determine how sulfur-metabolizing bacteria might contribute to CRC pathogenesis.

Associations between the gut microbiota and host responses to high altitude.

Hypobaric hypoxia and dietary protein and fat intakes have been independently associated with an altered gastrointestinal (GI) environment and gut microbiota, but little is known regarding host-gut microbiota interactions at high altitude (HA) and the impact of diet macronutrient composition. This study aimed to determine the effect of dietary protein:fat ratio manipulation on the gut microbiota and GI barrier function during weight loss at high altitude (HA) and to identify associations between the gut microbiota and host responses to HA. Following sea-level (SL) testing, 17 healthy males were transported to HA (4,300 m) and randomly assigned to consume provided standard protein (SP; 1.1 g·kg-1·day-1, 39% fat) or higher protein (HP; 2.1 g·kg-1·day-1, 23% fat) carbohydrate-matched hypocaloric diets for 22 days. Fecal microbiota composition and metabolites, GI barrier function, GI symptoms, and acute mountain sickness (AMS) severity were measured. Macronutrient intake did not impact fecal microbiota composition, had only transient effects on microbiota metabolites, and had no effect on increases in small intestinal permeability, GI symptoms, and inflammation observed at HA. AMS severity was also unaffected by diet but in exploratory analyses was associated with higher SL-relative abundance of Prevotella, a known driver of interindividual variability in human gut microbiota composition, and greater microbiota diversity after AMS onset. Findings suggest that the gut microbiota may contribute to variability in host responses to HA independent of the dietary protein:fat ratio but should be considered preliminary and hypothesis generating due to the small sample size and exploratory nature of analyses associating the fecal microbiota and host responses to HA. NEW & NOTEWORTHY This study is the first to examine interactions among diet, the gut microbiota, and host responses to weight loss at high altitude (HA). Observed associations among the gut microbiota, weight loss at HA, and acute mountain sickness provide evidence that the microbiota may contribute to variability in host responses to HA. In contrast, dietary protein:fat ratio had only minimal, transient effects on gut microbiota composition and bacterial metabolites which were likely not of clinical consequence.

Efficacy of a Sonicating Swab for Removal and Capture of Listeria monocytogenes in Biofilms on Stainless Steel.

Listeria monocytogenes is of great concern in food processing facilities because it persists in biofilms, facilitating biotransfer. Stainless steel is commonly used for food contact surfaces and transport containers. L. monocytogenes biofilms on stainless steel served as a model system for surface sampling, to test the performance of a sonicating swab in comparison with a standard cotton swab. Swab performance and consistency were determined using total viable counts. Stainless steel coupons sampled with both types of swabs were examined using scanning electron microscopy, to visualize biofilms and surface structures (i.e., polishing grooves and scratches). Laser scanning confocal microscopy was used to image and to quantitate the biofilms remaining after sampling with each swab type. The total viable counts were significantly higher (P ≤ 0.05) with the sonicating swab than with the standard swab in each trial. The sonicating swab was more consistent in cell recovery than was the standard swab, with coefficients of variation ranging from 8.9% to 12.3% and from 7.1% to 37.6%, respectively. Scanning electron microscopic imaging showed that biofilms remained in the polished grooves of the coupons sampled with the standard swab but were noticeably absent with the sonicating swab. Percent area measurements of biofilms remaining on the stainless steel coupons showed significantly (P ≤ 0.05) less biofilm remaining when the sonicating swab was used (median, 1.1%), compared with the standard swab (median, 70.4%). The sonicating swab provided greater recovery of cells, with more consistency, than did the standard swab, and it is employs sonication, suction, and scrubbing.IMPORTANCE Inadequate surface sampling can result in foodborne illness outbreaks from biotransfer, since verification of sanitization protocols relies on surface sampling and recovery of microorganisms for detection and enumeration. Swabbing is a standard method for microbiological sampling of surfaces. Although swabbing offers portability and ease of use, there are limitations, such as high user variability and low recovery rates, which can be attributed to many different causes. This study demonstrates some benefits that a sonicating swab has over a standard swab for removal and collection of microbiological samples from a surface, to provide better verification of surface cleanliness and to help decrease the potential for biotransfer of pathogens into foods.

Gut microbial metabolism of 5-ASA diminishes its clinical efficacy in inflammatory bowel disease.

For decades, variability in clinical efficacy of the widely used inflammatory bowel disease (IBD) drug 5-aminosalicylic acid (5-ASA) has been attributed, in part, to its acetylation and inactivation by gut microbes. Identification of the responsible microbes and enzyme(s), however, has proved elusive. To uncover the source of this metabolism, we developed a multi-omics workflow combining gut microbiome metagenomics, metatranscriptomics and metabolomics from the longitudinal IBDMDB cohort of 132 controls and patients with IBD. This associated 12 previously uncharacterized microbial acetyltransferases with 5-ASA inactivation, belonging to two protein superfamilies: thiolases and acyl-CoA N-acyltransferases. In vitro characterization of representatives from both families confirmed the ability of these enzymes to acetylate 5-ASA. A cross-sectional analysis within the discovery cohort and subsequent prospective validation within the independent SPARC IBD cohort (n = 208) found three of these microbial thiolases and one acyl-CoA N-acyltransferase to be epidemiologically associated with an increased risk of treatment failure among 5-ASA users. Together, these data address a longstanding challenge in IBD management, outline a method for the discovery of previously uncharacterized gut microbial activities and advance the possibility of microbiome-based personalized medicine.

Metatranscriptomics for the Human Microbiome and Microbial Community Functional Profiling.

Shotgun metatranscriptomics (MTX) is an increasingly practical way to survey microbial community gene function and regulation at scale. This review begins by summarizing the motivations for community transcriptomics and the history of the field. We then explore the principles, best practices, and challenges of contemporary MTX workflows: beginning with laboratory methods for isolation and sequencing of community RNA, followed by informatics methods for quantifying RNA features, and finally statistical methods for detecting differential expression in a community context. In thesecond half of the review, we survey important biological findings from the MTX literature, drawing examples from the human microbiome, other (nonhuman) host-associated microbiomes, and the environment. Across these examples, MTX methods prove invaluable for probing microbe-microbe and host-microbe interactions, the dynamics of energy harvest and chemical cycling, and responses to environmental stresses. We conclude with a review of open challenges in the MTX field, including making assays and analyses more robust, accessible, and adaptable to new technologies; deciphering roles for millions of uncharacterized microbial transcripts; and solving applied problems such as biomarker discovery and development of microbial therapeutics.

The Sulfur Microbial Diet and Risk of Colorectal Cancer by Molecular Subtypes and Intratumoral Microbial Species in Adult Men.

INTRODUCTION: We recently described the sulfur microbial diet, a pattern of intake associated with increased gut sulfur-metabolizing bacteria and incidence of distal colorectal cancer (CRC). We assessed whether this risk differed by CRC molecular subtypes or presence of intratumoral microbes involved in CRC pathogenesis (Fusobacterium nucleatum and Bifidobacterium spp.). METHODS: We performed Cox proportional hazards modeling to examine the association between the sulfur microbial diet and incidence of overall and distal CRC by molecular and microbial subtype in the Health Professionals Follow-Up Study (1986-2012). RESULTS: We documented 1,264 incident CRC cases among 48,246 men, approximately 40% of whom had available tissue data. After accounting for multiple hypothesis testing, the relationship between the sulfur microbial diet and CRC incidence did not differ by subtype. However, there was a suggestion of an association by prostaglandin synthase 2 (PTGS2) status with a multivariable adjusted hazard ratio for highest vs lowest tertile of sulfur microbial diet scores of 1.31 (95% confidence interval: 0.99-1.74, Ptrend = 0.07, Pheterogeneity = 0.04) for PTGS2-high CRC. The association of the sulfur microbial diet with distal CRC seemed to differ by the presence of intratumoral Bifidobacterium spp. with an adjusted hazard ratio for highest vs lowest tertile of sulfur microbial diet scores of 1.65 (95% confidence interval: 1.14-2.39, Ptrend = 0.01, Pheterogeneity = 0.03) for Bifidobacterium-negative distal CRC. We observed no apparent heterogeneity by other tested molecular markers. DISCUSSION: Greater long-term adherence to the sulfur microbial diet could be associated with PTGS2-high and Bifidobacterium-negative distal CRC in men. Additional studies are needed to further characterize the role of gut microbial sulfur metabolism and CRC.

Acute stressor alters inter-species microbial competition for resistant starch-supplemented medium.

Gut microbiome community dynamics are maintained by complex microbe-microbe and microbe-host interactions, which can be disturbed by stress. In vivo studies on the dynamics and manipulation of those interactions are costly and slow, but can be accelerated using in vitro fermentation. Herein, in vitro fermentation was used to determine how an acute stressor, a sudden change in diet, impacts inter-bacterial species competition for resistant starch-supplemented medium (RSM). Fermentation vessels were seeded with fecal samples collected from 10 individuals consuming a habitual diet or U.S. military rations for 21 days. Lactobacillus spp. growth in response to RSM was attenuated following ration consumption, whereas growth of Ruminococcus bromii was enhanced. These differences were not evident in the pre-fermentation samples. Findings demonstrate how incorporating in vitro fermentation into clinical studies can increase understanding of stress-induced changes in nutrient-microbiome dynamics, and suggest that sudden changes in diet may impact inter-species competition for substrates.

Metatranscriptome of human faecal microbial communities in a cohort of adult men.

The gut microbiome is intimately related to human health, but it is not yet known which functional activities are driven by specific microorganisms' ecological configurations or transcription. We report a large-scale investigation of 372 human faecal metatranscriptomes and 929 metagenomes from a subset of 308 men in the Health Professionals Follow-Up Study. We identified a metatranscriptomic 'core' universally transcribed over time and across participants, often by different microorganisms. In contrast to the housekeeping functions enriched in this core, a 'variable' metatranscriptome included specialized pathways that were differentially expressed both across participants and among microorganisms. Finally, longitudinal metagenomic profiles allowed ecological interaction network reconstruction, which remained stable over the six-month timespan, as did strain tracking within and between participants. These results provide an initial characterization of human faecal microbial ecology into core, subject-specific, microorganism-specific and temporally variable transcription, and they differentiate metagenomically versus metatranscriptomically informative aspects of the human faecal microbiome.