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Concept: Gut flora

237

Two recent studies have reanalyzed previously published data and found that when data sets were analyzed independently, there was limited support for the widely accepted hypothesis that changes in the microbiome are associated with obesity. This hypothesis was reconsidered by increasing the number of data sets and pooling the results across the individual data sets. The preferred reporting items for systematic reviews and meta-analyses guidelines were used to identify 10 studies for an updated and more synthetic analysis. Alpha diversity metrics and the relative risk of obesity based on those metrics were used to identify a limited number of significant associations with obesity; however, when the results of the studies were pooled by using a random-effect model, significant associations were observed among Shannon diversity, the number of observed operational taxonomic units, Shannon evenness, and obesity status. They were not observed for the ratio of Bacteroidetes and Firmicutes or their individual relative abundances. Although these tests yielded small P values, the difference between the Shannon diversity indices of nonobese and obese individuals was 2.07%. A power analysis demonstrated that only one of the studies had sufficient power to detect a 5% difference in diversity. When random forest machine learning models were trained on one data set and then tested by using the other nine data sets, the median accuracy varied between 33.01 and 64.77% (median, 56.68%). Although there was support for a relationship between the microbial communities found in human feces and obesity status, this association was relatively weak and its detection is confounded by large interpersonal variation and insufficient sample sizes.

Concepts: Bacteria, Gut flora, Taxonomy, Ratio, Diversity index, Shannon index, Measurement of biodiversity, Index numbers

227

Emerging evidence suggests that the in utero environment is not sterile as once presumed. Work in the mouse demonstrated transmission of commensal bacteria from mother to fetus during gestation, though it is unclear what modulates this process. We have previously shown in the nonhuman primate that, independent of obesity, a maternal high-fat diet during gestation and lactation persistently shapes the juvenile gut microbiome. We therefore sought to interrogate in a population-based human longitudinal cohort whether a maternal high-fat diet similarly alters the neonatal and infant gut microbiome in early life.

Concepts: Pregnancy, Childbirth, Infant, Human, Gut flora, Breastfeeding, Maternal bond, Mammal

227

The human intestinal microbiota is a crucial factor in the pathogenesis of various diseases, such as metabolic syndrome or inflammatory bowel disease (IBD). Yet, knowledge about the role of environmental factors such as smoking (which is known to influence theses aforementioned disease states) on the complex microbial composition is sparse. We aimed to investigate the role of smoking cessation on intestinal microbial composition in 10 healthy smoking subjects undergoing controlled smoking cessation.

Concepts: Immune system, Inflammation, Bacteria, Gut flora, Nutrition, Ulcerative colitis, Gastroenterology, Inflammatory bowel disease

218

In 1958, doctors in Denver administered feces by enema to their patients with fulminant, life-threatening pseudomembranous enterocolitis.(1) The goal of this infusion of donor feces (also termed fecal microbiota transplantation [FMT]) was to “re-establish the balance of nature” within the intestinal flora to correct the disruption caused by antibiotic treatment. They reported “immediate and dramatic” responses and concluded that “this simple yet rational therapeutic method should be given more extensive clinical evaluation.” During the ensuing 50 years, the association between Clostridium difficile infection and pseudomembranous enterocolitis was established, and effective antimicrobial treatments were identified. Despite these advances, C. difficile became . . .

Concepts: Bacteria, Gut flora, Antibiotic, Metronidazole, Probiotic, Clostridium difficile, Pseudomembranous colitis, Clostridium

214

OBJECTIVE: To characterise the influence of diet on abdominal symptoms, anal gas evacuation, intestinal gas distribution and colonic microbiota in patients complaining of flatulence. DESIGN: Patients complaining of flatulence (n=30) and healthy subjects (n=20) were instructed to follow their usual diet for 3 days (basal phase) and to consume a high-flatulogenic diet for another 3 days (challenge phase). RESULTS: During basal phase, patients recorded more abdominal symptoms than healthy subjects in daily questionnaires (5.8±0.3 vs 0.4±0.2 mean discomfort/pain score, respectively; p=<0.0001) and more gas evacuations by an event marker (21.9±2.8 vs 7.4±1.0 daytime evacuations, respectively; p=0.0001), without differences in the volume of gas evacuated after a standard meal (262±22 and 265±25 mL, respectively). On flatulogenic diet, both groups recorded more abdominal symptoms (7.9±0.3 and 2.8±0.4 discomfort/pain, respectively), number of gas evacuations (44.4±5.3 and 21.7±2.9 daytime evacuations, respectively) and had more gas production (656±52 and 673±78 mL, respectively; p<0.05 vs basal diet for all). When challenged with flatulogenic diet, patients' microbiota developed instability in composition, exhibiting variations in the main phyla and reduction of microbial diversity, whereas healthy subjects' microbiota were stable. Taxa from Bacteroides fragilis or Bilophila wadsworthia correlated with number of gas evacuations or volume of gas evacuated, respectively. CONCLUSIONS: Patients complaining of flatulence have a poor tolerance of intestinal gas, which is associated with instability of the microbial ecosystem.

Concepts: Bacteria, Gut flora, Fundamental physics concepts, Intestine, Flatulence, Plasma, Emergency evacuation, Bacteroides fragilis

202

Choosing the right nutrients to consume is essential to health and wellbeing across species. However, the factors that influence these decisions are poorly understood. This is particularly true for dietary proteins, which are important determinants of lifespan and reproduction. We show that in Drosophila melanogaster, essential amino acids (eAAs) and the concerted action of the commensal bacteria Acetobacter pomorum and Lactobacilli are critical modulators of food choice. Using a chemically defined diet, we show that the absence of any single eAA from the diet is sufficient to elicit specific appetites for amino acid (AA)-rich food. Furthermore, commensal bacteria buffer the animal from the lack of dietary eAAs: both increased yeast appetite and decreased reproduction induced by eAA deprivation are rescued by the presence of commensals. Surprisingly, these effects do not seem to be due to changes in AA titers, suggesting that gut bacteria act through a different mechanism to change behavior and reproduction. Thus, eAAs and commensal bacteria are potent modulators of feeding decisions and reproductive output. This demonstrates how the interaction of specific nutrients with the microbiome can shape behavioral decisions and life history traits.

Concepts: Protein, Gut flora, Amino acid, Acid, Amine, Nutrition, Ecology, Essential amino acid

202

The human gut harbors thousands of bacterial taxa. A profusion of metagenomic sequence data has been generated from human stool samples in the last few years, raising the question of whether more taxa remain to be identified. We assessed metagenomic data generated by the Human Microbiome Project Consortium to determine if novel taxa remain to be discovered in stool samples from healthy individuals. To do this, we established a rigorous bioinformatics pipeline that uses sequence data from multiple platforms (Illumina GAIIX and Roche 454 FLX Titanium) and approaches (whole-genome shotgun and 16S rDNA amplicons) to validate novel taxa. We applied this approach to stool samples from 11 healthy subjects collected as part of the Human Microbiome Project. We discovered several low-abundance, novel bacterial taxa, which span three major phyla in the bacterial tree of life. We determined that these taxa are present in a larger set of Human Microbiome Project subjects and are found in two sampling sites (Houston and St. Louis). We show that the number of false-positive novel sequences (primarily chimeric sequences) would have been two orders of magnitude higher than the true number of novel taxa without validation using multiple datasets, highlighting the importance of establishing rigorous standards for the identification of novel taxa in metagenomic data. The majority of novel sequences are related to the recently discovered genus Barnesiella, further encouraging efforts to characterize the members of this genus and to study their roles in the microbial communities of the gut. A better understanding of the effects of less-abundant bacteria is important as we seek to understand the complex gut microbiome in healthy individuals and link changes in the microbiome to disease.

Concepts: Archaea, Health, Bacteria, Gut flora, Microbiology, Full genome sequencing, Microorganism, Human flora

189

Honey bee pollination is a key ecosystem service to nature and agriculture. However, biosafety research on genetically modified crops rarely considers effects on nurse bees from intact colonies, even though they receive and primarily process the largest amount of pollen. The objective of this study was to analyze the response of nurse bees and their gut bacteria to pollen from Bt maize expressing three different insecticidal Cry proteins (Cry1A.105, Cry2Ab2, and Cry3Bb1). Naturally Cry proteins are produced by bacteria (Bacillus thuringiensis). Colonies of Apis mellifera carnica were kept during anthesis in flight cages on field plots with the Bt maize, two different conventionally bred maize varieties, and without cages, 1-km outside of the experimental maize field to allow ad libitum foraging to mixed pollen sources. During their 10-days life span, the consumption of Bt maize pollen had no effect on their survival rate, body weight and rates of pollen digestion compared to the conventional maize varieties. As indicated by ELISA-quantification of Cry1A.105 and Cry3Bb1, more than 98% of the recombinant proteins were degraded. Bacterial population sizes in the gut were not affected by the genetic modification. Bt-maize, conventional varieties and mixed pollen sources selected for significantly different bacterial communities which were, however, composed of the same dominant members, including Proteobacteria in the midgut and Lactobacillus sp. and Bifidobacterium sp. in the hindgut. Surprisingly, Cry proteins from natural sources, most likely B. thuringiensis, were detected in bees with no exposure to Bt maize. The natural occurrence of Cry proteins and the lack of detectable effects on nurse bees and their gut bacteria give no indication for harmful effects of this Bt maize on nurse honey bees.

Concepts: DNA, Bacteria, Gut flora, Insect, Escherichia coli, Honey bee, Beekeeping, Genetically modified food

187

Walnuts are rich in omega-3 fatty acids, phytochemicals and antioxidants making them unique compared to other foods. Consuming walnuts has been associated with health benefits including a reduced risk of heart disease and cancer. Dysbiosis of the gut microbiome has been linked to several chronic diseases. One potential mechanism by which walnuts may exert their health benefit is through modifying the gut microbiome. This study identified the changes in the gut microbial communities that occur following the inclusion of walnuts in the diet. Male Fischer 344 rats (n=20) were randomly assigned to one of two diets for as long as 10 weeks: (1) walnut (W), and (2) replacement ® in which the fat, fiber, and protein in walnuts were matched with corn oil, protein casein, and a cellulose fiber source. Intestinal samples were collected from the descending colon, the DNA isolated, and the V3-V4 hypervariable region of 16S rRNA gene deep sequenced on an Illumina MiSeq for characterization of the gut microbiota. Body weight and food intake did not differ significantly between the two diet groups. The diet groups had distinct microbial communities with animals consuming walnuts displaying significantly greater species diversity. Walnuts increased the abundance of Firmicutes and reduced the abundance of Bacteriodetes. Walnuts enriched the microbiota for probiotic-type bacteria including Lactobacillus, Ruminococcaceae, and Roseburia while significantly reducing Bacteroides and Anaerotruncus. The class Alphaproteobacteria was also reduced. Walnut consumption altered the gut microbial community suggesting a new mechanism by which walnuts may confer their beneficial health effects.

Concepts: Bacteria, Gut flora, Nutrition, Fatty acid, Microbiology, Ribosomal RNA, Omega-3 fatty acid, 16S ribosomal RNA

187

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembraneous colitis and is responsible for a large and increasing fraction of hospital-acquired infections. Fecal microbiota transplantation (FMT) is an alternate treatment option for recurrent C. difficile infection (RCDI) refractory to antibiotic therapy. It has recently been discussed favorably in the clinical and scientific communities and is receiving increasing public attention. However, short- and long-term health consequences of FMT remain a concern, as the effects of the transplanted microbiota on the patient remain unknown. To shed light on microbial events associated with RCDI and treatment by FMT, we performed fecal microbiota analysis by 16S rRNA gene amplicon pyrosequencing of 14 pairs of healthy donors and RCDI patients treated successfully by FMT. Post-FMT patient and healthy donor samples collected up to one year after FMT were studied longitudinally, including one post-FMT patient with antibiotic-associated relapse three months after FMT. This analysis allowed us not only to confirm prior reports that RCDI is associated with reduced diversity and compositional changes in the fecal microbiota, but also to characterize previously undocumented post-FMT microbiota dynamics. Members of the Streptococcaceae, Enterococcaceae, or Enterobacteriaceae were significantly increased and putative butyrate producers, such as Lachnospiraceae and Ruminococcaceae were significantly reduced in samples from RCDI patients before FMT as compared to post-FMT patient and healthy donor samples. RCDI patient samples showed more case-specific variations than post-FMT patient and healthy donor samples. However, none of the bacterial groups were invariably associated with RCDI or successful treatment by FMT. Overall microbiota compositions in post-FMT patients, specifically abundances of the above-mentioned Firmicutes, continued to change for at least 16 weeks after FMT, suggesting that full microbiota recovery from RCDI may take much longer than expected based on the disappearance of diarrheal symptoms immediately after FMT.

Concepts: Bacteria, Gut flora, Ribosomal RNA, Antibiotic resistance, 16S ribosomal RNA, Antibiotic, Clostridium difficile, Diarrhea