Composition Of Human Microbiota Research Articles

Associations of antibiotic use with risk of primary liver cancer in the Clinical Practice Research Datalink.

Background:Use of antibiotics could alter human microbiota composition and decrease bacterial diversity. Such microbial dysbiosis may have implications in hepatocarcinogenesis; however, the association between antibiotic use and liver cancer risk has been minimally examined in humans.Methods:We performed a nested case–control study (1195 primary liver cancer cases and 4640 matched controls) within the United Kingdom's Clinical Practice Research Datalink. Antibiotic use was obtained from prescription records. Multivariable-adjusted odds ratio (OR) and 95% confidence interval (CI) were estimated using conditional logistic regression.Results:Ever-use of prescription antibiotics was associated with a slightly increased risk of liver cancer, compared to non-use (OR=1.22, 95% CI=1.03−1.45). However, there was no clear dose–response relationship by the number of prescriptions or cumulative dose of antibiotic use, suggesting a non-causal association.Conclusions:Our results do not support a role of antibiotic use in liver cancer development.

Support for the Microgenderome: Associations in a Human Clinical Population.

The ‘microgenderome’ provides a paradigm shift that highlights the role of sex differences in the host-microbiota interaction relevant for autoimmune and neuro-immune conditions. Analysis of cross-sectional self-report and faecal microbial data from 274 patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) suggests that commensal gut microorganisms may play both protective and deleterious roles in symptom expression. Results revealed significant sex-specific interactions between Firmicutes (Clostridium, Streptococcus, Lactobacillus and Enterococcus) and ME/CFS symptoms (including neurological, immune and mood symptoms), regardless of compositional similarity in microbial levels across the sexes. Extending animal studies, we provide support for the microgenderome in a human clinical population. Applied and mechanistic research needs to consider sex-interactions when examining the composition and function of human microbiota.

Patterns of Antibiotic Exposure and Clinical Disease Activity in Inflammatory Bowel Disease: A 4-year Prospective Study.

Antimicrobial treatment is known to cause short- and long-term changes in the composition of normal human microbiota. The relationship between antibiotic use and overall clinical behavior in inflammatory bowel disease (IBD) has not been explored. We aim to prospectively characterize patterns of antibiotic use and clinical IBD activity in a large IBD cohort. Prospective observational study from a longitudinal IBD natural history registry between 2009 and 2012. Antibiotic prescriptions were identified and categorized using electronic medical record data. Cumulative rates over the 4-year study period were compared. Demographic, clinical, laboratory, health care utilization, and treatment data of the patients with IBD were collected and analyzed. Quality of life was measured by Short IBD Questionnaire data. Primary outcomes were markers of disease activity including Short IBD Questionnaire scores, C-reactive protein levels, health care utilization, and medication use. Seven hundred eighteen patients followed over 4 years were included (47.6% male; mean age, 46.7 ± 15.2 yr), 59.9% had Crohn's disease, whereas 38.6% had ulcerative colitis. Most patients (66.3%) were exposed to antibiotics during the study period. Antibiotic-exposed patients were more likely to have Crohn's disease (63% versus 53.7%; P = 0.05), require narcotics (43.7% versus 14.9%; P < 0.0001), receive antidepressants (43.1% versus 18.6%; P < 0.001), prednisone (52.7% versus 31%; P < 0.0001), or biological therapy (52% versus 36.5%; P < 0.0001). Antibiotic-exposed patients had a lower mean Short IBD Questionnaire (50.2 ± 11.5 versus 56.4 ± 9.5; P < 0.0001), higher rates of C-reactive protein elevation (49.2% versus 31.8%; P < 0.0001), and higher health care utilization compared with nonantibiotic-exposed patients. The majority of patients with IBD receive antibiotic treatment, and these individuals demonstrate a more severe clinical course.

Composition of human faecal microbiota in resistance to Campylobacter infection

In mice, specific species composition of gut microbiota enhances susceptibility to Campylobacter jejuni but little is known about the specific composition of the human gut microbiota in providing protection from infections caused by enteropathogens. Healthy adult individuals, who travelled in groups from Sweden to destinations with an estimated high risk for acquisition of Campylobacter infection, were enrolled. Faecal samples, collected before travelling and after returning home, were cultured for bacterial enteropathogens, and analysed for Campylobacter by PCR and for the species composition of the microbiota by 16S amplicon massive parallel sequencing. The microbiota compositions were compared between persons who became infected during their travel and those who did not. A total of 63 participants completed the study; 14 became infected with Campylobacter, two with Salmonella and 47 remained negative for the enteropathogens tested. After exclusion of samples taken after antimicrobial treatment, 49 individuals were included in the final analyses. Intra-individual stability of the microbiota was demonstrated for samples taken before travelling. The original diversity of the faecal microbiota was significantly lower among individuals who later became infected compared with those who remained uninfected. The relative abundances of bacteria belonging to the family Lachnospiraceae, and more specifically its two genera Dorea and Coprococcus, were significantly higher among those who remained uninfected. The travel-related infection did not significantly modify the faecal microbiota composition. Species composition of human gut microbiota is important for colonization resistance to Campylobacter infection. Especially individuals with a lower diversity are more susceptible to Campylobacter infection.

In vitro characterization of the impact of different substrates on metabolite production, energy extraction and composition of gut microbiota from lean and obese subjects.

The aim of this study was to investigate the effect of galacto-oligosaccharides, lactulose, apple fiber and sugar beet pectin on the composition and activity of human colonic microbiota of lean and obese healthy subjects using an in vitro model of the proximal colon: TIM-2. Substrate fermentation was assessed by measuring the production of short-chain and branched-chain fatty acids, lactate and ammonia and by studying the composition of the bacterial communities over time. The results suggest that energy harvest (in terms of metabolites) of lean and obese microbiotas is different and may depend on the fermentable substrate. For galacto-oligosaccharides and lactulose, the cumulative amount of short-chain fatty acids plus lactate produced in TIM-2 was lower in the fermentation experiments with the lean microbiota (123 and 155 mmol, respectively) compared to the obese (162 and 173 mmol, respectively). This was reversed for the pectin and the fiber. The absolute amount produced of short-chain fatty acids including lactate was higher after 72 h in the fermentation experiments with apple fiber-L (108 mmol) than with apple fiber-O (92 mmol). Sugar beet-L was also higher (130 mmol) compared to sugar beet-O (103 mmol). Galacto-oligosaccharides and lactulose boosted the balance of health-promoting over toxic metabolites produced by the microbiota from obese subjects. Firmicutes were more predominant in the inoculum prepared from feces of obese subjects compared to lean subjects. The average abundance at time zero was 92% and 74%, respectively. On the other hand, Bacteroidetes were more dominant in the microbiota prepared with homogenates from lean subjects with an average abundance of 22% compared with the microbiota prepared with homogenates from obese subjects (3.6%). This study brings evidence that different fermentable carbohydrates are fermented differently by lean and obese microbiotas, which contributes to the understanding of the role of diet and the microbiota in tackling obesity.

Smoking cessation alters intestinal microbiota: insights from quantitative investigations on human fecal samples using FISH.

There has been a dramatic increase in investigations on the potential mechanistic role of the intestinal microbiota in various diseases and factors modulating intestinal microbial composition. We recently reported on intestinal microbial shifts after smoking cessation in humans. In this study, we aimed to conduct further microbial analyses and verify our previous results obtained by pyrosequencing using a direct quantitative microbial approach. Stool samples of healthy smoking human subjects undergoing controlled smoking cessation during a 9-week observational period were analyzed and compared with 2 control groups, ongoing smoking and nonsmoking subjects. Fluorescence in situ hybridization was applied to quantify specific bacterial groups. Intestinal microbiota composition was substantially altered after smoking cessation as characterized by an increase in key representatives from the phyla of Firmicutes (Clostridium coccoides, Eubacterium rectale, and Clostridium leptum subgroup) and Actinobacteria (HGC bacteria and Bifidobacteria) as well as a decrease in Bacteroidetes (Prevotella spp. and Bacteroides spp.) and Proteobacteria (β- and γ-subgroup of Proteobacteria). As determined by fluorescence in situ hybridization, an independent direct quantitative microbial approach, we could confirm that intestinal microbiota composition in humans is influenced by smoking. The characteristics of observed microbial shifts suggest a potential mechanistic association to alterations in body weight subsequent to smoking cessation. More importantly, regarding previously described microbial hallmarks of dysbiosis in inflammatory bowel diseases, a variety of observed microbial alterations after smoking cessation deserve further consideration in view of the divergent effect of smoking on the clinical course of Crohn's disease and ulcerative colitis.

Correlation network analysis reveals relationships between diet-induced changes in human gut microbiota and metabolic health.

Background:Recent evidence suggests that the gut microbiota plays an important role in human metabolism and energy homeostasis and is therefore a relevant factor in the assessment of metabolic health and flexibility. Understanding of these host–microbiome interactions aids the design of nutritional strategies that act via modulation of the microbiota. Nevertheless, relating gut microbiota composition to host health states remains challenging because of the sheer complexity of these ecosystems and the large degrees of interindividual variation in human microbiota composition.Methods:We assessed fecal microbiota composition and host response patterns of metabolic and inflammatory markers in 10 apparently healthy men subjected to a high-fat high-caloric diet (HFHC, 1300 kcal/day extra) for 4 weeks. DNA was isolated from stool and barcoded 16S rRNA gene amplicons were sequenced. Metabolic health parameters, including anthropomorphic and blood parameters, where determined at t=0 and t=4 weeks.Results:A correlation network approach revealed diet-induced changes in Bacteroides levels related to changes in carbohydrate oxidation rates, whereas the change in Firmicutes correlates with changes in fat oxidation. These results were confirmed by multivariate models. We identified correlations between microbial diversity indices and several inflammation-related host parameters that suggest a relation between diet-induced changes in gut microbiota diversity and inflammatory processes.Conclusions:This approach allowed us to identify significant correlations between abundances of microbial taxa and diet-induced shifts in several metabolic health parameters. Constructed correlation networks provide an overview of these relations, revealing groups of correlations that are of particular interest for explaining host health aspects through changes in the gut microbiota.

Maillard reaction products modulate gut microbiota composition in adolescents.

Scarce data are available concerning effects of certain bioactive substances such as Maillard reaction products (MRP) on the gut microbiota composition, and the question of how a diet rich in MRP affects gut microbiota in humans is still open. Two experiments were conducted. In expt. 1, adolescents consumed diets either high or low in MRP in a two-period crossover trial; in expt. 2, rats were fed diets supplemented or not with MRP model-systems. Intestinal microbiota composition in fecal (adolescents) or cecal (rat) samples was assessed by qPCR analysis. Negative correlations were found in the human assay between lactobacilli numbers and dietary advanced MRP (r = -0.418 and -0.387, for hydroxymethylfurfural and carboxymethyl-lysine respectively, p < 0.05), whereas bifidobacteria counts were negatively correlated with Amadori compounds intake. In the rat assay, total bacteria and lactobacilli were negatively correlated with MRP intake (r = -0.674,-0.675 and -0.676, for Amadori compounds, hydroxymethylfurfural and carboxymethyl-lysine respectively, p < 0.05), but no correlations were found with bifidobacteria. Dietary MRP are able to modulate in vivo the intestinal microbiota composition both in humans and in rats, and the specific effects are likely to be linked to the chemical structure and dietary amounts of the different browning compounds.

Does the change on gastrointestinal tract microbiome affects host?

During the past decade, studies on the composition of human microbiota and its relation to the host became one of the most explored subjects of the medical literature. The development of high-throughput molecular technologies allowed a deeper characterization of human microbiota and a better understanding of its relationship with health and disease. Changes in human habits including wide use of antimicrobials can result in dysregulation of host–microbiome homeostasis, with multiple consequences. The purpose of this review is to highlight the most important evidence in the literature of host–microbiome interactions and illustrate how these intriguing relations may lead to new treatment and prevention strategies.

Research highlights for issue 5: the role of the microbiome in shaping evolution.

Research highlights for issue 5: the role of the microbiome in shaping evolution.

Xylooligosaccharide increases bifidobacteria but not lactobacilli in human gut microbiota

This study was conducted to determine the tolerance and effects of the prebiotic xylooligosaccharide (XOS) on the composition of human colonic microbiota, pH and short chain fatty acids (SCFA) in order to determine whether significant changes in the microbiota would be achievable without side effects. Healthy adult subjects (n = 32) were recruited in a double-blind, randomized, placebo-controlled study. Subjects received 1.4 g XOS, 2.8 g XOS or placebo in daily doses. The study consisted of a 2 week run-in, an 8 week intervention, and a 2 week washout phase. Stool samples were collected at baseline, after 4 and 8 weeks of intervention and 2 weeks after cessation of intervention. Samples were subjected to culture, pyrosequencing of community DNA, pH and SCFA analyses. Tolerance was evaluated by daily symptom charts. XOS was tolerated without significant gastrointestinal side effects. Bifidobacterium counts increased in both XOS groups compared to the placebo subjects, the 2.8 g per day group showed significantly greater increases than the 1.4 g per day group. Total anaerobic counts and Bacteroides fragilis group counts were significantly higher in the 2.8 g per day XOS group. There were no significant differences in the counts of Lactobacillus, Enterobacteriaceae and Clostridium between the three groups. XOS intervention had no significant effect on stool pH, SCFA or lactic acid. Pyrosequencing showed no notable shifts in bacterial diversity. XOS supplementation may be beneficial to gastrointestinal microbiota and 2.8 g per day may be more effective than 1.4 g per day. The low dose required and lack of gastrointestinal side effects makes the use of XOS as a food supplement feasible.

Association of dietary type with fecal microbiota in vegetarians and omnivores in Slovenia

The purpose of this study was to discover differences in the human fecal microbiota composition driven by long-term omnivore versus vegan/lacto-vegetarian dietary pattern. In addition, the possible association of demographic characteristics and dietary habits such as consumption of particular foods with the fecal microbiota was examined. This study was conducted on a Slovenian population comprising 31 vegetarian participants (11 lacto-vegetarians and 20 vegans) and 29 omnivore participants. Bacterial DNA was extracted from the frozen fecal samples by Maxwell 16 Tissue DNA Purification Kit (Promega). Relative quantification of selected bacterial groups was performed by real-time PCR. Differences in fecal microbiota composition were evaluated by PCR-DGGE fingerprinting of the V3 16S rRNA region. Participants' demographic characteristics, dietary habits and health status information were collected through a questionnaire. Vegetarian diet was associated with higher ratio (% of group-specific DNA in relation to all bacterial DNA) of Bacteroides-Prevotella, Bacteroides thetaiotaomicron, Clostridium clostridioforme and Faecalibacterium prausnitzii, but with lower ratio (%) of Clostridium cluster XIVa. Real-time PCR also showed a higher concentration and ratio of Enterobacteriaceae (16S rDNA copies/g and %) in female participants (p<0.05 and p<0.01) and decrease in Bifidobacterium with age (p<0.01). DGGE analysis of the 16S rRNA V3 region showed that relative quantity of DGGE bands from certain bacterial groups was lower (Bifidobacterium, Streptococus, Collinsella and Lachnospiraceae) or higher (Subdoligranulum) among vegetarians, indicating the association of dietary type with bacterial community composition. Sequencing of selected DGGE bands revealed the presence of common representatives of fecal microbiota: Bacteroides, Eubacterium, Faecalibacterium, Ruminococcaceae, Bifidobacterium and Lachnospiraceae. Up to 4% of variance in microbial community analyzed by DGGE could be explained by the vegetarian type of diet. Long-term vegetarian diet contributed to quantity and associated bacterial community shifts in fecal microbiota composition. Consumption of foods of animal origin (eggs, red meat, white meat, milk, yoghurt, other dairy products, fish and seafood) and vegetarian type of diet explained the largest share of variance in microbial community structure. Fecal microbiota composition was also associated with participants' age, gender and body mass.

PS14 - 68. Differential effects of antibiotics on bile acid metabolism, intestinal microbiota composition and insulin resistance in obese humans; a randomised controlled trial

Recent data in animal models reveal that obesity is associated with substantial changes in composition and metabolic function of gut microbiota.

Effects of administration of Bifidobacterium animalis subsp. lactis GCL2505 on defecation frequency and bifidobacterial microbiota composition in humans

The aim of this study was to evaluate the changes in endogenous bifidobacteria and administered Bifidobacterium animalis subsp. lactis (B. lactis) GCL2505 (GCL2505) in the intestine after administration of GCL2505 by means of a randomized, placebo-controlled double-blind, cross-over study. An increase in the number of total bifidobacteria (the sum of B. bifidum, B. breve, B. longum subsp. longum, B. adolescentis, B. anglatum, B. catenulatum, B. pseudocatenulatum, B. dentium, B. longum subsp. infantis and B. lactis) in the feces were observed after administration of GCL2505 using species- and subspecies-specific real-time polymerase chain reaction analysis. However, the number of endogenous bifidobacteria species (excluding B. lactis) remained unchanged. B. lactis also became the predominant bifidobacterial species. Taking into account the number of GCL2505 administered, the findings further suggested that GCL2505 proliferated in the intestine. In addition, the defecation frequency increased during GCL2505 administration compared with the placebo. Moreover, a single administration study (n=17) clearly demonstrated that GCL2505 successfully reached the intestine before proliferating at least 10-fold. This is the first report to show an increase in intestinal bifidobacteria, with no changes to the endogenous species, and improvements in constipation following proliferation of administered bifidobacteria.

Composition of Human Skin Microbiota Affects Attractiveness to Malaria Mosquitoes

The African malaria mosquito Anopheles gambiae sensu stricto continues to play an important role in malaria transmission, which is aggravated by its high degree of anthropophily, making it among the foremost vectors of this disease. In the current study we set out to unravel the strong association between this mosquito species and human beings, as it is determined by odorant cues derived from the human skin. Microbial communities on the skin play key roles in the production of human body odour. We demonstrate that the composition of the skin microbiota affects the degree of attractiveness of human beings to this mosquito species. Bacterial plate counts and 16S rRNA sequencing revealed that individuals that are highly attractive to An. gambiae s.s. have a significantly higher abundance, but lower diversity of bacteria on their skin than individuals that are poorly attractive. Bacterial genera that are correlated with the relative degree of attractiveness to mosquitoes were identified. The discovery of the connection between skin microbial populations and attractiveness to mosquitoes may lead to the development of new mosquito attractants and personalized methods for protection against vectors of malaria and other infectious diseases.

Resistant Starches Types 2 and 4 Have Differential Effects on the Composition of the Fecal Microbiota in Human Subjects

BackgroundTo systematically develop dietary strategies based on resistant starch (RS) that modulate the human gut microbiome, detailed in vivo studies that evaluate the effects of different forms of RS on the community structure and population dynamics of the gut microbiota are necessary. The aim of the present study was to gain a community wide perspective of the effects of RS types 2 (RS2) and 4 (RS4) on the fecal microbiota in human individuals.Methods and FindingsTen human subjects consumed crackers for three weeks each containing either RS2, RS4, or native starch in a double-blind, crossover design. Multiplex sequencing of 16S rRNA tags revealed that both types of RS induced several significant compositional alterations in the fecal microbial populations, with differential effects on community structure. RS4 but not RS2 induced phylum-level changes, significantly increasing Actinobacteria and Bacteroidetes while decreasing Firmicutes. At the species level, the changes evoked by RS4 were increases in Bifidobacterium adolescentis and Parabacteroides distasonis, while RS2 significantly raised the proportions of Ruminococcus bromii and Eubacterium rectale when compared to RS4. The population shifts caused by RS4 were numerically substantial for several taxa, leading for example, to a ten-fold increase in bifidobacteria in three of the subjects, enriching them to 18–30% of the fecal microbial community. The responses to RS and their magnitudes varied between individuals, and they were reversible and tightly associated with the consumption of RS.ConclusionOur results demonstrate that RS2 and RS4 show functional differences in their effect on human fecal microbiota composition, indicating that the chemical structure of RS determines its accessibility by groups of colonic bacteria. The findings imply that specific bacterial populations could be selectively targeted by well designed functional carbohydrates, but the inter-subject variations in the response to RS indicates that such strategies might benefit from more personalized approaches.

Mode of delivery affects the bacterial community in the newborn gut

The first colonisation of the intestine is one of the most profound immunological exposures faced by the newborn and it is influenced by external and internal factors. The early composition of human microbiota could have long-lasting metabolic effects and the initial composition of human intestinal bacteria is also known to affect postnatal immune system development, as we are already aware that reduced microbial stimulation during infancy would result in slower postnatal maturation of the immune system and development of an optimal balance between TH1 and TH2-like immunity. Mode of delivery has a major role on the composition of intestinal microbiota in early infancy, as it has been shown that infants born by Caesarean section (CS) have lower numbers of Bifidobacteria and Bacteroides compared with vaginally born infants. We designed a study to investigate the influence of mode of delivery (CS vs. vaginal delivery) on intestinal microbial composition on day 3 of life using PCR-denaturing gradient gel electrophoresis (DGGE) and PCR-temperature gradient gel electrophoresis (TGGE). Both DGGE and TGGE analyses have been used, together with the specific amplifications for 10 Bifidobacterium sp., 3 Ruminococcus sp., and Bacteroides that all have a highly relevant physiological role in the intestinal ecosystem of the newborn. A total of 46 term infants were enrolled in the study, consecutively recruiting all the CS-delivered babies (n = 23; 8 males and 15 females) and the immediately following spontaneously delivered babies (n = 23; 11 males and 12 females). DGGE analysis carried out with Bifidobacterium-specific primers revealed the presence of this genus in 13 of 23 (56.5%) samples derived from vaginally delivered newborns but in none of the samples obtained from newborns delivered by CS. PCR analysis with Bifidobacterium-species-specific primers showed that naturally delivered infants had a large number of bifidobacterial species, whereas in CS-delivered babies only two samples (8.7%) gave positive results, one for B. longum and another for B. gallicum. In all babies enrolled, micro-organisms belonging to Ruminococcus species were absent and Bacteroides was found in 8.7% of spontaneously delivered babies only. Based on our findings, it seems that newborn's intestinal bacteria during the first 3 days of life are strongly influenced by mode of delivery. The intestinal flora of CS and vaginally delivered infants appears to be very different; the former being altered and characterised by a substantial absence of Bifidobacteria sp., the latter characterised by subject-specific microbial profiles, although predominant groups such as B. longum and B. catenulatum could be identified. In summary, mode of delivery does affect the early stage of intestinal bacterial colonisation, which is altered in CS-delivered infants compared with vaginally delivered infants, with only a minor influence of the type of feeding. In addition, the importance of methodological aspects for determining intestinal microbiota in clinical trials requires emphasis if intestinal microbiota composition is to be considered a measure of postnatal adaptation.

Microbiota and SCFA in Lean and Overweight Healthy Subjects

Obesity has recently been linked to the composition of human microbiota and the production of short chain fatty acids (SCFAs). However, these findings rely on experimental studies carried out using rather small and defined groups of volunteers or model animals. Our aim was to evaluate differences within the human intestinal microbiota and fecal SCFA concentration of lean and obese subjects. A total of 98 subjects volunteered to take part in this study. The BMI in kg/m(2) of 30 volunteers was within the lean range, 35 were overweight and 33 were obese. The fecal microbiota was characterized by real-time PCR analyses. With the primers used herein we were able to cover 82.3% (interquartile range of 68.3-91.4%) of the total microbiota detectable with a universal primer. In addition, the concentration of SCFA was evaluated. The total amount of SCFA was higher in the obese subject group (P = 0.024) than in the lean subject group. The proportion of individual SCFA changed in favor of propionate in overweight (P = 0.019) and obese subjects (P = 0.028). The most abundant bacterial groups in faeces of lean and obese subjects belonged to the phyla Firmicutes and Bacteroidetes. The ratio of Firmicutes to Bacteroidetes changed in favor of the Bacteroidetes in overweight (P = 0.001) and obese subjects (P = 0.005). Our results are in line with previous reports suggesting that SCFA metabolism might play a considerable role in obesity. However, our results contradict previous reports with regard to the contribution of various bacterial groups to the development of obesity and this issue remains controversial.

Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures

Exopolysaccharides (EPS) isolated from two Bifidobacterium strains, one of human intestinal origin ( Bifidobacterium longum subsp. longum IPLA E44) and the other from dairy origin ( Bifidobacterium animalis subsp. lactis IPLA R1), were subjected to in vitro chemically simulated gastrointestinal digestion, which showed the absence of degradation of both polymers in these conditions. Polymers were then used as carbon sources in pH-controlled faecal batch cultures and compared with the non-prebiotic carbohydrate glucose and the prebiotic inulin to determine changes in the composition of faecal bacteria. A set of eight fluorescent in situ hybridisation oligonucleotide probes targeting 16S rRNA sequences was used to quantify specific groups of microorganisms. Growth of the opportunistic pathogen Clostridium histolyticum occurred with all carbohydrates tested similarly to that found in negative control cultures without added carbohydrate and was mainly attributed to the culture conditions used rather than enhancement of growth by these substrates. Polymers E44 and R1 stimulated growth of Lactobacillus / Enterococcus, Bifidobacterium, and Bacteroides / Prevotella in a similar way to that seen with inulin. The EPS R1 also promoted growth of the Atopobium cluster during the first 24 h of fermentation. An increase in acetic and lactic acids was found during early stages of fermentation (first 10–24 h) correlating with increases of Lactobacillus, Bifidobacterium, and Atopobium. Propionic acid concentrations increased in old cultures, which was coincident with the enrichment of Clostridium cluster IX in cultures with EPS R1 and with the increases in Bacteroides in cultures with both microbial EPS (R1 and E44) and inulin. The lowest acetic to propionic acid ratio was obtained for EPS E44. None of the carbohydrates tested supported the growth of microorganisms from Clostridium clusters XIVa+b and IV, results that correlate with the poor butyrate production in the presence of EPS. Thus, EPS synthesized by bifidobacteria from dairy and intestinal origins can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of short chain fatty acids.

Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults

In this paper we present the in silico assessment of the diversity of variable regions of the small subunit ribosomal RNA (SSU rRNA) gene based on an ecosystem-specific curated database, describe a probe design procedure based on two hypervariable regions with minimal redundancy and test the potential of such probe design strategy for the design of a flexible microarray platform. This resulted in the development and application of a phylogenetic microarray for studying the human gastrointestinal microbiota – referred as the human intestinal tract chip (HITChip). Over 4800 dedicated tiling oligonucleotide probes were designed based on two hypervariable regions of the SSU rRNA gene of 1140 unique microbial phylotypes (< 98% identity) following analysis of over 16 000 human intestinal SSU rRNA sequences. These HITChip probes were hybridized to a diverse set of human intestinal samples and SSU rRNA clones to validate its fingerprinting and quantification potential. Excellent reproducibility (median Pearson's correlation of 0.99) was obtained following hybridization with T7 polymerase transcripts generated in vitro from SSU rRNA gene amplicons. A linear dose–response was observed with artificial mixtures of 40 different representative amplicons with relative abundances as low as 0.1% of total microbiota. Analysis of three consecutively collected faecal samples from ten individuals (five young and five elderly adults) revealed temporal dynamics and confirmed that the adult intestinal microbiota is an individual-specific and relatively stable ecosystem. Further analysis of the stable part allowed for the identification of a universal microbiota core at the approximate genus level (90% sequence similarity). This core consists of members of Actinobacteria, Bacteroidetes and Firmicutes. Used as a phylogenetic fingerprinting tool with the possibility for relative quantification, the HITChip has the potential to bridge the gaps in our knowledge in the quantitative and qualitative description of the human gastrointestinal microbiota composition.