The species composition of the human intestinal microbiota differs between particle-associated and liquid phase communities

Many of the substrates available as energy sources for microorganisms in the human colon, including dietary plant fibre and secreted mucin, are insoluble. It seems likely that such insoluble substrates support a specialized microbiota, and in order to test this hypothesis, faecal samples from four healthy subjects were fractionated into insoluble (washed particulate) and liquid fractions. Analysis of 1252 PCR-amplified 16S rRNA sequences revealed a significantly lower percentage of Bacteroidetes (P = 0.021) and a significantly higher percentage of Firmicutes (P = 0.029) among bacterial sequences amplified from particle-associated (mean 76.8% Firmicutes, 18.5% Bacteroidetes) compared with liquid phase (mean 65.8% Firmicutes, 28.5% Bacteroidetes). Within the Firmicutes, the most significant association with solid particles was found for relatives of Ruminococcus-related clostridial cluster IV species that include Ruminococcus flavefaciens and R. bromii, which together accounted for 12.2% of particle-associated, but only 3.3% of liquid phase, sequences. These findings were strongly supported by microscopy, using group-specific FISH probes able to detect these species. This work suggests that the primary colonizers of insoluble substrates found in the gut are restricted to certain specialized groups of bacteria. The abundance of such primary degraders may often be underestimated because of the difficulty in recovering these bacteria and their nucleic acids from the insoluble substrate.     

In vitro anaerobic biofilms of human colonic microbiota

The human gastrointestinal tract hosts a complex community of microorganisms that grow as biofilms on the intestinal mucosa. These bacterial communities are not well characterized, although they are known to play an important role in human health. This study aimed to develop a model for culturing biofilms (surface-adherent communities) of intestinal microbiota. The model utilizes adherent mucosal bacteria recovered from colonic biopsies to create multi-species biofilms. Culture on selective media and confocal microscopy indicated the biofilms were composed of a diverse community of bacteria. Molecular analyses confirmed that several phyla were represented in the model, and demonstrated stability of the community over 96 h when cultured in the device. This model is novel in its use of a multi-species community of mucosal bacteria grown in a biofilm mode of growth.     

Formation of propionate and butyrate by the human colonic microbiota

The human gut microbiota ferments dietary non‐digestible carbohydrates into short‐chain fatty acids (SCFA). These microbial products are utilized by the host and propionate and butyrate in particular exert a range of health‐promoting functions. Here an overview of the metabolic pathways utilized by gut microbes to produce these two SCFA from dietary carbohydrates and from amino acids resulting from protein breakdown is provided. This overview emphasizes the important role played by cross‐feeding of intermediary metabolites (in particular lactate, succinate and 1,2‐propanediol) between different gut bacteria. The ecophysiology, including growth requirements and responses to environmental factors, of major propionate and butyrate producing bacteria are discussed in relation to dietary modulation of these metabolites. A detailed understanding of SCFA metabolism by the gut microbiota is necessary to underpin effective strategies to optimize SCFA supply to the host.     

Mathematical modelling of carbohydrate degradation by human colonic microbiota

The human colon is an anaerobic ecosystem that remains largely unexplored as a result of its limited accessibility and its complexity. Mathematical models can play a central role for a better insight into its dynamics. In this context, this paper presents the development of a mathematical model of carbohydrate degradation. Our aim was to provide an in silico approach to contribute to a better understanding of the fermentation patterns in such an ecosystem. Our mathematical model is knowledge-based, derived by writing down mass-balance equations. It incorporates physiology of the intestine, metabolic reactions and transport phenomena. The model was used to study various nutritional scenarios and to assess the role of the mucus on the system behavior. Model simulations provided an adequate qualitative representation of the human colon. Our model is complementary to experimental studies on human colonic fermentation, which, of course, is not meant to replace. It may be helpful to gain insight on questions that are still difficult to elucidate by experimentation and suggest future experiments.     

Dominant and diet-responsive groups of bacteria within the human colonic microbiota

The populations of dominant species within the human colonic microbiota can potentially be modified by dietary intake with consequences for health. Here we examined the influence of precisely controlled diets in 14 overweight men. Volunteers were provided successively with a control diet, diets high in resistant starch (RS) or non-starch polysaccharides (NSPs) and a reduced carbohydrate weight loss (WL) diet, over 10 weeks. Analysis of 16S rRNA sequences in stool samples of six volunteers detected 320 phylotypes (defined at >98% identity) of which 26, including 19 cultured species, each accounted for >1% of sequences. Although samples clustered more strongly by individual than by diet, time courses obtained by targeted qPCR revealed that ‘blooms'' in specific bacterial groups occurred rapidly after a dietary change. These were rapidly reversed by the subsequent diet. Relatives of Ruminococcus bromii (R-ruminococci) increased in most volunteers on the RS diet, accounting for a mean of 17% of total bacteria compared with 3.8% on the NSP diet, whereas the uncultured Oscillibacter group increased on the RS and WL diets. Relatives of Eubacterium rectale increased on RS (to mean 10.1%) but decreased, along with Collinsella aerofaciens, on WL. Inter-individual variation was marked, however, with >60% of RS remaining unfermented in two volunteers on the RS diet, compared to <4% in the other 12 volunteers; these two individuals also showed low numbers of R-ruminococci (<1%). Dietary non-digestible carbohydrate can produce marked changes in the gut microbiota, but these depend on the initial composition of an individual''s gut microbiota.     

The effects of the novel bifidogenic trisaccharide,neokestose, on the human colonic microbiota

The potential prebiotic effect of the fructo-trisaccharide, neokestose, on intestinal bacteria was investigated. Bifidobacterium sp. utilized neokestose to a greater extend and produced more biomass from neokestose than facultative anaerobes under anaerobic conditions in batch culture. Lactobacillus salivarius utilized glucose but negligible amounts of neokestose. L. salivarius and the facultative anaerobes produced significantly more biomass from glucose than from neokestose, whereas the biomass yields obtained with bifidobacteria on neokestose and glucose, respectively, were not significantly different. Static batch cultures inoculated with faeces supported the prebiotic effect of neokestose, which had been observed in the pure culture investigations. Bifidobacteria and lactobacilli were increased while potentially detrimental coliforms, clostridia and bacteroides, decreased after 24 h fermentation with neokestose. In addition, this effect was more pronounced with neokestose than with a commercial prebiotic fructo-oligosaccharide. It was concluded that neokestose has potential as a novel bifidogenic substance and that it might have advantages over the commercially available sources currently used.     

Modelling the emergent dynamics and major metabolites of the human colonic microbiota

We present here a first attempt at modelling microbial dynamics in the human colon incorporating both uncertainty and adaptation. This is based on the development of a Monod‐equation based, differential equation model, which produces computer simulations of the population dynamics and major metabolites of microbial communities from the human colon. To reduce the complexity of the system, we divide the bacterial community into 10 bacterial functional groups (BFGs) each distinguished by its substrate preferences, metabolic pathways and its preferred pH range. The model simulates the growth of a large number of bacterial strains and incorporates variation in microbiota composition between people, while also allowing succession and enabling adaptation to environmental changes. The model is shown to reproduce many of the observed changes in major phylogenetic groups and key metabolites such as butyrate, acetate and propionate in response to a one unit pH shift in experimental continuous flow fermentors inoculated with human faecal microbiota. Nevertheless, it should be regarded as a learning tool to be updated as our knowledge of bacterial groups and their interactions expands. Given the difficulty of accessing the colon, modelling can play an extremely important role in interpreting experimental data and predicting the consequences of dietary modulation.     

Amoxicillin treatment modifies the composition of Bifidobacterium species in infant intestinal microbiota

ObjectivesAmoxicillin is a beta-lactam antibiotic largely used in childhood. However only few studies described its impact on composition of children gut microbiota, in particular on Bifidobacterium populations considered as beneficial microorganisms. In this study, the impact on faecal Bifidobacterium species of a seven-day amoxicillin treatment was quantitatively and qualitatively assessed in infants during an episode of acute respiratory infection.MethodsFaecal samples from 31 infants were obtained on day 0 (just before amoxicillin therapy) and on day 7 (the end of therapy). Total DNA was extracted and bifidobacteria were quantified using real-time PCR. Predominant Bifidobacterium species were then identified using specific PCR-TTGE.ResultsBifidobacteria concentrations were not significantly altered by amoxicillin compared to the healthy group. However, amoxicillin treatment induced a complete disappearance of Bifidobacterium adolescentis species (occurrence rate of 0% versus 36.4% in healthy group, P < 0.001), a significant decrease in the occurrence rate of Bifidobacterium bifidum (23% versus 54.5% in healthy group, P < 0.05), but did not affect Bifidobacterium longum (93.5% versus 100% in healthy group) and Bifidobacterium pseudocatenulatum/B. catenulatum (about 55% in both groups). The number of Bifidobacterium species per microbiota significantly decreased from 2.5 ± 1 for healthy group to 1.8 ± 0.9 for treated infants (P < 0.05).ConclusionsThis study showed that a 7 day amoxicillin treatment did not alter the counts of Bifidobacterium. However amoxicillin can have an impact by changing the microbiota at the species level and decreased the diversity of this population.     

The role of behavioral dynamics in determining the patch distributions of interacting species

The effect of the behavioral dynamics of movement on the population dynamics of interacting species in multipatch systems is studied. The behavioral dynamics of habitat choice used in a range of previous models are reviewed. There is very limited empirical evidence for distinguishing between these different models, but they differ in important ways, and many lack properties that would guarantee stability of an ideal free distribution in a single-species system. The importance of finding out more about movement dynamics in multispecies systems is shown by an analysis of the effect of movement rules on the dynamics of a particular two-species-two-patch model of competition, where the population dynamical equilibrium in the absence of movement is often not a behavioral equilibrium in the presence of adaptive movement. The population dynamics of this system are explored for several different movement rules and different parameter values, producing a variety of outcomes. Other systems of interacting species that may lack a dynamically stable distribution among patches are discussed, and it is argued that such systems are not rare. The sensitivity of community properties to individual movement behavior in this and earlier studies argues that there is a great need for empirical investigation to determine the applicability of different models of the behavioral dynamics of habitat selection.     

Predominant role of host genetics in controlling the composition of gut microbiota

Background

The human gastrointestinal tract is inhabited by a very diverse symbiotic microbiota, the composition of which depends on host genetics and the environment. Several studies suggested that the host genetics may influence the composition of gut microbiota but no genes involved in host control were proposed. We investigated the effects of the wild type and mutated alleles of the gene, which encodes the protein called pyrin, one of the regulators of innate immunity, on the composition of gut commensal bacteria. Mutations in MEFV lead to the autoinflammatory disorder, familial Mediterranean fever (FMF, MIM249100), which is characterized by recurrent self-resolving attacks of fever and polyserositis, with no clinical signs of disease in remission.

Methodology/Principal Findings

A total of 19 FMF patients and eight healthy individuals were genotyped for mutations in the MEFV gene and gut bacterial diversity was assessed by sequencing 16S rRNA gene libraries and FISH analysis. These analyses demonstrated significant changes in bacterial community structure in FMF characterized by depletion of total numbers of bacteria, loss of diversity, and major shifts in bacterial populations within the Bacteroidetes, Firmicutes and Proteobacteria phyla in attack. In remission with no clinical signs of disease, bacterial diversity values were comparable with control but still, the bacterial composition was substantially deviant from the norm. Discriminant function analyses of gut bacterial diversity revealed highly specific, well-separated and distinct grouping, which depended on the allele carrier status of the host.

Conclusions/Significance

This is the first report that clearly establishes the link between the host genotype and the corresponding shifts in the gut microbiota (the latter confirmed by two independent techniques). It suggests that the host genetics is a key factor in host-microbe interaction determining a specific profile of commensal microbiota in the human gut.     

Standard colonic lavage alters the natural state of mucosal-associated microbiota in the human colon

Background & Aims

Past studies of the human intestinal microbiota are potentially confounded by the common practice of using bowel-cleansing preparations. We examined if colonic lavage changes the natural state of enteric mucosal-adherent microbes in healthy human subjects.

Methods

Twelve healthy individuals were divided into three groups; experimental group, control group one, and control group two. Subjects in the experimental group underwent an un-prepped flexible sigmoidoscopy with biopsies. Within two weeks, subjects were given a standard polyethylene glycol-based bowel cleansing preparation followed by a second flexible sigmoidoscopy. Subjects in control group one underwent two un-prepped flexible sigmoidoscopies within one week. Subjects in the second control group underwent an un-prepped flexible sigmoidoscopy followed by a second flexible sigmoidoscopy after a 24-hour clear liquid diet within one week. The mucosa-associated microbial communities from the two procedures in each subject were compared using 16S rRNA gene based terminal restriction fragment length polymorphism (T-RFLP), and library cloning and sequencing.

Results

Clone library sequencing analysis showed that there were changes in the composition of the mucosa-associated microbiota in subjects after colonic lavage. These changes were not observed in our control groups. Standard bowel preparation altered the diversity of mucosa-associated microbiota. Taxonomic classification did not reveal significant changes at the phylum level, but there were differences observed at the genus level.

Conclusion

Standard bowel cleansing preparation altered the mucosal-adherent microbiota in all of our subjects, although the degree of change was variable. These findings underscore the importance of considering the confounding effects of bowel preparation when designing experiments exploring the gut microbiota.     

Sources of variation in community composition of the hindgut microbiota in two tropical Kyphosus species

Gut microbiota play a fundamental role in the nutrition of many vertebrate herbivores through foregut and hindgut fermentation of plant carbohydrates. Some species of marine herbivorous fishes contain moderate to high levels of short-chain fatty acids in the hindgut, indicating the importance of hindgut fermentation. Herbivorous fish hindgut microbiota are diverse and can vary with geographic location, but data on the scale of geographic variation involving a few km of separation are limited. Here, we used the 16S rRNA gene to describe community composition of the gut microbiota of the herbivorous species Kyphosus vaigiensis and K. cinerascens collected in the vicinity of Lizard Island, northern Great Barrier Reef, Australia, in 2011 and 2017. Microbiota community structure differed between posterior hindgut sections, host species, sampling years and two mid-shelf and outer reef locations approximately 20 km apart. Hindgut bacterial community composition varied remarkably between mid-shelf and outer reef locations, and among individual fish on the mid-shelf reef. In both fish species, the most abundant phyla were Pseudomonadota, Bacillota and Bacteroidota, followed by Spirochaetota, Thermodesulfobacteriota and Verrucomicrobiota. There were no clear differences between the host species in terms of the relative abundance and composition of bacterial genera in outer reef samples. In contrast, the dominant genera differed between mid-shelf samples of K. cinerascens and K. vaigiensis, being Endozoicomonas-like (Pseudomonadota) and Brevinema (Spirochaetota), respectively. Endozoicomonas are emerging as important symbionts in many marine hosts worldwide and are thought to be important in the coral sulphur cycle. Differences in microbiota composition were not associated with variation in fish condition, suggesting that the different microbial taxa perform equivalent functional roles.

     

Long‐term monitoring of the human intestinal microbiota composition

The microbiota that colonizes the human intestinal tract is complex and its structure is specific for each of us. In this study we expand the knowledge about the stability of the subject‐specific microbiota and show that this ecosystem is stable in short‐term intervals (< 1 year) but also during long periods of time (> 10 years). The faecal microbiota composition of five unrelated and healthy subjects was analysed using a comprehensive and highly reproducible phylogenetic microarray, the HITChip. The results show that the use of antibiotics, application of specific dietary regimes and distant travelling have limited impact on the microbiota composition. Several anaerobic genera, including Bifidobacterium and a number of genera within the Bacteroidetes and the Firmicutes phylum, exhibit significantly higher similarity than the total microbiota. Although the gut microbiota contains subject‐specific species, the presence of which is preserved throughout the years, their relative abundance changes considerably. Consequently, the recently proposed enterotype status appears to be a varying characteristic of the microbiota. Our data show that the intestinal microbiota contains a core community of permanent colonizers, and that environmentally introduced changes of the microbiota throughout adulthood are primarily affecting the abundance but not the presence of specific microbial species.     

Root hydrocarbons as potential markers for determining species composition

Grasslands can be a complex mixture of plant species. A method is described to allow the identification of both roots and shoots of five different grass species, thus permitting greater knowledge about whole plant allocation and competition in mixed pastures. The five species were Lolium perenne, Festuca ovina, Festuca rubra, Poa trivialis and Agrostis capillaris. N‐alkanes with odd‐numbers of carbon atoms in the chains predominate in plants and in the five grass species studied, concentrations of alkanes of chain length of C₂₉, C₃₁ and C₃₃ were highest. Average concentrations of C₂₇‐C₃₃ alkanes in shoots and roots were 187 and 11 mg kg ^{? 1}, respectively. This wide range of values required considerable modifications to the method of analysis, including expressing concentrations on an organic matter basis and scaling‐down the procedure. The n‐alkane concentrations in roots are different from those in shoots and therefore values from shoots cannot be used to predict the composition in roots. Using a canonical variate analysis, all five grass species could be separated using concentrations of C₂₆, C₃₁ and C₃₃ values in the roots. The greatest difference occurred between A. capillaris and the others, whereas discrimination was least between the two Festuca species. Defoliation had contrasting effects on the concentration of a few n‐alkanes, but not in the n‐alkanes used to discriminate between grass species. Alkane analysis shows great potential as a method to quantify the species composition of the root biomass beneath mixed pasture species.     

Oligosaccharide structures of isolated human colonic mucin species

Purified human colonic mucin contains six distinct components which may be separated by DEAE-cellulose chromatography. Past studies defined the structure of oligosaccharide side chains from the most abundant species III, IV, and V which elute at intermediate salt concentrations. In these studies the structures of oligosaccharide side chains liberated from the remaining early and late eluting species I, II, and VI were determined after isolation by sequential conventional and high performance liquid chromatography through combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Mucin species I, II, and VI contained a less varied array of discrete oligosaccharide structures than that observed in the major mucin components. Mucin species I and II contained five and 10 structures, respectively, which account for 68 and 71% of total oligosaccharide content in these fractions. The predominant oligosaccharides of mucin species I included three neutral structures: a disaccharide GlcNAc beta (1-3)GalNAc-ol, a trisaccharide Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol, and a tetrasaccharide GlcNAc beta (1-4)Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol as well as two acidic components representing the sialylated forms of two of these oligosaccharides. Mucin species II contained these same oligosaccharides as well as four additional acidic structures, notably a disaccharide Neu alpha (2-6)GalNAc-ol and a hexasaccharide Gal beta (1-4)GlcNAc beta (1-3)Gal beta (1-4)GlcNAc beta (1-3) (NeuAc alpha (2-6))-GalNAc-ol, not identified in any other mucin species. The late eluting mucin species VI contained at least five discrete neutral oligosaccharides and six major acidic structures. While the majority of these structures had been previously isolated from the earlier eluting mucin species IV and V, species VI also contained di- and trisialylated oligosaccharides not identified in other mucin species. In conjunction with earlier studies of the major mucin species III, IV, and V, these data define the range of oligosaccharide structures present in human colonic mucin. These studies demonstrate that human colonic mucin possesses species with characteristic and distinguishable combinations of oligosaccharides which reflect variations of common core structures.     

The role of historical processes in determining tree species richness in the forests of Western Caucasus

To estimate the role of history in variation of tree species richness in the forests of the Western Caucasus we analyzed correlation between their local richness (S--the mean number of species per 300 m2) and size of actual species pool (N--the number of species per 1 ha). If compared communities are differently distant from the point of evolutionary equilibrium one should expect a significant variation in correlation between S and N (determined with the greater sensitivity of N than S in respect of historical factors). The lower value of N/S corresponds to less saturated level of historically determined species richness. A mean N/S ratio in Japana temperate broadleaved forests (Masaki et al., 1999) provided the basis for analysis. The present tree species richness of the forest communities in the 1 ha plots seem essentially determined by the historical processes. The mountain forest communities of Western Caucasus are characterized on the average with lower saturation level of the actual tree species pool in comparison with the Japan temperate broad-leaved forest communities. On the Western Caucasus the middle mountain beech and coniferous-broadleaved communities (400-1600 m a.s.l.) are characterized with the higher saturation level of the actual species pool in comparison with communities located lower and higher. These results confirm published historical reconstructions, according to which the middle mountain forest communities in the Western Caucasus are older than forests located higher or lower. Present low mountain forests of the southern (to Black Sea) and the northern macroslopes of the Western Caucasus are characterized with similar saturation level of the actual species pool. These data agree with the assumption of Dolukhanov (1980) that low mountain zone of the southern macroslope was not a refuge for tree species in Pleistocene.     

The role of microbiota in infectious disease

The intestine harbors an ecosystem composed of the intestinal mucosa and the commensal microbiota. The microbiota fosters development, aids digestion and protects host cells from pathogens - a function referred to as colonization resistance. Little is known about the molecular basis of colonization resistance and how it can be overcome by enteropathogenic bacteria. Recently, studies on inflammatory bowel diseases and on animal models for enteric infection have provided new insights into colonization resistance. Gut inflammation changes microbiota composition, disrupts colonization resistance and enhances pathogen growth. Thus, some pathogens can benefit from inflammatory defenses. This new paradigm will enable the study of host factors enhancing or inhibiting bacterial growth in health and disease.     

In vitro fermentation of broiler cecal content: the role of lactobacilli and pH value on the composition of microbiota and end products fermentation

Aim:  To assess the probiotic effects of Lactobacillus agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230 and the pH on the cecal microflora of chicken and metabolic end products.
Methods and Results:  An in vitro system, operated with batch bioreactor, was used for this assessment. Selected bacterial species were monitored at two pH values, over 24 h of batch culture incubation. The concentration of short chain fatty acids (SCFA) and lactate in the fermented material was also determined. The addition of L. agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230 into vessel 2 (Cc + P) increased the total anaerobes, lactobacilli and bifidobacteria after 24 h incubation. Moreover, lactobacilli supplementation decreased the total aerobes and streptococci, but it did not have any effects on coliforms. The supplementation of lactobacilli in vessel 2 (Cc + P) was found to significantly increase the production of lactate, propionate and butyrate. Furthermore, pH did not alter the formation of butyrate, whereas the production of acetate and propionate was significantly decreased at pH = 5·8.
Conclusions:  L. agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230, as probiotic bacteria, have the ability to re-establish proper microbial balance by the formation of lactate as well as propionate, and stimulate butyrate-producing bacteria to produce butyrate in the chicken cecum.
Significance and Impact of the Study:  This study was the first to report this under in vitro conditions, highlighting the probiotic roles of the two Lactobacillus strains in broiler cecal fermentation at different initial pH. These useful data can be helpful in improving the fermentation process in chicken cecum.     

Green kiwifruit modulates the colonic microbiota in growing pigs

Aims: To investigate whether green kiwifruit modulates the composition of colonic microbiota in growing pigs. Methods and Results: Thirty‐two pigs were fed the control diet or one of the three test diets containing either cellulose, freeze‐dried kiwifruit or kiwifruit fibre as the sole fibre source for 14‐day study. A Ward’s dendrogram of similarity cluster analysis on PCR‐DGGE gels revealed that inclusion of freeze‐dried kiwifruit and kiwifruit fibre into diets altered the bacterial community, indicating the presence of two distinct clusters. Quantification of different bacterial groups by qPCR demonstrated that pigs fed the freeze‐dried kiwifruit or kiwifruit fibre diets had a significantly higher number (P < 0·05) of total bacteria and Bacteroides group and a lower number of Enterobacteria and Escherichia coli group, as well as a greater ratio of Lactobacillus to Enterobacteria when compared to pigs fed the control or cellulose diets. Conclusions: Green kiwifruit, mainly because of fibre, modulated the colonic microbiota, leading to an improved intestinal environment in growing pigs. Significance and Impact of the Study: This is the first report regarding the effect of green kiwifruit on gut microbiota using the in vivo pig model. These results provide the first evidence of interaction between green kiwifruit and colonic microbiota.