Evaluation of non-lethal gut microbiome sampling methods in a passerine bird

Gut microbial communities play critical roles in the biological functions of their host, such as mediating nutrient absorption, digesting food components the host cannot, and offering protection against enteric pathogens. Extensive research on gut microbial communities has been conducted on mammals, including humans and rodents, but much less work has been done in birds. Furthermore, much of the research on host–microbe interactions make use of faecal samples and rectal/cloacal swabs as a proxy for intestinal samples, which can be difficult to obtain directly. However, little is known about the overlap between the microbial communities of the gut, faeces and swabs, which limits interpretability of results based on faecal samples and swabs. To address this gap in knowledge, we compared the microbiome from five sample types – proventriculus, small intestine, large intestine, cloacal swabs and faeces – across individual Zebra Finches Taeniopygia guttata housed in constant conditions with a standardized diet. We compared diversity and community composition through 16S rRNA gene sequencing. Our results show that microbial communities from both cloacal swabs and faeces were distinct from proventriculus and small intestinal samples, but generally indistinguishable from large intestinal samples, indicating that these non-lethal samples may be useful proxies for large intestinal bacterial communities. Gaining insight into non-invasive sampling techniques for passerines has implications for studies of gut microbial diversity and abundance in wild bird populations. Furthermore, reliable non-lethal sampling is necessary for experiments where repeated sampling is required.     

Effects of Dietary Linseed Oil and Propionate Precursors on Ruminal Microbial Community,Composition, and Diversity in Yanbian Yellow Cattle

The rumen microbial ecosystem is a complex system where rumen fermentation processes involve interactions among microorganisms. There are important relationships between diet and the ruminal bacterial composition. Thus, we investigated the ruminal fermentation characteristics and compared ruminal bacterial communities using tag amplicon pyrosequencing analysis in Yanbian yellow steers, which were fed linseed oil (LO) and propionate precursors. We used eight ruminally cannulated Yanbian yellow steers (510 ± 5.8 kg) in a replicated 4 × 4 Latin square design with four dietary treatments. Steers were fed a basal diet that comprised 80% concentrate and 20% rice straw (DM basis, CON). The CON diet was supplemented with LO at 4%. The LO diet was also supplemented with 2% dl-malate or 2% fumarate as ruminal precursors of propionate. Dietary supplementation with LO and propionate precursors increased ruminal pH, total volatile fatty acid concentrations, and the molar proportion of propionate. The most abundant bacterial operational taxonomic units in the rumen were related to dietary treatments. Bacteroidetes dominated the ruminal bacterial community and the genus Prevotella was highly represented when steers were fed LO plus propionate precursors. However, with the CON and LO diet plus malate or fumarate, Firmicutes was the most abundant phylum and the genus Ruminococcus was predominant. In summary, supplementing the diets of ruminants with a moderate level of LO plus propionate precursors modified the ruminal fermentation pattern. The most positive responses to LO and propionate precursors supplementation were in the phyla Bacteriodetes and Firmicutes, and in the genus Ruminococcus and Prevotella. Thus, diets containing LO plus malate or fumarate have significant effects on the composition of the rumen microbial community.     

The effect of dietary concentrate and soya oil inclusion on microbial diversity in the rumen of cattle

Aims: Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. Methods and Results: The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate–forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T‐RFLP and automated ribosomal intergenic spacer analysis) and real‐time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0·009) in high concentrate–forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. Conclusions: The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. Significance and Impact of the Study: Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.     

Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial,Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community''s bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.     

Phylogenetic Analysis of Bacterial Communities in Different Regions of the Gastrointestinal Tract of Agkistrodon piscivorus,the Cottonmouth Snake

Vertebrates are metagenomic organisms in that they are composed not only of their own genes but also those of their associated microbial cells. The majority of these associated microorganisms are found in the gastrointestinal tract (GIT) and presumably assist in processes such as energy and nutrient acquisition. Few studies have investigated the associated gut bacterial communities of non-mammalian vertebrates, and most rely on captive animals and/or fecal samples only. Here we investigate the gut bacterial community composition of a squamate reptile, the cottonmouth snake, Agkistrodon piscivorus through pyrosequencing of the bacterial 16S rRNA gene. We characterize the bacterial communities present in the small intestine, large intestine and cloaca. Many bacterial lineages present have been reported by other vertebrate gut community studies, but we also recovered unexpected bacteria that may be unique to squamate gut communities. Bacterial communities were not phylogenetically clustered according to GIT region, but there were statistically significant differences in community composition between regions. Additionally we demonstrate the utility of using cloacal swabs as a method for sampling snake gut bacterial communities.     

The symbiotic intestinal ciliates and the evolution of their hosts

The evolution of sophisticated differentiations of the gastro-intestinal tract enabled herbivorous mammals to digest dietary cellulose and hemicellulose with the aid of a complex anaerobic microbiota. Distinctive symbiotic ciliates, which are unique to this habitat, are the largest representatives of this microbial community. Analyses of a total of 484 different 18S rRNA genes show that extremely complex, but related ciliate communities can occur in the rumen of cattle, sheep, goats and red deer (301 sequences). The communities in the hindgut of equids (Equus caballus, Equus quagga), and elephants (Elephas maximus, Loxodonta africanus; 162 sequences), which are clearly distinct from the ruminant ciliate biota, exhibit a much higher diversity than anticipated on the basis of their morphology. All these ciliates from the gastro-intestinal tract constitute a monophyletic group, which consists of two major taxa, i.e. Vestibuliferida and Entodiniomorphida. The ciliates from the evolutionarily older hindgut fermenters exhibit a clustering that is specific for higher taxa of their hosts, as extant species of horse and zebra on the one hand, and Africa and Indian elephant on the other hand, share related ciliates. The evolutionary younger ruminants altogether share the various entodiniomorphs and the vestibuliferids from ruminants.     

Oral Samples as Non-Invasive Proxies for Assessing the Composition of the Rumen Microbial Community

Microbial community analysis was carried out on ruminal digesta obtained directly via rumen fistula and buccal fluid, regurgitated digesta (bolus) and faeces of dairy cattle to assess if non-invasive samples could be used as proxies for ruminal digesta. Samples were collected from five cows receiving grass silage based diets containing no additional lipid or four different lipid supplements in a 5 x 5 Latin square design. Extracted DNA was analysed by qPCR and by sequencing 16S and 18S rRNA genes or the fungal ITS1 amplicons. Faeces contained few protozoa, and bacterial, fungal and archaeal communities were substantially different to ruminal digesta. Buccal and bolus samples gave much more similar profiles to ruminal digesta, although fewer archaea were detected in buccal and bolus samples. Bolus samples overall were most similar to ruminal samples. The differences between both buccal and bolus samples and ruminal digesta were consistent across all treatments. It can be concluded that either proxy sample type could be used as a predictor of the rumen microbial community, thereby enabling more convenient large-scale animal sampling for phenotyping and possible use in future animal breeding programs aimed at selecting cattle with a lower environmental footprint.     

Comparative Survey of Rumen Microbial Communities and Metabolites across One Caprine and Three Bovine Groups, Using Bar-Coded Pyrosequencing and 1H Nuclear Magnetic Resonance Spectroscopy

Pyrosequencing of 16S rRNA genes (targeting Bacteria and Archaea) and (1)H nuclear magnetic resonance were applied to investigate the rumen microbiota and metabolites of Hanwoo steers in the growth stage (HGS), Hanwoo steers in the late fattening stage (HFS), Holstein-Friesian dairy cattle (HDC), and Korean native goats (KNG) in the late fattening stage. This was a two-part investigation. We began by comparing metabolites and microbiota of Hanwoo steers at two stages of husbandry. Statistical comparisons of metabolites and microbial communities showed no significant differences between HFS and HGS (differing by a dietary shift at 24 months and age [67 months versus 12 months]). We then augmented the study by extending the investigation to HDC and KNG. Overall, pyrosequencing of 16S rRNA genes showed that the rumens had highly diverse microbial communities containing many previously undescribed microorganisms. Bioinformatic analysis revealed that the bacterial sequences were predominantly affiliated with four phyla-Bacteroidetes, Firmicutes, Fibrobacteres, and Proteobacteria-in all ruminants. However, interestingly, the bacterial reads belonging to Fibrobacteres were present at a very low abundance (<0.1%) in KNG. Archaeal community analysis showed that almost all of these reads fell into a clade related to, but distinct from, known cultivated methanogens. Statistical analyses showed that the microbial communities and metabolites of KNG were clearly distinct from those of other ruminants. In addition, bacterial communities and metabolite profiles of HGS and HDC, fed similar diets, were distinctive. Our data indicate that bovine host breeds override diet as the key factor that determines bacterial community and metabolite profiles in the rumen.     

Association of Bitter Taste Receptor T2R38 Polymorphisms,Oral Microbiota,and Rheumatoid Arthritis

The association of taste genetics and the oral microbiome in autoimmune diseases such as rheumatoid arthritis (RA) has not been reported. We explored a novel oral mucosal innate immune pathway involving the bitter taste G protein-coupled receptor T2R38. This case–control study aimed to evaluate whether T2R38 polymorphisms associate with the buccal microbial composition in RA. Genomic DNA was obtained from buccal swabs of 35 RA patients and 64 non-RA controls. TAS2R38 genotypes were determined by Sanger sequencing. The buccal microbiome was assessed by Illumina MiSeq sequencing of the V4-16S rRNA gene. Bacterial community differences were analyzed with alpha and beta diversity measures. Linear discriminant analysis effect size identified taxa discriminating between RA versus non-RA and across TAS2R38 genotypes. TAS2R38 genotype frequency was similar between RA and non-RA controls (PAV/PAV; PAV/AVI; AVI/AVI: RA 42.9%; 45.7%; 11.4% versus controls 32.8%; 48.4%; 18.8%, chi-square (2, N = 99) = 2.1, p = 0.35). The relative abundance of Porphyromonas, among others, differed between RA and non-RA controls. The relative abundance of several bacterial species also differed across TAS2R38 genotypes. These findings suggest an association between T2R38 polymorphisms and RA buccal microbial composition. However, further research is needed to understand the impact of T2R38 in oral health and RA development.     

Microbiome analysis of dairy cows fed pasture or total mixed ration diets

Understanding rumen microbial ecology is essential for the development of feed systems designed to improve livestock productivity, health and for methane mitigation strategies from cattle. Although rumen microbial communities have been studied previously, few studies have applied next-generation sequencing technologies to that ecosystem. The aim of this study was to characterize changes in microbial community structure arising from feeding dairy cows two widely used diets: pasture and total mixed ration (TMR). Bacterial, archaeal and protozoal communities were characterized by terminal restriction fragment length polymorphism of the amplified SSU rRNA gene and statistical analysis showed that bacterial and archaeal communities were significantly affected by diet, whereas no effect was observed for the protozoal community. Deep amplicon sequencing of the 16S rRNA gene revealed significant differences in the bacterial communities between the diets and between rumen solid and liquid content. At the family level, some important groups of rumen bacteria were clearly associated with specific diets, including the higher abundance of the Fibrobacteraceae in TMR solid samples and members of the propionate-producing Veillonelaceae in pasture samples. This study will be relevant to the study of rumen microbial ecology and livestock feed management.     

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.Subject terms: Microbial ecology, Food webs     

High-grain diet feeding altered the composition and functions of the rumen bacterial community and caused the damage to the laminar tissues of goats

In the current intensive production system, ruminants are often fed high-grain (HG) diets. However, this feeding pattern often causes rumen metabolic disorders and may further trigger laminitis, the exact mechanism is not clear. This study investigated the effect of HG diet feeding on fermentative and microbial changes in the rumen and on the expression of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) in the lamellar tissue. In all, 12 male goats were fed a hay diet (0% grain; n=6) or an HG diet (56.5% grain; n=6). On day 50 of treatment, samples of blood, rumen content, and lamellar tissue of hooves of goats were collected. The data showed that compared with the hay group, HG-fed goats had lower (P<0.05) rumen pH but higher (P<0.05) total volatile fatty acids and lactate in the rumen and higher (P<0.05) lipopolysaccharide (LPS) levels in the rumen and blood. HG diet feeding altered the composition of rumen bacterial community, and correspondingly, the results suggested that their functions in the HG group were also altered. HG diet feeding increased (P<0.05) the expression of interleukin-1β, interleukin-6, tumour necrosis factor-α and MMP-2 mRNA in the lamellar tissues compared with the hay group. Correlation analysis indicated that the expression of pro-inflammatory cytokines were positively correlated with MMP-2 expression in lamellar tissues. Overall, these results revealed that HG feeding altered the patterns of rumen fermentation and the composition and functions of rumen bacterial community, and lead to higher levels of LPS in the peripheral blood, and further activated the inflammatory response in lamellar tissues, which may progress to the level of laminar damage.     

PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium

Bacteria attached to the rumen epithelium (or epimural community) are not well characterised and their role in rumen functioning is not totally understood. There is just one published report of a clone library from one cow that suggests that this epimural community differs from the bacteria associated with the rumen digestive contents. However, this time-consuming approach is not adapted for examining microbial population changes in groups of animals. In in vivo studies, when samples from several animals have to be analysed simultaneously, a simpler technique has to be used. In this study, a genetic fingerprinting technique, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterise the structure of the bacterial population attached to the rumen epithelium. This community was compared with that present in the solid and liquid phases of rumen content under two contrasting diets. Rumen samples were obtained from four forage-fed and four high-concentrate-fed (80 : 20, wheat grain : hay) 5-month-old lambs. After slaughter, samples from five epithelial sites and the solid and liquid digesta phases were taken for DNA extraction and analysis. Bacterial communities were profiled by PCR-DGGE using bacterial-specific 16S rDNA primers. Analysis of the fingerprint revealed that the epithelial community differed from those of rumen content in both diets. As expected, the nature of the feed influenced the bacterial communities from the solid and liquid rumen phases but no diet effect was observed in the rumen epithelial profiles suggesting a strong host effect on this bacterial population. Additionally, no differences were observed among the five epithelial sampling sites taken from each animal. The profile of the bacterial population attached to the rumen epithelium presented a high inter-animal variation, whether this difference has an influence in the function of this community remains to be determined.     

The Effect of Sampling and Storage on the Fecal Microbiota Composition in Healthy and Diseased Subjects

Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80°C, -20°C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80°C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p<0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80°C versus the other methods and -80°C samples (p<0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80°C, but was higher in fecal swabs (p<0.05). Storage up to 24 hours (at +4°C or room temperature) or freezing at -20°C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.     

Measuring the gut microbiome in birds: Comparison of faecal and cloacal sampling

The gut microbiomes of birds and other animals are increasingly being studied in ecological and evolutionary contexts. Numerous studies on birds and reptiles have made inferences about gut microbiota using cloacal sampling; however, it is not known whether the bacterial community of the cloaca provides an accurate representation of the gut microbiome. We examined the accuracy with which cloacal swabs and faecal samples measure the microbiota in three different parts of the gastrointestinal tract (ileum, caecum, and colon) using a case study on juvenile ostriches, Struthio camelus, and high‐throughput 16S rRNA sequencing. We found that faeces were significantly better than cloacal swabs in representing the bacterial community of the colon. Cloacal samples had a higher abundance of Gammaproteobacteria and fewer Clostridia relative to the gut and faecal samples. However, both faecal and cloacal samples were poor representatives of the microbial communities in the caecum and ileum. Furthermore, the accuracy of each sampling method in measuring the abundance of different bacterial taxa was highly variable: Bacteroidetes was the most highly correlated phylum between all three gut sections and both methods, whereas Actinobacteria, for example, was only strongly correlated between faecal and colon samples. Based on our results, we recommend sampling faeces, whenever possible, as this sample type provides the most accurate assessment of the colon microbiome. The fact that neither sampling technique accurately portrayed the bacterial community of the ileum nor the caecum illustrates the difficulty in noninvasively monitoring gut bacteria located further up in the gastrointestinal tract. These results have important implications for the interpretation of avian gut microbiome studies.     

Impact of subacute ruminal acidosis on the diversity of liquid and solid-associated bacteria in the rumen of goats

This study was aimed to investigate the impact of subacute ruminal acidosis (SARA) on the diversity of liquid (LAB) and solid-associated bacteria (SAB) following high-grain feeding. Six ruminally cannulated goats were divided into two groups: one group was fed a hay diet (COD), and the other group was fed a high grain diet (SAID). Rumen liquids and rumen solids were sampled after 2 weeks adaption. SARA was diagnosed with a pH below 5.8 for 8 h. SAID decreased ruminal pH (P < 0.001) and increased the acetate (P = 0.017), propionate (P = 0.001), butyrate (P < 0.001) and total volatile fatty acid (P < 0.001) concentration in rumen compared with the COD. Denaturing gradient gel electrophoresis fingerprints analysis revealed a clear separation between both the diet and the fraction of rumen digesta in bacterial communities. Pyrosequencing analysis showed that the proportion of phylum Bacteroidetes in the SAID-LAB and SAID-SAB communities was less than in the COD group, whereas the SAID group had a greater percentage of Firmicutes in both the LAB and SAB libraries. UniFrac analyses and a Venn diagram revealed a large difference between the two diets in the diversity of rumen bacterial communities. Overall, our findings revealed that SARA feeding did alter the community structure of rumen liquids and rumen solids. Thus, manipulation of dietary factors, such as ratio of forage to concentrate may have the potential to alter the microbial composition of rumen liquid and rumen solid.     

Variations in Diversity and Richness of Gut Bacterial Communities of Termites (Reticulitermes flavipes) Fed with Grassy and Woody Plant Substrates

Diets shape the animal gut microbiota, although the relationships between diets and the structure of the gut microbial community are not yet well understood. The gut bacterial communities of Reticulitermes flavipes termites fed on four individual plant biomasses with different degrees of recalcitrance to biodegradation were investigated by 16S rRNA pyrosequencing analysis. The termite gut bacterial communities could be differentiated between grassy and woody diets, and among grassy diets (corn stover vs. sorghum). The majority of bacterial taxa were shared across all diets, but each diet significantly enriched some taxa. Interestingly, the diet of corn stover reduced gut bacterial richness and diversity compared to other diets, and this may be related to the lower recalcitrance of this biomass to degradation.     

Ruminal Prevotella spp. May Play an Important Role in the Conversion of Plant Lignans into Human Health Beneficial Antioxidants

Secoisolariciresinol diglucoside (SDG), the most abundant lignan in flaxseed, is metabolized by the ruminal microbiota into enterolignans, which are strong antioxidants. Enterolactone (EL), the main mammalian enterolignan produced in the rumen, is transferred into physiological fluids, with potentially human health benefits with respect to menopausal symptoms, hormone-dependent cancers, cardiovascular diseases, osteoporosis and diabetes. However, no information exists to our knowledge on bacterial taxa that play a role in converting plant lignans into EL in ruminants. In order to investigate this, eight rumen cannulated cows were used in a double 4×4 Latin square design and fed with four treatments: control with no flax meal (FM), or 5%, 10% and 15% FM (on a dry matter basis). Concentration of EL in the rumen increased linearly with increasing FM inclusion. Total rumen bacterial 16S rRNA concentration obtained using Q-PCR did not differ among treatments. PCR-T-RFLP based dendrograms revealed no global clustering based on diet indicating between animal variation. PCR-DGGE showed a clustering by diet effect within four cows that had similar basal ruminal microbiota. DNA extracted from bands present following feeding 15% FM and absent with no FM supplementation were sequenced and it showed that many genera, in particular Prevotella spp., contributed to the metabolism of lignans. A subsequent in vitro study using selected pure cultures of ruminal bacteria incubated with SDG indicated that 11 ruminal bacteria were able to convert SDG into secoisolariciresinol (SECO), with Prevotella spp. being the main converters. These data suggest that Prevotella spp. is one genus playing an important role in the conversion of plant lignans to human health beneficial antioxidants in the rumen.     

Comparative analyses of foregut and hindgut bacterial communities in hoatzins and cows

Foregut fermentation occurs in mammalian ruminants and in one bird, the South American folivorous hoatzin. This bird has an enlarged crop with a function analogous to the rumen, where foregut microbes degrade the otherwise indigestible plant matter, providing energy to the host from foregut fermentation, in addition to the fermentation that occurs in their hindguts (cecum/colon). As foregut fermentation represents an evolutionary convergence between hoatzins and ruminants, our aim was to compare the community structure of foregut and hindgut bacterial communities in the cow and hoatzin to evaluate the influences of host phylogeny and organ function in shaping the gut microbiome. The approach used was to hybridize amplified bacterial ribosomal RNA genes onto a high-density microarray (PhyloChip). The results show that the microbial communities cluster primarily by functional environment (foreguts cluster separately from hindguts) and then by host. Bacterial community diversity was higher in the cow than in the hoatzin. Overall, compared with hindguts, foreguts have higher proportions of Bacteroidetes and Spirochaetes, and lower proportions of Firmicutes and Proteobacteria. The main host differences in gut bacterial composition include a higher representation of Spirochaetes, Synergistetes and Verrucomicrobia in the cow. Despite the significant differences in host phylogeny, body size, physiology and diet, the function seems to shape the microbial communities involved in fermentation. Regardless of the independent origin of foregut fermentation in birds and mammals, organ function has led to convergence of the microbial community structure in phylogenetically distant hosts.