Comparative Metagenomic Analysis of Chicken Gut Microbial Community, Function, and Resistome to Evaluate Noninvasive and Cecal Sampling Resources.
ABSTRACT: When conducting metagenomic analysis on gut microbiomes, there is no general consensus concerning the mode of sampling: non-contact (feces), noninvasive (rectal swabs), or cecal. This study aimed to determine the feasibility and comparative merits and disadvantages of using fecal samples or rectal swabs as a proxy for the cecal microbiome. Using broiler as a model, gut microbiomes were obtained from cecal, cloacal, and fecal samples and were characterized according to an analysis of the microbial community, function, and resistome. Cecal samples had higher microbial diversity than feces, while the cecum and cloaca exhibited higher levels of microbial community structure similarity compared with fecal samples. Cecal microbiota possessed higher levels of DNA replicative viability than feces, while fecal microbiota were correlated with increased metabolic activity. When feces were excreted, the abundance of antibiotic resistance genes like tet and ErmG decreased, but some antibiotic genes became more prevalent, such as fexA, tetL, and vatE. Interestingly, Lactobacillus was a dominant bacterial genus in feces that led to differences in microbial community structure, metabolism, and resistome. In conclusion, fecal microbiota have limited potential as a proxy in chicken gut microbial community studies. Thus, feces should be used with caution for characterizing gut microbiomes by metagenomic analysis.
Project description:<h4>Background</h4>Comprehensive studies of wild bird microbiomes are often limited by difficulties of sample acquisition. However, widely used non-invasive cloacal swab methods and under-explored museum specimens preserved in alcohol provide promising avenues to increase our understanding of wild bird microbiomes, provided that they accurately portray natural microbial community compositions. To investigate this assertion, we used 16S rRNA amplicon sequencing of Great tit (Parus major) gut microbiomes to compare 1) microbial communities obtained from dissected digestive tract regions and cloacal swabs, and 2) microbial communities obtained from freshly dissected gut regions and from samples preserved in alcohol for 2 weeks or 2 months, respectively.<h4>Results</h4>We found no significant differences in alpha diversities in communities of different gut regions and cloacal swabs (except in OTU richness between the dissected cloacal region and the cloacal swabs), or between fresh and alcohol preserved samples. However, we did find significant differences in beta diversity and community composition of cloacal swab samples compared to different gut regions. Despite these community-level differences, swab samples qualitatively captured the majority of the bacterial diversity throughout the gut better than any single compartment. Bacterial community compositions of alcohol-preserved specimens did not differ significantly from freshly dissected samples, although some low-abundant taxa were lost in the alcohol preserved specimens.<h4>Conclusions</h4>Our findings suggest that cloacal swabs, similar to non-invasive fecal sampling, qualitatively depict the gut microbiota composition without having to collect birds to extract the full digestive tract. The satisfactory depiction of gut microbial communities in alcohol preserved samples opens up for the possibility of using an enormous resource readily available through museum collections to characterize bird gut microbiomes. The use of extensive museum specimen collections of birds for microbial gut analyses would allow for investigations of temporal patterns of wild bird gut microbiomes, including the potential effects of climate change and anthropogenic impacts. Overall, the utilization of cloacal swabs and museum alcohol specimens can positively impact bird gut microbiome research to help increase our understanding of the role and evolution of wild bird hosts and gut microbial communities.
Project description:<h4>Background</h4>Feed contributes most to livestock production costs. Improving feed efficiency is crucial to increase profitability and sustainability for animal production. Host genetics and the gut microbiota can both influence the host phenotype. However, the association between the gut microbiota and host genetics and their joint contribution to feed efficiency in chickens is largely unclear.<h4>Results</h4>Here, we examined microbial data from the duodenum, jejunum, ileum, cecum, and feces in 206 chickens and their host genotypes and confirmed that the microbial phenotypes and co-occurrence networks exhibited dramatic spatial heterogeneity along the digestive tract. The correlations between host genetic kinship and gut microbial similarities within different sampling sites were weak, with coefficients ranging from - 0.07 to 0.08. However, microbial genome-wide analysis revealed that genetic markers near or inside the genes MTHFD1L and LARGE1 were associated with the abundances of cecal Megasphaera and Parabacteroides, respectively. The effect of host genetics on residual feed intake (RFI) was 39%. We further identified three independent genetic variations that were related to feed efficiency and had a modest effect on the gut microbiota. The contributions of the gut microbiota from the different parts of the intestinal tract on RFI were distinct. The cecal microbiota accounted for 28% of the RFI variance, a value higher than that explained by the duodenal, jejunal, ileal, and fecal microbiota. Additionally, six bacteria exhibited significant associations with RFI. Specifically, lower abundances of duodenal Akkermansia muciniphila and cecal Parabacteroides and higher abundances of cecal Lactobacillus, Corynebacterium, Coprobacillus, and Slackia were related to better feed efficiency.<h4>Conclusions</h4>Our findings solidified the notion that both host genetics and the gut microbiota, especially the cecal microbiota, can drive the variation in feed efficiency. Although host genetics has a limited effect on the entire microbial community, a small fraction of gut microorganisms tends to interact with host genes, jointly contributing to feed efficiency. Therefore, the gut microbiota and host genetic variations can be simultaneously targeted by favoring more-efficient taxa and selective breeding to improve feed efficiency in chickens. Video abstract.
Project description:Crohn's Disease and Ulcerative Colitis are chronic, inflammatory conditions of the digestive tract, collectively known as Inflammatory Bowel Disease (IBD). The combined influence of lifestyle factors, genetics, and the gut microbiome contribute to IBD pathogenesis. Studies of the gut microbiome have shown significant differences in its composition between healthy individuals and those with IBD. Due to the high inter-individual microbiome variation seen in humans, mouse models of IBD are often used to investigate potential IBD mechanisms and their interplay between host, microbial, and environmental factors. While fecal samples are the predominant material used for microbial community analysis, they may not be the ideal sample to use for analysis of the microbiome of mice with experimental colitis, such as that induced by 2, 4, 6 trinitrobenzesulfonic acid (TNBS). As TNBS is administered intrarectally to induce colitis and inflammation is confined to the colon in this model, we hypothesized that the microbiome of the colonic mucus would most closely correlate with TNBS colitis severity. Based on our previous research, we also hypothesized that sex would be associated with both disease severity and microbial differences in mice with chronic TNBS colitis. We examined and compared the fecal, cecal content, and colonic mucus microbiota of 8-week old male and female C57BL/6J wild-type mice prior to and after the induction of TNBS colitis via 16S rRNA gene sequencing. We found that the colonic mucus microbiome was more closely correlated with disease severity than were alterations in the fecal and cecal microbiomes. We also found that the microbiomes of the feces, cecum, and mucus were distinct, but found no significant differences that were associated with sex in either compartment. Our findings highlight the importance of sampling colonic mucus in TNBS-induced colitis. Moreover, consideration of the differential impact of sex on the microbiome across mouse strains may be critical for the appropriate application of TNBS colitis models and robust comparisons across studies in the future.
Project description:All animals carry specialized microbiomes, and their gut microbiota are continuously released into the environment through excretion of waste. Here we propose the meta-gut as a novel conceptual framework that addresses the ability of the gut microbiome released from an animal to function outside the host and alter biogeochemical processes mediated by microbes. We demonstrate this dynamic in the hippopotamus (hippo) and the pools they inhabit. We used natural field gradients and experimental approaches to examine fecal and pool water microbial communities and aquatic biogeochemistry across a range of hippo inputs. Sequencing using 16S RNA methods revealed community coalescence between hippo gut microbiomes and the active microbial communities in hippo pools that received high inputs of hippo feces. The shared microbiome between the hippo gut and the waters into which they excrete constitutes a meta-gut system that could influence the biogeochemistry of recipient ecosystems and provide a reservoir of gut microbiomes that could influence other hosts. We propose that meta-gut dynamics may also occur where other animal species congregate in high densities, particularly in aquatic environments.
Project description:Despite the convenience and non-invasiveness of fecal sampling, the fecal microbiota does not fully represent that of the gastrointestinal (GI) tract, and the efficacy of fecal sampling to accurately represent the gut microbiota in birds is poorly understood. In this study, we aim to identify the efficacy of feces as a gut proxy in birds using chickens as a model. We collected 1,026 samples from 206 chickens, including duodenum, jejunum, ileum, cecum, and feces samples, for 16S rRNA amplicon sequencing analyses. In this study, the efficacy of feces as a gut proxy was partitioned to microbial community membership and community structure. Most taxa in the small intestine (84.11-87.28%) and ceca (99.39%) could be identified in feces. Microbial community membership was reflected with a gut anatomic feature, but community structure was not. Excluding shared microbes, the small intestine and ceca contributed 34.12 and 5.83% of the total fecal members, respectively. The composition of Firmicutes members in the small intestine and that of Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria members in the ceca could be well mirrored by the observations in fecal samples (? = 0.54-0.71 and 0.71-0.78, respectively, P < 0.001). However, there were few significant correlations for each genus between feces and each of the four gut segments, and these correlations were not high (? = -0.2-0.4, P < 0.05) for most genera. Our results suggest that fecal microbial community has a good potential to identify most taxa in the chicken gut and could moderately mirror the microbial structure in the intestine at the microbial population level with phylum specificity. However, it should be interpreted with caution by using feces as a proxy to study associations for microbial structure at individual microorganism level.
Project description:Several associations have been made between characteristics of the resident gut microbiota and human health and disease susceptibility. Animal models provide the means to test these correlations prospectively and evaluate causality. Experimental fecal microbiota transfer (FMT), or the intentional transplantation of gut microbes into recipient mice depleted of their autochthonous microbes with antibiotics, is a commonly used method of testing these relationships. The true completeness of microbial transfer through such procedures is poorly documented in the literature, particularly in the context of reciprocal transfer of microbes between recipient and donor mice harboring microbial populations of differing richness and diversity. Moreover, it is unclear whether the use of frozen fecal contents or cecal contents would confer any difference in the outcomes of transfer. Herein, groups of mice colonized with distinct gut microbiota of differing richness and composition were used in a reciprocal FMT study, with different groups receiving transfer of material prepared from fresh cecal contents, fresh feces, or frozen feces. Targeted 16S rRNA gene amplicon sequencing was used at intervals throughout the study to characterize the microbiota. Notably, despite comparable depletion of the microbiota in recipient mice prior to transfer, donor-specific taxa reliably colonized recipients only when relatively rich donor material was transferred to mice originally colonized with a simpler microbiota. It is unclear whether these differences were due to differences in the endogenous recipient microbiota or host factors induced in early life by microbial factors. These findings are of practical import for researchers using FMT to prospectively assess the influence of the gut microbiota in mouse models, and to those studying host-microbial interactions and their influence on gut barrier function.
Project description:Microbiota transplant is becoming a popular process to restore or initiate "healthy" gut microbiota and immunity. But, the potential risks of the related practices need to be carefully evaluated. This study retrospectively examined the resistomes of donated fecal microbiota for treating intestinal disorders, vaginal microbiota of pregnant women, and infant fecal microbiota from rural and urban communities, as well as the impact of transplants on the fecal resistome of human and animal recipients. Antibiotic resistance (AR) genes were found to be abundant in all donor microbiota. An overall surge of resistomes with higher prevalence and abundance of AR genes was observed in the feces of all transplanted gnotobiotic pigs as well as in the feces of infant subjects, compared to those in donor fecal and maternal vaginal microbiota. Surprisingly, transplants using rural Amish microbiota led to more instead of less AR genes in the fecal microbiota of gnotobiotic pigs than did transplants using urban microbiota. New AR gene subtypes undetected originally also appeared in gnotobiotic pigs, in Crohn's Disease (CD) patients after transplant, and in feces of infant subjects. The data illustrated the key role of the host gastrointestinal tract system in amplifying the ever-increasing AR gene pool, even without antibiotic exposure. The data further suggest that the current approaches of microbiota transplant can introduce significant health risk factor(s) to the recipients, and newborn human and animal hosts with naïve gut microbiota were especially susceptible. Given the illustrated public health risks of microbiota transplant, minimizing massive and unnecessary damages to gut microbiota by oral antibiotics and other gut impacting drugs becomes important. Since eliminating risk factors including AR bacteria and opportunistic pathogens directly from donor microbiota is still difficult to achieve, developing microbial cocktails with defined organisms and functions has further become an urgent need, should microbiota transplantation become necessary.
Project description:Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to microbial infection. For decades, the potential role of gut microbiota in sepsis pathogenesis has been revealed. However, the systemic and functional link between gut microbiota and sepsis has remained unexplored. To address this gap in knowledge, we carried out systematic analyses on clinical stool samples from patients with sepsis, including 16S rDNA sequencing, metabolomics, and metaproteomics analyses. In addition, we performed fecal microbiota transplantation from human to mice to validate the roles of gut microbiota on sepsis progression. We found that the composition of gut microbiota was significantly disrupted in patients with sepsis compared with healthy individuals. Besides, the microbial functions were significantly altered in septic feces as identified by metabolomics and metaproteomics analyses. Interestingly, mice that received septic feces exhibited more severe hepatic inflammation and injury than mice that received healthy feces after cecal ligation and puncture. Finally, several strains of intestinal microbiota and microbial metabolites were corelated with serum total bilirubin levels in patients with sepsis. Taken together, our data indicated that sepsis development is associated with the disruption of gut microbiota at both compositional and functional levels, and such enteric dysbiosis could promote organ inflammation and injury during sepsis.-Liu, Z., Li, N., Fang, H., Chen, X., Guo, Y., Gong, S., Niu, M., Zhou, H., Jiang, Y., Chang, P., Chen, P. Enteric dysbiosis is associated with sepsis in patients.
Project description:Ulcerative colitis (UC), a subtype of inflammatory bowel disease, is characterized by repetitive remission and relapse. Gut microbiome is critically involved in pathogenesis of UC. The shifts in microbiome profile during disease remission remain under-investigated. Recent studies revealed that UC pathogenesis is likely to originate in the mucosal barrier. Therefore, we investigated the effectiveness of mucosal tissue microbiomes to differentiate patients with subclinical UC from healthy individuals. The microbiomes of cecal and rectal biopsies and feces were characterized from 13 healthy individuals and 45 patients with subclinical UC. Total genomic DNA was extracted from the samples, and their microbial communities determined using next-generation sequencing. We found that changes in relative abundance of subclinical UC were marked by a decrease in Proteobacteria and an increase in Bacteroidetes phyla in microbiome derived from rectal tissues but not cecal tissue nor feces. Only in the microbiome of rectal tissue had significantly higher community richness and evenness in subclinical UC patients than controls. Twenty-seven operational taxonomic units were enriched in subclinical UC cohort with majority of the taxa from the Firmicutes phylum. Inference of putative microbial functional pathways from rectal biopsy microbiome suggested a differential increase in interleukin-17 signaling and T-helper cell differentiation pathways. Rectal biopsy tissue was suggested to be more suitable than fecal samples for microbiome assays to distinguish patients with subclinical UC from healthy adults. Assessment of the rectal biopsy microbiome may offer clinical insight into UC disease progression and predict relapse of the diseases.
Project description:Fermentation differs between the proximal and distal gut but little is known regarding how the bacterial communities differ or how they are influenced by diet. In order to investigate this, we compared community diversity in the cecum and feces of rats by 16S rRNA gene content and DNA shot gun metagenomics after feeding purified diets containing different fermentable substrates. Gut community composition was dependent on the source of fermentable substrate included in the diet. Cecal communities were dominated by Firmicutes, and contained a higher abundance of Lachnospiraceae compared to feces. In feces, community structure was shifted by varying degrees depending on diet towards the Bacteroidetes, although this change was not always evident from 16S rRNA gene data. Multi-dimensional scaling analysis (PCoA) comparing cecal and fecal metagenomes grouped by location within the gut rather than by diet, suggesting that factors in addition to substrate were important for community change in the distal gut. Differentially abundant genes in each environment supported this shift away from the Firmicutes in the cecum (e.g., motility) towards the Bacteroidetes in feces (e.g., Bacteroidales transposons). We suggest that this phylum level change reflects a shift to ammonia as the primary source of nitrogen used to support continued microbial growth in the distal gut.