The Attwater’s prairie-chicken (Tympanuchus cupido attwateri; APC) is a species of grouse native to Texas coastal prairies and is on the critically endangered species list as a result of habitat destruction and overhunting. All of the current populations were captively bred and released into the wild. Survivorship for released APCs is very low, and individuals seldom survive to reproduce in the wild. One factor contributing to this may be an alteration in the gut microbiota as a result of captivity. Factors potentially influencing the gut microbial composition in captivity include antibiotic therapy, stress, and a predominantly commercially formulated diet. Recent studies have begun to shed light on the importance of the host microbial endosymbionts. Antibiotic administration, stress, diet, age, genotype and other factors have been shown to influence microbial populations in the gastrointestinal tracts of many different vertebrates. Sequencing of 16S rRNA gene amplicons on the Ion Torrent™ platform was used in this study to identify groups of bacteria in the cloacas as a surrogate for the gut microbiota in the APC. Antibiotic-treated and untreated birds, wild-hatched and captive-bred birds, and individuals sampled before and after release to the wild were examined. Significant differences were found between wild-hatched and captive raised birds both pre- and post release. In addition, there was extensive variation among the populations at the lower taxonomic ranks between individuals for each group of APCs. Principal coordinate analysis based on the weighted UniFrac distance metric further exhibited some clustering of individuals by treatment. These data suggest that captive breeding may have long-term effects on the cloacal microbiota of APCs with unknown consequences to their long-term health and survivorship.

Mule deer are an important rangeland grazer, large prey species, and game animal for the state of Utah, so herd size is monitored and managed actively. A significant cause of population decline is poor overwintering survival, including from the lack of available forage during winter months. Mule deer energy storage is correlated with greater overwintering success, so physical estimates of energy storage including body condition score (BCS), rump fat (RF), and loin thickness (LT), can be used to track and predict a herd's health. Current methods of collecting deer information are costly, time consuming, and cause physical stress to deer, so here we sought to test if a microbiome analysis could be used to predict deer overwintering success. We analyzed nearly 1000 fecal samples collected from deer in Utah over a five-year period. We found that the microbiome composition of these samples shared characteristics with published reports of other reported ruminant species. Also, the location and time when the samples were collected significantly influenced mule deer microbiota composition and abundance. We found that there is a relationship between microbes and health measures (BCS, RF, LT), including some microbial abundances that could predict the health measures of mule deer several months ahead of time. There were also microbial groups whose abundances were significantly correlated with the latitude and elevation of the deer. Finally, a longitudinal analysis on a subset of sampled deer produced slightly different results than the broad analysis of all samples, including suggesting that some of the differences in microbiota composition with time may have been related more to sampling distinct deer at different time points, rather than that the deer microbiota composition changed with time. . These results suggest possible candidate microbial taxa for use in developing assays to replace current methods of measuring and tracking deer health.

The human gut microbiome is home to many hundreds of different microbes which play a crucial role in human physiology. For most of them, little is known about how their genetic diversity translates into functional traits and how they interact with their host, which is to some extent due to the lack of isolate genomes. Cultivation-free metagenomic approaches yield extensive amounts of bacterial genetic data, and recently developed algorithms allow strain-level resolution and reconstruction of bacterial genomes from metagenomes, yet bacterial within-species diversity and transmission dynamics after fecal microbiota transplantation remain largely unexplored over cohorts and using these technological advances. To investigate bacterial within-species diversity I first undertook large-scale exploratory studies to characterize the population-level genomic makeup of the two key human gut microbes Eubacterium rectale and Akkermansia muciniphila , leveraging many hundreds of bacterial draft genomes reconstructed from short-read shotgun metagenomics datasets from all around the planet. For E. rectale , I extended previous observations about clustering of subspecies with geography, which suggested isolation by distance and the putative ancestral loss of four distinct motility operons, rendering a subspecies specifically found in Europe immotile. For A. muciniphila, I found that there are several closely related but undescribed Akkermansia spp. in the human gut that are all likely human-specific but are differentially associated with host body mass index, showcasing metabolic differences and distinct co-abundance patterns with putative cognate phages . For both species, I discovered distinct subspecies-level genetic variation in structural polysaccharide synthesis operons. Next, utilizing a complementary strain-resolved approach to track strains between individuals, I undertook a fecal microbiota transplantation (FMT) meta-analysis integrating 24 distinct clinical metagenomic datasets. I found that patients with an infectious disease or those who underwent antibiotic treatment displayed increased donor strain uptake and that some bacterial clades engraft more consistently than others. Furthermore, I developed a machine-learning framework that allows optimizing microbial parameters - such as bacterial richness - in the recipient after FMT based on donor microbiome features, representing first steps towards making a rational donor choice. Taken together, in my work I extended the strain-level understanding of human gut commensals and showcased that genomes from metagenomes can be suitable to conduct large-scale bacterial population genetics studies on other understudied human gut commensals. I further confirmed that strain-resolved metagenomics allows tracking of strains and thus inference of strain engraftment characteristics in an FMT meta-analysis, revealing important differences in engraftment over cohorts and species and paving the way towards better designed FMTs. I believe that my work is an important contribution to the field of microbiome research, showcasing the power of shotgun metagenomics, modern algorithms and large-scale data analysis to reveal previously unattainable insights about the human gut microbiome.

The CpG Island Methylator Phenotype (CIMP) is a distinct form of aberrant DNA methylation in cancer, and it is seen in 20-40% of colorectal cancers (CRC) where its causes remain elusive. Intestinal microbiota represents an important environmental component implicated in CRC development. Interestingly, microbiota have been shown to modulate DNA methylation in preclinical models but the relationship between tumor-infiltrating bacteria and CIMP status in currently unknown. Our hypothesis is that the gut microbiota affects colonic neoplasia through modulating aberrant DNA methylation in host epigenome. To test this hypothesis, we analyzed CIMP status in CRC patient tumor samples. We used a genome-wide approach to determine the CIMP status by filtering cancer-related sites. A total of 1317 CpG sites were filtered and used to determine distinct CIMP classifications that aligned with well-known characteristics of CIMP cases, including localization in the proximal colon, a higher prevalence in female patients, and a higher frequency of MLH1 hypermethylation. To study the association between CIMP and the gut microbiota, we analyzed the enrichment of four bacterial species associated with CRC, including Bacteroides fragilis, Escherichia coli, Fusobacterium nucleatum, and Klebsiella pneumoniae. Notably, they exhibited higher enrichment in CIMP-Positive tumor samples, except for E. coli. This analysis also identified a group of samples referred to as bacterial "Superhigh," characterized by remarkably high abundances of these three bacterial species. The bacterial Superhigh cases displayed a significant association with CIMP status and MLH1 methylation. We validated the association between the CRC-associated bacteria and CIMP by analyzing the Cancer Genome Atlas (TCGA) 450K methylation array data and whole exome sequencing data. The analysis demonstrated that bacterial Superhigh cases in the TCGA datasets also had significantly higher odds of being CIMP-Positive and having MLH1 methylation. To expand our investigation, we conducted 16S rRNA gene sequencing to identify additional bacterial taxa linked to CIMP. Numerous bacterial genera and species were found to be enriched in CRC tumor tissues, with specific enrichments in CIMP-Positive and CIMP-High groups. Notably, Bergeyella, Campylobacter concisus, and Fusobacterium canifelinum were significantly enriched in CIMP-Positive tumors. Additionally, I studied the causal relationship between gut microbiota and CpG island methylation by colonizing germ-free mice ApcMinΔ850/+;Il10–/– with E. coli NC101 & K. pneumoniae, specific pathogen free bacteria, and the mouse bacterial Consortium. Differential methylation analyses of adjacent normal colon tissue revealed a pronounced tissue side-specific difference, particularly in non-CpG island regions. The tissue specificity diminished with the increasing tumorigenic potential of the microbiota group. Comparisons between microbiota groups and germ-free mice indicated a more significant increase in methylation within CpG islands when gut microbiota with higher tumorigenic potential was present. In conclusion, our study underscores the association between CIMP in CRC and the gut microbiota and the causal relationship between the cut microbiota and CpG island methylation. It highlights specific bacterial taxa that may impact DNA methylation especially in CpG islands and contribute to the development ang progression of CIMP in colorectal cancer. / Biomedical Sciences

Woody breast (WB) is a meat quality problem that has caused significant economic losses for the poultry industry. Ross × Ross 708 chicks were randomly assigned to a 3 (diet) × 2 (cocci challenge) × 2 (sex) factorial arrangement of treatments. The three diets included the control diet (corn-soybean meal basal diet), antibiotic diet (basal diet + 6.075 mg bacitracin /kg feed), and probiotic diet (basal diet + 2.2 × 108 CFU Bacillus subtilis PB6 /kg feed). Birds in the cocci challenge treatment group received 20 × the live cocci vaccine as an inoculum on d 14. Growth performance and WB score were measured to understand the effects of management factors (diet and coccidiosis) on broiler growth and WB development. Results indicated that dietary bacitracin and Eimeria spp. increased WB incidence, body weight, and growth rate. Bacillus subtilis increased WB incidence in male broilers without affecting body weight and growth rate. The association of the development of the internal organs and skeletal muscle with WB myopathy incidence in broilers were evaluated. The digestion organs (proventriculus and gizzard) and the skeletal muscles (drumsticks, thighs, and wings) developed at lower rates in birds with WB. In addition, the effects of the dietary and challenge interventions on the gut microbiota diversity and composition associated with WB in broilers were investigated. Results showed that cocci challenge altered gut microbiota composition and various biosynthetic pathways. Maintaining a healthy gut ecosystem is critical for the reduction of WB incidence in broilers. Gene expression related to oxidative stress, gut barrier function, and inflammation in jejunal mucus was investigated. Results showed that WB is related to decreased mucin expression (MUC6) in mucus, indicating a correlation between WB incidence and a lessening of the secretion of gel-forming mucin. In conclusion, dietary antibiotic and probiotic and challenge intervention strategies increased WB incidence, and microbiota composition and gut health gene expression differed in broilers exhibiting WB myopathy.

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