Effects of environmental factors on monogastric gut microbial community and functional dynamics

Abstract

Fundamental knowledge, for understanding establishment and disturbance of gut microbiota during both health and disease, is the composition and function of the gut microbiome. However, a healthy gut microbiota has not been defined at any profound taxonomic resolution, and even less on a functional level. Previous aims of monogastric microbiome, or comprehensive gut microbial membership, relied greatly on marker gene sequencing, which sequenced less than 0.01% of the microbial genome, limiting our knowledge on microbial functions and strain-level dynamics. The research on understanding the gut microbiota impact on the host is still at a juvenile stage, and much still needs to be learned in understanding the microbiota dynamics in healthy hosts and disturbances on the short- and long-term. To achieve such goals and understand the implications of environmental changes, associated with age development and antibiotic treatment, I utilized two distinct monogastric swine populations. With these swine, I evaluated their gut microbiota for microbial membership, function, and genetic variation. In my first study, I elucidated the dynamics of bacteria, archaea and fungi populations in the swine gut for the duration of the host lifetime. My objective was to provide a foundational understanding of healthy gut microbiome during long-term development. I collected 234 fecal samples, across 31 time points, from 10 swine from birth through 156 days of age. Samples were collected during the three swine development stages (preweaning, nursery, and growth adult). Next, I performed bacterial 16S rRNA amplicon sequencing for bacteria and fungal qPCR for the dominating fungus of the swine gut, Kazachstania slooffiae. My results demonstrated a highly volatile bacteriome, with low K. slooffiae presence, in the young, preweaning host. Following weaning, bacterial populations became relatively established with a peak in K. slooffiae abundance. Finally, I determined multiple negative, competitive interactions between bacterial and K. slooffiae fungi during the nursery and growth adult stages. I provided evidence for previously unknown competitive interactions which occur throughout the weaned and adult periods. This first study indicated a need for future genetic support of microbial functions pertaining to establishment and competitive dynamics. My second objective was a thorough investigation into the functions of methanogenic archaea during the host lifetime. Archaea of the monogastric gut are historically understudied relative to bacteria. I performed shotgun metagenome sequencing on a subset of the hosts (n=7) and samples (n=112) from my first objective. I resolved 1,130 microbial genomes termed metagenome assembled genomes (MAGs). Within these genomes were 8 methanogenic archaea MAGs which fell into two orders: Methanomassiliicoccales (5) and Methanobacteriales (3). I discovered the first US swine MAGs for two archaea, while describing novel evidence of acetoclastic methanogenesis. Furthermore, I described age-associated detection and methanogenic functions. My second objective provided a comprehensive, gene-supported analysis of monogastric-associated methanogens which furthered our understanding of microbiome development and functions. The focus of my final objective was to determine genetic variation and function of microbes following antibiotic treatments. A distinct swine population, relative to the first study, of 648 weaned swine were assigned to one of three treatments: control (no antibiotic ever), chlortetracycline (CTC) for 14 days, or tiamulin (TMU) for 14 days. Pigs were housed in pens where there were 8 pens/treatment and 27 pigs/pen (i.e. 216 pigs/treatment). Fecal samples were collected from 5 random swine from each of 2 random pens per treatment every collection. Collections occurred 7 days prior to treatment (i.e. day of weaning), and every 7 days until 14 days past antibiotic treatment with one final collection at 28 days post treatment. Samples were pooled according to pen and collection day, followed by gDNA extraction, library preparation, and shotgun metagenomic sequencing. I curated 81 MAGs and analyzed genetic variation according to pre- and post-treatment. I found 11 MAGs with no statistical difference in detection and statistically consistently high variation in the form of genetic entropy (SDHSE [sustained detection and high sustained entropy] MAGs). The SDHSE MAGs were suggested to be multidrug resistant (MDR) due to their continued detection throughout CTC and TMU treatments. Even though I identified 22 unique antimicrobial resistance genes in SDHSE MAGs, less than a third contained genes with TMU resistance. There are likely additional TMU resistance genes contributing to the SDHSE MAGs detention throughout TMU treatment. Together, this investigation described how MDR microbial populations harbor genetic variation, with potential for additional resistance, and highlighted the need for further antimicrobial investigations into gene AMR functions. In conclusion, this dissertation offers a comprehensive, functional understanding of the many microbiome members, including bacteria, archaea and fungi. These studies are critical for understanding how monogastric microbes act through the host lifetime and in response to antibiotic treatments, which will aid future endeavors for monogastric health as it pertains to the gut microbiome.