Development of gut microbiota in pigs and the effect of diet, antibiotics and other environmental factors

The intestinal tract of humans and animals is colonized by trillions of microorganisms that constitute a community or ecosystem known as the gut microbiota. The gut microbiota undergoes remarkable alterations during early age, reaches a relative stable state in adulthood, and is driven by internal and external factors such as genotype of the host, diet and antibiotics. The objective of this research was to determine the effects of antibiotic treatment, microbial exposure and diet on the development of intestinal microbiota, focusing on the pig as an important production animal as well as a model for human. To achieve this objective, a series of intervention experiments were performed both in piglets and adult pigs. To determine the impact of antibiotic treatment on the development of intestinal microbiota of piglets, two experiments were performed. The first experiment aimed to determine the effect of perinatal maternal antibiotic treatment on the intestinal microbiota of piglets. In this experiment, the sows received amoxicillin orally around parturition, and their offspring was serially sacrificed up to 42 days of age for analysis of ileal and colonic microbiota. It was observed that amoxicillin treatment drastically impacted the sows’ faecal microbiota, and furthermore influenced specific microbial groups in the ileum and colon of the piglets before and after weaning. These findings indicated that maternal amoxicillin treatment may indirectly affect the gut microbiota of offspring through disturbing the maternal microbiota and the transfer of maternal microbiota to the offspring. In a second experiment, we determined the effect of early antibiotic treatment on intestinal microbial colonization and immune development of piglets. Additionally, the effect of stress factors associated with routine farm practice was investigated. Antibiotic treatment affected the composition and diversity of jejunal microbiota, and reduced the expression of a large number of genes involved in immune-related processes. The cumulative effect of management procedures on top of the use of an antibiotic was limited. This study reinforced the notion that the early phase of life is critical for intestinal immune development, also under regular production circumstances. Apart from antibiotic treatment, the effect of early microbial association on the development of intestinal microbiota and immune system of piglets was also studied in this thesis. One group of caesarean derived piglets was inoculated with a mixture of three microbial species (<em>Lactobacillus amylovorus</em>, <em>Clostridium glycolicum</em>, and <em>Parabacteroides </em>sp<em>.</em> ASF519) at day 1 and 2 after birth (the simple microbial association group), whereas a second group of piglets was inoculated with the above mixture at day 1 and 2 after birth as well as diluted adult sow faeces at day 3 and 4 after birth as the complex microbial association (CA) group. CA caused an increase of faecal microbial diversity and accelerated the faecal microbiota to develop into a stable and diverse microbiota. CA significantly affected luminal microbial composition and gene expression in jejunal and ileal mucosa, albeit in different ways. In the pig ileum, CA led to an increased relative abundance of microbial groups known to have beneficial effects, whereas it reduced the relative contribution of potential pathobionts. CA also induced the enrichment of immune-related gene sets in the ileal mucosa. Another research goal of this thesis was to determine the influence of diet on the microbiota in the large intestine of adult pigs. To this end, the effect of resistance starch (RS) was evaluated in two studies. In the first study, pigs were either assigned to an RS diet or a digestible starch (DS) diet for two weeks. Samples from along the intestine were collected for measuring luminal microbiota composition, short chain fatty acid (SCFA) concentrations and the expression of host genes involved in SCFA uptake, SCFA signalling, and satiety regulation in mucosal tissue. In both the caecum and colon, differences in microbiota composition and SCFA concentrations were observed between DS- and RS-fed pigs. Caecal tissue expression of genes encoding monocarboxylate transporter 1 and glucagon was induced by RS. Based on these results, an additional experiment was performed. In this study, ten pigs, fitted with a cannula in the proximal colon for repeated collection of tissue biopsies and luminal content, were fed a DS diet, or a diet high in RS (34%) for two consecutive periods of 14 days in a crossover design. RS increased the relative abundance of several butyrate-producing microbial groups and reduced that of potentially pathogenic members of the genus <em>Leptospira</em> and the phylum of Proteobacteria. Concentrations of acetate, propionate and butyrate in carotid plasma were significantly higher after RS consumption. Upon RS feeding, oxidative metabolic pathways, such as TCA cycle and beta-oxidation, were induced, whereas many immune response pathways, including adaptive and innate immune system, as well as cell division were suppressed. The nuclear receptor PPARG was identified as a potential key upstream regulator. In conclusion, this thesis provides direct evidence that maternal antibiotic treatment, early antibiotic admistration and microbial exposure affect the development of intestinal microbiota of the piglets. Moreover, both early antibiotic admistration and microbial exposure affected piglet mucosal tissue gene expression. These findings reinforce the notion that the early phase of life is critical for the development of intestinal microbiota and immune system. Furthermore, it is proposed that manipulation of the microbial association at early age may be a way of supporting functional gut development. In addition to the above discussed early life envents, a diet with RS can also affect the microbiota in the large intestine of adult pigs. This thesis provides an enhanced understanding of the interaction between diet, microbiota and host in a number of complementary pig models and revealed the impact of antibiotics in early life microbial colonization. The gained insight is expected to be instrumental in improving sustainable pig management. Moreover, it may also be useful in understanding similar processes in the human gut.