Alpha diversity
An overall increasing trend in alpha community diversity and richness of the gut microbiome was observed during the pre-harvest lifespan of the pigs, consistent with previous studies [10, 12]. Increased richness and diversity from 10-day pre-weaning to 21-day post-weaning were shown in commercial pigs [12]. Community diversity (Shannon index) plateaued on d 146, whereas richness indices (number of observed features) kept increasing until the end of the experiment when the pigs were shipped for slaughter. The high alpha diversities on d 174 were very comparable to those of the sows, indicating a fully developed swine gut microbiome before market. In our study, we did not observe decreased gut microbiome diversity on the last sampling dates before market, which is inconsistent with previous reports. Han observed reduced alpha diversities starting on d 63 when antibiotics were supplied in diets [13]. In a recent study, De Rodas and colleagues [11] reported increased alpha diversity in different locations along the GI tract from birth to d 84. Interestingly, they also observed decreased diversity in market samples [11]. Of note, pigs in their study were fed antibiotic-free diets, like those in our trial, but were supplemented with pharmaceutical levels of zinc during the nursery stage. Therefore, the differences between these studies on alpha diversity could be due to high zinc levels. Human microbiome diversity increases from infancy to adulthood when the community matures and stays stable before decreasing as people age, likely as a result of changes in diet, dentition, medication, and physiology of gut ecosystems [20]. Although domestic pigs can live as long as 20 years, the pre-harvest pigs raised in this study and in most commercial farms are mainly grown for food production and are reared for 6–7 months only before slaughter. Therefore, it is difficult to determine when the swine gut microbiome plateaus and how the swine gut microbiome changes during aging. Nonetheless, gut microbiome diversity of endpoint pigs was comparable with those of the sows, suggesting that the swine gut microbiome matures after the finishing stage.
Beta diversity: factors shaping the swine gut microbiome
This study provides a comprehensive view of the succession of the swine gut microbiome from birth to market by longitudinally collecting fecal samples from the same set of pigs across different growth stages. Such a study design allowed us to address several important ecological questions regarding the swine gut microbiome: (1) How does the swine gut microbiome change over time across different growth stages? (2) What are the underlying determinants of these changes?
Our study showed consistent patterns of succession of the swine gut microbiome along different growth stages among the three groups of pigs from two different animal trials. At birth, meconium samples showed greater community diversity than the other two lactation samples. Dramatic decreases in community diversity and significant changes in community structure were observed on d 11 and d 20 when the pigs were fed sow milk. Whether the in utero environment is sterile has been a controversial issue in the field of human microbiome area. The meconium samples were collected within 6 h after farrowing with possibilities for the pigs to suckle colostrum. Therefore, we cannot rule out the possibility of postnatal colonization of bacteria from the colostrum, sow teat skin, or the environment. However, given the fact that meconium samples were black and sticky, very different from other lactation fecal samples, and the meconium microbiomes were remarkably different from the day 11 and 20 fecal microbiomes, it is more likely that the meconium microbiomes were vertically transmitted from the sows rather than rapidly colonized by bacteria from other sources. Our study shows that, although the meconium bacterial biomass is low, with very low DNA concentration (less than 10 ng/μl), the meconium microbiomes are unlikely a result of contamination as they were distinct from those of the negative controls, and mock communities (Additional file 1: Figure S14). Thus, our data show that meconium samples harbor a diverse microorganisms (although with low bacterial load) that might serve as seeding bacteria to prime the development of the swine gut microbiome at subsequent time points or to educate postnatal innate and adaptive immune responses [21]. Colostrum and milk consumption is critical for the development of GI tract morphology, immune function, and the gut microbiome. Nutrients in milk such as oligosaccharides, amino acids, and fat activate digestive enzymes and chemical secretions, which alter the gut ecosystem for microbiome colonization [22]. The dramatic decrease of community diversity on d 11 indicates that the gut ecosystem during the first 10 days of life does not accommodate a highly diverse microbial colonization.
Another significant change in community structure occurred between d 20 (end of lactation) and d 27 (7 days postweaning), which might be attributed to weaning stress and/or the introduction of solid food. Weaning stress includes dietary and environmental transitions, and separation from the dam typically results in reduced feed intake and growth performance as well as a high incidence of diarrhea [23]. Stress has been reported to contribute to various degrees of microbial dysbiosis that affect immune and endocrine systems [24]. Diet serves as a major challenge during weaning transitions, causing both physical and metabolic reconstructions in the GI tract. Sow milk is highly palatable and digestible, whereas feed is rough, solid, less tasty, and not as easily digested [25]. Abrupt transitions to a solid feed diet induce short-term villus atrophy and crypt hyperplasia, which in turn impair digestive efficiency and gut integrity. A “leaky” gut could cause increased penetration of pathogens and nutrient loss.
Dramatic changes in both community structure and composition were observed 7 days postweaning. PCoA plots based on both Bray-Curtis and Jaccard showed distinct clusters completely separating the lactation and nursery microbiomes. Interestingly, such huge changes in microbiome did not happen in 1 day. In our validation trial, the microbiomes collected from the first 2 days (d 22 and 23) were not distinguishable from the end of lactation (d 21) samples. Given the fact that the d 27 samples in trial 1 and the day 29 samples in trial 2 were distinct from the d 20 end of lactation samples, we posit that it takes 7 to 9 days for the swine gut microbiome to adapt to a new diet and gut physiology.
The swine gut microbiome is driven by multiple factors such as host genetics, age, diet, environment, body weight, health, and antibiotics. Longitudinal studies are powerful given that animals serve as their own controls and many of the confounders are taken into consideration. However, it is also challenging in such studies to pinpoint which one is the major driver of the swine gut microbiome given that many of these factors are correlated. For instance, as pigs age, their body weights, rearing environments, and diet types also change accordingly. Therefore, we only selected age as a variable in the PERMANOVA models together with diet, sex, sow origin, and PigID. Diet was arguably the most important factor shaping the swine gut microbiome. Corn NDF in particular had the strongest effect in shaping the swine gut microbiome. NDF contains most of the structural components in plant cells such as lignin, hemicellulose, and cellulose that cannot be digested by the pigs and are consequently passed to the colon for fermentation by the swine gut microbiota. Our data is consistent with previous studies. Frese et al. reported that the GI tract catabolic pathways shifted from milk-derived glycan metabolism to plant glycan deconstruction and consumption after a solid feed diet was introduced to the pigs [26]. Our previous findings suggested that neonatal pigs provided with milk replacer along with solid diets during lactation had microbial community structures distinct from those of their sow-fed littermates, suggesting the significant effect of diet on the gut microbiomes of pigs of similar age [6]. Similarly, Bian et al. also pointed out that the impact of age and diet on gut microbiome succession surpasses that of sow genetics [27].
Age is another factor affecting the swine gut microbiome. Age is an indicator for physical maturation, which is accompanied by comprehensive functional transformations in metabolism, immunity, hormone secretion, muscle and bone development, and the nervous system [28, 29]. All these age-dependent biological alterations give rise to changes in microbiome structures [30,31,32].
“Core” members, residents, passengers, and origins of the swine gut microbiome
This study also enabled us to address some other important biological questions regarding the swine gut microbiome, including (1) What is the core gut microbiome? (2) Which bacterial taxa are residents, persisting in the whole pre-harvest section from birth to market? (3) Which bacteria are passengers, present only at a certain point in time? (4) What were the origins of the swine gut microbiome (e.g., sows, diet, or environment)?
A total of 69 core microbiome members were shared between the three groups of pigs in the two animal trials, based on the definition that these bacterial taxa were present at least in one pig of each group at all the time points. Notably, a subset of these members (13 out of 69) was present in at least 50% of the pigs for at least 150 days from birth to market. These members include features 1, 5, 8, 9, 13, 17, 23, 46, 50, 62, 77, 112, and 132. Among these features, five (features 1, 5, 17, 62, and 132) were detected at all time points including d 0 (meconium), indicating vertical transmission of these bacterial taxa from the sows. These features were early colonizers of the swine gut and persisted throughout the entire pre-harvest lifespan, from sow milk-based lactation stage to the solid feed-based nursery, growing, and finishing stages.
Some new colonizers appeared and persisted after the introduction of solid feed. These features include features 3, 6, 12, 52, 63, and 153. Of note, F3 and F52 were detected in the d 0 samples as well, disappeared during lactation, then re-appeared after the solid feed supplementation in the nursery stage. Therefore, these taxa were likely vertically transmitted as well, but were suppressed during the lactation stage to an undetectable level, and proliferated when the nutrient and environment became more favorable. Many of these features belong to the genus of Prevotella, which was the largest genus in the swine gut microbiome at most of the time points during the solid feed stages. Members of Prevotella are associated with plant food-based diet and fiber digestion [33]. Interestingly, significant sub-OTU level differences in abundance and dynamics within this genus were observed. For example, members of Prevotella copri (features 3, 6, 14, and 36) proliferated during the nursery phase and gradually decreased at subsequent stages, whereas features of the unclassified Prevotella (e.g., F9) were one of the residents of the swine GI tract, present from lactation until the end of the finishing stage. The roles that P. copri plays in human health have been debatable. In a recent study, De Filippis et al. detected distinct strains of P. copri by metagenome studies and showed that diet might select distinctive P. copri populations [34]. Genes were enriched for drug metabolism in individuals on a Western diet, whereas genes in people consuming fiber-rich diets were enriched for complex carbohydrate degradation [34]. Introduction of solid fiber-rich feed during the nursery stage explains, at least partially, the increased abundance of P. copri. Similar to P. copri, members of Megasphaera (F1) and Blautia (F16) also increased postweaning, which is in agreement with previous reports [12]. Like Prevotella, members of Megasphaera and Blautia can also degrade carbohydrate efficiently [35,36,37]. Therefore, these microbes proliferated postweaning when pigs were provided with plant carbohydrate diets.
Later colonizers appeared during the late growing stage and persisted throughout the entire finishing stage. These late colonizers include features 4, 10, 18, and 19. Passengers refer to those bacterial taxa that showed up early or at the middle of the pre-harvest section but disappeared or faded out at later stages. Members associated with E. coli (F7) belong to the passenger category. In line with previous swine weaning [38,39,40] and human infant gut microbiome studies [41, 42], E. coli was abundant at birth (d 0) and during lactation stage but phased out after weaning, which could be due to the maturation of the immune system or suppression by other bacteria. Mucus presents a critical role for binding and preventing food-borne pathogens away from the host, and the physical structure of the mucosa is age-dependent [43, 44]. Pathogen-binding affinity of the mucus in immature animals is lower than in mature animals [45].
Potential probiotics
The metabolic property of bacteria directly correlates with feed conversion rate and contributes to the host’s nutrient supply. Modulation of the gut microbiome to improve feed efficiency has become a novel strategy in the livestock industry. In our study, we identified top bacterial taxa that are most positively related to BW in adult pigs. Feature 26, associated with Turicibacter, was positively correlated with BW on d 90, 104, 116, 130, 159, and 174. Of note, Turicibacter is related to host immunity and is sensitive to host GI tract physiological conditions. Turicibacter populations were fewer in immunodeficient mice compared with their wildtype counterparts [46, 47]. Moreover, Turicibacter could reduce susceptibility to Salmonella infection in mice deficient in the expression of blood group glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase 2 (B4galnt2), which is responsible for the synthesis of blood antigens. Hence, Turicibacter might play some positive roles in swine-microbiome immune interactions, consequently promoting an enhanced growth performance.
Feature 27 is a member of Clostridium butyricum (C. butyricum), with positive correlations with BW on d 130, 159, and 174; C. butyricum produces butyric acid, which serves as the most efficient energy source for livestock and GI epithelium maintenance. Dietary C. butyricum supplementation on weaning piglets has been reported to reduce the diarrhea score and enhance intestinal villus height [48]. Supplementation with Butyricum in grow-finishing pigs showed enhanced energy conversion rate [49]. Furthermore, C. butyricum is also involved in GI immunosuppressive modulation. Chen and colleagues showed that C. butyricum supplementation during weaning suppressed pro-inflammatory response indicated by increased mucosa IL-10 and reduced plasma tumor necrosis factor (TNF)-α [48]. Therefore, C. butyricum could enhance growth performance by providing more energy and/or improve the immune system. Features 4 and 18 are all associated with Clostridiaceae. These taxa proliferated in later stages (growing-finishing) and were positively correlated with BW. Of note, F4 was remarkably abundant (about 8% on d 174) at these stages. Features 4 and 18 were positively correlated with BW at almost all the last seven sample collection dates.
Features 2 (Streptococcus) and 454 (Lactobacillus mucosae) were identified as growth-related taxa during the nursery phase in the first animal trial. In the second validation trial, FMT did not significantly change the overall community structure but did improve animal growth performance. Interestingly, abundance of both of these two features was increased by FMT, suggesting the colonization of these features and their possible roles in promoting animal growth. Lactobacillus mucosae was first isolated from pigs with mucus-binding activity [50]. Members of this group have been reported to decrease epithelial permeability and improve barrier function. In another independent study, we detected improved growth performance in a group of pigs raised in an isolator with creep feed. In that study, F2 was also enriched in the high-performance group (Additional file 1: Figure S15) [6]. Although studies wherein these F2 strains are isolated and fed back to pigs would be necessary to prove their function in growth performance, our data in all these three trials corroborate F2 as a strong probiotic candidate.