In this study, we present a comprehensive description of the microbial ecosystems in the oropharyngeal cavity, proximal colon, and vaginal canal of a relatively understudied native breed of pigs on the Korean island of Jeju. These body sites were selected because their microbial communities potentially influence function and health of the respiratory, digestive, and reproductive system in these animals [23]. Additionally, because of a putative cross talk that occurs between microbiota and their host, in ways that sometimes transcend body site [24, 25], we not only explored core membership of each community but also ubiquitous features across these three environments.
Brief comparison of the body sites
The habitat provided within a body site is a strong determinant of microbial composition [26]. Despite the behavior of suids that includes interacting with groupmates and exploring their environments using their snout, diversity within oropharyngeal microbial communities was lower than that in the proximal colon and vagina. This implies that conditions within the oropharynx favor colonization by only a select group of microbes from the great range of environments that the animal gets exposed to. In the proximal colon, microbial communities tended to cluster closely and exhibited high diversities and evenness among them. On the other hand, although highly diverse, the vaginal communities had a low evenness and tended to cluster less closely relative to the oropharyngeal and proximal colonic samples. This variation within the vaginal samples is probably due to varying stages of the estrus cycle among the studied gilts. In the study design, we did not collect data on the estrus cycles of the gilts and there were no managemental attempts to synchronize estrus. We therefore assumed that the gilts were at various stages of their individual estrus cycles. Factors such as the increased immunoglobulin levels during the follicular phase of the cycle [27] are likely to influence bacterial attachment and overall vaginal microbial ecosystem [28]. In contrast to this hypothesis, Lorenzen and colleagues [29] found no variation in the vaginal microbiota in prepubertal and sexually mature minipigs. This contrast might be a peculiarity of the breed of pig studied in their experiment.
Microbial composition in the oropharyngeal region
Within the oropharyngeal microbiota, Proteobacteria, Bacteroidetes, Fusobacteria and Firmicutes were the dominant phyla among our 7-month-old Jeju Black pig gilts making up 93.24% of the ASV’s. The phyla occupied 36.85%, 31.08%, 14.85%, and 10.46% respectively. The JBP gilts had a slight difference in oropharyngeal composition at the phylum level in comparison to findings from other breeds of domestic pigs (Table S11). In previous studies, Firmicutes occupied a more significant proportion of the oropharyngeal community, comparable only to Proteobacteria within these environments [30, 31]. The reason for this discrepancy could be attributed to the difference in breed, and age of the study animals since piglets and weaners were involved in the above-mentioned studies compared to the 7-month-old gilts.
Twenty-five genera were found in the oropharyngeal microbiota in at least 8 of the 9 pigs sampled and collectively made up a relative abundance of 75.01% within the oropharyngeal ecosystem. Most of the core features were contributed by families that were not only dominant within the oropharynx but also responsible for differences with the other body sites. For instance, Moraxellaceae, Leptotrichiaceae, Neisseriaceae, Flavobacteriaceae and an unclassified genus from the phylum OD1 had significantly higher relative abundances in oropharyngeal samples than in any of the other two body sites. Pasteurellaceae and Fusobacteriaceae, were also significantly higher in the oropharyngeal than in the proximal colon although not significantly higher than in the vaginal communities. Interestingly, some of the core members were contributed by families occurring at relatively low abundances such as Streptococcaceae, Prevotellaceae, Campylobacteraceae, Bacillaceae, Erysipelotrichaceae, Peptostreptococcaceae, Alcaligenaceae, Porphyromonadaceae, Ruminococcaceae as well as an unclassified genus from the phylum, SR1.
For the core features that were classified at the genus level, we found overlaps with core genera of the swine tonsillar microbiome including genera such as Actinobacillus, Alkanindiges, Streptococcus, Prevotella, Campylobacter and Porphyromonas [32, 33]. Annotating the features to the family level, revealed stronger similarities to the core porcine tonsillar microbiome. Families such as Pasteurellaceae, Moraxellaceae, Fusobacteriaceae, Neisseriaceae, Streptococcaceae, Peptostreptococcaceae, Prevotellaceae, Campylobacteraceae, and Porphyromonadaceae were common to both our JBP oropharyngeal core and the swine tonsillar microbiota [32, 33]. The core community, however, did not include members of the families Veillonellaceaea, and Treponemataceae as reported in the core tonsillar community by the above studies. Our results are therefore an extension of this earlier work as our sample are taken from a broader community that encompasses and probably serves as a source of the tonsillar microbial community.
These core genera included some well-known members of the human oral microbiome such as Prevotella, Streptococcus, Neisseria, Porphyromonas, as well as members of the family, Fusobacteriaceae, and the phylum, SR1 [34, 35]. Some of the genera Streptococcus, Neisseria, Corynebacterium, Prevotella, Porphyromonas and Fusobacterium have been linked with a ‘healthy oral microbiome’ in humans [36].
We looked for some commonly used probiotic genera such as Bacillus, Lactobacillus, Bifidobacterium, Enterococcus, Pediococcus, and Streptococcus [37]. We did find all these genera except Bifidobacterium within the oropharyngeal communities sampled. And of these, only Bacillus, and Streptococcus featured among the core community. This suggests an ability of these probiotic bacteria to attach and colonize in the oropharyngeal region.
Microbial composition in the proximal colon
Firmicutes, and Bacteroidetes were the most abundant phyla in the proximal colons of the JBP gilts (~ 87% of all sequences). In this environment, the core features belonged to 20 genera including Streptococcus, Prevotella, Lactobacillus, Clostridium, Ruminococcus, Bacillus, Gemmiger, Faecalibacterium, Anaerorhabdus, Roseburia, Succinivibrio, Coprococcus, Propionispira. Also included were unclassified genera from the families Ruminococcaceae, S24-7(Muribaculaceae), Erysipelotrichaceae, and Lactobacillaceae as well as unclassified genera from the orders Clostridiales, and Bacteroidales.
While several studies have described the core members of the intestinal tract [15, 38] (Table S12), our study is the first to focus on the proximal segment of the swine colon. Similar to these previous reports, we found core features from the genera Prevotella, Lactobacillus, Clostridium, Ruminococcus, and Roseburia [15, 38]. This therefore suggests that these genera are shared with the core of the entire swine gastro-intestinal tract. Our findings are therefore an extension of these studies and brings a higher resolution focus on the proximal colon.
Microbial composition in the vaginal canal
Bacteroidetes, Firmicutes, Proteobacteria, and Fusobacteria were the most abundant phyla in the vaginal canal of the Jeju black pig gilts accounting for 86.75% of the sequences. A notable finding in our study was the dominance of Bacteroidetes over the Firmicutes. This was in contrast to findings from studies among other breeds of pigs [39,40,41] (Table S13). The glaring dominance of the Bacteroidetes were a peculiar feature in the JBP vaginal microbiota. Among other breeds of pigs, Firmicutes feature as the most dominant phylum followed by Proteobacteria and Bacteroidetes especially among sows [40, 41]. In their study, Wang et. al [40], link the proliferation of Bacteroidetes and Proteobacteria to endometritis among postpartum sows. The difference between the vaginal microbiota in our studied gilts and previous reports is likely due to difference in age group studied [42] as well as due to breed differences. The vaginal microbiota is known to be markedly influenced by the reproductive phase of a pig given that hormonal levels and reproductive tract secretions significantly alter the vaginal environment [28].
The core features within the vaginal microbial environment of our JBP gilts were annotated to the genera Clostridium, Bacteroides, Prevotella, Campylobacter, Streptococcus, Peptostreptococcus, Lactobacillus, Escherichia, Bacillus, Peptoniphilus. Others belonged to unclassified genera from the families Fusobacteriaceae, Pasteurellaceae, Ruminococcaceae, and Flavobacteriaceae as well as genera from the orders Bacteroidales, and Clostridiales. Despite differences at phylum level, the vaginal microbiome of our JBP had similarities with that of commercial pig breeds at the genus level. Some of the core features in this community belonged to Clostridium, Streptococcus, Lactobacillus, Bacillus, and an unclassified genus of Fusobacteriaceae which show up as core members of the vaginal microbiota in gilts of commercial breeds [42]. As with other breeds of domestic pigs and other non-human mammals the abundance of Lactobacillus was relatively low within the vaginal microbiota of the JBP gilts compared to that in humans [43].
We were also interested in identifying features with a possible influence on reproductive performance within the core of our JBP gilt vaginal microbiome. Our data revealed some taxa, such as Bacteroides, Prevotella, Campylobacter, and Ruminococcaceae that had been identified as potential biomarkers of performance based on certain reproductive parameters [44].
Ubiquitous features within the body sites
Microbial features belonging to 6 genera occupied all the three body sites. This was interesting considering the variability in physiology, function and biochemical characteristics of the oropharyngeal cavity, proximal colon, and vaginal canal. The features were annotated to Firmicutes and Bacteroidetes phyla which dominated all the three body sites among these JBP gilts. The ubiquitous nature of these microbial features suggests an ability to colonize multiple body sites.
Several factors enable intermingling and transfer of microbiota across body sites and between individuals in domestic pigs. One is the social interaction among grouped pigs which is a common rearing practice at this stage of the pig’s life. The high degree of interaction increases the transferability of microbiota between these three body sites as pigs groom each other and interact with manure-smeared floors, surfaces, and various objects within their environment [4]. In such interactions, microbial organisms from the gut could ascend from the rectum into the vaginal canal, and grooming groupmates could lead to ingestion of gut microbes from fecal matter, vagina/ vulva as well skin and other parts of the pig’s body. Moreover, a key managemental practice among this age group is acclimatization of replacement gilts on introduction into a breeding farm. This involves, among many practices, exposure of replacement gilts to fresh fecal and placental material from older breeding sows as a means of inducing immunity against endemic pathogens on the receiving farm [45, 46].
Ubiquitous members of these ecosystems are also important to understand since they are likely to enable a vertical transfer of microbiota to newborn piglets. Following recruitment of gilts into the breeding herd, their vaginal and fecal microbiota will be the first encountered by neonate piglets and is believed to seed the microbiota of the new pigs. Evidence supporting this is found in the fact that the pharyngeal microbiota of neonates tends to look more like that within the vaginal tract of their dams [47, 48]. These phenomena among others, make it interesting to understand the commonality that might exist within these body sites, among gilts at a period when they enter the reproductive cycle.
Predicted functionality
In addition to knowledge on phylogenetic composition, it is important to understand the potential contribution that a microbial community brings to a host’s physiology and health. As expected, our analysis showed that pathway abundances generally clustered by body sites, corroborating the strong effect of body site on the microbiome of Jeju black pigs. Our exploration of the microbial pathways revealed several common metabolic reactions (260 pathways in 30 super pathways) in these 3 body sites. These ubiquitous pathways serve several functions including those involved in energy generation as well as biosynthesis of cofactors, carriers, and vitamins. They also provided capacity for biosynthesis and degradation of nutrient sources such as amines and polyamines, amino acids, carbohydrates, fatty acid and lipids, nucleosides and nucleotides as well as secondary metabolites. Pathways that enabled bacteria to degrade unlikely compounds such as certain alcohols, C1 compounds, carboxylates, inorganic nutrients, and polysaccharides were also shared across these body sites.
A notable finding in this analysis was that body-site-associated, microbial, functional capacity consisted of diverging reaction pathways within shared super classes across the three environments. This was embodied in 10 of the super pathway classes to which some of the shared pathways belonged. These super classes also contributed pathways whose patterns of enrichment showed significant association with body sites. They include pathways of Amine and Polyamine Biosynthesis, Amino Acid Biosynthesis, Carbohydrate Biosynthesis, Carbohydrate Degradation, Cell Structure Biosynthesis, Cofactor, Carrier, and Vitamin Biosynthesis, Fatty Acid and Lipid Biosynthesis, Inorganic Nutrient Metabolism, as well as Secondary Metabolite Degradation. It is typical for many, taxonomically distinct microorganisms within an ecosystem to have the potential to perform similar metabolic functions. This phenomenon is described as ‘functional redundancy’ [49, 50]. However, their pathways and strategies of metabolism are dynamic and can vary with availability of, and quality of resources as well as competition within the community [51]. Therefore, it is not surprising that the distinguishing pathways in these communities, also belong to similar super pathway classes. However, it is also worth noting that presence of a predicted microbial pathway within a community does not necessarily signify its importance in the ecosystem. Unless necessary, many genes/enzymes are not expressed since gene expression is costly and comes as a trade-off with an organism’s fitness [52, 53]. This gap in interpretation can be closed, at least in part, by applying further “omics” techniques. Techniques that probe for proteins/enzymes (proteomics) and metabolites (metabolomics) being produced and therefore, what reactions are occurring within a microbial ecosystem.