Introduction
The bacterial content of the mammalian gastrointestinal tract is estimated to be composed of approximately 1014 bacteria (Luckey, Reference Luckey1972; Savage, Reference Savage1977). This is at least ten times greater than the total number of eukaryotic cells that make up most mammals. The collective genome of the bacterial population that inhabits mammals is estimated to contain 3 million genes (Qin et al. , Reference Qin, Li, Raes, Arumugam, Burgdorf, Manichanh, Nielsen, Pons, Levenez, Yamada, Mende, Li, Xu, Li, Li, Cao, Wang, Liang, Zheng, Xie, Tap, Lepage, Bertalan, Batto, Hansen, Le Paslier, Linneberg, Nielsen, Pelletier, Renault, Sicheritz-Ponten, Turner, Zhu, Yu, Li, Jian, Zhou, Li, Zhang, Li, Qin, Yang, Wang, Brunak, Doré, Guarner, Kristiansen, Pedersen, Parkhill, Weissenbach, Bork, Ehrlich and Wang2010), while the human genome contains approximately 23,000 genes (International Human Genome Sequencing Consortium, 2004). Therefore, the genetic diversity of the microbiota in the gastrointestinal tract is immense and has the potential to provide numerous biological activities that the host lacks. The indigenous microbiota within the gastrointestinal tract is known to provide important benefits to its mammalian host (Savage, Reference Savage1977; Roediger, Reference Roediger1980; Berg, Reference Berg1996; Backhed et al. , Reference Backhed, Ley, Sonnenburg, Peterson and Gordon2005). However, the composition and distribution of the microbiota are only now being extensively described. Prior to the development of new molecular tools to describe taxonomic compositions and distributions, what we knew about the gastrointestinal microbiota was based on culture-based systems. While culture-based systems have been important in determining many of the major microbial constituents in the gastrointestinal tract, the vast majority of the specific bacteria comprising indigenous microflora have never been grown in the laboratory and, therefore, have not been described through phenotypic characterization. Extensive descriptions of the intestinal microbiota are being facilitated by the recognition that the sequence of the 16S rRNA gene of bacteria can be used to determine taxonomic identities and, when coupled with high throughput DNA sequencing, provides a means to completely describe the bacteria present in a highly diverse community (Woese and Fox, Reference Woese and Fox1977; Fox et al. , Reference Fox, Stackebrandt, Hespell, Gibson, Maniloff, Dyer, Wolfe, Balch, Tanner, Magrum, Zablen, Blakemore, Gupta, Bonen, Lewis, Stahl, Luehrsen, Chen and Woese1980; Schuster, Reference Schuster2008). Thus, these technical developments have provided the tools to comprehensively study the composition of microbial populations in almost any environment.
It has long been recognized that the indigenous microflora of the mammalian gastrointestinal tract is important for the health and development of animals. Given the large and highly diverse genome of the collective microflora this would be expected. For example, the rumen of cattle is known to contain large numbers of cellulolytic bacteria that are essential for the breakdown of cellulose. This is essential for ruminants because mammals lack enzymes to degrade plant cellulose. Since cellulose is a major component of their diets, the absence of cellulolytic bacteria to assist in metabolism would result in the animal's wasting this abundant energy source. Bacteria are known to provide other metabolic activities for the host including the stimulation of water transport in the colon (stimulated by volatile fatty acids produced by bacteria) (Yolton and Savage, Reference Yolton and Savage1976), re-cycling of bile salts (Shimada et al. , Reference Shimada, Bricknell and Finegold1969; Gilliland and Speck, Reference Gilliland and Speck1977), production of vitamin K (Ramotar et al. , Reference Ramotar, Conly, Chubb and Louie1984), and providing exogenous alkaline phosphatases (Yolton and Savage, Reference Yolton and Savage1976). The gastrointestinal microflora also is an essential stimulus in the development of the animal's immune system (Sellon et al. , Reference Sellon, Tonkonogy, Schultz, Dieleman, Grenther, Balish, Rennick and Sartor1998; Rakoff-Nahoum et al. , Reference Rakoff-Nahoum, Paglino, Eslami-Varzaneh, Edberg and Medzhitov2004; Mazmanian et al. , Reference Mazmanian, Liu, Tzianabos and Kasper2005). Work with germ-free animals has shown that the indigenous microflora stimulates the immune system by promoting the development and expansion of the lamina propria in the intestines (Savage, Reference Savage1977). More recent studies have shown that the gut microbiota is responsible for the selective loss of invariant natural killer T cell (iNKT) subsets and that, in their absence, the host can become more prone to autoimmune diseases including colitis and asthma (Leslie, Reference Leslie2012).
Observational and experimental studies by Rene Dubos led to the hypothesis that the microbes of mammals living in intimate contact with each other co-evolved with animals (Dubos et al. , Reference Dubos, Schaedler, Costello and Hoet1965; Yolton and Savage, Reference Yolton and Savage1976). Dubos stated, ‘It is to be expected, therefore, that anatomical structures and physiological needs have been determined in part by the microbiota (microbiome) which prevailed during evolutionary development, and that many manifestations of the body at any given time are influenced by the microbiota now present’. Thus, during the co-evolution of the microflora and the host, a set of mutualistic or even symbiotic relationships developed between the host and microbes.
The recognition that bacteria make important metabolic contributions to the health and well being of animals and humans and the limited detail we have of the microbial populations in mammals, led to the initiation of the human microbiome project. The term microbiome, which was coined by Joshua Lederberg (Lederberg and McCray, Reference Lederberg and McCray2001), is used to describe the entire microbial content of an environment including bacteria, viruses, protozoa, and fungi. The aims of the human microbiome project are to ‘characterize the microbial communities found at several different sites on the human body, including nasal passages, oral cavities, skin, gastrointestinal tract, and urogenital tract, and to analyze the role of these microbes in human health and disease’ (Peterson et al. , Reference Peterson, Garges, Giovanni, McInnes, Wang, Schloss, Bonazzi, McEwen, Wetterstrand, Deal, Baker, Di Francesco, Howcroft, Karp, Lunsford, Wellington, Belachew, Wright, Giblin, David, Mills, Salomon, Mullins, Akolkar, Begg, Davis, Grandison, Humble, Khalsa, Little, Peavy, Pontzer, Portnoy, Sayre, Starke-Reed, Zakhari, Read, Watson and Guyer2009). In particular, there is a specific goal to understand the relationships between changes in the composition of the microbiome and its bearing on health and disease. However, implicit in this work is the understanding of how the microbiome contributes to the metabolic needs of the host.
The objectives of this review are to provide a summary of what we know about the microbiome of the gastrointestinal tract of pigs and to then describe how the microbiome changes in response to perturbations such as changes in diet, production stresses, and diseases. While the major focus of this review is to provide details afforded to us by using contemporary molecular sequence-based tools, a limited review of data collected using culture-based and non-sequence-based molecular techniques will also be presented.
The gastrointestinal tract is an open tube with inputs through the mouth. Microbial populations that eventually establish themselves in the gastrointestinal tract do so by entry at the anterior end of the gastrointestinal tract. Thus, the system is constantly subjected to new microbial populations via the oral route. Animals are born into the world in a pristine state devoid of microbial contamination, particularly within the gastrointestinal tract. However, during birth and thereafter, they are barraged by constant exposure to microbes (Turnbaugh and Gordon, Reference Turnbaugh and Gordon2008). It is well established that there is a succession of microbial populations that change as the intestinal tract moves to an anaerobic state and with changes in diet (Savage, Reference Savage1977). The succession continues till the establishment of what has been described as a climax community. The climax community comprises bacteria that remain in stable association with the host and have a relative population composition that is stable. However, even after the climax community has been established, there is continual change in microbial composition that occurs in response to new microbes in the individual's environment, the disease and stress levels of the individual, and diet. Following perturbations the climax community usually returns to the ‘normal’ state after the cause of the disruption is removed (Dethlefsen et al. , Reference Dethlefsen, Huse, Sogin and Relman2008).
Two types of microbial populations have been described in mammals: autochthonous and allochthonous (Dubos et al. , Reference Dubos, Schaedler, Costello and Hoet1965). Autochthonous bacteria are the resident stable microbes that were described by DuBos as co-evolving within a mammalian habitat. The allochthonous bacteria are considered to be non-resident microbes that are ‘passing through’ a habitat or that represent microbes that are opportunistic colonizers of a habitat and may be associated with disease states or other perturbations. However, some microbes might be allochthonous in one habitat and autochthonous in another. This is a result of the gastrointestinal tract being open-ended and containing numerous unique habitats. Thus, not only is there a succession of microbes during maturation of an animal, but also there are significant site predilections. Site predilections can be linked to the nutrients present in a specific site, the availability of preferred tissue receptors that provide a means to colonize that site, pH, and what metabolic activities are needed in that site. In the aggregate, the data that are accumulating about the composition and function of the microbiome have led to a new view that the microbiome should be considered as a multicellular, complex organ that is important in the health and well being of animals (Backhed et al. , Reference Backhed, Ley, Sonnenburg, Peterson and Gordon2005; Turnbaugh and Gordon, Reference Turnbaugh and Gordon2008).
The microbiome based on culture-dependent techniques
Prior to the development of the current molecular tools, investigations of the microbiome of the gastrointestinal tract employed culture-dependent tools. Culture methods were used to describe the composition of the gastrointestinal microbiome and most studies were focused more on function. However, because these studies depended on microbial growth in vitro, they employed the use of specific media (sometimes selective media) known to promote the growth of certain groups of bacteria. That is, media were used which allowed investigators to grow things they knew about. Because the lumen of the gastrointestinal tract is anaerobic, methods were developed to grow strict anaerobes. Technologies pioneered by Hungate, including the use of roll tube, gassing of media with nitrogen gas, media containing rumen fluid, and then the anaerobe chambers, were major breakthroughs that enhanced characterization of the gastrointestinal microflora (Bryant, Reference Bryant1972).
A high percentage of all species of bacteria present in the mammalian gastrointestinal tract have not been cultured in vitro. This observation is supported by some of the early, culture-dependent studies. Comparisons of the number of bacteria observed using microscopes when compared with the results from growing these microbes showed a huge discrepancy between the two. For example, Russell (Reference Russell1979) was able to culture only 52% of the bacterial species observed by microscopic observation in gastrointestinal luminal contents and only 20% of the tissue-associated bacteria. Observations of this type demonstrated that any characterization of the microbiome using culture-dependent procedures was incomplete. Since Russell observed only the high abundance bacteria using microscopy, on a taxon basis, he grossly underestimated the diversity of bacteria present in the pig gastrointestinal tract and the percentage that could not be grown in culture.
Estimates of the size of microbial populations within the gastrointestinal tract have been fairly consistent: approximately 1011 per gram of tissue, approximately 1010 for luminal contents, and 1010 per gram of feces (Allison et al. , Reference Allison, Robinson, Bucklin and Booth1979; Russell, Reference Russell1979). However, there have been few studies that have estimated concentrations of bacteria at different sites within the same animal. One of the difficulties in comparing data between different laboratories is that they frequently used different culture techniques. This problem was demonstrated by Allison et al. (Reference Allison, Robinson, Bucklin and Booth1979) in a study in which they compared the cecal and colonic microflora of pigs using media with different carbon sources. They found major differences in what microbes grew and their proportions relative to each other. Using sets of different media, they also compared the microflora of littermates and found that while they were similar, they were not identical. This is a conclusion that has been confirmed using molecular tools as well.
In general, the majority of the bacteria grown from gastrointestinal tissues have been Gram positive. Major genera identified include: Streptococcus, Eubacterium, Lactobacillus, Clostridium, and Propionobacterium. As expected, results vary by media used and the site sampled. Thus, Robinson et al. (Reference Robinson, Allison and Bucklin1981) found that 78% of bacteria found in the ceca of pigs were Gram negative and contained Bacteroides, Selenomonas, and Butyrivibrio. The Gram positive bacteria included Lactobacillus, Peptostreptococcus, and Eubacterium. The composition of the gastrointestinal microbiome also varies by health status. For example, pigs challenged with Brachyspira hyodysenteria, a pathogen that causes severe dysentery in pigs, contained mainly Gram negative bacteria (88%) in their colons, while healthy pigs had mainly Gram positive bacteria (71%) (Robinson et al. , Reference Robinson, Whipp, Bucklin and Allison1984).
The microbiome based on culture-independent techniques
Carl Woese's pioneering work on the sequences of bacterial 16S rRNA led to the understanding that this remarkable molecule could be used to infer taxonomic designations for bacteria (Woese and Fox, Reference Woese and Fox1977; Fox et al. , Reference Fox, Stackebrandt, Hespell, Gibson, Maniloff, Dyer, Wolfe, Balch, Tanner, Magrum, Zablen, Blakemore, Gupta, Bonen, Lewis, Stahl, Luehrsen, Chen and Woese1980; Olsen and Woese, Reference Olsen and Woese1993). This system of taxonomic designation is based on unique sequences within these molecules and provides for a genotypic rather than a phenotypic means to classify bacteria. The 16S rRNA is unique in that it is conserved in all bacteria and is structurally composed of multiple conserved sequences that are maintained in all species and unique hyper variable regions that correlate with species type. In its simplest implementation, physical methods that can distinguish differences in the sequence of the 16S rRNA gene have been used to assign taxonomic designations. Nocker et al. (Reference Nocker, Burr and Camper2007) have provided an excellent review of the ways in which such measurements can be made.
Methods to characterize the pig microbiome have been divided into three categories: physical methods based on 16S rRNA gene sequence differences, PCR amplification of the 16S rRNA gene followed by cloning and full-length sequencing, and PCR amplification of specific variable regions of the 16S rRNA gene sequence followed by high throughput next generation DNA sequencing (i.e. sequencing by synthesis using Roche 454 sequencers or Illumina sequencers). Simpson and co-workers (Simpson et al. , Reference Simpson, McCracken, White, Gaskins and Mackie1999, Reference Simpson, Kocherginskaya, Aminov, Skerlos, Bradley, Mackie and White2002) used the technique of denaturing gradient gel electrophoresis (DGGE) applied to the V3 region of bacteria obtained from pig feces to measure bacterial diversity. While this technique does not provide direct taxonomic assignments, it can be used to compare the compositions and relative concentrations of microbes from groups of pigs. The major conclusions from these studies were that, as expected, the gastrointestinal microbiome of the pig was complex, there was variability in the concentrations of each component, there was similarity in microbial contents within a single gastrointestinal compartment within the pig, but that the gastrointestinal microbiomes of littermates were not identical.
Use of full-length sequencing of 16S rRNA gene
Pryde et al. (Reference Pryde, Richardson, Stewart and Flint1999) published one of the first studies describing the gastrointestinal microbiome of the pig, using sequence data of the 16S rRNA gene. They compared their results with those obtained from bacteriologic culture from the same animals. The approach they used was to extract total DNA from the samples, PCR amplify using universal 16S rRNA gene primers, clone, and perform DNA sequencing. The number of clones sequenced, however, was quite small. Nevertheless, they obtained good concordance between the culture results and sequencing results though they mainly identified Lactobacillus, streptococci, and Selenomonas. Interestingly, 59% of the DNA sequences did not match at the 95% identity level (equivalent to a genus designation; Schloss and Handelsman, Reference Schloss and Handelsman2005) to currently known bacteria. This suggests that many bacteria detected using this technique, were previously unknown. Furthermore, classification at the genus level does not estimate the richness of species and particularly the discovery of new species. The findings also lead to the question of whether deeper sequencing would yield even greater compositional diversity.
Leser et al. (Reference Leser, Amenuvor, Jensen, Lindecrona, Boye and Moller2002) reported data from a study that provided greater depth of sequencing. They employed PCR amplification coupled with gene cloning and full-length sequencing of the 16S rRNA gene. A total of 3.5 Mb of DNA was sequenced, representing 4270 clones from ileum, cecum, and colon. They used a 97% identity level to define an operational taxonomic unit (OTU). Using this criterion, they found 375 unique OTUs. This number of OTUs is much greater than what Pryde and co-workers found. Ninety-four OTUs were found only once. The five most common phylogenetic lineages found in order of dominance were: Eubacterium, Clostridium, Bacillus–Lactobacillus–Streptococcus subdivision, Flexibacter–Cytophaga-Bacteroides group, and bacteria in the phylum Proteobacterium. This study confirmed the assumption that a greater depth of sequencing would yield greater diversity.
Use of variable region sequencing of 16S rRNA gene with next generation DNA sequencing
Several recent studies have been published on the microbiome of the pig using sequencing of PCR-amplified variable regions of the 16S rRNA gene coupled with pyrosequencing. Because of the nature of the sequencing technology, the depth of coverage is much greater compared to all of the previous studies combined. Dowd et al. (Reference Dowd, Sun, Wolcott, Domingo and Carroll2008) examined the ilea of 24 comingled pigs at approximately 26 days of age. Two of the pigs had been challenged with Salmonella enterica serovar Typhimurium. The 10 most frequently identified genera were (in order of frequency): Clostridium, Lactobacillus, Streptococcus, Helicobacter, Ruminococcus, Veillonella, Candidatus, Actinobacillus, Bacillus, and Turicibacter. In addition, Salmonella was detected in eight pigs but was cultured from only four pigs. However, the authors did not mention differences in the microbiomes of the pigs challenged with Salmonella Typhimurium compared with the non-challenged pigs.
Longitudinal studies on microbiome composition
Kim et al. (Reference Kim, Borewicz, White, Singer, Sreevatsan, Tu and Isaacson2011) published one of the most comprehensive studies of the pig gastrointestinal microbiome. They described the fecal microbiome of 20 commercial pigs from two farms and how it changed over time. From a phylum level perspective, most bacteria were classified in five phyla: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Spirochaetes. Bacteria that were in the phylum Firmicutes represented the largest proportion of the total population followed by Bacteroidetes. These two phyla accounted for approximately 90% of all bacteria present. In the same study, data were collected longitudinally with sampling of pigs beginning at 10 weeks of age and proceeding at 3-week intervals until the pigs were 22 weeks of age. Over time, the proportion of bacteria in the phylum Firmicutes increased, while the proportion of bacteria in the phylum Bacteroidetes decreased. In addition, over the same time frame, the number of bacteria that fell into the non-classified group increased. The non-classified group consists of sequences that do not show sufficient homology to other sequences to allow for taxonomic classification. The most predominant genus was Prevotella, which is in the phylum Bacteroidetes. Prevotella represented up to 30% of all classifiable bacteria when the pigs were 10 weeks of age. However, by the time these pigs were 22 weeks of age, Prevotella accounted for only 3.5–4.0% of the bacteria. As the levels of Prevotella decreased, there was a pronounced increase in Anaerobacter (in the phylum Firmicutes). The 16 most abundant species over all five time points included Prevotella and Anaerobacter along with Streptococcus, Lactobacillus, Coprococcus, Sporacetigenium, Megasphaera, Subdoligranulum, Blautia, Oscillibacter, Faecalibacterium, Pseudobutyrivibrio, Dialister, Sarcina, Roseburia, and Butyricoccus.
Using a non-taxonomic picture of the microbiome, Kim et al. (Reference Kim, Borewicz, White, Singer, Sreevatsan, Tu and Isaacson2011) found that there was a clear shift in the composition of the pig fecal microbiome in OTUs of animals as they aged. OTUs represent unique DNA sequences with a single OTU being defined as having 97% identity with other sequences. Using a heat map to visualize the distribution of OTUs in pigs over time, a defined set of less abundant OTUs completely disappeared from fecal samples and were replaced by another set. Correlated with this observation was the tight clustering of microbiome components between pigs at the same age as measured by principal coordinate analysis. The microbiome compositions varied by age. In comparison of different pigs in this study, it was found that some variation between animals occurred, but groups of animals of the same age were more similar to each other compared to pigs of different ages. The results of this study demonstrated the dynamic process that occurred as the microbiome of young animals mature to a climax community. It is presumed that these shifts in composition correspond to changes in the animal's diet and the need to contribute essential metabolic functions to the host.
Looft et al. (Reference Looft, Johnson, Allen, Bayles, Alt, Stedtfeld, Chai, Cole, Hashsham, Tiedje and Stanton2012) reported on the fecal microbiome of 3 pigs sampled at 18 and 20 weeks of age. Similar to the results of Kim et al., they found that the majority of the bacteria were classified in the phyla Bacteroidetes and Firmicutes. However, unlike the report of Kim et al. (Reference Kim, Borewicz, White, Singer, Sreevatsan, Tu and Isaacson2011), where commercial pigs were used, the most predominant phylum was Bacteroidetes and the concentration of bacteria in this phylum did not decrease from week 18 to week 20. The most predominant genera were Prevotella, Anaerovibrio, Succinivibrio, Oscillibacter, Parabacteroides, Hallella, and Coprococcus, which is similar to the findings of Kim et al. (Reference Kim, Borewicz, White, Singer, Sreevatsan, Tu and Isaacson2011).
Non-bacterial components of the microbiome
While the major focus of microbiome studies has been on bacteria, Urubschurov et al. (Reference Urubschurov, Janczyk, Souffrant, Freyer and Zeyner2011) examined the composition of yeasts in the feces of pigs. This group used the 26S rRNA gene coupled with DGGE and DNA sequencing to identify the genera associated with specific gel bands. The only yeast that was detected was Kazachstania slooffiae. In contrast, K. slooffiae, Galactomyces geotrichum, Candida catenulate, and Candida glabrata were detected by culture (Urubschurov et al. , Reference Urubschurov, Janczyk, Souffrant, Freyer and Zeyner2011). In this case, culture-dependent tools were superior to molecular detection.
Recently an analysis of the pig virome was undertaken using a metagenomic approach (i.e. sequencing of the total extracted DNA rather than just the 16S rRNA gene) (Shan et al. , Reference Shan, Li, Simmonds, Wang, Moeser and Delwart2011). Feces from 24 healthy pigs and 12 pigs with diarrhea were examined. Viruses were collected by differential centrifugation followed by membrane filtration. Viral nucleic acids were extracted, and the total viral community nucleic acid sequenced using high throughput pyrosequencing. On average 4.2 different mammalian viruses were identified in the fecal samples of healthy pigs and 5.4 unique viruses in the pigs with diarrhea. Most of the viruses identified (99%) were RNA viruses in the families Picornaviridae, Astroviridae, Coronaviridae, and Caliciviridae. The remaining viruses were DNA viruses in the families Circoviridae and Parvoviridae.
Microbiome composition from anterior to posterior
A pilot study to discover the differences in composition of the microbiomes in jejunum, ileum, cecum, and colon was performed by Isaacson et al. (2011, unpublished work). At the phylum level, the compositions of the microbiomes of the colon and cecum were very similar to those previously described with most bacteria being in the phyla Firmicutes or Bacteroidetes. These two phyla represented greater than 90% of the bacteria detected. However, the compositions of the microbiota of the jejunum and ileum were quite different. In the jejunum, bacteria in the phylum Firmicutes were the most dominant (>90%) followed by bacteria in the phyla Proteobacteria, Cyanobacteria, and Actinobacteria. In the ileum, Firmicutes and Proteobacteria were the two dominant phyla with the Protebacteria representing between 5% and 40% of the bacteria detected. These data confirm the reports of culture-dependent work where there were site-specific differences in the composition of microbial communities.
Effects of antibiotics on microbiome composition
Several studies have been performed to determine the effect of antibiotics on the composition of the pig gastrointestinal microbiome. These studies were performed using antibiotics used as growth promoters. This area of research has become important because of the growing concern about the over usage of antibiotics in agriculture and in particular the usage of antibiotic as growth promoters. These antibiotics are used at ‘sub therapeutic’ levels, but are likely to be one of the many contributors to the selection of antibiotic resistance, particularly in important bacterial pathogens (Phillips et al. , Reference Phillips, Casewell, Cox, De Groot, Friis, Jones, Nightingale, Preston and Waddell2004). While the mechanism by which antibiotics promote animal growth is not known, it is likely that these antibiotics alter the composition of the gastrointestinal microbiome in pigs. Several reports have appeared describing the effects of growth promoting antibiotics on the gastrointestinal microbiome of pigs. Rettedal et al. (Reference Rettedal, Vilain, Lindblom, Lehnert, Scofield, George, Clay, Kaushik, Rosa, Francis and Brözel2009) measured the effects of chlortetracycline on the ileal microbiome. They found that chlortetracycline resulted in decreases in Lactobacillus johnsonii and Turicibacter and an increase in Lactobacillus amylovorus. That study employed cloning of full-length 16S rRNA genes from ileal tissues, and full sequencing of the entire gene. Their conclusions were drawn from the sequencing of a limited number of clones (2050). Collier et al. (Reference Collier, Smiricky-Tjardes, Albin, Wubben, Gabert, Deplancke, Bane, Anderson and Gaskins2003) compared the microbiomes of pig feces treated with the antibiotic tylosin in comparison with non-treated controls. They used DGGE and made taxonomic assignments to specific electrophoretic bands by cutting them from the gels and directly sequencing them. They found a decrease in three species of Lactobacillus, one species of Streptococcus, and one species of Bacillus and an increase of Lactobacillus gasseri in response to tylosin.
In two other publications, high throughput DNA sequencing was employed to measure the effects of growth promoting antibiotics on the pig fecal microbiome. Looft et al. (Reference Looft, Johnson, Allen, Bayles, Alt, Stedtfeld, Chai, Cole, Hashsham, Tiedje and Stanton2012) used a study design that employed six pigs: three pigs were treated with a combination of chlortetracycline, sulfamethazine, and penicillin (ASP250) and three served as untreated controls. Pigs were treated with antibiotics at 18 weeks of age and sampled at 18, 20, and 21 weeks of age. At 20 weeks of age (2 weeks of treatment) there were decreases in bacteria in the phylum Bacteroidetes. Specific changes were decreases in Anaerobacter, Barnesiella, Papillibacter, Sporacetigenium, and Sarcina. Members of the phylum Proteobacteria were increased. In particular, the increase in Proteobacteria was correlated with increases in Escherichia coli. Using the same pigs, a metagenomic analysis was used to determine whether there was enrichment in specific sets of genes following treatment with ASP250. It was observed that genes associated with resistance to antibiotics increased. There was an increase in genes such as aminoglycoside O-phosphotransferase, which is involved in resistance to aminoglycoside antibiotics. It is interesting that ASP250 does not contain an aminoglycoside antibiotic and therefore this selection occurred by an unknown and probably indirect process.
In a related study, the effect of ASP250 on the intestinal virome also was studied (Allen et al. , Reference Allen, Looft, Bayles, Humphrey, Levine, Alt and Stanton2011). ASP250 had an effect by increasing the number of bacteriophages in the sample. When this occurred there was a decrease in the abundance of the host bacteria. In particular, ASP250 caused a decrease in bacterial members of the phylum Firmicutes and specifically members of the Streptococcus genus. Correlated with this decrease was an increase in the number of streptococcal bacteriophages. It was assumed that lysogenic bacteriophages were induced by treatment with ASP250 and the drop in the concentration of Streptococcus was the result of cell lysis. The results of these studies are in contrast to those performed in humans with therapeutic doses of antibiotics. In those situations large changes in the fecal microbiome were detected (Dethlefsen et al. , Reference Dethlefsen, Huse, Sogin and Relman2008). These results suggest that antibiotic growth promoters exert their effects in more subtle ways probably because of the low but chronic administration.
Metabolic capacity of the gastrointestinal microbiome
One of the objectives of the analysis of microbiomes is to understand the metabolic contributions that the microbiome makes to the physiology and metabolism in the host's gastrointestinal tract. Lamendella et al. (Reference Lamendella, Domingo, Ghosh, Martinson and Oerther2011) performed a comparative analysis of the fecal metagenome to identify functional capacity of the microbiome. They obtained 130 Mb of sequence data from DNA extracted from pig feces. The data led to several important conclusions. Firstly, by identifying the microbial composition in feces they confirmed previous observations that most of the bacteria identified were in two phyla: Firmicutes and Bacteroidetes. They also confirmed that Prevotella was the most abundant genus. These results were obtained using molecular detection, but did not rely on sequencing of the 16S rRNA gene. Instead they created libraries from total DNA, sequenced, and identified sequence linkages to specific bacteria by comparison with DNA sequence data in GenBank. Thus, similar results on microbial composition of the pig gastrointestinal microbiome were obtained and helped to validate the results based on the sequencing of the 16S rRNA gene. The metagenomic sequence data were compared with sequences deposited in GenBank in an attempt to assign gene functions. Gene functions were assigned to SEED subsystem classifications. The largest number of genes was related to carbohydrate metabolism. This suggests that the bacteria in the gastrointestinal tract were important for the host to obtain energy. However, that conclusion must be placed in the context that metagenomic analysis only measures theoretical capacity and not when those functions were expressed. A third interesting conclusion was on hierarchical clustering based on taxonomy and functional activities. Their conclusion was that the intestinal microbiomes of pigs most closely clustered with the intestinal microbiomes of chickens and the cow rumen. This close clustering is consistent with these animals having mainly plant-based diets. The fourth observation was that there was a high prevalence of genes associated with antibiotic resistance. Since the diets of most swine include antibiotics, this was not an unexpected result.
Effects of diet on the pig microbiome
The importance of the pig gastrointestinal microbiome in the conversion of feed substances to more readily digestible forms has been implied from studies that have employed germ-free animals. The results from the study described above by Lamendella et al. (Reference Lamendella, Domingo, Ghosh, Martinson and Oerther2011) demonstrated that the most abundant microbial genes in the pig microbiome were related to carbohydrate metabolism. This observation is consistent with the hypothesis that the microbiome is important in digestion of animal feeds in the gastrointestinal tract. This hypothesis suggests that changes in diet should result in differences in the composition of the microbiome, such that the proper metabolic capacities are provided by the bacteria to digest different substrates. Numerous studies have been published measuring specific microbial changes occurring based on differences in diets. For example, using culture-dependent methods, xylanolytic and cellulolytic bacteria have been shown to increase in pigs fed high fiber content feeds (Varel et al. , Reference Varel, Robinson and Jung1987). High oil diets also have been shown to result in statistically significant changes in microbial populations.
Using culture-independent techniques, several new studies have been published showing effects of diet on the microbiome. For example, Zhu et al. (Reference Zhu, Williams, Konstantinov, Tamminga, De Vos and Akkermans2003) reported on the effect of adding sugar beet pulp to swine feces. In this in vitro study, differences in microbial components were assessed using DGGE. The authors found that Eubacterium eligen and Lachnospira pectinoschiza increased in numbers. Since this study was in vitro, it was difficult to correlate the results to in vivo effects. However, this study does demonstrate the importance of substrates on microbiome composition.
Leser et al. (Reference Leser, Lindecrona, Jensen, Jensen and Moller2000) compared the microbiomes in the colon of pigs fed two diets: a conventional diet and a diet containing cooked rice supplement with added fiber. They used a procedure employing terminal restriction length polymorphisms (T-RFLP) of PCR amplified 16S rRNA gene. They found that while the pigs fed a conventional diet maintained a conserved gel band pattern over time, pigs fed the cooked rice supplement had an altered banding pattern. Thus, while the specific microbes that were altered in abundance were not known, changes did occur. This suggests that a shift in the composition of the microbiome occurred to aid in the digestion of the rice supplement.
Zinc oxide (ZnO) is a feed supplement that is used to prevent diarrhea in pigs. Vahjen et al. (Reference Vahjen, Pieper and Zentek2010) analyzed the microbiomes of 40- to 42-day-old pigs to determine whether ZnO caused changes in the ileal microbiome. No statistically significant differences were detected at the phylum or order levels. However, statistically significant differences were measured at the genus level. In particular, Weissella, Leuconostoc, and Streptococcus were increased while there was a decrease in Sarcina. The researchers also observed a consistent, but not statistically significant increase in members of the family Enterobacteriacae, including increases in Enterobacter, Microbacterium, Citrobacter, and Acinetobacter. There also was an increase in Neisseria.
On a related topic, the question of whether probiotic bacteria in the diet of pigs altered the microbiome was asked. Pieper et al. (Reference Pieper, Janczyk, Urubschurov, Korn, Pieper and Souffrant2009) fed weaned pigs Lactobacillus plantarum and used DGGE to detect microbiome changes. They found increases in DGGE bands that correlated with Clostridium glycolicum, Lactobacillus sobrius, Eubacterium rectale, and Roseburia faecalis in the L. plantarum-treated pigs.
Microbiome changes due to stress
Stresses are known to have effects on pigs which increase their susceptibility to certain pathogens. In particular, the stresses associated with weaning or transporting pigs and feed withdrawal are known to increase shedding of S. enterica in feces (Isaacson et al. , Reference Isaacson, Firkins, Weigel, Zuckermann and DiPietro1999). Therefore, investigations looking for changes in the microbiome of pigs that were stressed have been performed. In one study, weaning was correlated with changes in the microbiome in the small intestines (Janczyk et al. , Reference Janczyk, Pieper, Smidt and Souffrant2007). Upon weaning, there was a large increase in Lactobacillus and a reduction in Bifdobacterium and E. coli. L. sobrius and L. amylovorus became the dominant lactobacilli, while Lactobacillus salivarius and L. gasseri declined. This work, which was based on culture-dependent procedures, was confirmed using 16S rRNA gene amplification and DGGE. However, since weaning is accompanied by changes in diet, it is not clear whether the changes observed in this study were due to stresses caused by weaning or diet. Dowd et al. (Reference Dowd, Callaway and Morrow-Tesch2007) showed that weaning caused an increase in the shedding of E. coli.
Microbiome change due to infectious diseases
One of the important functions ascribed to the microbiome is resistance to infection. It has been suggested that the normal microbiota play important roles in excluding pathogens either by occupying space or by directly interfering with specific pathogens (Berg, Reference Berg1996). In humans, it is well known that perturbations of the gastrointestinal microbiome by therapeutic use of antibiotics can result in the emergence of pathogens such as Clostridium difficile. However, little data exist to support this concept in the pig. Leser et al. (Reference Leser, Lindecrona, Jensen, Jensen and Moller2000) compared the microbiomes of healthy pigs with those with infections with B. hyodysenteriae using T-RFLP analysis of 16S rRNA gene products. They found numerous changes and suggested that this was evidence that B. hyodysenteriae de-stabilized the microbiome. However, another interpretation is that a de-stabilized microbiome resulted in susceptibility to the infection. Isaacson et al. (Reference Isaacson, Borewicz, Kim, Vannucci, Gebhart, Singer, Sreevatsan and Johnson2011) obtained preliminary data that demonstrated experimental challenges of pigs with Salmonella Typhimurium or Lawsonia intracellularis or both caused specific and consistent changes in the colonic and cecal microbiome measured by sequencing of 16S rRNA genes. Furthermore, co-infection of pigs with both pathogens resulted in increased shedding of Salmonella Typhimurium over time and at much higher concentrations. This could be the result of increased inflammation in the gastrointestinal tract caused by L. intracellularis allowing S. enterica to more readily colonize and proliferate in these sites.
Conclusions
The objective of this review was to describe what is known about the microbiome in the gastrointestinal tract of the pig. To date, there is limited data available that is based on the newer high throughput molecular protocols. Data are now being accumulated and better descriptions and understanding of the pig gastrointestinal microbiome are now emerging. To a large extent, the new methods corroborate data accumulated over several decades using culture-dependent systems. For example, the molecular techniques confirm the high preponderance of anaerobes in the gastrointestinal tract of the pig. Furthermore, the data also confirm observations that have emerged from the human microbiome project, namely that the bacteria in the gastrointestinal tracts of pigs are mainly in two phyla: Firmicutes and Bacteroidetes. Of interest is that in humans, bacteria in the phylum Bacteroidetes tend to be the most abundant microbes, while in pigs bacterium in the phylum Firmicutes are the most abundant. However, in the pig Prevotella, a member of the phylum Bacteroidetes, is the most abundant bacterium. The gastrointestinal microbiome of the pig is highly diverse, more so than what was inferred from culture-dependent studies. Much of the diversity is found at the genus level where many bacteria still remain unclassified. The diversity also is greater among the bacteria that do not represent the high abundance bacteria. Most of the current studies of microbiomes focus on the most abundant bacteria. Therefore, it is likely that studies that employ deeper sequencing or methods that enrich for the low abundance bacteria will be of great value in understanding the functions of the microbiome. Furthermore, the microbiome changes over time and is subject to changes through numerous perturbations including diet, stress, antibiotics, and diseases. Many of the observed changes occur in composition of the less abundant bacterial population.
One of the most important aspects of the microbiome that needs further investigation is an understanding of how the microbiome's composition is translated to functions and how these functions contribute to animal health and well-being. Current data are primarily descriptive and does not get at whether compositional correlations are causes or effects linked to changes in the animal. Whether components of the microbiome are important in metabolism is accepted as true, but which microbes do what needs to be defined. Furthermore, can the same metabolic functions be provided by different sets of bacteria that happen to possess similar metabolic activities? Descriptions of the microbiome are essential first steps in understanding the functions they provide to the host, and therefore, future work must be focused on functional analysis. Techniques such as metatranscriptomics and metabolomics will be essential tools in dissecting function. The microbiome plays important developmental roles in the maturation of the animal. For example, the microbiome is an important contributor to the development of the host's immune system. By analyzing further the gastrointestinal microbiome of the pig, specific microbes will be identified that are involved in host maturation. This will require better understanding of which bacteria are present and at what specific sites within the gastrointestinal tract and then teasing out the functional role that each plays. It is expected that while some bacteria will directly affect the host, it is also likely that other bacteria interact only with other bacteria. In other words, they contribute to the host by stabilizing the microbial community structure or by providing conditions necessary for other specific microbes to persist in the gastrointestinal tract.
It has been hypothesized that the normal or ‘healthy’ microbiome contributes to animal health by reducing the potential for pathogens to colonize the gastrointestinal tract and thus prevents infectious diseases (Berg, Reference Berg1996). Work cited above has shown that experimental oral challenges with specific pathogens led to alterations of the pig gastrointestinal microbiome (Isaacson et al. , Reference Isaacson, Borewicz, Kim, Vannucci, Gebhart, Singer, Sreevatsan and Johnson2011). Understanding the mechanisms of disease pathogenesis used by infectious pathogens has been the basis for much work that has led to products and practices to control many infectious diseases of pigs. Included are vaccines and antibiotics and new specific management practices. We are beginning to understand that infectious diseases often are multi-factorial and that interactions between sets of microbes can be just as important in disease causation as specific virulence gene products. The understanding of microbial interactions in diseases has led to the concept of polymicrobic infections (Brogden et al. , Reference Brogden, Guthmiller and Taylor2005). This understanding of polymicrobic infections links with the new focus on molecular characterization of the gastrointestinal microbiome has the potential to lead to new approaches to control infectious diseases. A more in depth understanding of the roles of the microbiome in infectious diseases is likely to lead to a better understanding of disease pathogenesis, the definition of what constitutes a healthy microbiome, potentially new strategies to control disease, and new diagnostic/predictive tools.
Emerging studies on human health have revealed interesting associations between the gastrointestinal microbiome and diverse sets of conditions including certain chronic and autoimmune diseases including obesity and asthma (Leslie, Reference Leslie2012). However, most of that work has been performed using mouse models. The pig provides an interesting large animal model that can be used to further establish these correlations. Because of the similarity of the pig gastrointestinal tract to the human gastrointestinal tract and because there is limited genetic diversity between pigs, the pig as a model has advantages over mouse models. What is learned from studies using pigs also will have direct application to animal production and could be the basis of new production practices for swine. For example, current work on antibiotic growth promoters and accompanying changes in the composition of the microbiome might be the basis to identify functionally important microbes that contribute to animal growth. Those functionally important microbes could then be the basis of probiotic approaches to improve pig growth in the absence of antibiotics. Understanding the functional contributions of specific bacteria to the metabolism of the pig also could be used to develop unique feeds that shift digestion of feeds in a manner that promotes growth.
