Changes in the Microbiome Profile in Different Parts of the Intestine in Piglets with Diarrhea

Simple Summary The most common genera in the piglet microbiome were Lactobacillus, Escherichia-Shigella, Enterococcus, Bacteroides, and Fusobacterium. Bacteria of the Lactobacillus genus dominated in healthy piglets. An increased number of Escherichia-Shigella and Enterococcus was detected in diarrheal pigs. This indicates an important role of these bacteria in the pathogenesis of diarrhea. A decreased number of Bacteroides was detected in diarrheal pigs. According to the assessment of the microbiome composition in different sections of the intestine, bacteria of the Lactobacillus genus were the most common in the ileum, while Fusobacterium and Bacteroides were more common in the rectum. Our results show that the gut microbiome may make a significant contribution to the pathogenesis of diarrhea. Abstract Determining the taxonomic composition of microbial consortia of the piglet intestine is of great importance for pig production. However, knowledge on the variety of the intestinal microbiome in newborn piglets is limited. Piglet diarrhea is a serious gastrointestinal disease with a high morbidity and mortality that causes great economic damage to the pig industry. In this study, we investigated the microbiome of various sections of the piglet intestine and compared the microbiome composition of healthy and diarrheal piglets using high-throughput sequencing of the 16S rRNA gene. The results showed that bacteria of the Lactobacillus genus were the most common in the ileum, while Fusobacterium and Bacteroides dominated in the rectum. Comparing the microbiome composition of healthy and diarrheal piglets revealed a reduced number of Lactobacillus bacteria as a hallmark of diarrhea, as did an increased content of representatives of the Escherichia-Shigella genus and a reduced number of Bacteroides, which indicates the contribution of these bacteria to the development of diarrhea in piglets. The relative abundance of Enterococcus bacteria was higher in the diarrhea group. Although some bacteria of this genus are commensals, a small number of species may be associated with the development of diarrhea in piglets. Therefore, our results indicate that the gut microbiome may be an important factor in the development of diarrhea in piglets.


Introduction
The formation of the gut microbiome at an early age is of particular importance for piglets' health. Microbiome composition is a perspective predictive tool for health and disease assessment; however, it stays poorly described in terms of predisposition to diarrhea. Here, we aim to assess whether the composition of the gut microbiome is associated with differences in the susceptibility of pigs to diarrhea. During DNA isolation from samples, we added the sample that served as a negative control for data analysis and contained only the Milli-Q water which is used in the laboratory. This sample underwent sample preparation completely identically to the test samples to exclude the contamination of the test samples in the laboratory. During the bioinformatics analysis of the obtained data we used the decontam R package to identify and subsequently remove any laboratory contaminants. This additional step makes it possible to obtain more accurate data on the composition of the studied bacterial communities, which is based on the analysis of the V3 hypervariable region of the 16S rRNA marker gene and metagenomic data.

Amplification of the 16S rRNA Gene
In our work, to study the gut microbiome of piglets using sequencing on the Ion Torrent PGM platform we selected the hypervariable region V3 of the 16S rRNA gene. For the amplification of bacterial DNA we used universal primers 337F and 518R ( Table 1).
The amplification was performed with a 5 × ScreenMix-HS Master Mix kit (Evrogen, Moscow, Russia) in the following regime: 94 • C for 4 min; 37 cycles of 94 • C for 30 s, 53 • C for 30 s, and 72 • C for 30 s; and final elongation at 72 • C for 5 min.

Ion Torrent PGM Sequencing
PCR products were purified using AMPureXP magnetic particles (Beckman Coulter, Brea, CA, USA). The preparation of libraries for sequencing was performed using a NEB-Next Fast DNA Library Prep kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's protocol. After that, the obtained libraries were mixed in equimolar amounts for emulsion PCR on a OneTouch 2 System (Thermo Fisher Scientific, Madison, WI, USA). Sequencing was performed with an Ion PGM Hi-Q View Sequencing Kit (Thermo Fisher Scientific, Madison, WI, USA) using an Ion Torrent PGM system.

Statistical Data Analysis
The sets of sequences for each sample were obtained in a BAM format. Next, the files were converted into a FastQ format using SAMtools v.1.2 software and analyzed using the R programming language in the RStudio environment. The raw reads were filtered by length and the quality was controlled by using the functions of VSEARCH v.2.8.2 software. The samples were pooled and unique sequences were identified before searching for operational taxonomic units (OTUs). To find the OTUs, we used the UNOISE2 algorithm, which reduces noise by correcting errors. To generate the OTUs and make the OTU table, we combined all the reads for all the samples. After that, the processed reads were consistent with the reference readings in the SILVA v.123 databases (https://www.arb-silva.de, accessed on 26 October 2021) using the DADA2 package. The DADA2 package provides a native implementation of the naive Bayesian classifier method for this purpose.
The statistical analysis was performed using GraphPad Prism 9 software (GraphPad, Sand Diego, CA, USA). The differences in the bacterial composition of the microbiomes from the intestine sections were analyzed using two-way analysis of variance (ANOVA). The results are expressed as mean ± standard error of the mean (SEM).

Results
In this study, we investigated the bacterial profiles (at the level of genera) of the ileum, cecum, colon, and rectum from twelve piglets. After filtering the reads obtained by sequencing of 41 studied samples, a total of 307,977 unique sequences were identified, which corresponded to 166 genera (99% identity) ( Figure 1).  Figure 1 shows that 38% of the sequences belonged to members of the Lactobacillus genus. The next in numbers were Escherichia-Shigella and Enterococcus (11% each), Bacteroides (9%), Fusobacterium (8%), Streptococcus and Prevotella (3% each), and Blautia, Clostridium sensu stricto 1, Rikenellaceae RC9 gut group, and Sphaerochaeta (1% each). The gen-  Figure 1 shows that 38% of the sequences belonged to members of the Lactobacillus genus. The next in numbers were Escherichia-Shigella and Enterococcus (11% each), Bacteroides (9%), Fusobacterium (8%), Streptococcus and Prevotella (3% each), and Blautia, Clostridium sensu stricto 1, Rikenellaceae RC9 gut group, and Sphaerochaeta (1% each). The genera with the content below 1% were combined into the "Others" group.
The results revealed the differences between the microbial profiles of different sections of piglet gastrointestinal tract, as well as between sick and healthy piglets (the 40 most common genera identified for each intestinal section are shown in Figure 2). The bacterial composition of individual samples can be found in Supplementary Material Figure S1.  Figure 1 shows that 38% of the sequences belonged to members of the Lactobacillus genus. The next in numbers were Escherichia-Shigella and Enterococcus (11% each), Bacteroides (9%), Fusobacterium (8%), Streptococcus and Prevotella (3% each), and Blautia, Clostridium sensu stricto 1, Rikenellaceae RC9 gut group, and Sphaerochaeta (1% each). The genera with the content below 1% were combined into the "Others" group.
The results revealed the differences between the microbial profiles of different sections of piglet gastrointestinal tract, as well as between sick and healthy piglets (the 40 most common genera identified for each intestinal section are shown in Figure 2). The bacterial composition of individual samples can be found in Supplementary Material Figure S1.    Figure 2 shows that the Lactobacillus genus prevailed over other genera in all sections of the intestine; however, a greater number of these bacteria was found in healthy piglets (51% on average) vs. animals with diarrhea (30% on average). Bacteria of the Escherichia-Shigella genus were also found in large numbers (24%) in sick piglets compared to 1% in healthy animals. Representatives of the Bacteroides genus were more common in the healthy group (14%) vs. 4% in sick piglets. The abundance of Streptococcus in the healthy animals was 6%, while in piglets with diarrhea, the content of these bacteria was approximately 1%. In the healthy group, the amount of Sphaerochaeta was 2%; in the sick group, these bacteria were absent. The relative content of representatives of the Enterococcus genus in the healthy and sick groups was 1% and 20%, respectively. Figure 3 shows the bacterial genera, the intestinal content of which differed statistically in the healthy and diseased groups.
Among all identified bacterial genera, statistically significant differences were found for the representatives of Enterococcus, Lactobacillus, Escherichia-Shigella, and Bacteroides. The content of Enterococcus and Escherichia-Shigella bacteria in the sick group increased (to 21 and 23%, respectively) compared to the healthy animals, in which their relative abundance was 1% for both genera. At the same time, in sick piglets, the content of Lactobacillus (31%) and Bacteroides (3%) was reduced compared to 50% and 14%, respectively, in the healthy group.
We also carried out a comparative analysis of the bacterial composition in different sections of the intestine from healthy piglets and revealed statistically significant differences in the relative abundance of bacteria belonging to the Lactobacillus, Fusobacterium, and Bacteroides genera (Figures 4-6). Figure 4 shows that the amount of Lactobacillus bacteria in the ileum (72%) was significantly higher than in the cecum (50%) and rectum (29%).
Among all identified bacterial genera, statistically significant differences were found for the representatives of Enterococcus, Lactobacillus, Escherichia-Shigella, and Bacteroides. The content of Enterococcus and Escherichia-Shigella bacteria in the sick group increased (to 21 and 23%, respectively) compared to the healthy animals, in which their relative abundance was 1% for both genera. At the same time, in sick piglets, the content of Lactobacillus (31%) and Bacteroides (3%) was reduced compared to 50% and 14%, respectively, in the healthy group. We also carried out a comparative analysis of the bacterial composition in different sections of the intestine from healthy piglets and revealed statistically significant differences in the relative abundance of bacteria belonging to the Lactobacillus, Fusobacterium, and Bacteroides genera (Figures 4-6). Figure 4 shows that the amount of Lactobacillus bacteria in the ileum (72%) was significantly higher than in the cecum (50%) and rectum (29%).  The relative number of representatives of the Fusobacterium genus increases in the direction from the small to the large intestine ( Figure 5). However, a statistically significant difference was observed only between the content of Fusobacterium bacteria in the ileum and rectum (3% and 12%, respectively), i.e., in the most distant sections. The relative number of representatives of the Fusobacterium genus increases in the direction from the small to the large intestine ( Figure 5). However, a statistically significant difference was observed only between the content of Fusobacterium bacteria in the ileum and rectum (3% and 12%, respectively), i.e., in the most distant sections. Figure 6 demonstrates a statistically significant difference in the content of Bacteroides genus between the ileum (3%) and cecum (15%), as well as between the ileum (3%) and colon (21%).
Next, we compared the bacterial composition of each intestinal section in the healthy and sick groups (Figures 7-10).
In the ileum of sick piglets, the content of Enterococcus and Escherichia-Shigella bacteria (14% each) was approximately 7 and 20 times higher than in the healthy animals (2 and 0.7%, respectively). The relative number of representatives of the Fusobacterium genus increases in the direction from the small to the large intestine ( Figure 5). However, a statistically significant difference was observed only between the content of Fusobacterium bacteria in the ileum and rectum (3% and 12%, respectively), i.e., in the most distant sections.  Figure 6 demonstrates a statistically significant difference in the content of Bacteroides genus between the ileum (3%) and cecum (15%), as well as between the ileum (3%) and colon (21%). Next, we compared the bacterial composition of each intestinal section in the healthy and sick groups (Figures 7-10).
In the ileum of sick piglets, the content of Enterococcus and Escherichia-Shigella bacteria (14% each) was approximately 7 and 20 times higher than in the healthy animals (2 and 0.7%, respectively). In the cecum, a significant difference in the relative content was found only for the Escherichia-Shigella genus (36% in sick animals vs. 0.6% in healthy piglets).
The relative content of Enterococcus (9.5%) and Escherichia-Shigella (28%) bacteria was increased in the colon and ileum of sick piglets (compared to 0.5% and 1%, respectively, in the healthy group).
In the rectum, a significant difference in the relative abundance was observed for the three genera. The content of Enterococcus and Escherichia-Shigella bacteria was increased in the sick group as compared to the healthy animals (27% and 23% vs. 0.5% and 1%, respectively). However, the number of representatives of the Bacteroides genus was reduced in sick piglets (5%) in comparison with the healthy ones (21%).
Next, we compared the bacterial composition of each intestinal section in the healthy and sick groups (Figures 7-10).
In the ileum of sick piglets, the content of Enterococcus and Escherichia-Shigella bacteria (14% each) was approximately 7 and 20 times higher than in the healthy animals (2 and 0.7%, respectively). In the cecum, a significant difference in the relative content was found only for the Escherichia-Shigella genus (36% in sick animals vs. 0.6% in healthy piglets). The relative content of Enterococcus (9.5%) and Escherichia-Shigella (28%) bacteria was increased in the colon and ileum of sick piglets (compared to 0.5% and 1%, respectively, in the healthy group). In the rectum, a significant difference in the relative abundance was observed for the The relative content of Enterococcus (9.5%) and Escherichia-Shigella (28%) bacteria was increased in the colon and ileum of sick piglets (compared to 0.5% and 1%, respectively, in the healthy group). In the rectum, a significant difference in the relative abundance was observed for the three genera. The content of Enterococcus and Escherichia-Shigella bacteria was increased in the sick group as compared to the healthy animals (27% and 23% vs. 0.5% and 1%, respectively). However, the number of representatives of the Bacteroides genus was reduced in sick piglets (5%) in comparison with the healthy ones (21%).

Discussion
In this study, we discovered that Lactobacillus was one of the major genera of the pig gastrointestinal tract (Figure 1). Lactobacillus representatives are common in both proximal and distal sections of pig digestive tract, which they colonize shortly after birth [31].
Lactobacillus bacteria possess several probiotic properties necessary for resistance to infections and diseases of the gastrointestinal tract. They exhibit antipathogenic activity [32,33] and antioxidant activity [34,35] and are involved in the regulation of immune system [36]. All these traits affect gut microbial populations, such that propagation of opportunistic pathogens, such as Salmonella, Clostridia, and Enterobacteriaceae, is controlled, resulting in the prevention of infections and intestinal disorders [37][38][39]. It has been shown that Lactobacillus species colonize piglet intestine shortly after birth and are stable members of the gut microbiome throughout the entire intestinal tract [40]. The low abundance of Lactobacillus representatives, which are considered beneficial, may be an indicator of gastrointestinal problems in pigs. In our study (Figure 3), we observed a reduced amount of Lactobacillus bacteria in piglets with diarrhea, which confirms the probiotic properties of the Lactobacillus genus.
Physiological variations along the length of the intestine include chemical and nutrient gradients, as well as compartmentalized host immune activity. All these factors influence bacterial community composition [41]. It is well known that piglets experience extreme stress when they are weaned from the sow. This can lead to the development of intestinal dysfunction and immune system disorders, which ultimately leads to a deterioration in the health of piglets, especially during the first week after weaning. We would like to point out that only suckling piglets were involved in this study. This made it possible to exclude the influence of stress endured during weaning on changes in the microbiome and, as a consequence, the development of diarrhea [42].
According to the results of our study, Lactobacillus dominated in the distal part of the small intestine (ileum) (Figure 4). Lactobacillus species grow in an oxygen-free or low-oxygen atmosphere. Oxygen present in the small intestine is gradually depleted by aerobic bacteria, and only a small amount of it remains in the ileum, where most Lactobacillus bacteria are found. Some Lactobacillus species produce acetic acid, which has a fairly strong antipathogenic effect [43].

Discussion
In this study, we discovered that Lactobacillus was one of the major genera of the pig gastrointestinal tract (Figure 1). Lactobacillus representatives are common in both proximal and distal sections of pig digestive tract, which they colonize shortly after birth [31].
Lactobacillus bacteria possess several probiotic properties necessary for resistance to infections and diseases of the gastrointestinal tract. They exhibit antipathogenic activity [32,33] and antioxidant activity [34,35] and are involved in the regulation of immune system [36]. All these traits affect gut microbial populations, such that propagation of opportunistic pathogens, such as Salmonella, Clostridia, and Enterobacteriaceae, is controlled, resulting in the prevention of infections and intestinal disorders [37][38][39]. It has been shown that Lactobacillus species colonize piglet intestine shortly after birth and are stable members of the gut microbiome throughout the entire intestinal tract [40]. The low abundance of Lactobacillus representatives, which are considered beneficial, may be an indicator of gastrointestinal problems in pigs. In our study (Figure 3), we observed a reduced amount of Lactobacillus bacteria in piglets with diarrhea, which confirms the probiotic properties of the Lactobacillus genus.
Physiological variations along the length of the intestine include chemical and nutrient gradients, as well as compartmentalized host immune activity. All these factors influence bacterial community composition [41]. It is well known that piglets experience extreme stress when they are weaned from the sow. This can lead to the development of intestinal dysfunction and immune system disorders, which ultimately leads to a deterioration in the health of piglets, especially during the first week after weaning. We would like to point out that only suckling piglets were involved in this study. This made it possible to exclude the influence of stress endured during weaning on changes in the microbiome and, as a consequence, the development of diarrhea [42].
According to the results of our study, Lactobacillus dominated in the distal part of the small intestine (ileum) (Figure 4). Lactobacillus species grow in an oxygen-free or lowoxygen atmosphere. Oxygen present in the small intestine is gradually depleted by aerobic bacteria, and only a small amount of it remains in the ileum, where most Lactobacillus bacteria are found. Some Lactobacillus species produce acetic acid, which has a fairly strong antipathogenic effect [43].
In our study, the content of Escherichia-Shigella bacteria was much higher in piglets with diarrhea ( Figure 3). Several Escherichia-Shigella species are believed to play an important role in the development of diarrhea in piglets and to have a serious effect on the barrier function of the animal intestine [44]. An increased number of Escherichia-Shigella bacteria in sick piglets vs. healthy ones was observed in all sections of the intestine (Figures 7-10). However, we found no differences in the number of these bacteria in the intestine sections in the healthy group. This suggests that Escherichia-Shigella bacteria are distributed approximately evenly in the investigated sections of the intestine.
Members of the Bacteroides genus are often found in the digestive tract of mammals. They are early colonizers of the intestines of healthy piglets [19]. Bacteroides species play an important role in health promotion by producing butyrate, which activates T cell-mediated immune response, thus limiting colonization of the digestive tract by potentially pathogenic bacteria [45]. In the healthy group, bacteria of this genus were more common in the rectum ( Figure 6). The number of Bacteroides representatives in the rectum of piglets with diarrhea was lower than in the control group ( Figure 10).
We also found an increased number of Enterococcus bacteria in sick piglets ( Figure 3). Information on the enterococcal flora of healthy newborn piglets is scanty, but a small number of representatives of this genus can be a part of the normal gut microbiota [46,47]. However, Enterococcus is sometimes associated with piglet diarrhea [48,49]. Although many members of the Enterococcus genus are believed to be commensals of the intestinal tract, some representatives cause diarrhea in suckling animals of various species [50][51][52][53][54][55].
We also found an increased number of Enterococcus bacteria in the ileum (Figure 7), colon (Figure 9), and rectum ( Figure 10) of piglets with diarrhea compared to healthy animals. This may indicate that pathogenic Enterococcus representatives are prevalent in the intestinal microbiome, which may be associated with the diarrhea of newborns piglets.
Fusobacterium species are anaerobic, Gram-negative, non-spore-forming, non-motile, rod-shaped bacteria. The main metabolite produced by these bacteria is butyric acid [56]. According to numerous data, Fusobacterium is considered to be a normal representative of the oropharyngeal, gastrointestinal and genital microbiota. At the same time, this genus is the second most commonly isolated anaerobic microbial group from clinical samples of both humans and animals, especially in the case of purulent-necrotic infections [57]. Fusobacterium is involved in various clinical anaerobic infections and can cause intestinal inflammation [58]. Even though many studies have shown increased levels of Fusobacterium in piglets with various intestinal disorders compared to the healthy ones [59][60][61][62][63], we did not find statistically significant differences in the content of this genus in the studied groups. However, we found an increased content of Fusobacterium members in the rectum compared to the ileum of healthy pigs. The content of Fusobacterium bacteria in newborn piglets requires further research.
We studied the bacterial composition inhabiting the intestines of healthy newborn piglets, as well as piglets with diarrhea. Thus, our study, using high-throughput sequencing, showed that microbial communities in the studied samples included many commensal and opportunistic microorganisms. Compared to classical microbiological and immunological approaches, as well as PCR, the advantage of this method is the ability to identify all bacteria contained in the test sample, including non-culturable microorganisms [64]. We used the universal primer pair 337F/518R (see Section 2.3. Amplification of the 16S rRNA gene) to amplify a fragment of the 16S rRNA gene that includes the V3 region, which is considered one of the most effective hypervariable regions for phylogenetic analysis and taxonomic classification of bacterial species [65,66].
The results of this study can be used to predict bacterial taxa indicative of healthy development of gut microbiome in suckling piglets, as well as to identify taxa that can be used as probiotics to prevent post-weaning diarrhea.

Conclusions
The most common genera in the piglet microbiome were Lactobacillus, Escherichia-Shigella, Enterococcus, Bacteroides, and Fusobacterium. Bacteria of the Lactobacillus genus dominated in healthy piglets, which once again proves their probiotic effect. An increased number of Escherichia-Shigella representatives in diarrheal pigs indicates the contribution of these bacteria to the development of diarrhea. A decreased number of Bacteroides may also indicate the development of diarrhea. The content of Enterococcus bacteria was higher in sick piglets. According to the assessment of the microbiome composition in different sections of the intestine, bacteria of the Lactobacillus genus were most common in the ileum, while Fusobacterium and Bacteroides were more common in the rectum.
Further studies are needed to understand the protective mechanisms of the gut microbial community and to develop clinical interventions to improve gut health in piglets.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12030320/s1, Figure S1: The abundance of microorganisms for the samples.

Conflicts of Interest:
The authors declare no conflict of interest.