Dietary supplementation with Bacillus mixture promotes the gut health of weaned piglets

This study was conducted to investigate the effects of dietary supplementation with a mixture of Bacillus on the intestinal health of weaned piglets. We randomly assigned 120 piglets to three groups; a control group (basal diet), a probiotics group (supplemented with 4 × 10 9 CFU/g Bacillus licheniformis - B. subtilis mixture; BLS mix), and an antibiotics group (supplemented with 0.04 kg/t virginiamycin, 0.2 kg/t colistin, and 3000 mg/kg zinc oxide). All groups had five replicates with eight piglets per replicate. On days 7, 21, and 42 of the trial, intestine and digesta samples were collected to determine the intestinal morphology, gut microbiota and metabolites, and the expression of genes related to gut health. The results showed that the BLS mix decreased the jejunum crypt depth, increased the ileum villus height, and increased the jejunum and ileum villus height to crypt depth ratio. The BLS mix also increased the expression levels of E-cadherin and Occludin in the colon and pro-inflammatory cytokines and TLR4 in ileum and colon. The BLS mix also increased Simpson’s diversity index in the gut microbiota and the relative abundances of o_ Bacteroidetes and f_ Ruminococcaceae , but it decreased the relative abundances of Blautia , and Clostridium . Collectively, these findings suggested that dietary BLS mix supplementation efficaciously promotes intestinal health through the modulation of gut microbiota in weaned piglets.


Introduction
In commercial swine production, early weaning contributes to intestinal development, digestive, or immune dysfunction in weaned piglets, which leads to reduced feed intake and growth inhibition (Campbell et al., 2013). Antibiotics are used to control the incidence of infectious diseases and improve growth in weaned piglets (Kim et al., 2012). However, many countries banned antibiotics in the livestock due to the increasing resistance of pathogens and antibiotic residues in food (Toutain et al., 2016;Allen et al., 2014). Therefore, effective antibiotic alternatives are urgently required to reduce the dependence of the animal industry on antibiotics. Previous studies have focused on the development of novel alternatives to antibiotics, including probiotics (Hu et al., 2018), prebiotics (Tran et al., 2018), and synbiotics (Zhang et al., 2018). Among these alternatives, probiotics have a higher potential to act pathogen exclusion (Azad et al., 2018).
also produces various digestive enzymes that stimulate peristalsis of the intestine, thereby improving nutrient digestion (Giang et al., 2011). Presently, Bacillus spp.
are widely used as commercialized probiotic products for humans and animals (Cutting, 2011). A previous study showed that a mix of B. subtilis and B.
licheniformis can improve the growth and immunological status of farmed tilapia (Abarike et al., 2018). Moreover, dietary supplementation with B. subtilis DSM32315 played a beneficial role in maintaining the intestinal barrier function and microflora balance of weaned piglets, and it improved their growth performance (Tang et al., 2019). Lan and Kim (2019) suggested that dietary supplementation with a B. licheniformis and B. subtilis complex in growing-finishing pigs increased digestibility and the fecal Lactobacillus counts and decreased the fecal NH 3 and total mercaptan emissions.
However, the Bacillus licheniformis and B. subtilis mixture (BLS mix), as with other strains of Bacillus, have rarely been studied as probiotics for weaned piglets. Furthermore, little is known about the effects of the BLS mix on the intestinal morphology and microflora composition of weaned piglets. Our previous study indicated that dietary supplementation with Bacillus subtilis decreased the diarrhea rate and improved body weight gain in piglets (Wang et al., 2019a). Thus, we hypothesized that dietary BLS mix supplementation will improve the intestinal development and health of weaned piglets, through the modulation of the gut microbiota. Therefore, the present study investigated the changes in intestinal morphological, microbiota communities, and bacterial metabolites in weaned piglets fed different diets with the Bacillus licheniformis and B. subtilis mixture by Illumina HiSeq 2500 sequencing and biochemical analysis, to provide new evidence for the mechanism of action of these probiotics.

Experimental design and dietary treatments
A total of 120 healthy crossbred piglets (Landrace × Large white, 7.00 ± 0.5 kg body weight) were weaned at 25 days of age and fed a corn and soybean mealbased diet. After 3 days of adaptation, the piglets were randomly assigned to one of three groups: a blank control group (basal diet), probiotics group (supplemented with 4 × 10 9 CFU/g Bacillus licheniformis-B. subtilis mixture; BLS mix), or an antibiotic group (supplemented with 0.04 kg/t virginiamycin, 0.2 kg/t colistin, and 3 000 mg/kg zinc oxide). The composition and nutrient levels of the basal diet met the nutritional requirements for nursey piglets established by the National Research Council (2012), which are shown in Table S1. The experiments lasted for 42 days.
The probiotic mixture, B. licheniformis (B26) and B. subtilis DSM32315 (B21), were produced by fermentation; the conditions were 200 r/min stirring speed and 350 L/h throughput. The fermentation products were filtered with an organic ceramic membrane filtration system and mixed with mineral adsorbent at a ratio of 1:1. The final product was a dry powder that was counted by the plate counting method to determine the viable number and then was mixed in the diet.

Sample collection and preparation
On days 7, 21, and 42 of the trial and 12 h after the last feeding, piglets from each replicate close to average weight were slaughtered by electric shock (120 V, 200 Hz). The intestinal contents from each colon (10 cm from the posterior to the ileocecal valve) were collected and stored at -20 °C for analyses of the short-chain fatty acids (SCFAs), indoles, skatoles, bioamines, and the composition of the microbiota. Samples of the jejunum, ileum, and colon tissue (approximately 2 cm) were collected, washed with cold physiological saline, immediately frozen in liquid nitrogen, and stored at -80 °C for further analyses. The jejunum and ileum from all piglets were fixed with 4 % paraformaldehyde-PBS overnight, and then dehydrated and embedded in paraffin blocks. A 5 μm section was cut from each sample for histological analysis.

Intestinal histological examination
The sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H.E.). From each intestinal sample, the villus length, villus width, and crypt depth were measured at 10 visual fields, and the villus height/crypt depth ratio was calculated.

16S sequencing and bioinformatics analysis
Microbial genomic DNA was extracted from all samples using a HiPure Stool DNA Kit (Magen, Guangzhou, China) following the manufacturer's instructions. A multiplexed amplicon library covering the V3-V4 region of the 16S rDNA gene was PCR-amplified with the optimized primer sets for the Illumina HiSeq 2500 sequencing instrument.
Each paired-end read was then spliced using the FLASH (Magoc and Salzberg, 2011) software (version 1.2.1) to obtain the original spliced sequence (Raw contigs). Raw tags were mass filtered using the Trimmomatic software (version 0.33) to obtain high-quality clean data. All chimeric sequences were removed by Uchime (Edgar et al., 2011) (version 4.2). The chimera-free sequences were processed with a standard QIIME 1.91 pipeline (Bokulich et al., 2013) and clustered into operational taxonomic units (OTUs) at a 97 % similarity threshold using an "Open-Reference" approach.
Alpha rarefaction was analyzed by Chao1, Shannon, and Simpson indexes (Chao and Lee, 1992). To decipher the difference in microbiota structure between groups, LEfSe (linear discriminant analysis effect size) was performed, and the cladogram was graphed with default parameters (Segata et al., 2011). To probe the microbial metabolism and predict metagenome functional content from the marker gene, PICRUSt was utilized to explore differences in the KEGG pathway between groups (Langille et al., 2013). Spearman correlation coefficients were calculated for the correlation between gut health and the change in microbiota to establish suitable microbial compositions for better gut health.

Analysis of gene expression related to gut health
Gene expression was quantitated by real-time polymerase chain reaction (RT-PCR), as described by Su et al. (2018). Briefly, total RNA was isolated from colonic tissues using TRIzol (Invitrogen, Carlsbad, CA, United States) and reverse transcribed with a Prime Script RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). RT-PCR was conducted with primers of the target genes (Table S2) initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95°C for 5 s, and annealing at 60 °C for 30 s. Relative gene expression was calculated by the 2 -ΔΔCt method (Schmittgen and Livak, 2008).

Statistical analysis
Intestinal morphology index, colonic metabolite, and genes expression were analyzed with a one-way ANOVA in SPSS 17.0 software (SPSS, Inc., Chicago, IL, United States). The data are presented as means ± SE and P < 0.05 indicates statistical significance. The alpha diversity indices, relative species abundances, and overall composition of gut microbiota were analyzed using the Kruskal Wallis test. Spearman correlation coefficient was used to assess the relationships between health parameters and relative abundances of genera. LEfSe were used to identify different taxa microbes using default parameters.

Effect of BLS Mix on the intestinal morphology of weaned piglets
The intestinal morphology data are summarized in Table 1. On day 7 of the trial, dietary supplementation with antibiotics or BLS mix decreased the crypt depth (P < 0.05) and increased the ratio of villus height to crypt depth in the jejunum compared with the control group (P < 0.05). On day 7 and day 42, the villus height and ratio of villus height to crypt depth in the ileum increased in both the antibiotics and BLS mix groups compared with the control group (P < 0.05).
Interestingly, an increase in the crypt depth was observed in the BLS mix group relative to the antibiotics group on day 21 (P < 0.05).

Effect of BLS Mix on the microbiota diversity of weaned piglets
The 16S rDNA V3-V4 region amplicon sequencing generated 843 766 high-quality reads, with an average of 56 251 reads (range 45 822 to 67 947) per sample. Alpha diversity was measured to detect the diversity and structure of the colonic microbial communities in the different treatment groups (Figure 1). The Simpson index did not differ throughout the experimental period (P > 0.05). However, compared to the control group, the Chao1 index of the BLS mix group was lower on day 7 (P<0.05), whereas the Shannon index was higher on day 21 (P<0.05). This illustrates that dietary supplementation with the BLS mix increased the colonic microbial diversity of the piglets. The differences in the microbial communities at the phylum and genus levels are shown in Figure 3. On day 7, Ruminococcaceae occurred at higher amounts in the BLS mix group than in the control group (P < 0.05). Blautia occurred at lower levels in the antibiotics and BLS mix groups than in the control group (P < 0.05). On day 21 of the trial, Antibiotics led to a decrease in Lactobacillus and an increase in Tenericutes and RF39 compared to their relative abundances in the control group (P < 0.05). The BLS mix led to an increase in Bacteroidales compared to their relative abundances in the control group (P < 0.05). On day 42, Prevotella occurred at a lower level in the BLS mix group than in the control group (P < 0.05). Clostridium occurred at a higher level in the antibiotics and BLS mix groups than in the control group (P < 0.05).

Effect of BLS Mix on the microbial function of weaned piglets
The LefSe analysis was performed to confirm the different effects of the BLS mix on the intestinal microbiota in piglets ( Figure 4). Significant differences in the colonic microbiota were found between the control, antibiotic, and BLS mix groups for the entire experimental period. These results confirmed a significant enrichment of

Effect of BLS Mix on the gut metabolites of weaned piglets
As shown in Table S3, the concentrations of SCFA and bioamines in the colonic contents of the three treatment groups did not differ on day 7 of the trial (P > 0.05).
The SCFA concentration on day 21 also did not differ (Table 2). On day 42, the concentration of isobutyrate and isovalerate decreased in the BLS mix group relative to the control group (P < 0.05), while the acetate concentration increased (P < 0.05; Table 2). The bioamines concentration of the colonic contents is shown in Table 2. On day 21, the spermine concentration increased in the BLS mix group relative to the control and antibiotics groups (P < 0.05). The skatoles concentration increased in the antibiotics group relative to the control and BLS mix groups (P < 0.05). However, the skatoles concentration decreased in the antibiotics and BLS mix groups relative to the control group on day 42 (P < 0.05).

Effect of BLS Mix on the intestinal health-related genes of weaned piglets
On day 7 of the trial, dietary supplementation with the BLS mix increased the mRNA level of IL-2, IL-6, IL-1β, and TLR-4 ( P < 0.05) and decreased the level of TNF-α (P < 0.05) in the ileum compared to the antibiotics group. BLS mix supplementation also increased the mRNA level of IL-2 in the jejunum compared to the antibiotics group (P < 0.05). On day 21, dietary supplementation with the BLS mix increased the mRNA level of TLR4 (P < 0.05) and decreased the mRNA level of TNF-α (P < 0.05) in the ileum compared to the antibiotics group. On day 42 of the trial, an increase in the mRNA level of TLR-4 in the ileum was observed in the BLS mix group relative to the control group (P < 0.05).
The mRNA levels in the colon contents of the piglets from different dietary treatments are shown in Table 3. Compared to the control group, dietary supplementation with the BLS mix increased the mRNA level of Occludin, IL-6, IL-1β, and TLR-4 on day 7, and the level of E-cadherin and IL-1β on day 42 (P < 0.05).

Relationships between gut microbiota, metabolite, and intestinal healthrelated genes
A spearman's rank correlation analysis was performed to evaluate the potential link between alterations in gut microbiota and health parameters of the weaned piglets ( Figure 6). The genus Prevotella was positively correlated with spermidine and spermine (P < 0.01) and negatively correlated with acetate and propionate (P < 0.05). The phylum Bacteroidetes and genus CF231 were positively correlated with tryptamine, spermidine, and spermine (P < 0.01) but negatively correlated with acetate (P < 0.01). The genus Anaerovibrio was positively correlated with spermine but negatively correlated with acetate, propionate, and butyrate. The order Bacteroidetes was positively correlated with spermine (P < 0.05). The phylum Treponema was positively correlated with tyramine, tryptamine, putrescine, and spermidine but negatively correlated with skatole (P < 0.05). The family Erysipelotrichaceae was positively correlated with spermidine, TLR-4, and IL-1β (P < 0.05). The genus Clostridium was positively correlated with spermidine and negatively correlated with acetate (P < 0.05). The order Clostridiales was positively correlated with the level of Occludin (P < 0.05). The phylum Firmicutes was negatively correlated with spermidine (P < 0.05). The family Ruminococcaceae was positively correlated with the levels of Occludin and TLR-4 (P < 0.05) but negatively correlated with tyramine, tryptamine, and putrescine (P < 0.05). The order RF39 and phylum Tenericutes were negatively correlated with tyramine, tryptamine, and putrescine (P < 0.05). The genus Lactobacillus was positively correlated with acetate (P < 0.05) but negatively correlated with spermidine, E-cadherin, and IL-1β (P < 0.05). The genus Blautia was negatively correlated with IL-1β (P < 0.05).

Discussion
Bacillus spp. is widely used as a commercialized probiotic product for humans and animals (Cutting, 2011)  The intestinal mucosal barrier is an important modulator of intestinal homeostasis, and intestinal morphology reflects mucosal integrity and injury (Blikslager et al., epithelial cells to digest and absorb nutrients (Yin et al., 2019). Our results showed that dietary BLS mix supplementation can decrease the jejunum crypt depth, increase the ileum villus height, and change the ratio of villus height to crypt depth in the jejunum and ileum. These findings suggest that the intestinal mucosal barrier is intact and has strong nutrient digestibility and absorption capacity . Recent studies suggested that the gut physiological barrier is formed by the epithelial cells, tight junction proteins, and intestinal secretions (Suzuki, 2013;Yan and Ajuwon, 2017), in which the E-cadherin, Occludin, and ZO-1 are indicators for tight junction assembly, stability, and barrier function (Zihni et al., 2016). The results presented here showed that the BLS mix upregulated the expression of Ecadherin and Occludin in the colon, suggesting that the BLS mix enhanced the repair of intestinal damage and preserved the integrity of the intestinal mucosa.
Alpha diversity reflects the species diversity in a single sample by incorporating indices for species richness (Chao and Ace) and diversity (Shannon and Simpson) (DeSantis et al., 2006). Our findings are consistent with those of Wang et al. (2019b), who found that probiotics increase the Simpson's diversity index of the microbial ecosystem in piglets. Firmicutes and Bacteroidetes are regarded as the main microbiota phyla in the pig gut, regardless of probiotic or antibiotic use (Wang et al., 2019b). The present study showed that dietary supplementation with the BLS mix increases the relative abundances of Bacteroidale and Ruminococcaceae and decreases the abundances of Blautia, and Clostridium. Bacteroidales can degrade proteins and carbohydrates (Thomas et al., 2011) and activate the host's immune system (Mazmanian et al., 2008). Clostridium is closely related to protein fermentation and can increase the risk of diarrhea (Rist et al., 2014). The lower abundance of Clostridium in the probiotics group may explain our previous finding that dietary supplementation with Bacillus Subtilis decreased the diarrhea rate in the piglets (Wang et al., 2019a). Ruminococcaceae ferments cellulose and hemicellulose and produces SCFAs used for energy (Biddle et al., 2013). These findings suggest that the BLS mix may regulate gut community composition and improve gut health.
PICRUSt (Langille et al., 2013) can be used to investigate the functional differences in microbiota to determine the metabolic alterations caused by antibiotics or probiotics (Wang et al., 2019b). In this study, dietary BLS mix supplementation increased the metabolic pathways of cofactors/vitamin metabolism and carbohydrate metabolism on day 21 of trial, as well as the metabolic pathways of carbohydrate, lipid, amino acid metabolism, cofactors and vitamins, and glycan biosynthesis and metabolism on day 42. Vitamins and cofactors are critical for the bioconversion of nutrients to energy and for maintaining homeostasis (Hu et al., 2016;Sharma et al., 2019). The glycan biosynthesis and metabolism pathway is important for carbohydrate metabolism (Hu et al., 2016;Varki, 2017). Gao et al (2017) showed that feed-additive probiotics accelerate intestinal microbiota maturation. Therefore, our results suggest that dietary BLS mix supplementation might accelerate intestinal microbiota maturation by enrichment of the important metabolic pathways.
The SCFAs produced by the colonic microbes via the fermentation of indigestible fiber are important for gut integrity, glucose homeostasis, and immune function (Morrison and Preston, 2016). In our study, acetate and propionate were the major SCFAs produced in the colon, which was consistent with previous findings in pregnant Huanjiang mini-pigs (Kong et al., 2016). Acetate can inhibit pathogenic bacteria, and butyrate acts as a major energy source for colonic epithelial cells (Morrison and Preston, 2016). BCFAs are produced by microbes through the deamination and decarboxylation of amino acids (Le Roy et al., 2013). In this study, dietary BLS mix supplementation increased the acetate concentration and decreased the concentrations of isobutyrate and isovalerate. Rist et al (2013) suggested that BCFAs might function as indicators of the extent of protein fermentation and exert detrimental effects on the host. These findings suggest that the BLS mix may inhibit pathogenic bacteria, decrease protein fermentation, and improve gut health.
The colonic microbiota catabolizes nitrogenous compounds to putrefactive catabolites, such as bioamines, indoles, and skatoles (Kong et al., 2016). Bioamines are markers of potentially harmful metabolic pathways. However, spermine is essential for somatic cell growth (Davila et al., 2013). Our results showed that dietary BLS mix supplementation can increase the spermine concentration and decrease the skatole concentration. High levels of bioamines and skatole might be toxic to gut health . Thus, decreasing the concentration of these compounds via a dietary BLS mix supplementation may exert beneficial effects on gut health.
Cytokines play crucial roles in the regulation of the immune and inflammatory responses and the barrier integrity of the gut (Andrews et al., 2018). LPS-mediated induction of the TLR4 signaling pathway results in the activation of nuclear factor κB (NF-κB) and, therefore, the expression of pro-and anti-inflammatory cytokines.
The piglets challenged with LPS upregulated their gene expression of proinflammatory cytokines in the jejunal mucosa, including TNF-α,  Our findings showed that dietary BLS mix supplementation increases the level of pro-inflammatory cytokine and TLR4 in the ileum and colon, suggesting that the BLS mix might activate the inflammatory pathway and improve autoimmunity. Shi et al (2019) showed that moderate activation of the TLR4 signaling pathway promotes inflammation and repair of the intestinal epithelium in DSS-induced colitis. Thus, we suggest that the BLS mix might alter the composition of the gut microbiota and accelerate intestinal microbiota maturation by increasing the metabolic pathways, enhancing the repair of intestinal damage, preserving the integrity of the intestinal mucosa, and providing better mucosal immunity.
In conclusion, dietary supplementation with the BLS mix had beneficial effects in weaned piglets, improving intestinal integrity and providing better mucosal immunity. These effects may be regulated by the activation of metabolic pathways and by the alteration of the gut microbiota community composition.

Ethics approval and consent to participate
The experimental design and procedures used in this study were reviewed and approved by the Animal Care and Use Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. The animal experiments and sample collection strictly followed the relevant guidelines.

Consent for publication
Not applicable.

Availability of data and material
All sequences analyzed in this study can be accessed in the SRA database under the accession number PRJNA589726 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA589726?reviewer=jscimf1vmv52kf9 c5236l4jve).

Competing interests
Author Wenming Zhang was employed by the company Evonik Degussa (China). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Yin, J., Li, Y., Han, H., Liu, Z., Zeng, X., Li, T., and Yin, Y. (2018). Long-term effects of lysine concentration on growth performance, intestinal microbiome, and metabolic profiles in a pig model. Food Funct. 9,[4153][4154][4155][4156][4157][4158][4159][4160][4161][4162][4163] Zhang, J., Sun, J., Chen, X., Nie, C., Zhao, J., Guan, W., Lei, L., He, T., Chen, Y.,     Figure 1 Alpha diversity of the colonic bacterial community of weaned piglets with different treatmen Figure 1 Alpha diversity of the colonic bacterial community of weaned piglets with different treatmen Comparison of the colonic microbial community in weaned piglets from control, antibiotic, an Comparison of the colonic microbial community in weaned piglets from control, antibiotic, an Figure 4 LefSe analysis of the colonic microbial community in weaned piglets from control, antibiotic, Figure 4 LefSe analysis of the colonic microbial community in weaned piglets from control, antibiotic,

Figure 5
PICRUSt and KEGG analysis of the colonic microbial community in weaned piglets from contro PICRUSt and KEGG analysis of the colonic microbial community in weaned piglets from contro Figure 6 Correlations between the microbiota, health parameters, and colonic metabolite concentratio Figure 6 Correlations between the microbiota, health parameters, and colonic metabolite concentratio

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