Growth performance and intestinal morphology
The effects of synbiotics administration on growth performance of weaned piglets is shown in Supplementary Table 1. Final body weight, average daily gain, and gain to feed ratio were significantly increased in SYN1 and SYN2 group compared to the CONT, PRO and PRE groups, suggesting that supplementation of synbiotics may improve growth performance of weaned piglets. The effects of prebiotic, probiotic and synbiotic supplementation on fecal index is shown in Supplementary table 2. Piglets of the SYN1 and SYN2 group significantly decreased the fecal score (P < 0.05), while no differences were observed between PRE and PRO as compared to the CONT group suggesting that synbiotics administration decreased the diarrhea incidence in weaned piglets.
Histological examinations of ileal tissue revealed that STEC-challenged CONT group increased the inflammatory cells including neutrophils and macrophages in the lamina propria as compared to the healthy CONT group, which were given PBS only. Interestingly, oral administration of PRE, PRO, SYN1 and SYN2 group decreased these inflammatory cells (Supplementary Fig. 1). In the cecum tissue, STEC-challenged CONT group expanded the mucosal crypt and increased the number of plasma cells as compared to the healthy CONT group. However, these observations were lower in STEC-challenged pig groups fed PRO, PRE, SYN1 and SYN2 (Supplementary Fig. 2). In the colon tissue, neutrophils and plasma cells were increased in the STEC-challenged CONT group compared to the healthy CONT group. Similarly, PRO, PRE, SYN1 and SYN2 decreased these inflammatory cells with higher reduction in the SYN2 (Supplementary Fig. 3).
DNA sequencing data
Total DNA was extracted from fecal samples of pigs and the extracted community DNA was PCR amplified and sequenced using primers specific for the V5 to V6 hypervariable regions of the 16S rRNA genes. The 16S rRNA gene sequencing produced a total of 4,875,951 raw sequence reads from 48 fecal samples ranging from 66,397 to 165,469 reads. The average quality score (Phred scores) across all the samples ranged from 32 to 36. Phred scores greater than Q30 indicated that that there was less than 0.1% chance that a base was called incorrectly. Further data filtering was performed in the OTU table to remove low quality sequences and to improve downstream statistical analysis (Supplementary Fig. 4). The total number of reads after quality filtering was 3,409,184 ranging from 46,296 to 118,099 with an average counts per sample of 71,024. For alpha and beta diversity analyses, all samples were rarefied to the minimum number of sequences to account for unequal sequencing depth. A total of 1688 OTUs were obtained after data filtering.
Alpha diversity
Alpha diversity indices were compared between the treatment groups (PRE, PRO, SYN1, and SYN2) as compared to the CONT. Also, the alpha diversity between the STEC-challenged treatment groups and their non-challenged counterpart were compared to determine the effects of STEC on alpha diversity and the protective effects of PRE, PRO, SYN1, and SYN2. The rarefaction curves for 16S rRNA gene sequences of all the samples with an OTU definition at 97% identity cut-off were shown in (Supplementary Fig. 5), indicating that sampling depth was sufficient for downstream OTU-based analysis. The number of observed OTUs and Chao1 were used to measure species richness, whereas Shannon and Simpson diversity indices were used to measure species diversity.
Interestingly, Chao1 and observed OTUs were significantly lower in the CONT challenged with STEC compared to those of CONT without STEC challenge, indicating that STEC challenge significantly decreased the microbial species richness (Fig. 1a and Fig. 1b). However, no significant differences in Chao1 and number of observed OTUs were observed between the PRE, PRO, SYN1 and SYN2 and their STEC-challenged counterparts, suggesting that probiotics, prebiotics or their combination may play a role in maintaining the balance of microbial communities in the gut of piglets against STEC infection (Fig. 1a and Fig. 1b).
In addition, oral challenge of STEC significantly decreased the Shannon diversity in weaned piglets as compared to the non-challenged CONT group, indicating that STEC infection altered the species diversity of microbial communities (Fig. 1c). Furthermore, no significant difference in Simpson diversity was observed between the treatment groups compared to the non-challenged healthy CONT group, and between those groups infected with STEC compared to the STEC-challenged CONT group (P > 0.05) (Fig. 1d).
Beta diversity
Beta diversity, which is defined as the diversity among the treatment groups, was measured using the weighted and unweighted UniFrac distances with the former takes into account the relative abundance of species and the latter considers the presence or absence of OTUs in the community. Principal coordinate analysis (PCoA) was used to visualize the separation of microbial community among the treatment groups (CONT, PRE, PRO, SYN1, SYN) and effects of STEC infection. The PCoA plots based on both unweighted (Fig. 2a) and weighted UniFrac (Fig. 2b) distances showed significant differences in the separation of microbial communities in pigs in response to the different treatment groups (PRE, PRO, PRO, SYN1 and SYN2) and oral challenge of STEC as measured using ANOSIM (P < 0.05). PCoA results indicated that fecal microbial communities differ in pigs between treatment group (PRE, PRO, SYN1, and SYN2) and CONT groups. Moreover, separation of microbiota obtained were also significantly different between the unchallenged treatment groups and pigs treatment groups orally challenged with STEC based on ANOSIM (P < 0.05). These results suggest that prebiotics, probiotics and their synbiotic combination had individual effects on the intestinal microbial community structure in pigs.
Microbial compositions associated with the administration of prebiotics, probiotics and synbiotics in weaned piglets
We examined the bacterial compositions associated with oral administration of probiotics, prebiotics and synbiotics and STEC oral challenge in weaned piglets. At the phylum level, a total of 11 phyla were identified and the top 5 most abundant phyla were Bacteroidetes (42.63–51.10%), Firmicutes (32.94–46.78%), Proteobacteria (3.11–13.82%), Spirochaetes (0.19–4.48%), and Tenericutes (0.05–2.61%). Phylum Bacteroidetes and Firmicutes collectively ranged from 83.70–94.58% of the total sequences among the groups (Fig. 3a).
At the genus level, 50 unique genera were identified from at least one sample in each group. Regardless of the treatment group, the top 5 most abundant genera were Prevotella (25.73–37.52%), Lactobacillus (1.59–9.97%), Oscillospira (1.60–6.24%), Succinivibrio (0.27–5.63%), and Roseburia (0.26–3.72) (Fig. 3b).
Without STEC challenge, differential abundance analysis revealed that each additive resulted in different alterations of the pig intestinal microbiota at the genus level (Fig. 4). Relative bundance of Lactobacillus was not different among PRO, SYN1 and SYN2 groups (Fig. 4a). One of the most interesting observations of the present study is the significant increase in the abundance of Prevotella in all the dietary treatments (PRE, PRO, SYN1 and SYN2) as compared to the CONT (P < 0.05) (Fig. 4b). Moreover, abundance of Roseburia was significantly elevated by oral administration of PRE, PRO and SYN2 as compared to the negative CONT group (P < 0.05) (Fig. 4c). Another interesting finding of the study is the significant increase of Succinivibrio in the PRO group as compared to the CONT group (P < 0.05) (Fig. 4d). These findings indicate that prebiotics, probiotics and synbiotics have unique effects on gut microbial composition in weaned piglets.
Differential abundance in microbial composition associated with STEC infection in weaned piglets receiving prebiotics, probiotics and synbiotics
Differences in microbial composition at the genus level between non-challenged and STEC-challenged CONT groups were compared to determine the effects of STEC infection in weaned piglets (Fig. 5). STEC-challenge resulted to significant increase in the population of Phascorlarctobacterium and Prevotella while there was a significant decrease in abundance of Lactobacillus in comparison to the CONT pigs fed PBS (P < 0.05). No significant differences were observed between STEC-challenged CONT and PRE group challenged with STEC (P > 0.05). On the other hand, a significant increase in the abundance of Prevotella and Lactobacillus and significant reduction of Phascolarctobacterium were observed in PRO group challenged with STEC (P < 0.05). However, in SYN1 group challenged with STEC, significant depletion in the abundance of Prevotella and Phascolarctobacterium were observed (P < 0.05). In the SYN2 group challenged with STEC, we observed a significant increase in the relative abundance of Prevotella and a significant depletion of Phascolarctobacterium similar to those observations in the PRO group with STEC (P < 0.05). These findings suggest that STEC infection significantly altered the composition of the pig gut microbiota, however, prebiotics, probiotics or their synbiotic combination have inhibitory effects to fight against STEC.
Core microbiota in weaned piglets orally administered with prebiotics, probiotics and synbiotics
The core microbiome analysis was performed at the genus level based on the sample prevalence and relative abundance cut-off value at 20% and 0.02%, respectively. Regardless of the treatment group, the 6 core bacterial genera were identified as Prevotella, Lactobacillus, Oscillospira, Succinivibrio, Roseburia and Parabacteroides, which were shown in descending order according to prevalence (Fig. 6). Our results were similar to a previous study on meta-analysis of the swine gut microbiota using published data sets from 16S rRNA gene sequences to define a core microbiota in the pig gut [13]. These findings suggest that pig gut microbiota may be used for future gut microbiota manipulation studies for potential health benefits.