A total of 50 healthy weanling pigs [Duroc x (Landrace x Yorkshire)] with average body weight of 5.33 ± 0.60 kg weaned at the age of 28 days were used in this study at experimental research center of Dankook University, Cheonan, South Korea. All pigs in this study were selected from one delivery room and had similar husbandry practices. Each pen was equipped with a one-sided self-feeder and a nipple water-feeder for ad libitum access to feed and water throughout the experiment. The experimental procedures used in this study were approved by the Animal Care and Use Committee of Dankook University (No. DK-1-1645).

On the day of weaning, piglets were divided into five groups consisting of ten pigs per treatment and housed in pens of five animals per pen using a randomized complete block design for the 7 weeks trial. Basal diet was provided in a mash form and formulated to meet or exceed the nutrient requirements (Table 1). The 5 dietary treatments were: (i) CONT, basal diet without any antibiotics or feed supplements, (ii) PRE, basal diet + 0.05% prebiotics, (iii) PRO, basal diet + 0.1% probiotics (Pediococcus acidilactici), (iv) SYN1, basal diet + 0.05% synbiotics, (v) SYN2, basal diet + 0.1% synbiotics. The synbiotics was formulated with a prebiotic lactulose at a concentration of 10 g/kg feed combined with Pediococcus acidilactici GB-U15 KCCM 11856P at a concentration of 5.0 x 10⁹ colony forming units (CFU)/mL. The synbiotics used in the study were provided by Genebiotech Co., Ltd. (Seoul, Korea). We selected lactulose as the prebiotics, the low doses of lactulose can help to stimulate the growth of health-promoting bacteria in the gastrointestinal tract.

At week 5 of the experiment (56-day-old), five pigs from each group were inoculated with the pathogenic STEC to determine the impact of the dietary treatments on intestinal microbiota of piglets challenged with STEC. STEC strain used in this study was isolated from the sick pig and had the virulent genes, such as F18, F6, heat-labile enterotoxin (LT), Shiga toxin type 2 (stx2), Shiga toxin type 2e (stx2e) genes. The STEC, which was isolated from piglet feces, was grown in fresh LB broth and incubated at 37°C with shaking for 24 h. The final concentration of E. coli used was ~2 x 10⁹ CFU/mL. Five piglets from each group orally inoculated with 5 mL of E. coli (2 x 10⁹ CFU/mL) diluted in PBS. PBS was orally administered in the remaining five piglets from each group. The health status of piglets during the experiments was assessed by fecal consistency scoring using a five-grade system. The scoring system for stool consistency that indicate stool hardness or softness is as follows: 1 = hard, dry pellets in a small, hard mass; 2 = hard, formed stool that remains firm and soft; 3 = soft, formed and moist stool that retains its shape; 4 = soft, unformed stool that assumes the shape of the container; 5 = watery, liquid stool that can be poured.

Fresh fecal samples were collected individually from the rectum of each piglet at week 7 of the experiment. Two pigs from each group were euthanized at week 7 of the experiment (2 weeks after E. coli oral challenge) for intestinal morphological analysis. The proximal segments of ileum, colon and cecum were sampled for histological examination. For histology, 3-cm sections from the ileum, cecum and colon were removed, opened longitudinally, and fixed in a 10% neutral formalin solution. Tissue samples were dehydrated and embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin and eosin. Morphological measurements were performed with a light microscope.

Total DNA from the feces was extracted from 200 mg of feces per sample using QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Cell lysis was performed by bead-beating the samples twice for 2 min at 300 rpm, with an incubation period of 5 min in a water bath at 70°C between beatings. The concentrations of DNA were measured using a Colibri Microvolume Spectrometer (Titertek Berthold, Pforzheim, Germany) and samples with OD260/280 ratios of 1.80–2.15 were processed further.

The PCR primers 799F-mod6 (5′ CMGGATTAGATA CCCKGGT-3′) and 1114R (5′-GGGTTGC GCTCGTTGC-3′) were used to amplify the V5 to V6 hypervariable regions of the 16S rRNA genes. The amplification mix contained 5 X PrimeSTAR Buffer (Mg²⁺) (Takara Bio, Inc., Shiga, Japan), 2.5 mM concentrations of each deoxynucleotide triphosphates, 2.5 U/μL of PrimeSTAR HS DNA Polymerase, 10 pmol of each primer, and 25 ng of DNA in a reaction volume of 50 μL. The thermal cycling parameters were as follows: initial denaturation was at 98°C for 3 min, followed by 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 30 s, and a final 3-min extension at 72°C. PCR products were purified using PCR a Wizard^® SV Gel and PCR Clean-Up System purification kit, (Promega, Wisconsin, USA).

After sample preparation and quality control, 16S rRNA gene amplicons were sequenced using the Illumina MiSeq platform at Macrogen Inc. (Seoul, Republic of Korea) according to the manufacturer's instructions. Briefly, the sequencing library was prepared by random fragmentation of the DNA samples followed by 5′ and 3′ adapter ligation. This step attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit. The final products were normalized and pooled using the PicoGreen, and the size of libraries were verified using the TapeStation DNA ScreenTape D1000 (Agilent). The PCR conditions were as follows: initial denaturation (3 min at 95°C) 8 amplification cycles (95°C for 30s, 55°C for 30s, 72°C for 30s) and final elongation (72°C for 5 min).

All the raw sequence data from Illumina MiSeq platform were checked for quality using FastQC. Then, Mothur software was used to remove low-quality sequences (12). Briefly, sequences that did not match the PCR primers were eliminated from demultiplexed sequence reads. Sequences containing ambiguous base calls and sequences with a length of <200 bp were also removed to minimize the effects of random sequencing errors. Chimeric sequences were identified and excluded for downstream analysis using the UCHIME algorithm implemented in Mothur. Next, the QIIME (Quantitative Insights into Microbial Ecology) pipeline (version 1.9.1) was used to perform operational taxonomic unit (OTU) picking using the open-reference OTU picking workflow with the SortMeRNA and SUMACLUST methods for reference OTU and de novo OTU picking, respectively. Taxonomy was assigned using the naïve Bayesian Ribosomal Database Project (RDP) classifier based on GreenGenes taxonomy reference database version 13_8. Low-abundance OTUs and singletons were filtered from the OTU table for downstream analysis with minimum count of 4 and low-count filter based on 20% prevalence in samples. Then, data normalization was performed by rarefying the data to the minimum library size and by data scaling using the total sum scaling before any statistical comparison to address the variability in sampling depth and the sparsity of the data.

Statistical analysis was performed using the R package MicrobiomeAnalystR and GraphPad Prism v7.00 (La Jolla, CA, USA). Alpha diversity measures including observed OTUs, Chao1, Shannon and Simpson indices were computed using the MicrobiomeAnalystR. Significant differences in alpha diversity among the groups and pairwise comparisons were calculated based on analysis of variance tests. Significant difference level was set at P < 0.05. The principal coordinate analysis (PCoA) plots at the OTU level based on the weighted and unweighted UniFrac distances. Significant differences in beta-diversity were performed using the Analysis of Similarities (ANOSIM) based on the unweighted and weighted UniFrac distances. The heatmap of core microbiota was performed with the default parameters at 20% sample prevalence and 0.2% relative abundance.

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 Figure 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 Figure 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 Figure 3).

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 >Q30 indicated that that there was <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 Figure 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 1,688 OTUs were obtained after data filtering.

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 Figure 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 (Figures 1A, B). 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 (Figures 1A, B).

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 (Figure 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) (Figure 1D).

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 (Figure 2A) and weighted UniFrac (Figure 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.

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 to 94.58% of the total sequences among the groups (Figure 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) (Figure 3B).

Without STEC challenge, differential abundance analysis revealed that each additive resulted in different alterations of the pig intestinal microbiota at the genus level (Figure 4). Relative bundance of Lactobacillus was not different among PRO, SYN1 and SYN2 groups (Figure 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) (Figure 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) (Figure 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) (Figure 4D). These findings indicate that prebiotics, probiotics and synbiotics have unique effects on gut microbial composition in weaned piglets.

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 (Figure 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.

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 six core bacterial genera were identified as Prevotella, Lactobacillus, Oscillospira, Succinivibrio, Roseburia, and Parabacteroides, which were shown in descending order according to prevalence (Figure 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.