2.1 Overview of the metatranscriptomes
After removing the host-related sequences from clean reads, on the average, 100 million raw sequence reads were obtained from the metatranscriptome of each sample, and a total of 210.09 Gbp of high-quality sequences were generated from 16 samples after removing the adapters and quality filtering. The Q20 and Q30 base percentages of each sample were above 98.95% and 96.30%, respectively.
A total of 1,081,814 contigs were identified after de novo assembling using MEGAHIT in the 16 samples (four samples per group). The length of these contigs ranged from 546 to 1,004,658 bp, with an average length of 1,623.05 bp. A total of 712,210 unigenes were clustered with CD-HIT (http://www.bioinformatics.org/cd-hit/) (95% identity and 90% coverage). Up to 254,331 core genes were found in all four groups, and each group had its own unique genes (Fig. 1). In general, the PEC group had the highest numbers of specific genes compared with the other groups. Principal coordinate analysis (PCoA) based on unweighted UniFrac distances showed that the colonic luminal digesta samples in the PEC group were clustered distinctly from those in the other groups, and INU samples was clearly segregated from control samples. While samples in the RPS group were more similar to controls, RPS-3 acting as an outlier, it is impossible to deny the influence of individual differences, but the main reason is the effect of RPS, as the other three samples in the RPS group are still relatively clustered (Fig. 2).
2.2 Effect of different dietary fibers on colonic microbiota composition
The distribution of dominant bacteria in each group is shown in Fig. 3. In the CON group, the bacteria detected in the proximal colonic luminal digesta samples belonged to 55 different phyla. The most abundant phylum was Bacteroidetes, followed by Firmicutes and Proteobacteria. Up to 1,002 bacteria genera were observed in this group, with Prevotella, Bacteroides and Clostridium as the most abundant. The INU group contained 968 genera belonging to 57 phyla, the RPS group had 49 phyla made up of 918 genera and the PEC group contained 60 phyla and 1131 genera.
At the phylum level, the abundance of Verrucomicrobia in the RPS group was lower (fold change >2 or < 0.5; FDR < 0.05) than that in the CON group. The abundance of Verrucomicrobia in the INU group was also lower (fold change >2 or < 0.5; FDR < 0.05) than that in the CON group, while the abundance of Fusobacteria, Actinobacteria and Cyanobacteria were greater (fold change >2 or < 0.5; FDR < 0.05) than those in the CON group. Meanwhile, the populations of Proteobacteria, Spirochaetes and Verrucomicrobia phyla were greater (fold change >2 or < 0.5; FDR < 0.05) in the PEC group than in the CON group colonic digesta samples (Additional File 1).
At the genus level, compared with the CON group, significant shifts were detected (p < 0.05) in 13 genera in the RPS group, while 15 and 23 genera changed significantly in the INU and PEC groups, respectively (Fig. 4). The abundance of Parabacteroides, Faecalibacterium, Ruminococcus and Alloprevotella increased but Sutterella decreased in the RPS group. Inulin supplement increased the abundance of Fusobacterium and Rhodococcus but decreased Bacillus. The abundance of Streptococcus and Bacteroidetes_norank increased but Clostridium, Clostridioides, Intestinibacter, Ruminococcaceae_unclassified, Gemmiger, Muribaculum, Enterococcus and Vibrio decreased in the PEC group.
2.3 Effect of different dietary fibers on the activities of colonic CAZymes
In terms of CAZyme profiles, 222 CAZyme families were detected, including seven auxiliary activities (AAs), 36 carbohydrate-binding modules (CBMs), 15 carbohydrate esterases (CEs), 94 glycoside hydrolases (GHs), 57 glycosyl transferases (GTs) and 13 polysaccharide lyases (PLs). As shown in Fig. 5, GHs were the most abundant class in all four groups, but the distribution of CAZymes at the class level did not exhibit significant change among the four groups.
Compared with the CON group at the family level, some changes were found in the dietary fiber groups (Additional File 2). The gene expressions of the CAZymes that were significantly affected by the different dietary fibers in the pigs’ colon are shown in Fig. 6. Thirty CAZyme families changed significantly (fold change >2 or < 0.5; FDR < 0.05) in the RPS group. The specific changes were as follows: nine CAZymes (CBM21, CBM74, GH128, GH77, GH85, GH97, GT10, GT27 and GT3) were upregulated in the mRNA expression while 21 CAZymes (AA7, CBM26, CBM41, GH101, GH112, GH14, GH15, GH24, GH27, GH35, GH38, GH8, GH89, GT14, GT25,GT31, GT49, GT77, GT8, GT84 and GT91) were downregulated. In the INU group, 14 CAZyme families, namely, AA4, AA7, CBM26, CBM41, GH101, GH14, GH15, GH24, GH26, GH27, GH38, GT49, GT77 and GT84 were downregulated significantly. Meanwhile, 35 CAZyme families changed significantly in the PEC group, 13 CAZymes (AA12, AA3, CBM61, CBM9, CE14, GH102, GH103, GH16, GH5, GH85,GH88, GT1 and GT21) manifested higher abundance while 22 CAZymes (AA1, AA2, AA6, CBM21, CBM26, CBM41, GH101, GH112, GH132, GH14, GH17, GH18, GH24, GH37, GH38, GT15, GT32, GT39, GT49, GT77, GT91 and PL4) were lower than in the CON group. Among the altered CAZyme families, four CAZymes, namely, AA7, GH15, GH27 and GT84, were downregulated in both INU and RPS groups. GH112 and GT91 decreased while GH85 increased in both RPS and PEC groups while CBM21 increased in the RPS group but decreased in the PEC group. Two altered CAZymes (AA4 and GH26) were specific in the INU group, 14 CAZymes (CBM74, GH128, GH35, GH77, GH8, GH89, GH97, GT10, GT14, GT25, GT27, GT3, GT31 and GT8) were specific in the RPS group and 23 CAZymes (AA1, AA12, AA2, AA3, AA6, CBM61, CBM9, CE14, GH102, GH103, GH132, GH16, GH17, GH18, GH37, GH5, GH88, GT1, GT15, GT21, GT32, GT39 and PL4) were specific in the PEC group.
2.4 Correlation between CAZymes and colonic microbiota
One of the most critical roles of the microbiota is their ability to utilise complex carbohydrate sources. The network of correlation analyses between CAZyme classes and the microbiota (at the genus level) showed that Prevotella and Tannerella primarily contributed the CAZyme-encoding gene fragments of the GHs, the GTs were mainly produced by Prevotellamassilia and Prevotella, Prevotellamassilia and Roseburia primarily contributed to CBMs, Lachnotalea and Butyricimonas primarily contributed to CEs, Butyricimonas and Mediterranea primarily contributed to PLs and AAs were mainly produced by Turicibacter and Chlamydia in the growing pigs’ colon metatranscriptome among the significantly changed bacterial genera (Additional File 3).
To explore the potential association between the microbiota and CAZymes, Spearman’s rank correlations were constructed between the 51 bacterial genera and 79 CAZyme families that were significantly affected by the three dietary fiber treatments. The results revealed a strong association with a threshold of Spearman’s correlation coefficient > 0.5 or <-0.5 and p < 0.05. As shown in Fig. 7, the productions of CBM21 and GT91 were negatively correlated with the abundance of Sutterella. Parabacteroides had a negative correlation with the production of GH14, GH24, GH8, GT14, GT31 and GT77 but contributed proportions of GT10 and GT3. Alloprevotella and Ruminococcus contributed proportions of GH77, GH97, GT10, GT27 and GT3, while Faecalibacterium was a contributor of CBM74 and GH38. Streptococcus, Clostridioides, Intestinibacter, Vibrio, Clostridium, Gemmiger, Muribaculum and Enterococcus altered significantly specific to the PEC vs. CON group. Streptococcus had a positive correlation with the production of AA12, AA3, CBM61, GH102, GH103, GH16 and GH5. Clostridioides contributed proportions of AA1, CBM21 and PL4 but was negatively correlated with the production of AA3, CBM61, GH102 and GH103. Intestinibacter had a negative correlation with the production of AA3, CE14 and GH102. Vibrio had a negative correlation with the production of AA12, AA3, CBM61, GH102 and GH103. Clostridium was a contributor of GH18 and GT32 but was negatively correlated with the production of AA3. Gemmiger contributed proportions of GH101 and GH112 but was negatively correlated with the production of AA3 and CE14. Muribaculum was a contributor of CE14 and GH37.
According to the taxonomic distribution (top 10 genera) of the predicted CAZymes identified from the metatranscriptomes in the PEC group, Prevotella, Bacteroides, Mesorhizobium and Parabacteroides were the largest genera in the predicated AAs, GHs, GTs, CEs, PLs and CBMs. It is worth noting that Streptococcus was also the major microbial origin of the predicted CBMs (Fig. 8). Focusing on the contributions of the CAZymes from the major microbial communities in growing pigs’ colon in the PEC group compared with the CON group, it is worth noting that Prevotella was the main contributor of GH5, Mesorhizobium was the main contributor of GH16 and Streptococcus was the main contributor of CBM61 in the PEC group (Fig. 9).