Effect of Trp and LPS on Alpha Diversity of Ileum and Cecum Microbiota
We assessed the effects of Trp on the gut microbiota composition by sequencing the bacterial 16S rRNA. A total of 1,638,794 and 1,675579 high-quality sequences were obtained in the ileum and cecum contents, respectively (Table 1). Overall, in the ileum and cecum, 369 and 859 operational taxonomic units (OTUs) were detected based on the nucleotide sequence identity of 97% between reads, respectively.
Table 1
The alpha diversity in each group of pig. A: non-challenged control group; B: LPS-challenged control group; C: control + 0.2% Trp treatment group; D: LPS + 0.2% Trp treatment group; E: LPS + 0.4% Trp treatment group. ACE: abundance-based coverage estimator.
Item | A | B | C | D | E | SEM | P |
Ileum | | | | | | | |
Sequens | 46167 | 44891 | 40861 | 45021 | 44694 | 656.33 | 0.076 |
Shannon | 2.02ab | 1.71a | 2.38b | 1.98 ab | 1.69 ab | 0.11 | 0.259 |
Simpson | 0.299ab | 0.342a | 0.207b | 0.305ab | 0.389a | 0.03 | 0322 |
Chao1 | 178.03ab | 116.76a | 242.92b | 182.24ab | 126.92a | 17.46 | 0.081 |
ACE | 191.69ab | 124.43a | 276.52b | 192.69ab | 136.21a | 19.04 | 0.151 |
Cecum | | | | | | | |
Sequens | 48619 | 49257 | 47698 | 46192 | 39484 | 931.77 | 0.006 |
Shannon | 4.02 | 4.09 | 3.90 | 4.09 | 4.04 | 0.06 | 0.814 |
Simpson | 0.045 | 0.058 | 0.064 | 0.052 | 0.055 | 0.004 | 0.701 |
Chao1 | 494.32 | 516.73 | 482.57 | 521.71 | 509.53 | 12.00 | 0.768 |
ACE | 491.85 | 513.88 | 474.5 | 519.88 | 499.96 | 11.85 | 0.839 |
The alpha diversity was estimated by diversity (Shannon and Simpson index) and richness (Chao1 and ACE indexes). In ileal samples, the diversity and richness of gut microbiota were decreased in LPS-challenged control group, but increased in the Trp treatment group. Moreover, fed LPS-challenged piglets with a 0.2% Trp diet restored the diversity and richness of gut microbiota to the level of control group. But fed LPS-challenged piglets with a 0.4% Trp diet showed no effect on diversity and richness of gut microbiota. The LPS and Trp showed no effect on the alpha diversity of cecum microbiota.
Effect of Trp and LPS on the Composition of Ileum and Cecum Microbiota
The relative abundance of gut microbiota and the principal components analysis (PCA) are showed in Fig. 1. The Trp treatment group had significantly deviated from three LPS-challenged groups in ileum microbiota. The composition of control group was similar with the Trp treatment group both in ileum and cecum (Fig. 1C, F). After filtering the relative abundance of phyla lower than 0.1% in all groups, 5 and 7 phyla were identified in the ileum and cecum microbiota, respectively (Fig. 1A, D). Firmicutes, Proteobacteria and Actinobacteria were three major phyla in ileum microbiota, and Firmicutes, Bacteroidetes and Proteobacteria were three major phyla in cecum microbiota. LPS increased the level of Firmicutes and decreased the level of Proteobacteria and Actinobacteria. However, 0.2% Trp diet decreased the level of Firmicutes and increased the level of Proteobacteria and Actinobacteria in ileum. But in the cecum, the LPS and Trp had little effect on the relative abundance of microbiota at phyla level.
At the genus level, a total of 13 and 27 taxa were defined as the most abundant in ileum and cecum, respectively (༞1% of the total sequences). Lactobacillus and Clostridium_sensu_stricte_1 were two major genera in ileum, Lactobacillus and Prevotella_9 were two major genera in cecum (Fig. 1B, E). We found that the relative abundance of microbiota was significantly influenced by LPS and Trp in ileum and cecum. In the ileum content, compared with control group, 2 genera were significantly affected by LPS (P༜0.05), and 4 genera were little affected by LPS (P༜0.1), Olsenella was little affected by 0.2% dietary Trp (P = 0.09). Compared with LPS-challenged group, Actinobacillus was little decreased in LPS + 0.2%Try group (P = 0.07), Blautia was little increased in LPS + 0.4% Trp group (P = 0.08) (Figure S1 A-D). In the cecum content, compared with control group, 3 genera were significantly affected by LPS (P༜0.05), Terrisporobacter was little decreased by LPS (P = 0.07); Blautia was significantly increased by 0.2% dietary Trp (P༜0.05), Prevotellaceae_NK3B31_group was little decreased by 0.2% dietary Trp (P = 0.05). Compared with LPS-challenged group, norank_f__Veillonellaceae was significantly decreased in LPS + 0.2% Trp or LPS + 0.4%Trp groups (P༜0.05) (Figure S2 A-D).
Bacterial Interaction Network Analysis
Co-occurrence networks in ileum and cecum were generated for all piglets and each group individually, based on their 50 most abundant genera (Fig. 3A and Figure S3). The overall topological properties of these network were calculated in order to distinguish the genus correlations at different groups. For the number of nodes, edges and correlations, the LPS + 0.4%Trp group showed the highest values, the LPS and 0.2%Trp groups showed lower values. This result indicating that the network topology existed more complex in ileum microbiota of piglets after the treated with LPS and Trp.
Bacterial core members were easily found in network interactions among five groups, including Lactobacillus, Clostridium_sensu_strecto, Terrisporobacter, Strepotococcus, Actinobacillus (Fig. 3A). Interestingly, in the control group, the Clostridium_sensu_strecto is the key genera negatively correlated with the dominant genera (Fig. 3B). However, in the LPS, LPS + 0.2%Trp and LPS + 0.4%Trp groups, Lactobacillus is the key genus negatively correlated with the relative abundance of the dominant genera (Fig. 3C, 3E and 3F).
Effect of LPS and Trp on the abundance ofLactobacillusin Ileum
As the results of Fig. 2 and Fig. 3 shown, the relative abundance of Lactobacillus of piglets was significantly increased by LPS, and the Lactobacillus is a key genus negatively correlated with the dominant genus. Then, we analyzed the relative abundance of Lactobacillus of ileum at the species level (Fig. 4). As the results shown that, the relative abundance of Lactobacillus_amylovorus, s_unclassified_g__Lactobacillus, s_uncultured_bacterium_g__Lactobacillus and s_Lactobacillus_pontis were increased by the challenge of LPS. Compared with the 0.2%Trp group, the relative abundance of Lactobacillus_amylovorus and s_uncultured_bacterium_g__Lactobacillus were significantly increased in LPS, LPS + 0.2%Trp and LPS + 0.4%Trp groups (P༜0.05).
Correlation of gut microbiota with growth performance, serum and liver parameters of piglets
In our previous study, we analyzed the performance, serum and liver parameters of piglets. Then, a spearman correlation analysis was performed among the top 50 genera according the relative abundance in all piglets. As shown in Fig. 5, the relative abundance of Bacillus, Turicibacter, Clostridium_sensu_strecto-1, Terrisporobacter, Syntrophococcus and Blautia were significant positive correlated with the average daily gain (ADG) of piglets. However, the relative abundance of Lactobacillus was significant negative correlation with the ADG and average daily feed intake (ADFI) of piglets. It is interesting to note that, the relative abundance of Lactobacillus was significant positive correlated with the level of SOD, GSH-Px and T-AOC in liver.