Dietary Tryptophan Modulate the Composition of the Ileum and Cecum microbiota in Weaned Piglets after Lipopolysaccharide Challenge

Background: The gut microbiota plays a critical role in metabolism and growth of piglets, and was modiable by dietary tryptophan (Trp) in previous study. However, no studies focused on the investigation of whether additional dietary Trp supplementation would modulate the composition of ileum and cecum microbiota of piglets challenged by lipopolysaccharide (LPS). We conducted to investigate whether dietary tryptophan could alleviate dysbacteriosis of piglets after challenged with LPS. Methods: A total of 40 28 days old male weaning piglets were randomly allotted to ve groups, include Con group (basal diet), LPS group (basal diet), 0.2% Trp group (0.2% Trp diet), LPS+0.2% Trp group (0.2% Trp diet) and LPS+0.4% group (0.4% Trp diet). On day 10, 20 and 29, Con and 0.2% Trp groups were injected with saline, LPS, LPS+0.2% Trp and LPS+0.4% Trp groups were injected with LPS respectively. The experiment lasted for 30 days. Results: These results showed that the major three phyla in ileum were Firmicutes, Proteobacteria and Actinobacteria, in cecum were Firmicutes, Bacteroidetes and Proteobacteria. Dietary with 0.2% Trp could attenuate the effect of LPS on the alpha diversity (diversity and richness) in ileum (P (cid:0) 0.05). The alpha diversity of cecum microbiota was not affected by LPS or Try. The relative abundance of Turicibacte (P (cid:0) 0.05) and unclassied_f__Peptostreptococccaceae (P (cid:0) 0.05) were decreased but Lactobacillu (P (cid:0) 0.05) was increased in ileum by LPS. Compared with LPS group, the relative abundance of Actinobacillus (P=0.07) was decreased in LPS+0.2% Trp group, and the level of Blautia (P=0.08) was increased in LPS+0.4% Trp group. The complexity of ileum microbiota was decreased by LPS. But the complexity of ileum microbiota was increased in LPS+0.2% and LPS+0.4% groups. The relative abundance of Lactobacillu was negative correlation with the majority of genus in ileum and positive correlation with the antioxidant ability of liver (P (cid:0) 0.05).


Background
Early-weaning-induced stress cause diarrhea, thereby increasing mortality and reducing growth performance in piglets [1]. It caused the pig industry great economic loss and also represent a serious threat to farm animal welfare [2]. One of the main causes of diarrhea is gastrointestinal disorder. Several hypotheses have been put forth regarding the role of dysbiosis. One possibility is that dysbiosis disrupts production of microbial metabolites that are utilized by the intestinal epithelial cells for maintaining barrier integrity, which could elevate bacterial endotoxins in circulation, and trigger a pro-in ammatory cytokine cascade in the liver [3]. Recently, it is gradually realized that dietary transition and environmental changes during weaning period are linked to modi cations in piglets intestinal microbiota which could be involved in the etiology of post-weaning diarrhea and enteric infections [4,5].
The mammalian intestinal microbiota is composed of trillions of microbes that facilitate host health, including colonization resistance against gastrointestinal disorders [6]. Growing evidence suggests the potential of utilizing targeted reconstitution of the gut microbiota against gastrointestinal disorders [1,7].
Recent studies have showed that probiotics, such as Lactobacillus frumenti and Lactobacillus reuteri could enhance the intestinal barrier and defense diarrhea of piglets [1,8]. The gut microbiota could also increase the performance of pig through regulating the feed intake [9], feed e ciency [10] and oxidative stress [11] of host. Thus, maintenance and restoration of host intestinal microbiota homeostasis has shown bene cial effect in health and performance of pig.
The intestinal ora can actively utilize dietary amino acids for protein synthesis and modify the amino acid pro le in the plasma of the host [12]. Dietary supplementation with amino acids could in uence the composition and diversity of the intestinal microbiota, thus improving intestinal function [13]. Trp is considered as the second-limiting amino acid in most corn-based diets of swine [14]. Some previous studies have shown that dietary Trp level could regulate feed intake [15], growth [16], intestinal integrity [17] and oxidative stress [14] of pigs. Interestingly, recent studies reported that dietary Trp altered intestinal microbial composition and diversity, improved intestinal barrier and downregulated expression of in ammatory cytokines in the intestine of weaned piglets [18,19]. In addition, increasing dietary tryptophan level could improve feed intake and growth performance of weanling pigs orally challenged with Escherichia coli K88 [16]. However, few studies focused on the investigation of ileum and cecum microbiota of weaning piglets at the same time and it still remains largely unknown how the intestinal microbiota is modi ed by Trp supplementation in weaning piglets challenged by LPS. Therefore, the present study was conducted to investigate the effect of dietary Trp on gut microbiota composition through using an LPS-challenged piglet model.

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. 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 LPSchallenged 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 signi cantly 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 ltering the relative abundance of phyla lower than 0.1% in all groups, 5 and 7 phyla were identi ed 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 de ned 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 signi cantly in uenced by LPS and Trp in ileum and cecum. In the ileum content, compared with control group, 2 genera were signi cantly 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 signi cantly affected by LPS (P 0.05), Terrisporobacter was little decreased by LPS (P = 0.07); Blautia was signi cantly 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 signi cantly 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 ve 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 signi cantly 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_unclassi ed_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 signi cantly 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 signi cant positive correlated with the average daily gain (ADG) of piglets. However, the relative abundance of Lactobacillus was signi cant 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 signi cant positive correlated with the level of SOD, GSH-Px and T-AOC in liver.

Discussion
Host homeostasis with respect to issues of physiology and metabolism is crucially underpinned by the gut microbiota and its metabolites [20]. Researches indicated that the gut microbiota can facilitate metabolite production in two ways, rst, the resident species of the gut microbiota use the amino acids produced from food or the host as elements for protein synthesis, and second, conversion or fermentation are used to drive nutrient metabolism [21]. Previous study reported that, the tryptophanderived bacterial metabolite indole attenuates indicators of in ammation in epithelial cells and liver of host [3]. LPS has been widely used to mimic features of endotoxin-induced acute intestinal injury. LPS irritates the intestine, causing mucosal injury, metabolic disorder, and bacterial translocation. Recent study reported that LPS could induce the disorder of gut [22] and ruminal bacterial [23].
To evaluate the effect of Trp on gut microbiota disorder induced by LPS, ileum and cecum microbiota was extracted for 16S rRNA analysis. Gut microbial community is a mini-ecosystem whose diversity is regarded as a key health indicator of healthy individuals and affected by the health status of the hosts [24]. It has been reported that reduced biodiversity of the gut microbiota is associated with disease status [24,25]. In the study, we found that the alpha diversity of gut microbiota in cecum is not affected by Trp and LPS. However, in the ileum, it was increased with the supplementation of 0.2% Trp, and decreased with the challenge of LPS in piglets. This may be due to the small-intestine is the main target organ of LPS [26], and amino acids are absorbing in small-intestine [27]. Interesting, we found dietary 0.2% Trp alleviated the decrease of alpha diversity in ileum microbiota challenged by LPS, but dietary 0.4% has no effect on the reduction of diversity in ileum microbiota caused by LPS. This result consistent with a previous study, which reported the richness and diversity of gut microbiota in 0.2% Trp group higher than that in control and 0.4% Trp groups [19]. These results indicated that the addition of 0.2% Trp to the diet has improved regulation of ileum microbiota diversity than 0.4% Trp treatments regardless of whether the piglets are challenged by LPS.
As previous studies reported, LPS altered bacterial composition at genus level in ileum and cecum [28]. We found that Turicibacter in ileum were lower in LPS-challenged pig. In our previous study, we found the abundance of Turicibacter was higher in sows with higher performance and low oxidative stress [11]. In agreement, Suchodolski study reported signi cantly decrease of Turicibacter in dogs with Acute Hemorrhagic Diarrhea (AHD) [29]. But the molecular mechanism about Turicibacter regulated the health of intestinal remains to be resolve. It is curious that the relative abundance of Lactobacillus was signi cantly increased in LPS, LPS + 0.2%Trp and LPS + 0.4%Trp groups. Lactobacillus has been identi ed as one of the core probiotics in the gastrointestinal tract of pigs [30]. Bu it has been reported that when the absence of epithelial reactive oxygen species (ROS), compensatory adaptation of the Lactobacillus confers protection against enteric pathogens by fostering H 2 O 2 and downregulating virulence factors [31]. Moreover, in the present study, we found Lactobacillus is the key genus negatively correlated with the abundance of the dominant genera in LPS, LPS + 0.2%Trp and LPS + 0.4%Trp groups (Fig. 3). Yang also found that Lactobacillus could modulate the intestinal injury induced by LPS in piglets [8]. It suggested that Lactobacillus may play a key role in inhibiting the growth of pathogens and maintaining the homeostasis of the intestinal ora when the intestinal injury was induced by LPS of piglets.
Trp also altered bacterial composition at genus level in ileum and cecum. Olsenella is a kind of lactic acid bacterium [32], which is up-regulated by Trp in ileum in our study. Lactic acid is not only an intermediate metabolite of short-chain fatty acid (SCFA) produced by intestinal ora metabolism [33], but also playing an important role in maintaining homeostasis within the gut community, by preventing colonization and infection of incoming bacterial pathogens [34,35]. Comparing with the control group, the relative abundance of Prevotellaceae_NK3B31_group decrease in the cecum of Trp group. A recent study reported the Prevotellaceae_NK3B31_group was dominant in the low feed conversion ratios pigs cecum [36]. In addition, Trp supplementation is associated with reduced relative abundances of Actinobacillus in ileum and increased relative abundance of norank_f__Veillonellaceae in cecum of piglets challenged with LPS. Lower proportions of Actinobacillus was reported with the higher weight gain of piglets [37]. Interestingly, the average daily gain of piglets was increase with the addition of tryptophan in our study (unpublished data). Veillonellaceae, a family with known lactate utilizing species [38], which is associated with conversion of lactate to propionate [34]. In our study, lactic acid bacterium, such as Lactobacillus and Olsenella were signi cantly enriched in piglets challenged with LPS. These results suggested that the increase of Veillonellaceae may be induced the elevating of source of nutrients.
In general, the greater the stability of the microbiome in a given environment, the greater complexity of these relationships, since these organisms and environments are less susceptible to microbial invasion [39]. Our results revealed that the complexity of ileum microbiota in LPS group were reduced, but increased by the treated with 0.2% and 0.4% Trp. It suggested that Trp might increase the complexity and stability of ileum microbiota in pig challenged with LPS, and inhibit the invasion of pathogenic bacterium. Moreover, we observed that Lactobacillus is the key genus negatively correlated with the abundance of the dominant genus in piglets challenged with LPS, and the relative level of Lactobacillus in ileum was increased after the challenge of LPS. It is worth noting that the Lactobacillus_amylovorus is the most abundant strain of Lactobacillus in the ileum of piglets. Previous study had reported that Lactobacillus_amylovorus inhibited the in ammatory signaling triggered by enterotoxigenic Escherichia coli in intestinal Caco-2 and pig explants [40]. It also demonstrated that Lactobacillus_amylovorus could improve host metabolism and relieve disorder of gut microbiota [41]. These indicate that Lactobacillus_amylovorus might proliferate to inhibited the in ammation and alleviates intestinal ora disorders of ileum induced by LPS.
Spearman correlation analysis was used to reveal the relationship between gut microbiota and the growth performance or antioxidant ability of piglets. These results showed that the relative abundance of Lactobacillu in ileum was negatively with growth performance but positively with antioxidant parameters of piglets. Lactobacillu is probiotics which is bene cial to growth of piglets [42]. In addition, the growth performance of piglets was reduced due to the challenge of LPS (unpublished data), but the relative abundance of Lactobacillu was increased to alleviate dysbacteriosis of ileum induced by LPS. Therefore, the negative correlation between Lactobacillu and growth performance do not mean that Lactobacillu are detrimental to the health of piglets. Liver injury and oxidative stress were induced by LPS in previous report [43]. Furthermore, previous study had shown Lactobacillu could protective from oxidative and metabolic hepatic injury [44]. A possible positive correlation between Lactobacillu and antioxidant ability is that Lactobacillu was proliferate to alleviate oxidative damage of liver when piglets challenge with LPS.

Conclusions
These studies suggested that Trp could relieve the toxic effect of LPS through improving the diversity and complexity of ileum microbiota in piglets. And the Lactobacillu might proliferate to inhibit the growth of pathogenic bacteria and alleviate oxidative damage of liver when piglets were challenged with LPS.
These nding indicated that ileum microbiota might have a self-protective mechanism to resist the injury induced by LPS through enriching Lactobacillu.

Animals
Piglets were obtained from the Hunan New Wellful Coompany Yongan farm and the study was carried out in the Union Experimental Center of Institute of Subtropical Agriculture, Chinese Academy of Sciences and Hunan New Wellful Company (Liuyang, China). A total of 40 28 days old male piglets (Yorkshire × Landrace, initial body weight 7.79 ± 0.75 kg) were randomly divided into ve groups with eight replicate pens for each treat group and one pig for each pen. Piglets were individually caged in 1.80 × 1.10 m pens and allowed ad libitum access to feed and water in an environmentally controlled house.
The experiment included ve treatments: (1)  All raw reads were screened according to barcode and primer sequences with Quantitative Insights Into Microbial Ecology (QIIME, version 1.17), the following criteria: 1) The 300 bp reads were truncated at any site receiving an average quality score < 20 over a 10 bp sliding window; 2) the truncated reads that were < 50 bp were abandoned; 3) Sequences that overlap shorter than 10 bp, or reads containing ambiguous characters, or > 2 nucleotide mismatch in primer matching were removed. Operational taxonomic units (OTUs) were clustered according to the standard of cut-off of 97% similarity using UPARSE (version 7.1, http://drive5. com/uparse/). The chimeric sequences was identi ed and removed with UCHIME. RDP Classifer (http://rdp. cme.msu.edu/) was used to analyze the phylogenetic a iation of each 16S rRNA gene sequence, against the silva (SSU115) 16S rRNA data base with con dence threshold of 70%. A principal components analysis (PCA) was performed to nd clustering patterns of piglets in ve groups.

Statistical Analyses
The normal distribution of the data was calculated with the Kolmogorov-Smirnov test. Principal components analysis (PCA) was performed to nd the clustering patterns among the ve groups of OTUlevel microbiota abundance data. The differences of gut microbiota relative abundance among ve groups was evaluated using the Wilcoxon rank-sum test. Network analysis was plotted using Networkx. The Spearman correlation analysis was used to determine the relationship between relative abundance of genus and growth performance, serum and liver parameters of piglets. These results were considered statistically signi cant at P < 0.05.

Additional Files
Additional le 1: Figure S1. Ileum microbiota affected by LPS or Trp.
Additional le 3: Figure S3. The network analysis on genus level in Cecum. Figure 1 The

Supplementary Files
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