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LC Supplementation Increases the Population of Prevotella in(2)
Release date:2018-01-28 17:26    Author:admin    Click: 

DNA Sequence Analysis
Raw sequences were filtered with the average base quality lower than Q20, the quality of the head or tail bases lower than Q20, sequence lengths shorter than 150 bp, or reads with N. Then FLASH (v1.2.7) was used to assemble the paired-end sequences (Magoc and Salzberg, 2011). Reads with homopolymer runs exceeding 8 bp, primer mismatches, ambiguous bases and sequence lengths shorter than 150 bp were further removed in QIIME (v1.9.0) (Caporaso et al., 2010). The UCHIME (Chiu et al., 2017) of mothur (v1.31.2) (Edgar et al., 2011) was used to remove the chimera sequences.
  Operational taxonomic units (OTUs) were counted for all samples with a cutoff of 97% identity using the UCLUST function (Schloss et al., 2009) in QIIME. Any reads <7 times (0.001%) were removed to minimize the impact of rare OTUs on our data analysis (Edgar and Baterman, 2010). The representative sequences of each clustered OTU were selected according to the maximum length, aligned to Greengenes 16S rRNA gene database (v13.8) (Bokulich et al., 2013) and classified by RDP classifier (v.2.2) (Mc Donald et al., 2012). The alpha-diversity indices (PD, Shannon, observed species and Chao1) were calculated for each sample, and beta-diversity (non-metric multidimensional scaling, NMDS) analysis was performed to show the group differences in microbial community structure. Betadiversity statistical analyses were also tested using permutational multivariate analysis of variance (PERMANOVA) based on Bray– Curtis dissimilarities and 999 permutations in the vegan package (v. 2.3.2) (Wang et al., 2007).

Microbial Function Prediction and Statistical Analysis
The microbial function was predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) (v1.1.0) (Oksanen et al., 2015). Based on the pre-calculated Greengenes (v13.5) database (Langille et al.,
2013), PICRUSt was performed on the abundance predictions of Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs and KEGG pathways of bacterial communities. The functional differences among groups were compared using Statistical Analysis of Metagenomic Profiles (STAMP) (Parks et al., 2014). Statistical analysis of two groups was conducted for Two-sided Welch’s t-test and Benjamini-Hochberg FDR correction. Multiple groups were conducted for ANOVA with Tukey-Kramer test and Benjamini-Hochberg correction. Differences were considered significant at P < 0.05. Heatmap diagrams were plotted in R environment (v3.1.2) (http://www.r-project.org).

Sequence Data Accession Number
Raw paired-end reads were submitted to the Sequence Read Archive of the NCBI (accession number: SRP104359).

Effects of LC or AZ on the pH in Cecal Samples
The cecal pH value in the piglets fed with LC was determined to be 6.19, which was significantly lower than that in the control (6.55) (P < 0.05). Similarly, compared to that in the control, the cecal pH (6.33) was significantly lower in the piglets fed with AZ (P < 0.05). The pH between LC and AZ treatments was not significantly different (P > 0.05) (Supplementary Table S2).

Characteristics of Midgut Bacteria Community Libraries
At least 47,083 sequence reads were obtained per sample and a total of 706,251 high-quality sequences were used for later analysis (Supplementary Table S3). Based on 97% sequence similarity, these sequences were assigned to 17,890 OTUs. After removing the rare OTUs (lower than 0.001% of total sequences), 2,242, 2,134, and 2,129 OTUs were retained from CTR group, LC group, and AZ group, respectively (Supplementary Table S4). From the Veen analysis of OTU, out of total 2,624 OTUs, 1,585 (∼60% of the total OTUs) existed in three groups (Figure 1A). Instead, 148, 72, and 108 unique OTUs were identified in CTR, LC, and AZ groups, respectively.

Effects of Dietary LC and AZ on Alpha and Beta Bacterial Diversity
We compared bacterial diversity (PD and Shannon index) and richness (observed species and Chao index) indices for alphadiversity. PD index in the LC group or AZ group was significantly lower than that in the CTR group (P < 0.05), showing that the bacterial diversity in cecum was decreased by AZ or LC (Figure 1B). Shannon index between the two treatments did not show statistical difference while the value in the treatments was lower than the control (Supplementary Table S5). However, there was no significant difference in diversity index (PD and Shannon index) between LC and AZ (P > 0.05), indicating both of the treatment strategies decreased bacterial diversity under our testing conditions. About community richness indices (Observed species and Chao), there was no significant difference between LC and CTR, indicating that LC did not affect bacterial richness. However, the number of observed species or Chao index in the AZ group was significantly lower than that in CTR (P < 0.05) (Figures 1C,D), indicating that AZ decreased bacterial richness in the cecum. Collectively, our data suggested that piglets fed AZ had a decreased bacterial richness and diversity. Additionally, supplementary LC only decreased the gut bacterial diversity rather than bacterial richness.
  For beta diversity analysis, we examined the relationships in cecal microbiome between the control, AZ supplement and LC addition using NMDS. The distribution of microbiota at each group was distinctly clustered separately along principal coordinate (Figure 1E). Moreover, PERMANOVA was used to test the Beta-diversity statistical analysis. The results showed that three groups were distinctly different in the distribution of microbiota (P < 0.05) (Table 1), indicating that LC and AZ significantly affected the cecal bacterial structure when compared to CTR.

Effects of Dietary LC and AZ on the Composition of the Cecal Microbiota
Twenty-two bacterial phyla were assigned in the LC, AZ, and CTR groups and 14 of them were shared among them (Supplementary Table S6). Bacteroidetes, Firmicutes, and Proteobacteria accounted for more than 98% of the total
sequence reads (Figure 1F). Approximately 1% of sequences in the cecum samples could not be assigned to a certain rank (unclassified) (Supplementary Table S6), suggesting that there were uncharacterized bacterial taxa existing in the cecal
microbiome. The proportion of Bacteroidetes in LC and AZ was 1.5- and 1.9-fold higher than that in the CTR group (P < 0.05), respectively (Figure 1G). Instead, the proportion of Firmicutes in the LC and AZ treatments was around 0.8- and 0.6-fold lower than that in the CTR group (P < 0.05), respectively (Figure 1G). The proportion of Proteobacteria in the AZ treatment was 0.2- fold lower than that in the CTR group (P < 0.05), but in the LC treatment, it was in accordance with CTR group (Figure 1G).
  Moreover, 41 bacterial classes (Supplementary Table S7), 68 orders (Supplementary Table S8), 96 families (Supplementary Table S9), and 124 genera (Supplementary Table S10) were assigned to all the three treatments. On the genus level, the relative abundance of Prevotella in the LC group (30% of the total bacterial community) and AZ group (29%) was significantly higher than that in the CTR group (10%) (P < 0.05) (Figure 1H). Compared to CTR and AZ groups, the relative abundance of Succinivibrio was significantly increased by LC treatment (P < 0.05) (Figure 1H). Similarly, the relative abundance of Anaerovibrio in the LC group was significantly higher than that in AZ group (P < 0.05) (Figure 1H). However, the relative abundance of CF231 in AZ group was significantly higher than that in CTR as well as LC (P < 0.05) (Figure 1H). The relative abundance of Desulfovibrio in treatments LC and AZ was significantly lower than that in the control group (P < 0.05) (Figure 1H).

Function Prediction of the Microbial Community in the Cecum
Given the distinct microbiome changes within the LC and AZ pigs compared to CTR pigs, we tested whether the different treatments would lead to functional changes in each microbiome. Prediction of metagenome functional contents was conducted by applying PICRUSt to insight our 16S rDNA sequences. Compared to CTR, both LC and AZ had significantly higher function enrichments of energy metabolism, metabolism of terpenoids and polyketides, digestive systems and cell growth and death (P < 0.05) (Figures 2A,B). However, both treatments dramatically reduced the function enrichment of environmental information processing such as membrane transport (P < 0.05) (Figures 2A,B). In addition, LC group significantly increased the abundance of glycan biosynthesis and metabolism when compared to that in CTR group (P < 0.05) (Figure 2A). The relative abundance in the metabolism of cofactors and vitamin in LC treatment was higher than that in CTR or AZ group (P < 0.05) (Figures 2A,C). Furthermore, we also analyzed the microbial genes connected to KEGG Orthology (KO) terms. 1,373 of 6,014 KO terms (23%) were found to be significantly different among AZ, LC, and CTR groups based on TukeyKramer’s ANOVA Test (P < 0.05). Remarkably, out of 1,373, 462 KO terms were assigned to bacteria Prevotella. It is worthy of highlighting that most of them (462 different KO terms from any two-group comparisons) were enriched on carbohydrate metabolism (15% different KO terms), metabolism of cofactors and vitamins (9%), energy metabolism (8%), glycan biosynthesis and metabolism (6%) (Figure 3), suggesting that the drastically increased Prevotella by LC and AZ played an important function among the intestinal microbiota. Further, 79 different KO terms whose relative abundance were >0.1% in any group were screened out, and most of them including iron complex were increased in LC than in CTR group (Figure 4). In the metabolism pathways, there were 62 and 29 different KO terms, whose relative abundance was >0.01% in any group, involved in the metabolism of cofactors and vitamins (Supplementary Figure S1) and glycan biosynthesis and metabolism (Supplementary Figure S2), respectively.

The gut microbiota plays a critical role in animal growth and production; a different influence on microbiome profiles resulting from different treatments is expected to impact host digestion efficiency and intestinal function (Frese et al., 2015). LC, a growth promoter as the alternative supplement in animals, possibly promoted the animal growth performance by modulating the microbiota in the host digestion tract. Our study showed that supplementation of LC (50 mg/kg) to piglets decreased the cecal bacterial diversity without affecting bacterial richness while utilization of antibiotics and ZnO as antimicrobials decreased the cecal bacterial community richness and the bacterial diversity (Supplementary Table S5 and Figures 1B–D). Our early study also observed that in-feed antibiotics or high dietary ZnO supplementation to weaned piglets increased the microbiota diversity and richness of ileal digesta whereas decreased the microbiota diversity of the colonic digesta (Yu et al., 2017). Further, microbial community structure in piglets fed with LC was remarkably different from that in AZ group or CTR (Table 1, Figure 1E). Collectively, our results demonstrated LC differently modulated the intestinal microbiota in piglets using a different mechanism from that in AZ.

  In this study, the supplementation of LC (50 mg/kg) to weaned piglets significantly increased the cecal relative abundance of Bacteroidetes (Figure 1G). Bacteroidetes are wellknown excellent degraders for plant polysaccharides and other recalcitrant organic carbon and nitrogen sources (Salyers, 1984). It is also reported that abundant Bacteroidetes may prevent diarrhea because members of phylum Bacteroidetes are known to interact with the gut immune system, suggesting a link between early microbiota colonization and gut immune maturation after weaning (Jakobsson et al., 2013). Particularly, the abundance of genus Prevotella within phylum Bacteroidetes was acutely
increased from 10.41% in control group to 30.17% in LC group (Supplementary Table S10 and Figure 1H). Prevotella are saccharolytic and produce acetate and succinic acids as end fermentation products (Downes et al., 2007). Evidence showed
a strong association between Prevotella and carbohydrates from fiber-rich diets or from long-term carbohydrates diets (De Filippo et al., 2010; Wu et al., 2011). It was also reported  that a higher abundance of Prevotellaceae dominated in fecal microbiota of healthy piglets when compared to post-weaning diarrheic piglets (Dou et al., 2017). Thus, diets supplementation of LC, as oligosaccharides that belong to carbohydrates, stimulated the amount increase of Bacteroidetes and Prevotella to help the breakdown of carbohydrate and maybe improve the intestinal immune and decrease diarrhea. Further investigations are needed to establish the relation between the abundance of Bacteroidetes or Prevotella and diarrhea. Kong et al. (2014) indicated that dietary supplementation of COS at 500 mg/kg increased the number of Prevotella in ileal contents (Kong et al., 2014). Our study here showed that LC supplementation (50 mg/kg) could increase the gastrointestinal Prevotella population. Moreover, antibiotics and ZnO supplement also increased the relative abundance of Prevotella. The weaned pigs fed with chlortetracycline had an increased Prevotella in fecal samples (Li et al., 2017). Collectively, Prevotella could be a potential microbial marker for modulation effects of gut microflora by LC or AZ supplementation.
  An interesting finding is that the relative abundance of Succinivibrio was increased 38-fold in LC group while it was decreased 0.17-fold in AZ group compared to that in CTR (Figure 1H). The relative abundance of Succinivibrio in faece was
decreased in the weaned pigs fed with chlortetracycline, or zinc bacitracin, or colistin sulfate, or a mixture of them, indicating that Succinivibrio was sensitive to antibiotics or zinc (Li et al., 2017). Bacteria Succinivibrio are known to degrade starch and produce acetic and succinic acids (Wang et al., 2017). Our results further demonstrated that Succinivibrio involving in polysaccharide biodegradation can be increased by some oligosaccharides (i.e., COS). Moreover, the relative abundance of Enterobacteriaceae and Staphylococcus in the cecum presented a decreased trend in LC group and AZ group (Supplementary Tables S9, S10). However, dietary supplementation with LC (50mg/kg) and AZ group had no effects on increasing Lactobacillus and Bifidobacterium counts in the cecum (Table S10). The decreased intestinal pH may contribute to inhibit intestinal pathogen propagation and alleviate post-weaning diarrhea in young animals (Mourao et al., 2006). The cecal pH in LC group was the lowest compared to AZ and CTR (Supplementary Table S2). It is reasonable to assume the decreased pH is attributed to the increased Prevotella and Succinivibrio in LC because both produce acetic and succinic acids as the end fermentationproducts (Downes et al., 2007; Wang et al., 2017).
  Prediction of metagenome functional contents showed that several metabolic pathways were enriched in piglets with LC supplementation including energy metabolism, metabolism of terpenoids and polyketides, digestive systems, cell growth and death, glycan biosynthesis and metabolism as well as metabolism of cofactors and vitamin (Figure 2). It is worthy of highlighting that “digestive systems,” particularly those involved in biodegradation of plant materials (Figure 4), are very important for early-weaned piglets because of the sudden switching from protein-rich milk to plant fiber-rich diets. Abbreviation of such pathways in animal digestion tracts will cause digestive disorders, nutrient malabsorption and a high incidence of diarrhea. Terpenoids and polyketides serve numerous biochemical functions such as quinones in electron transport chains, membrane components, hormones, anti-microbial, and anti-parasites.
  Moreover, LC increased the abundances of riboflavin (VB2) kinase and iron complex related proteins (Figure 4). LC effectively chelates some vitamins and heavy metals (such as Fe3+) (Varma et al., 2004; Sun et al., 2010), which may contribute to solubilize these cofactors and transport them to hosts. 

  In conclusion, low-molecular-weight chitosan modulated the gut microbial diversity and altered the microbial community in the cecum of weaned piglets, and showed comparable effects to in-feed antibiotics and ZnO additive, especially increased the population of Prevotella microbiota in the cecal digesta. The understanding on effects of low-molecular-weight chitosan on intestinal bacterial communities may provide insights into future application of the alternative strategy for treating diarrhea in piglets.

CZ and ZC conceived and designed the experiments; YW performed the experiments; TY and SC analyzed the data; MH, ZW and XM provided the experimental materials; GW funded the sequencing cost. TY and SC wrote the manuscript. All authors reviewed the manuscript.

This project was supported by the National Science-technology Support Plan (2012BAD39B01-5) and National modern agriculture industry technology system (CARS-36), Natural Science Foundation of Guangdong Province (2015A030310368), Presidential Foundation of the Guangdong Academy of Agricultural Sciences (201427). Science and Technology Project of Guangdong Province (2009B090200067, 2013A061401020, 2016A020210041, 2016B070701013).

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2017.02182/full#supplementary-material

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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Yu, Wang, Chen, Hu, Wang, Wu, Ma, Chen and Zheng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums
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