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The dynamics of the piglet gut microbiome during the weaning transition in association with health and nutrition

The dynamics of the piglet gut microbiome during the weaning transition in association with... Background: Understanding the composition of the microbial community and its functional capacity during weaning is important for pig production as bacteria play important roles in the pig’s health and growth performance. However, limited information is available regarding the composition and function of the gut microbiome of piglets in early-life. Therefore, we performed 16S rRNA gene and whole metagenome shotgun sequencing of DNA from fecal samples from healthy piglets during weaning to measure microbiome shifts, and to identify the potential contribution of the early-life microbiota in shaping piglet health with a focus on microbial stress responses, carbohydrate and amino acid metabolism. Results: The analysis of 16S rRNA genes and whole metagenome shotgun sequencing revealed significant compositional and functional differences between the fecal microbiome in nursing and weaned piglets. The fecal microbiome of the nursing piglets showed higher relative abundance of bacteria in the genus Bacteroides with abundant gene families related to the utilization of lactose and galactose. Prevotella and Lactobacillus were enriched in weaned piglets with an enrichment for the gene families associated with carbohydrate and amino acid metabolism. In addition, an analysis of the functional capacity of the fecal microbiome showed higher abundances of genes associated with heat shock and oxidative stress in the metagenome of weaned piglets compared to nursing piglets. Conclusions: Overall, our data show that microbial shifts and changes in functional capacities of the piglet fecal microbiome resulted in potential reductions in the effects of stress, including dietary changes that occur during weaning. These results provide us with new insights into the piglet gut microbiome that contributes to the growth of the animal. Keywords: Metagenomics, Microbiome,Piglets,16S rRNA,Weaning Background in the health and growth of animals including the reduc- The mammalian gastrointestinal tract (GIT) harbors 500– tion in the incidence of infectious, inflammatory, and 1000 bacterial species that play important roles in the other immune diseases [2, 3] and contributing to the over- health and disease of the host [1]. It is known that early all metabolism and, therefore, the growth of the animal. bacterial gut colonizers are important in the initial estab- Weaning is a stressful event in a pig’s life and can lishment of the complex gut microbial community. The disrupt the piglet gut microbiome, which can lead to poor gut microbiome is thought to play many important roles health and growth performance [4]. Piglets experience a wide variety of stresses such as physiological, environmen- tal and social challenges during the weaning transition [4]. * Correspondence: mhsong@cnu.ac.kr; hbkim@dankook.ac.kr This is important to the swine industry since the changes Robin B. Guevarra, Sang Hyun Hong and Jin Ho Cho contributed equally to in the composition of the gut microbiota after weaning this work. Division of Animal and Dairy Science, Chungnam National University, can lead to an increased susceptibility of piglets to Daejeon, South Korea post-weaning diarrhea. Consequently, this can lead to an Department of Animal Resources Science, Dankook University, Cheonan, economic burden for pig farmers [5]. During the weaning South Korea Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 2 of 9 transition, the diet of piglets abruptly shifts from a Table 1 Composition of basal diet for weaned pigs (as-fed basis) high-fat, low-carbohydrate milk to a high-carbohydrate Item Content and low-fat feed. This change may lead to reduced prolif- Ingredient, % eration of intestinal epithelial cells [6]. Previously, it has Corn 56.09 been reported that the diet shapes the gut microbiome of Soybean meal, 44% 26.00 piglets during nursing and weaning periods [7]. More re- Soy protein concentrate 12.00 cently, the relationship between body weight and intestinal Soybean oil 3.00 microbiota in weaned piglets has also been investigated [8]. It has been shown that the introduction of solid feed Limestone 1.30 and the weaning process are important driving forces in Monocalcium phosphate 1.20 the succession of gut bacteria in piglets [9]. Even though, 1 Vit-Min premix 0.04 these studies have emphasized the great importance of L-Lysine HCl 0.24 early-life microbiota to growth, immune system develop- DL-Methionine 0.09 ment and the health of piglets, there is limited information L-Threonine 0.04 available regarding the structure and function of the gut microbiome of piglets in early-life in association with Total 100 health and growth performance. Calculated energy and nutrient contents Antimicrobial growth promoters (AGPs) have been ME, Mcal/kg 3.48 used in swine production for several decades. However, CP, % 24.17 their use has been banned in many countries worldwide Calicum, % 0.84 because of potential side effects, such as emergence of Phosphorus, % 0.66 resistance to antimicrobials. Thus, efforts to develop al- ternatives to AGPs with the goal of preserving the effi- Lysine, % 1.54 cacy of AGPs are being implemented. In this sense, Methionine, % 0.45 developing alternate ways to promote growth makes it Cysteine, % 0.39 more important to understand microbial and functional Threonine, % 0.96 succession of the piglet gut microbiome because one of Tryptophan, % 0.28 the specific mechanisms of AGPs is to alter gut micro- Arginine, % 1.60 bial population composition [10, 11]. Therefore, the work described in this study was de- Histidine, % 0.67 signed to better document the changes that occur in Isoleucine, % 1.03 the composition of the fecal microbiome in piglets Leucine, % 2.05 before and after weaning and to use metagenomics to Phenylalanine, % 1.21 identify metabolic functions that may be changed dur- Valine, % 1.09 ing these time points. It is a goal of this study to Provided per kilogram of diet: vitamin A, 12,000 IU; vitamin D , 2500 IU; bring new insights into the potential contribution of vitamin E, 30 IU; vitamin K , 3 mg; D-pantothenic acid, 15 mg; nicotinic acid, early-life microbiota in shaping the host metabolism 40 mg; choline, 400 mg; and vitamin B ,12 μg; Fe, 90 mg from iron sulfate; Cu, 8.8 mg from copper sulfate; Zn, 100 mg from zinc oxide; Mn, 54 mg from and health with focus on stress response, carbohy- manganese oxide; I, 0.35 mg from potassium iodide; Se, 0.30 mg from drate and amino acid metabolism. sodium selenite Methods Fecal sampling DNA extraction Fecal samples were collected from the rectum of 10 Total DNA from the feces was extracted from piglets just prior to weaning (21 d of age) and again 1 200 mg of feces per sample using QIAamp Fast DNA wk after weaning (28 d of age). The fecal samples were Stool Mini Kit (QIAGEN, Hilden, Germany) accord- placed in sterile test tubes and stored at − 80 °C. After ing to the manufacturer’s instructions. Cell lysis was weaning, the piglets were fed a typical nursery diet performed by bead-beating the samples twice for basedoncornand soybeanmeal.Thediet was 2 min at 300 r/min, with an incubation period of formulated to meet the National Research Council [12] 5 min in a water bath at 70 °C between beatings. The estimates of nutrient requirements of weaned piglets concentrations of DNA were measured using a (Table 1). Piglets were allowed free access to feed and Colibri Microvolume Spectrometer (Titertek Berthold, water. No antibiotics or supplementary additives were Pforzheim, Germany) and samples with OD260/280 administered to the piglets throughout the experiment. ratios of 1.80–2.15 were processed further. Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 3 of 9 16S rRNA gene and whole metagenome sequencing generated based on the weighted and unweighted UniFrac For the 16S rRNA gene sequencing, the primers distance metrics. Analysis of similarities (ANOSIM) was 799F-mod6 (5´-CMGGATTAGATACCCKGGT-3′)and used to determine whether the microbial compositions be- 1114R (5´-GGGTTGCGCTCGTTGC-3′)were used to tween the two groups were significantly different using amplify the V5 through V6 hypervariable regions of the QIIME and was based on the weighted and unweighted 16S rRNA gene [13]. The amplification mix contained 5× UniFrac distance metrics. 2+ PrimeSTAR Buffer (Mg ) (Takara Bio, Inc., Shiga, Japan), 2.5 mmol/L concentrations of each of deoxynucleotide tri- Whole metagenome sequence analysis phosphates (dNTPs), 2.5 IU/μL of PrimeSTAR HS DNA Whole metagenome shotgun sequencing was performed Polymerase, a 10 pmol of each primer, and 25 ng of DNA on a subset of eight samples selected randomly (four sam- in a reaction volume of 50 μL. The thermal cycling param- ples from the same piglets at 21 and 28 d of age) to inves- eters were as follows: initial denaturation at 98 °C for tigate the fecal microbial functions present in the fecal 3 min, followed by 35 cycles of 98 °C for 10 s, 55 °C for microbes of the samples. To analyze whole metagenomic 15 s, and 72 °C for 30 s, and a final 3-min extension at sequence data from nursing and weaned piglets, the raw 72 °C. PCR products were purified using PCR purification sequence data in FASTQ format were imported to the kit, Wizard® SV Gel and PCR Clean-Up System (Promega, CLC Genomics Workbench (version 10) with CLC Micro- Wisconsin, USA). The barcoded16S rRNA gene ampli- bial Genomics Module (version 1.2) (Qiagen Bioinformat- cons were sequenced using the Illumina MiSeq platform ics, Aarhus, Denmark). The quality of the sequences was at Macrogen Inc. (Seoul, Republic of Korea). For the assessed and high quality sequences were assembled using whole metagenome shotgun sequencing, DNA represent- CLC’s De Novo assembly algorithm. The contigs were ing the fecal microbial communities extracted from the submitted to the Metagenomics Rapid Annotation using feces was sequenced using paired-end shotgun sequencing Subsystem Technology (MG-RAST) pipeline for microbial using the Illumina Hi-Seq 2000 platform at Macrogen Inc. functional analysis. All the sequence reads were normal- (Seoul, Republic of Korea). ized in MG-RAST. The MG-RAST pipeline uses DESeq to analyse sequence count data, and to remove aspects of 16S rRNA gene sequence analysis inter-sample variability caused by differences in sequen- The 16S rRNA gene sequences were processed using the cing depth of samples [18]. Removal of artificial duplicate Mothur software to remove low-quality sequences [14]. reads and pig genomic DNA reads were performed using Briefly, sequences that did not match the PCR primers the MG-RAST pipeline [19]. The MG-RAST pipeline uses were eliminated from demultiplexed sequence reads. We bowtie to remove sequence reads that match to the gen- also trimmed sequences containing ambiguous base calls ome of the host [20]. To filter out host-derived metage- and sequences with a length less than 100 bp to minimize nomic reads, we used the reference swine genome (Sus the effects of random sequencing errors. Chimeric scrofa, NCBI v10.2) readily available in MG-RAST [19]. sequences were further deleted using the UCHIME The functional annotation of the sequence reads was per- algorithm implemented in Mothur. QIIME (Quantitative formed using the SEED Subsystems database, a collection Insights into Microbial Ecology) software package (version of functionally related protein families [21]. Similarity 1.9.1) was used for de novo operational taxonomic unit search between sequence reads and the SEED databases − 5 (OTU) clustering with an OTU definition at an identity was conducted by using an E-value of less than 1 × 10 , cutoff of 97% [15]. Taxonomic assignment was performed minimum identity of 60%, and a minimum alignment using the naïve Bayesian RDP classifier and the Greengenes length of 15 amino acids for protein. Multiple t-tests were reference database. Microbial alpha diversity including used to identify significant differences in functional pro- Chao1, observed OTUs, phylogenetic diversity (PD) whole files between nursing and weaned pigs using STAMP and tree, Shannon index and Simpson index were calculated GraphPad Prism version 7.00 (La Jolla, CA, USA). using QIIME. A two-sided Welch’s t-test in Statistical Ana- lysis of Metagenomic Profiles (STAMP) software v2.1.3 Results [16] was used to identify significant differences in relative Microbial diversity of nursing and weaned piglets based abundance of microbial taxa of the two groups. A P value on 16S rRNA gene data < 0.05 was considered to be significant. Beta-diversity was Sequencing of the 16S rRNA genes in the fecal samples measured using both weighted and unweighted UniFrac produced a total of 1,947,836 reads after quality-filtering, distance metrics using QIIME. The unweighted UniFrac with a mean sequence number of 97,392 ± 49,139 reads takes into account the community membership (presence per sample (Additional file 1: Table S1). The diversity of or absence of OTUs), whereas the weighted UniFrac con- the microbial communities in the fecal samples decreased siders the relative abundance of OTUs in the community after weaning as measured using Shannon, Simpson, and [17]. Principal coordinate analysis (PCoA) plots were Chao1 diversity indices (Table 2). However, the differences Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 4 of 9 Table 2 Alpha diversity of the piglet gut microbiota using 16S distances also showed distinct clustering between nurs- rRNA gene sequences ing and weaned piglets (Fig. 1b). Diversity index Nursing Weaned P-value Taxonomic classification of the bacteria using 16S rRNA PD whole tree 17.58 ± 2.94 13.03 ± 2.45 0.003 genes Shannon 5.13 ± 0.80 4.58 ± 1.06 0.223 Comparisons of the relative abundances of the gut micro- Simpson 0.89 ± 0.07 0.82 ± 0.13 0.189 biota compositions between nursing and weaned piglets at Chao1 1192.99 ± 324.63 1090.64 ± 287.64 0.463 the phylum, family and genus levels are shown in Fig. 2. Observed OTUs 631.56 ± 159.71 543.72 ± 134.26 0.237 At the phylum level, the bacterial sequences from the Values are presented as mean ± SD (n = 10 per group) nursing piglet samples were composed predominantly of OTU operational taxonomic unit the phyla Bacteroidetes (44.14%), Firmicutes (41.01%) Spirochaetes (9.87%), Proteobacteria (2.94%), Tenericutes observed between the two groups were not statistically (1.07%) and 14 other phyla that collectively comprised significant with the exception of the phylogenetic diversity 0.61% of the total sequences analyzed (Fig. 2a). By com- (PD) whole tree index. The mean number of observed parison, weaned piglets consisted largely of phyla Bacter- OTUs identified in the nursing group was 631.60 ± 168.36 oidetes (63.14%), Firmicutes (34.27%), Proteobacteria and 543.80 ± 141.54 for the weaned piglet samples. The (1.79%), Spirochaetes (0.29%), Tenericutes (0.26%) and Shannon-Weaver index values showed highly diverse mi- other 14 phyla which collectively comprised of 0.05% of crobial communities in nursing (5.13 ± 0.85) and weaned the total sequences analyzed in the weaned piglet samples piglets (4.58 ± 1.11). The PD whole tree index, a measure (Fig. 2a). After weaning, the populations of the phylum of biodiversity that assimilates phylogenetic difference be- Bacteroidetes significantly increased from an average of tween taxa, was significantly higher in nursing piglets 44.14% in nursing pigs to 63.14% in weaned animals compared to the weaned piglets (P <0.05). (P < 0.05). This coincided with a significant decrease Analysis of similarities (ANOSIM) of unweighted Uni- in the populations of phyla Spirochaetes, Tenericutes, Frac distances indicated that nursing and weaned pigs Actinobacteria, and Lentisphaerae (P < 0.05) (Fig. 2a). were significantly different (P = 0.001) with relatively At the family level, the three most abundant bacterial high R-value of 0.7373 suggesting that the microbiota of families in nursing pig microbiota primarily consisted of the two groups were significantly different. The un- Ruminococcaceae (20.43%), Prevotellaceae (12.93%) and weighted UniFrac PCoA plot visually confirmed the dis- Spirochaetaceae (9.84%). After weaning, populations of tinct separation of microbial communities between the Prevotellaceae and Lactobacillaceae significantly in- nursing and weaned piglets (Fig. 1a). The ANOSIM of creased by 44.31% and 4.78%, respectively (Fig. 2b). weighted UniFrac distances were similar to the un- At the genus level, Prevotella and Lactobacillus were weighted UniFrac distances, which showed a significant the top 2 most significantly enriched genera in the difference between the microbial communities of nurs- weaned piglets while Bacteroides was the most abundant ing and weaned pigs (P = 0.001) with an R-value of genera in nursing piglet fecal samples (P < 0.05) (Figs. 2c 0.7158. The PCoA plot of the weighted UniFrac & 3). While Prevotella represented the most abundant Fig. 1 Principal coordinates analysis (PCoA) plots based on (a) unweighted and (b) weighted UniFrac distance metrics Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 5 of 9 that differ between nursing and weaned piglets are shown in Additional file 2: Figure S1. The similar profiles of bacterial communities were ob- served when comparing the taxa between results obtained using the 16S rRNA gene data and whole metagenome shotgun sequencing (Additional file 2:Fig.S2). Microbial functional characteristics of the piglet gut metagenome associated with “stress response” and “virulence, disease and defense” Overall, the whole metagenome shotgun sequencing using HiSeq Illumina platform produced a total of 50,440,732 sequences. After quality trimming, a total of 1,120,421 contigs were assembled from eight fecal samples (Additional file 1: Table S2). In the level 1 SEED subsys- tems, we identified 28 SEED Subsystems in both nursing and weaned piglet metagenome (Additional file 2:Figure S3), and we focused on the differences of functional gene groups associated with “carbohydrates”, “amino acids and derivatives”, “stress response”,and “virulence, disease, and defense” (Additional file 2:Figure S4). Stresses and disturbances of the composition of the fecal microbiome during the weaning transition have been Fig. 2 Taxonomic classification of the 16S rRNA gene sequences at demonstrated to cause diarrhea and growth reduction [5]. the (a) phylum, (b) family, and (c) genus levels for the Nursing and Therefore, we investigated the impact of weaning on the Weaned piglets functional profiles of the bacterial communities to evalu- ate counter responses of piglet gut microbiome against genus in both groups, its relative abundance significantly the stresses caused by weaning. At the level 2 SEED sub- increased (P < 0.001) from an average of 12.93% in nurs- systems, within the “stress response”, gene families related ing piglets to 57.24% in weaned piglets (Fig. 3). Similar to “oxidative stress” and “heat shock” were significantly to a previous report on the piglet gut microbiome, Pre- enriched (P < 0.05) in the weaned piglets (Fig. 4a). votella was present in nursing piglets with a relatively At the level 4 SEED subsystems within the “heat shock low abundance and increased in weaned piglets when a and oxidative stress”, numerous proteins and enzymes plant-based diet was introduced [7]. The other genera that were involved in bacterial heat shock and oxidative stress were significantly enriched (P < 0.05) in weaned piglets including translation elongation factor LepA, Chaperone protein DnaJ, signal peptidase-like protein, heat shock protein GrpE (Fig. 4b), superoxide reductase (EC1.15.1.2) and cytochrome c551 peroxidase (EC1.11.1.5) (Fig. 4c). Diversity analysis of SEED subsystems retrieved from piglet gut microbial metagenome associated with “viru- lence, disease and defense” covered 2.45% of the total se- quences assigned to SEED subsystems. Interestingly, at level 2 SEED subsystems, the most abundant gene family within the virulence, disease and defense was “resistance to antibiotics” while other functional gene groups such as “adhesion”, “detection” and “invasion and intracellular Fig. 3 The Box plot identifying the significantly different taxa resistance” were less abundant (Additional file 2: Figure between Nursing and Weaned piglets at the genus level. The interquartile range is indicated by the outer bounds of the boxes, S5a). At the level 3 SEED subsystems, we observed that and the median is indicated by the black midline. The whiskers gene families associated with resistance to antibiotics represent the minimum and maximum values. The [P < 0.001], [P < 0.01] have the tendency to be higher in weaned piglets includ- and [P < 0.05] were indicated as [***], [**] and [*], respectively ing multidrug resistance efflux pumps, resistance to Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 6 of 9 Fig. 4 Comparison of the functional capacities of the gut microbiomes between Nursing and Weaned piglets associated with “stress response”. Normalized abundance of the level 2 SEED subsystem classified reads associated with stress response (a). Normalized abundance of proteins at the level 4 SEED subsystem associated with heat shock (b) and oxidative stress (c). The error bars show the calculated standard deviation of four replicates, and the [P < 0.001], [P < 0.01] and [P < 0.05] were indicated as [***], [**] and [*], respectively fluoroquinolones and beta-lactamase (Additional file 2: composition and the functional capacity of the microbiota Figure S5b). shift when such complex plant-derived glycans enter the gut. Similar to previous reports on the swine fecal metagen- Microbial functional characteristics of the piglet gut ome [22, 23], we observed that the abundance of genes metagenome associated with carbohydrate and amino mapping to carbohydrates metabolism associated with acid metabolism components of plant-derived polysaccharides significantly We performed a hierarchical clustering-based analysis of more prevalent in weaned pigs including “xylose the SEED subsystem and found that level 1 SEED sub- utilization”, “mannose metabolism” and “L-rhamnose systems associated with “carbohydrates” and “amino utilization” (Fig. 5a). These sugars are products of the hy- acids and derivatives” were significantly enriched in the drolysis of non-starch polysaccharides (NSP) that are weaned pigs (P < 0.05) (Additional file 2: Figures. S3 and mainly found in many feed ingredients including soybean S4). At the level 3 SEED subsystems, gene families map- meal, wheat bran and oats. The microbiome of the nursing ping to carbohydrate and amino acid metabolism were piglet had a significant enrichment of gene families associ- significantly higher in the weaned piglets (Fig. 5). ated with “lactose and galactose uptake and utilization” with The carbohydrate composition of the porcine diet lactose being the principal sugar in porcine milk (Fig. 5a). abruptly changes when the pigs are separated from the sow The relative abundance of genes associated with amino acid and complex plant-based feeds are introduced. The metabolism was also higher in weaned piglets than nursing Fig. 5 Comparison of the functional capacities of the gut microbiomes between Nursing and Weaned piglets associated with nutrition. Normalized abundance of the level 3 SEED subsystem classified reads associated with carbohydrate metabolism (a) and amino acid metabolism (b). The error bars show the calculated standard deviation of four replicates, and the [P < 0.001], [P < 0.01] and [P < 0.05] were indicated as [***], [**] and [*], respectively Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 7 of 9 piglets. Four of these pathways were significantly enriched understanding the microbial community structure and in weaned piglets including “histidine biosynthesis”, “argin- functional capacity of the microbiome during the weaning ine biosynthesis”, “glutamine, glutamate, aspartate and as- transition is substantial to pig production as it plays import- paragine biosynthesis” and “methionine biosynthesis” (P < ant roles in pig health and diseases. In this study, we found 0.05) (Fig. 5b). that there were significant enrichments of genes associated with bacterial heat shock responses in weaned pigs. The Discussion heat shock responses in bacteria are a result of a stress and We performed this study to better understand microbial are important for successfully adapting to changes in the succession and changes in functional capacity of the piglet physiological state, as well as to changes in the environment fecal microbiome during the weaning transition. One of of the bacterial habitat [32]. Heat shock responses involve the most striking observations in this study was the signifi- the induction of heat shock proteins (HSP) that are com- cant increase in the genus Prevotella after weaning. It has prised of a set of well-conserved proteins with molecular been reported that Prevotella is linked to the fermentation mass ranging from 27 to > 100 kDa, and are produced by of plant-derived non-starch polysaccharides to short-chain bacteria [33, 34]. HSP play a major role in the protection of fatty acids [24]. In humans, Prevotella spp. have also been cells by functioning as intra-cellular chaperones for other reported to produce enzymes, such as β-glucanase, man- proteins under different kinds of stressors [34]. Even though nase, and xylanase that can degrade polysaccharides in the exact mechanisms of the HSP are yet to be determined, plant cell wall [25]. The relative abundance of Lactobacillus our data suggest that bacterial heat shock response in also increased in weaned animals. Lactobacillus has been weaned piglets may play a role in mitigating the negative ef- recently identified as bacteria with the ability to consume fects associated with weaning. plant-derived monosaccharides and disaccharides [26]. The present study also showed a significantly higher The high abundance of Lactobacillus in the microbial abundance of functional gene groups associated with oxida- community of weaned piglets is consistent with other tive stress response in the bacterial metagenome of the studies on carbohydrate utilization in other mammalian weaned piglet. Oxidative stress is defined as a disturbance species [27]. Generally, Lactobacillus is recognized as a in the balance between the production of reactive oxygen carbohydrate-utilizing bacterium with numerous genes en- species (ROS) and antioxidant defenses [35]. The increase coding a wide range of functional capacities associated in production of ROS can cause damage to biological mole- with carbohydrate transport and utilization [28]. Our re- cules including DNA, protein and lipids and can even lead sults suggest that Lactobacillus mayalsoplayapivotal role to cell death [36]. A previous study indicated that weaning in the utilization of complex carbohydrates. Overall, the causes oxidative stress and the exposure of bacterial cells to present study reported higher abundances of Prevotella oxidative stress can have damaging effects on protein activ- and Lactobacillus in the weaned piglets that may allow ities and can contribute to death [37, 38]. As such, oxidative them to adapt to the dietary conditions after weaning. stress caused by weaning eventually induced enterocyte It was noteworthy that the nursing piglet microbiota had apoptosis and cell cycle arrest in the small intestine of significantly higher relative abundance of genus Bacteroides, post-weaning piglets [37]. A recent report on the impact of which are well-known bacteria that utilize milk oligosac- the gut microbiota on the development of metabolic dis- charides as carbon sources [7]. Our analysis of the whole eases revealed that Lactobacillus spp. have developed metagenome of nursing piglet microbiota showed signifi- defense mechanisms against oxidative stress [39]. The cant enrichments of genes associated with lactose and gal- higher proportion of sequences that mapped to oxidative actose uptake and utilization. In comparison to human stress genes in weaned piglets appear to be reflected in the breast milk, porcine milk is primarily composed of lactose increase of the relative abundance of Lactobacillus ob- (Lac), glucose (Glc), galactose (Gal), N-acetyl-glucosamine served in this study. However, potential roles of Lactobacil- (GlcNAc), fucose (Fuc), and sialic acids (NeuAc/ NeuGc) lus spp. to regulate oxidative stress in weaned piglets [29]. Milk oligosaccharides are composed of repeating units should be validated through evidence-based experiments. of lactose or N-acetyl-lactosamine that are usually bonded Amino acids play crucial physiological roles in young with sialic acid and fucose monosaccharides [30]. These piglets to support their maximum production performance complex milk oligosaccharides are not digested by the host [40]. In this study, SEED subsystems related to amino acid during the passage through the GIT suggesting that they metabolism were significantly elevated in the weaned pig- may play a role as natural prebiotics [31]. Our results sug- lets. These findings can be attributed to the increased use gest that both microbial composition and the metabolic of amino acids in feed for protein accretion in livestock functions of the nursing piglet microbiome is oriented to production. Although glucose is widely accepted as the pri- the utilization of milk oligosaccharides. mary nutrient for the maintenance and promotion of cell Weaning is a stressful event in a pig’s life and can contrib- function, it has been reported that glutamate, glutamine ute to intestinal and immune system dysfunctions [4]. Thus, and aspartate are the major contributors to the oxidative Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 8 of 9 fuel for the intestine and cells of the immune system [41, Additional file 2: Figure S1. Extended error bar plot identifying the 42]. Moreover, the present study showed that arginine bio- significantly different taxa between nursing and weaned piglets at the phylum (a), family (b) and genus (c) levels. Corrected P values are shown at synthesis was significantly enriched in the microbiome of right. The differences in the microbial community structure were measured weaned piglets. In addition, a recent study showed that using a two-sided Welch’s t-test, and P < 0.05 was considered significant. arginine supplementation in weaned piglets has beneficial Figure S2. Comparison of the taxonomic profiles obtained using 16S rRNA gene and whole metagenome sequencing between nursing and weaned effects against oxidative stress in the jejunum through the piglets. Stacked bar plots show the relative abundance of bacteria at the a suppression of inflammatory cytokine expression [43]. phylum, b family and c genus levels. Figure S3. Heatmap of relative abun- While the small intestine is the major site for amino acid dance of SEED level 1 subsystems based on whole metagenome sequen- cing data. The e-value cutoff for metagenomics sequence matches to the absorption, the absorption of amino acids in the large − 5 SEED subsystem database was 1 × 10 with a minimum alignment length intestine is limited [44]. However, amino acids in the colon, of 15 amino acids. The two-way hierarchical cluster analysis was performed including lysine, arginine, glycine, leucine, valine and isoleu- using unweighted pair group method with arithmetic mean (UPGMA) method. The side colors in the heatmap depicts the clustering of the sub- cine can be used by the colonic bacteria to generate a com- systems based on the relative abundance. The yellow cluster indicate the plex mixture of metabolic products, such as short-chain SEED Subsystem with relative abundance above 7% while the green cluster fatty acids (SCFA), which are available energy source to the represent the SEED Subsystem with relative abundance below 5%. Figure S4. Differences in the relative abundance of level 1SEED subsystems that pigs [45]. Our results have provided us with new insights were mapped to “Carbohydrates”, “Amino Acids and Derivatives”, “Stress Re- into the functional aspect of the microbiome to help us bet- sponse” and “Virulence, Disease and Defense”.Corrected P-values are calcu- ter understand the interplay between amino acid metabol- lated using the Benjamini-Hochberg false discovery rate approach (P <0.05). Figure S5. Comparison functional categories assigned to a “Virulence, Dis- ism in bacteria and pig health in response to an abrupt ease and Defense” SEED subsystem level 2 and b “Resistance to Antibiotics” dietary change that occurs during the weaning transition. SEED subsystem level 3 between nursing and weaned piglets based on Nevertheless, further studies are required to elucidate the whole metagenome shotgun sequences analyzed using MG-RAST. The error bars show the calculated standard deviation of four replicates, and the [P < exact roles of the functional gene groups of the weaned pig- 0.001], [P < 0.01] and [P < 0.05] were indicated as [***], [**] and [*], respect- let gut microbiome associated with the amino acid metab- ively. (PPTX 911 kb) olism for the swine performance. Abbreviations 16S rRNA: 16S ribosomal ribonucleic acid; ANOSIM: Analysis of similarities; Conclusions DNA: Deoxyribonucleic acid; dNTPs: Deoxynucleotide triphosphates; We observed distinct microbial communities and func- GIT: Gastrointestinal tract; HSP: Heat shock protein; MGRAST: Metagenomics tional capacities of the piglet gut microbiome between rapid annotation using subsystem technology; NRC: National research council; OTU: Operational taxonomic unit; PCoA: Principal co-ordinates nursing and weaned piglets. The weaning process signifi- analysis; PCR: Polymerase chain reaction; PD: Phylogenetic diversity; cantly altered the composition and functional capacities of QIIME: Quantitative insights into microbial ecology; ROS: Reactive oxygen the gut microbiome. As such, our data suggest that the species; STAMP: Statistical analysis of metagenomic profiles early-life stressors caused by dietary change could be an im- Acknowledgements portant driver to lead to these microbiome shifts. Even The authors thank Mo Re Kim (Westborough High School, MA, USA) for the though further studies are required to elucidate the effect initial English editing. of microbiome shifts on piglet health, our data suggest that Funding the microbiome shift and changes in functional capacities This work was supported by the fund (Project No. PJ012615), Rural of the pig gut microbiome were oriented to deal with heat Development Administration, Republic of Korea. shock and oxidative stress during the weaning transition. Availability of data and materials Overall, our results suggest that piglets overcome stresses All raw 16S rRNA gene data used in this study were deposited in National Center caused by dietary change during the weaning transition for Biotechnology Information (NCBI) under Sequence Read Archive (SRA) through a gut microbiome shift, and these results accession number SRP133974, and the whole metagenome datasets generated forthisstudy areavailablein theMG-RAST server with theproject IDmgp80424. emphasize the importance of the early-life microbiota. We believe that our results provide us with substantial Authors’ contributions insights into the piglet gut microbiome that contributes to JHC, BNK, YHK, SW, MS, and HBK designed the research. SHH, JHC, BK, JS, JHL, MS, and HBK performed research and generated data. RBG, SHH, JHC, the growth of the animal, and help us to better understand MS and HBK analyzed the data and RBG, SHH, JHC, REI, MS and HBK wrote the important roles of the essential gut microbiome for the manuscript. All authors read and approved the final manuscript. later studies aiming to develop pig gut modulators, such Ethics approval and consent to participate as feed additives. Not applicable. Additional files Competing interests The authors declare that they have no competing interests. Additional file 1: Table S1. Number of 16S rRNA gene sequence reads Author details of nursing and weaned piglet fecal microbiota before and after quality Department of Animal Resources Science, Dankook University, Cheonan, control. Table S2. Summary of whole metagenome sequence data South Korea. Division of Food and Animal Sciences, Chungbuk National before and after quality control and annotation. (DOCX 18 kb) University, Cheongju, South Korea. Abbvie Bioresearch Center, Abbvie, Guevarra et al. 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The dynamics of the piglet gut microbiome during the weaning transition in association with health and nutrition

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Springer Journals
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Copyright © 2018 by The Author(s).
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Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
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2049-1891
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10.1186/s40104-018-0269-6
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Abstract

Background: Understanding the composition of the microbial community and its functional capacity during weaning is important for pig production as bacteria play important roles in the pig’s health and growth performance. However, limited information is available regarding the composition and function of the gut microbiome of piglets in early-life. Therefore, we performed 16S rRNA gene and whole metagenome shotgun sequencing of DNA from fecal samples from healthy piglets during weaning to measure microbiome shifts, and to identify the potential contribution of the early-life microbiota in shaping piglet health with a focus on microbial stress responses, carbohydrate and amino acid metabolism. Results: The analysis of 16S rRNA genes and whole metagenome shotgun sequencing revealed significant compositional and functional differences between the fecal microbiome in nursing and weaned piglets. The fecal microbiome of the nursing piglets showed higher relative abundance of bacteria in the genus Bacteroides with abundant gene families related to the utilization of lactose and galactose. Prevotella and Lactobacillus were enriched in weaned piglets with an enrichment for the gene families associated with carbohydrate and amino acid metabolism. In addition, an analysis of the functional capacity of the fecal microbiome showed higher abundances of genes associated with heat shock and oxidative stress in the metagenome of weaned piglets compared to nursing piglets. Conclusions: Overall, our data show that microbial shifts and changes in functional capacities of the piglet fecal microbiome resulted in potential reductions in the effects of stress, including dietary changes that occur during weaning. These results provide us with new insights into the piglet gut microbiome that contributes to the growth of the animal. Keywords: Metagenomics, Microbiome,Piglets,16S rRNA,Weaning Background in the health and growth of animals including the reduc- The mammalian gastrointestinal tract (GIT) harbors 500– tion in the incidence of infectious, inflammatory, and 1000 bacterial species that play important roles in the other immune diseases [2, 3] and contributing to the over- health and disease of the host [1]. It is known that early all metabolism and, therefore, the growth of the animal. bacterial gut colonizers are important in the initial estab- Weaning is a stressful event in a pig’s life and can lishment of the complex gut microbial community. The disrupt the piglet gut microbiome, which can lead to poor gut microbiome is thought to play many important roles health and growth performance [4]. Piglets experience a wide variety of stresses such as physiological, environmen- tal and social challenges during the weaning transition [4]. * Correspondence: mhsong@cnu.ac.kr; hbkim@dankook.ac.kr This is important to the swine industry since the changes Robin B. Guevarra, Sang Hyun Hong and Jin Ho Cho contributed equally to in the composition of the gut microbiota after weaning this work. Division of Animal and Dairy Science, Chungnam National University, can lead to an increased susceptibility of piglets to Daejeon, South Korea post-weaning diarrhea. Consequently, this can lead to an Department of Animal Resources Science, Dankook University, Cheonan, economic burden for pig farmers [5]. During the weaning South Korea Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 2 of 9 transition, the diet of piglets abruptly shifts from a Table 1 Composition of basal diet for weaned pigs (as-fed basis) high-fat, low-carbohydrate milk to a high-carbohydrate Item Content and low-fat feed. This change may lead to reduced prolif- Ingredient, % eration of intestinal epithelial cells [6]. Previously, it has Corn 56.09 been reported that the diet shapes the gut microbiome of Soybean meal, 44% 26.00 piglets during nursing and weaning periods [7]. More re- Soy protein concentrate 12.00 cently, the relationship between body weight and intestinal Soybean oil 3.00 microbiota in weaned piglets has also been investigated [8]. It has been shown that the introduction of solid feed Limestone 1.30 and the weaning process are important driving forces in Monocalcium phosphate 1.20 the succession of gut bacteria in piglets [9]. Even though, 1 Vit-Min premix 0.04 these studies have emphasized the great importance of L-Lysine HCl 0.24 early-life microbiota to growth, immune system develop- DL-Methionine 0.09 ment and the health of piglets, there is limited information L-Threonine 0.04 available regarding the structure and function of the gut microbiome of piglets in early-life in association with Total 100 health and growth performance. Calculated energy and nutrient contents Antimicrobial growth promoters (AGPs) have been ME, Mcal/kg 3.48 used in swine production for several decades. However, CP, % 24.17 their use has been banned in many countries worldwide Calicum, % 0.84 because of potential side effects, such as emergence of Phosphorus, % 0.66 resistance to antimicrobials. Thus, efforts to develop al- ternatives to AGPs with the goal of preserving the effi- Lysine, % 1.54 cacy of AGPs are being implemented. In this sense, Methionine, % 0.45 developing alternate ways to promote growth makes it Cysteine, % 0.39 more important to understand microbial and functional Threonine, % 0.96 succession of the piglet gut microbiome because one of Tryptophan, % 0.28 the specific mechanisms of AGPs is to alter gut micro- Arginine, % 1.60 bial population composition [10, 11]. Therefore, the work described in this study was de- Histidine, % 0.67 signed to better document the changes that occur in Isoleucine, % 1.03 the composition of the fecal microbiome in piglets Leucine, % 2.05 before and after weaning and to use metagenomics to Phenylalanine, % 1.21 identify metabolic functions that may be changed dur- Valine, % 1.09 ing these time points. It is a goal of this study to Provided per kilogram of diet: vitamin A, 12,000 IU; vitamin D , 2500 IU; bring new insights into the potential contribution of vitamin E, 30 IU; vitamin K , 3 mg; D-pantothenic acid, 15 mg; nicotinic acid, early-life microbiota in shaping the host metabolism 40 mg; choline, 400 mg; and vitamin B ,12 μg; Fe, 90 mg from iron sulfate; Cu, 8.8 mg from copper sulfate; Zn, 100 mg from zinc oxide; Mn, 54 mg from and health with focus on stress response, carbohy- manganese oxide; I, 0.35 mg from potassium iodide; Se, 0.30 mg from drate and amino acid metabolism. sodium selenite Methods Fecal sampling DNA extraction Fecal samples were collected from the rectum of 10 Total DNA from the feces was extracted from piglets just prior to weaning (21 d of age) and again 1 200 mg of feces per sample using QIAamp Fast DNA wk after weaning (28 d of age). The fecal samples were Stool Mini Kit (QIAGEN, Hilden, Germany) accord- placed in sterile test tubes and stored at − 80 °C. After ing to the manufacturer’s instructions. Cell lysis was weaning, the piglets were fed a typical nursery diet performed by bead-beating the samples twice for basedoncornand soybeanmeal.Thediet was 2 min at 300 r/min, with an incubation period of formulated to meet the National Research Council [12] 5 min in a water bath at 70 °C between beatings. The estimates of nutrient requirements of weaned piglets concentrations of DNA were measured using a (Table 1). Piglets were allowed free access to feed and Colibri Microvolume Spectrometer (Titertek Berthold, water. No antibiotics or supplementary additives were Pforzheim, Germany) and samples with OD260/280 administered to the piglets throughout the experiment. ratios of 1.80–2.15 were processed further. Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 3 of 9 16S rRNA gene and whole metagenome sequencing generated based on the weighted and unweighted UniFrac For the 16S rRNA gene sequencing, the primers distance metrics. Analysis of similarities (ANOSIM) was 799F-mod6 (5´-CMGGATTAGATACCCKGGT-3′)and used to determine whether the microbial compositions be- 1114R (5´-GGGTTGCGCTCGTTGC-3′)were used to tween the two groups were significantly different using amplify the V5 through V6 hypervariable regions of the QIIME and was based on the weighted and unweighted 16S rRNA gene [13]. The amplification mix contained 5× UniFrac distance metrics. 2+ PrimeSTAR Buffer (Mg ) (Takara Bio, Inc., Shiga, Japan), 2.5 mmol/L concentrations of each of deoxynucleotide tri- Whole metagenome sequence analysis phosphates (dNTPs), 2.5 IU/μL of PrimeSTAR HS DNA Whole metagenome shotgun sequencing was performed Polymerase, a 10 pmol of each primer, and 25 ng of DNA on a subset of eight samples selected randomly (four sam- in a reaction volume of 50 μL. The thermal cycling param- ples from the same piglets at 21 and 28 d of age) to inves- eters were as follows: initial denaturation at 98 °C for tigate the fecal microbial functions present in the fecal 3 min, followed by 35 cycles of 98 °C for 10 s, 55 °C for microbes of the samples. To analyze whole metagenomic 15 s, and 72 °C for 30 s, and a final 3-min extension at sequence data from nursing and weaned piglets, the raw 72 °C. PCR products were purified using PCR purification sequence data in FASTQ format were imported to the kit, Wizard® SV Gel and PCR Clean-Up System (Promega, CLC Genomics Workbench (version 10) with CLC Micro- Wisconsin, USA). The barcoded16S rRNA gene ampli- bial Genomics Module (version 1.2) (Qiagen Bioinformat- cons were sequenced using the Illumina MiSeq platform ics, Aarhus, Denmark). The quality of the sequences was at Macrogen Inc. (Seoul, Republic of Korea). For the assessed and high quality sequences were assembled using whole metagenome shotgun sequencing, DNA represent- CLC’s De Novo assembly algorithm. The contigs were ing the fecal microbial communities extracted from the submitted to the Metagenomics Rapid Annotation using feces was sequenced using paired-end shotgun sequencing Subsystem Technology (MG-RAST) pipeline for microbial using the Illumina Hi-Seq 2000 platform at Macrogen Inc. functional analysis. All the sequence reads were normal- (Seoul, Republic of Korea). ized in MG-RAST. The MG-RAST pipeline uses DESeq to analyse sequence count data, and to remove aspects of 16S rRNA gene sequence analysis inter-sample variability caused by differences in sequen- The 16S rRNA gene sequences were processed using the cing depth of samples [18]. Removal of artificial duplicate Mothur software to remove low-quality sequences [14]. reads and pig genomic DNA reads were performed using Briefly, sequences that did not match the PCR primers the MG-RAST pipeline [19]. The MG-RAST pipeline uses were eliminated from demultiplexed sequence reads. We bowtie to remove sequence reads that match to the gen- also trimmed sequences containing ambiguous base calls ome of the host [20]. To filter out host-derived metage- and sequences with a length less than 100 bp to minimize nomic reads, we used the reference swine genome (Sus the effects of random sequencing errors. Chimeric scrofa, NCBI v10.2) readily available in MG-RAST [19]. sequences were further deleted using the UCHIME The functional annotation of the sequence reads was per- algorithm implemented in Mothur. QIIME (Quantitative formed using the SEED Subsystems database, a collection Insights into Microbial Ecology) software package (version of functionally related protein families [21]. Similarity 1.9.1) was used for de novo operational taxonomic unit search between sequence reads and the SEED databases − 5 (OTU) clustering with an OTU definition at an identity was conducted by using an E-value of less than 1 × 10 , cutoff of 97% [15]. Taxonomic assignment was performed minimum identity of 60%, and a minimum alignment using the naïve Bayesian RDP classifier and the Greengenes length of 15 amino acids for protein. Multiple t-tests were reference database. Microbial alpha diversity including used to identify significant differences in functional pro- Chao1, observed OTUs, phylogenetic diversity (PD) whole files between nursing and weaned pigs using STAMP and tree, Shannon index and Simpson index were calculated GraphPad Prism version 7.00 (La Jolla, CA, USA). using QIIME. A two-sided Welch’s t-test in Statistical Ana- lysis of Metagenomic Profiles (STAMP) software v2.1.3 Results [16] was used to identify significant differences in relative Microbial diversity of nursing and weaned piglets based abundance of microbial taxa of the two groups. A P value on 16S rRNA gene data < 0.05 was considered to be significant. Beta-diversity was Sequencing of the 16S rRNA genes in the fecal samples measured using both weighted and unweighted UniFrac produced a total of 1,947,836 reads after quality-filtering, distance metrics using QIIME. The unweighted UniFrac with a mean sequence number of 97,392 ± 49,139 reads takes into account the community membership (presence per sample (Additional file 1: Table S1). The diversity of or absence of OTUs), whereas the weighted UniFrac con- the microbial communities in the fecal samples decreased siders the relative abundance of OTUs in the community after weaning as measured using Shannon, Simpson, and [17]. Principal coordinate analysis (PCoA) plots were Chao1 diversity indices (Table 2). However, the differences Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 4 of 9 Table 2 Alpha diversity of the piglet gut microbiota using 16S distances also showed distinct clustering between nurs- rRNA gene sequences ing and weaned piglets (Fig. 1b). Diversity index Nursing Weaned P-value Taxonomic classification of the bacteria using 16S rRNA PD whole tree 17.58 ± 2.94 13.03 ± 2.45 0.003 genes Shannon 5.13 ± 0.80 4.58 ± 1.06 0.223 Comparisons of the relative abundances of the gut micro- Simpson 0.89 ± 0.07 0.82 ± 0.13 0.189 biota compositions between nursing and weaned piglets at Chao1 1192.99 ± 324.63 1090.64 ± 287.64 0.463 the phylum, family and genus levels are shown in Fig. 2. Observed OTUs 631.56 ± 159.71 543.72 ± 134.26 0.237 At the phylum level, the bacterial sequences from the Values are presented as mean ± SD (n = 10 per group) nursing piglet samples were composed predominantly of OTU operational taxonomic unit the phyla Bacteroidetes (44.14%), Firmicutes (41.01%) Spirochaetes (9.87%), Proteobacteria (2.94%), Tenericutes observed between the two groups were not statistically (1.07%) and 14 other phyla that collectively comprised significant with the exception of the phylogenetic diversity 0.61% of the total sequences analyzed (Fig. 2a). By com- (PD) whole tree index. The mean number of observed parison, weaned piglets consisted largely of phyla Bacter- OTUs identified in the nursing group was 631.60 ± 168.36 oidetes (63.14%), Firmicutes (34.27%), Proteobacteria and 543.80 ± 141.54 for the weaned piglet samples. The (1.79%), Spirochaetes (0.29%), Tenericutes (0.26%) and Shannon-Weaver index values showed highly diverse mi- other 14 phyla which collectively comprised of 0.05% of crobial communities in nursing (5.13 ± 0.85) and weaned the total sequences analyzed in the weaned piglet samples piglets (4.58 ± 1.11). The PD whole tree index, a measure (Fig. 2a). After weaning, the populations of the phylum of biodiversity that assimilates phylogenetic difference be- Bacteroidetes significantly increased from an average of tween taxa, was significantly higher in nursing piglets 44.14% in nursing pigs to 63.14% in weaned animals compared to the weaned piglets (P <0.05). (P < 0.05). This coincided with a significant decrease Analysis of similarities (ANOSIM) of unweighted Uni- in the populations of phyla Spirochaetes, Tenericutes, Frac distances indicated that nursing and weaned pigs Actinobacteria, and Lentisphaerae (P < 0.05) (Fig. 2a). were significantly different (P = 0.001) with relatively At the family level, the three most abundant bacterial high R-value of 0.7373 suggesting that the microbiota of families in nursing pig microbiota primarily consisted of the two groups were significantly different. The un- Ruminococcaceae (20.43%), Prevotellaceae (12.93%) and weighted UniFrac PCoA plot visually confirmed the dis- Spirochaetaceae (9.84%). After weaning, populations of tinct separation of microbial communities between the Prevotellaceae and Lactobacillaceae significantly in- nursing and weaned piglets (Fig. 1a). The ANOSIM of creased by 44.31% and 4.78%, respectively (Fig. 2b). weighted UniFrac distances were similar to the un- At the genus level, Prevotella and Lactobacillus were weighted UniFrac distances, which showed a significant the top 2 most significantly enriched genera in the difference between the microbial communities of nurs- weaned piglets while Bacteroides was the most abundant ing and weaned pigs (P = 0.001) with an R-value of genera in nursing piglet fecal samples (P < 0.05) (Figs. 2c 0.7158. The PCoA plot of the weighted UniFrac & 3). While Prevotella represented the most abundant Fig. 1 Principal coordinates analysis (PCoA) plots based on (a) unweighted and (b) weighted UniFrac distance metrics Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 5 of 9 that differ between nursing and weaned piglets are shown in Additional file 2: Figure S1. The similar profiles of bacterial communities were ob- served when comparing the taxa between results obtained using the 16S rRNA gene data and whole metagenome shotgun sequencing (Additional file 2:Fig.S2). Microbial functional characteristics of the piglet gut metagenome associated with “stress response” and “virulence, disease and defense” Overall, the whole metagenome shotgun sequencing using HiSeq Illumina platform produced a total of 50,440,732 sequences. After quality trimming, a total of 1,120,421 contigs were assembled from eight fecal samples (Additional file 1: Table S2). In the level 1 SEED subsys- tems, we identified 28 SEED Subsystems in both nursing and weaned piglet metagenome (Additional file 2:Figure S3), and we focused on the differences of functional gene groups associated with “carbohydrates”, “amino acids and derivatives”, “stress response”,and “virulence, disease, and defense” (Additional file 2:Figure S4). Stresses and disturbances of the composition of the fecal microbiome during the weaning transition have been Fig. 2 Taxonomic classification of the 16S rRNA gene sequences at demonstrated to cause diarrhea and growth reduction [5]. the (a) phylum, (b) family, and (c) genus levels for the Nursing and Therefore, we investigated the impact of weaning on the Weaned piglets functional profiles of the bacterial communities to evalu- ate counter responses of piglet gut microbiome against genus in both groups, its relative abundance significantly the stresses caused by weaning. At the level 2 SEED sub- increased (P < 0.001) from an average of 12.93% in nurs- systems, within the “stress response”, gene families related ing piglets to 57.24% in weaned piglets (Fig. 3). Similar to “oxidative stress” and “heat shock” were significantly to a previous report on the piglet gut microbiome, Pre- enriched (P < 0.05) in the weaned piglets (Fig. 4a). votella was present in nursing piglets with a relatively At the level 4 SEED subsystems within the “heat shock low abundance and increased in weaned piglets when a and oxidative stress”, numerous proteins and enzymes plant-based diet was introduced [7]. The other genera that were involved in bacterial heat shock and oxidative stress were significantly enriched (P < 0.05) in weaned piglets including translation elongation factor LepA, Chaperone protein DnaJ, signal peptidase-like protein, heat shock protein GrpE (Fig. 4b), superoxide reductase (EC1.15.1.2) and cytochrome c551 peroxidase (EC1.11.1.5) (Fig. 4c). Diversity analysis of SEED subsystems retrieved from piglet gut microbial metagenome associated with “viru- lence, disease and defense” covered 2.45% of the total se- quences assigned to SEED subsystems. Interestingly, at level 2 SEED subsystems, the most abundant gene family within the virulence, disease and defense was “resistance to antibiotics” while other functional gene groups such as “adhesion”, “detection” and “invasion and intracellular Fig. 3 The Box plot identifying the significantly different taxa resistance” were less abundant (Additional file 2: Figure between Nursing and Weaned piglets at the genus level. The interquartile range is indicated by the outer bounds of the boxes, S5a). At the level 3 SEED subsystems, we observed that and the median is indicated by the black midline. The whiskers gene families associated with resistance to antibiotics represent the minimum and maximum values. The [P < 0.001], [P < 0.01] have the tendency to be higher in weaned piglets includ- and [P < 0.05] were indicated as [***], [**] and [*], respectively ing multidrug resistance efflux pumps, resistance to Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 6 of 9 Fig. 4 Comparison of the functional capacities of the gut microbiomes between Nursing and Weaned piglets associated with “stress response”. Normalized abundance of the level 2 SEED subsystem classified reads associated with stress response (a). Normalized abundance of proteins at the level 4 SEED subsystem associated with heat shock (b) and oxidative stress (c). The error bars show the calculated standard deviation of four replicates, and the [P < 0.001], [P < 0.01] and [P < 0.05] were indicated as [***], [**] and [*], respectively fluoroquinolones and beta-lactamase (Additional file 2: composition and the functional capacity of the microbiota Figure S5b). shift when such complex plant-derived glycans enter the gut. Similar to previous reports on the swine fecal metagen- Microbial functional characteristics of the piglet gut ome [22, 23], we observed that the abundance of genes metagenome associated with carbohydrate and amino mapping to carbohydrates metabolism associated with acid metabolism components of plant-derived polysaccharides significantly We performed a hierarchical clustering-based analysis of more prevalent in weaned pigs including “xylose the SEED subsystem and found that level 1 SEED sub- utilization”, “mannose metabolism” and “L-rhamnose systems associated with “carbohydrates” and “amino utilization” (Fig. 5a). These sugars are products of the hy- acids and derivatives” were significantly enriched in the drolysis of non-starch polysaccharides (NSP) that are weaned pigs (P < 0.05) (Additional file 2: Figures. S3 and mainly found in many feed ingredients including soybean S4). At the level 3 SEED subsystems, gene families map- meal, wheat bran and oats. The microbiome of the nursing ping to carbohydrate and amino acid metabolism were piglet had a significant enrichment of gene families associ- significantly higher in the weaned piglets (Fig. 5). ated with “lactose and galactose uptake and utilization” with The carbohydrate composition of the porcine diet lactose being the principal sugar in porcine milk (Fig. 5a). abruptly changes when the pigs are separated from the sow The relative abundance of genes associated with amino acid and complex plant-based feeds are introduced. The metabolism was also higher in weaned piglets than nursing Fig. 5 Comparison of the functional capacities of the gut microbiomes between Nursing and Weaned piglets associated with nutrition. Normalized abundance of the level 3 SEED subsystem classified reads associated with carbohydrate metabolism (a) and amino acid metabolism (b). The error bars show the calculated standard deviation of four replicates, and the [P < 0.001], [P < 0.01] and [P < 0.05] were indicated as [***], [**] and [*], respectively Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 7 of 9 piglets. Four of these pathways were significantly enriched understanding the microbial community structure and in weaned piglets including “histidine biosynthesis”, “argin- functional capacity of the microbiome during the weaning ine biosynthesis”, “glutamine, glutamate, aspartate and as- transition is substantial to pig production as it plays import- paragine biosynthesis” and “methionine biosynthesis” (P < ant roles in pig health and diseases. In this study, we found 0.05) (Fig. 5b). that there were significant enrichments of genes associated with bacterial heat shock responses in weaned pigs. The Discussion heat shock responses in bacteria are a result of a stress and We performed this study to better understand microbial are important for successfully adapting to changes in the succession and changes in functional capacity of the piglet physiological state, as well as to changes in the environment fecal microbiome during the weaning transition. One of of the bacterial habitat [32]. Heat shock responses involve the most striking observations in this study was the signifi- the induction of heat shock proteins (HSP) that are com- cant increase in the genus Prevotella after weaning. It has prised of a set of well-conserved proteins with molecular been reported that Prevotella is linked to the fermentation mass ranging from 27 to > 100 kDa, and are produced by of plant-derived non-starch polysaccharides to short-chain bacteria [33, 34]. HSP play a major role in the protection of fatty acids [24]. In humans, Prevotella spp. have also been cells by functioning as intra-cellular chaperones for other reported to produce enzymes, such as β-glucanase, man- proteins under different kinds of stressors [34]. Even though nase, and xylanase that can degrade polysaccharides in the exact mechanisms of the HSP are yet to be determined, plant cell wall [25]. The relative abundance of Lactobacillus our data suggest that bacterial heat shock response in also increased in weaned animals. Lactobacillus has been weaned piglets may play a role in mitigating the negative ef- recently identified as bacteria with the ability to consume fects associated with weaning. plant-derived monosaccharides and disaccharides [26]. The present study also showed a significantly higher The high abundance of Lactobacillus in the microbial abundance of functional gene groups associated with oxida- community of weaned piglets is consistent with other tive stress response in the bacterial metagenome of the studies on carbohydrate utilization in other mammalian weaned piglet. Oxidative stress is defined as a disturbance species [27]. Generally, Lactobacillus is recognized as a in the balance between the production of reactive oxygen carbohydrate-utilizing bacterium with numerous genes en- species (ROS) and antioxidant defenses [35]. The increase coding a wide range of functional capacities associated in production of ROS can cause damage to biological mole- with carbohydrate transport and utilization [28]. Our re- cules including DNA, protein and lipids and can even lead sults suggest that Lactobacillus mayalsoplayapivotal role to cell death [36]. A previous study indicated that weaning in the utilization of complex carbohydrates. Overall, the causes oxidative stress and the exposure of bacterial cells to present study reported higher abundances of Prevotella oxidative stress can have damaging effects on protein activ- and Lactobacillus in the weaned piglets that may allow ities and can contribute to death [37, 38]. As such, oxidative them to adapt to the dietary conditions after weaning. stress caused by weaning eventually induced enterocyte It was noteworthy that the nursing piglet microbiota had apoptosis and cell cycle arrest in the small intestine of significantly higher relative abundance of genus Bacteroides, post-weaning piglets [37]. A recent report on the impact of which are well-known bacteria that utilize milk oligosac- the gut microbiota on the development of metabolic dis- charides as carbon sources [7]. Our analysis of the whole eases revealed that Lactobacillus spp. have developed metagenome of nursing piglet microbiota showed signifi- defense mechanisms against oxidative stress [39]. The cant enrichments of genes associated with lactose and gal- higher proportion of sequences that mapped to oxidative actose uptake and utilization. In comparison to human stress genes in weaned piglets appear to be reflected in the breast milk, porcine milk is primarily composed of lactose increase of the relative abundance of Lactobacillus ob- (Lac), glucose (Glc), galactose (Gal), N-acetyl-glucosamine served in this study. However, potential roles of Lactobacil- (GlcNAc), fucose (Fuc), and sialic acids (NeuAc/ NeuGc) lus spp. to regulate oxidative stress in weaned piglets [29]. Milk oligosaccharides are composed of repeating units should be validated through evidence-based experiments. of lactose or N-acetyl-lactosamine that are usually bonded Amino acids play crucial physiological roles in young with sialic acid and fucose monosaccharides [30]. These piglets to support their maximum production performance complex milk oligosaccharides are not digested by the host [40]. In this study, SEED subsystems related to amino acid during the passage through the GIT suggesting that they metabolism were significantly elevated in the weaned pig- may play a role as natural prebiotics [31]. Our results sug- lets. These findings can be attributed to the increased use gest that both microbial composition and the metabolic of amino acids in feed for protein accretion in livestock functions of the nursing piglet microbiome is oriented to production. Although glucose is widely accepted as the pri- the utilization of milk oligosaccharides. mary nutrient for the maintenance and promotion of cell Weaning is a stressful event in a pig’s life and can contrib- function, it has been reported that glutamate, glutamine ute to intestinal and immune system dysfunctions [4]. Thus, and aspartate are the major contributors to the oxidative Guevarra et al. Journal of Animal Science and Biotechnology (2018) 9:54 Page 8 of 9 fuel for the intestine and cells of the immune system [41, Additional file 2: Figure S1. Extended error bar plot identifying the 42]. Moreover, the present study showed that arginine bio- significantly different taxa between nursing and weaned piglets at the phylum (a), family (b) and genus (c) levels. Corrected P values are shown at synthesis was significantly enriched in the microbiome of right. The differences in the microbial community structure were measured weaned piglets. In addition, a recent study showed that using a two-sided Welch’s t-test, and P < 0.05 was considered significant. arginine supplementation in weaned piglets has beneficial Figure S2. Comparison of the taxonomic profiles obtained using 16S rRNA gene and whole metagenome sequencing between nursing and weaned effects against oxidative stress in the jejunum through the piglets. Stacked bar plots show the relative abundance of bacteria at the a suppression of inflammatory cytokine expression [43]. phylum, b family and c genus levels. Figure S3. Heatmap of relative abun- While the small intestine is the major site for amino acid dance of SEED level 1 subsystems based on whole metagenome sequen- cing data. The e-value cutoff for metagenomics sequence matches to the absorption, the absorption of amino acids in the large − 5 SEED subsystem database was 1 × 10 with a minimum alignment length intestine is limited [44]. However, amino acids in the colon, of 15 amino acids. The two-way hierarchical cluster analysis was performed including lysine, arginine, glycine, leucine, valine and isoleu- using unweighted pair group method with arithmetic mean (UPGMA) method. The side colors in the heatmap depicts the clustering of the sub- cine can be used by the colonic bacteria to generate a com- systems based on the relative abundance. The yellow cluster indicate the plex mixture of metabolic products, such as short-chain SEED Subsystem with relative abundance above 7% while the green cluster fatty acids (SCFA), which are available energy source to the represent the SEED Subsystem with relative abundance below 5%. Figure S4. Differences in the relative abundance of level 1SEED subsystems that pigs [45]. Our results have provided us with new insights were mapped to “Carbohydrates”, “Amino Acids and Derivatives”, “Stress Re- into the functional aspect of the microbiome to help us bet- sponse” and “Virulence, Disease and Defense”.Corrected P-values are calcu- ter understand the interplay between amino acid metabol- lated using the Benjamini-Hochberg false discovery rate approach (P <0.05). Figure S5. Comparison functional categories assigned to a “Virulence, Dis- ism in bacteria and pig health in response to an abrupt ease and Defense” SEED subsystem level 2 and b “Resistance to Antibiotics” dietary change that occurs during the weaning transition. SEED subsystem level 3 between nursing and weaned piglets based on Nevertheless, further studies are required to elucidate the whole metagenome shotgun sequences analyzed using MG-RAST. The error bars show the calculated standard deviation of four replicates, and the [P < exact roles of the functional gene groups of the weaned pig- 0.001], [P < 0.01] and [P < 0.05] were indicated as [***], [**] and [*], respect- let gut microbiome associated with the amino acid metab- ively. (PPTX 911 kb) olism for the swine performance. Abbreviations 16S rRNA: 16S ribosomal ribonucleic acid; ANOSIM: Analysis of similarities; Conclusions DNA: Deoxyribonucleic acid; dNTPs: Deoxynucleotide triphosphates; We observed distinct microbial communities and func- GIT: Gastrointestinal tract; HSP: Heat shock protein; MGRAST: Metagenomics tional capacities of the piglet gut microbiome between rapid annotation using subsystem technology; NRC: National research council; OTU: Operational taxonomic unit; PCoA: Principal co-ordinates nursing and weaned piglets. The weaning process signifi- analysis; PCR: Polymerase chain reaction; PD: Phylogenetic diversity; cantly altered the composition and functional capacities of QIIME: Quantitative insights into microbial ecology; ROS: Reactive oxygen the gut microbiome. As such, our data suggest that the species; STAMP: Statistical analysis of metagenomic profiles early-life stressors caused by dietary change could be an im- Acknowledgements portant driver to lead to these microbiome shifts. Even The authors thank Mo Re Kim (Westborough High School, MA, USA) for the though further studies are required to elucidate the effect initial English editing. of microbiome shifts on piglet health, our data suggest that Funding the microbiome shift and changes in functional capacities This work was supported by the fund (Project No. PJ012615), Rural of the pig gut microbiome were oriented to deal with heat Development Administration, Republic of Korea. shock and oxidative stress during the weaning transition. Availability of data and materials Overall, our results suggest that piglets overcome stresses All raw 16S rRNA gene data used in this study were deposited in National Center caused by dietary change during the weaning transition for Biotechnology Information (NCBI) under Sequence Read Archive (SRA) through a gut microbiome shift, and these results accession number SRP133974, and the whole metagenome datasets generated forthisstudy areavailablein theMG-RAST server with theproject IDmgp80424. emphasize the importance of the early-life microbiota. We believe that our results provide us with substantial Authors’ contributions insights into the piglet gut microbiome that contributes to JHC, BNK, YHK, SW, MS, and HBK designed the research. SHH, JHC, BK, JS, JHL, MS, and HBK performed research and generated data. RBG, SHH, JHC, the growth of the animal, and help us to better understand MS and HBK analyzed the data and RBG, SHH, JHC, REI, MS and HBK wrote the important roles of the essential gut microbiome for the manuscript. All authors read and approved the final manuscript. later studies aiming to develop pig gut modulators, such Ethics approval and consent to participate as feed additives. Not applicable. Additional files Competing interests The authors declare that they have no competing interests. Additional file 1: Table S1. Number of 16S rRNA gene sequence reads Author details of nursing and weaned piglet fecal microbiota before and after quality Department of Animal Resources Science, Dankook University, Cheonan, control. Table S2. Summary of whole metagenome sequence data South Korea. Division of Food and Animal Sciences, Chungbuk National before and after quality control and annotation. (DOCX 18 kb) University, Cheongju, South Korea. Abbvie Bioresearch Center, Abbvie, Guevarra et al. 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Published: Jul 30, 2018

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