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Dietary Phytase- and Lactic Acid-Treated Cereals Caused Greater Taxonomic Adaptations than Functional Adaptations in the Cecal Metagenome of Growing Pigs.

ABSTRACT: Phosphorus (P) is an essential nutrient for the gut bacteria and the host. Nevertheless, little information exists that indicates to what extent an improved level of P availability in the small intestine leads to functional adaptations in bacterial metabolic pathways in the large intestine. Therefore, we investigated the changes in the taxonomic and functional bacterial metagenome in cecal digesta of growing pigs fed diets containing phytase and/or cereals treated with 2.5% lactic acid (LA) for 19 days (n = 8/diet) using shotgun metagenome sequencing. The phytase supplementation resulted in strikingly distinct bacterial communities, affecting almost all major bacterial families, whereas functional changes were less dramatic among the feeding groups. While phytase treatment decreased predominant Prevotellaceae levels, it seemed that Clostridiaceae, Ruminococcaceae, and Lachnospiraceae filled the opening metabolic niches (P < 0.05). The LA-treated cereals mediated reduced levels of Bacteroidaceae and increased levels of Veillonellaceae, but those results were mainly seen when the cereals were fed as a single treatment (P < 0.05). In association with the taxonomic alterations, phytase caused changes within the major functional pathways corresponding to amino acid metabolism; translation; membrane transport; folding, sorting, and degradation; and energy metabolism, whereas the LA treatment of cereals resulted in decreased enzymatic capacities within the carbohydrate metabolism and energy metabolism pathways (P < 0.05). Metabolic dependencies corresponding to the starch and sucrose metabolism, glycolysis/gluconeogenesis, and citrate cycle pathways were indicated by diet-associated changes in enzymatic capacities related to short-chain fatty acid, methane, vitamin, and bacterial antigen synthesis. Accordingly, the present results support the idea of the importance of the availability of intestinal P for bacterial metabolism. However, the functional profiles were less different than the taxonomic profiles among the dietary treatment results, indicating a certain degree of metabolic plasticity within the cecal metagenome.IMPORTANCE Dietary strategies (e.g., phytase supplementation and lactic acid [LA] treatment of cereals) used to improve the availability of phytate-phosphorus (P) from pig feed reduce the amount of P flowing into the large intestine, whereas LA treatment-induced changes in nutrient fractions alter the substrate being available to the microbiota. In ruminants, lower intestinal P availability compromises the fibrolytic activity of the microbiome. Here, we report that the functional capacities were less dramatically affected than the taxonomic composition by phytase-supplemented and LA-treated cereals. The bacterial community appeared to be partly capable of functionally compensating for the altered flow of P by replacing taxa with higher P needs by those with lower P needs. Therefore, by acting as mucosal immune stimulants, alterations in microbiota-associated molecular patterns (MAMPs) due to the taxonomic shifts may play a greater role for host physiology and health than functional differences caused by differing intestinal P availabilities, which merits further research.

SUBMITTER: Klinsoda J

PROVIDER: S-EPMC7755247 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Besides the major nutrients, phosphorus (P) is an essential mineral for both the host animal and the porcine gut microbiota. Different strategies including phytase supplementation and more recently lactic acid (LA) are used to enhance the P availability from cereals in pig diets; however, their impact on the gut microbiota has been rarely related to fecal shedding of opportunistic pathogens. The present study investigated the effect of phytase supplementation and the treatment of dietary cereals with 2.5% LA on the fecal microbiome composition of metabolic active bacteria and expression of virulence factor genes of enterotoxigenic Escherichia coli and Clostridium perfringens in growing pigs. Phytase supplementation reduced the fecal abundance of the most abundant Lactobacillaceae family, whereas the LA-treatment of cereals had a stronger impact on the bacterial community, reducing amylolytic, pullulanolytic and hemicellulolytic Lactobacillaceae, Lachnospiraceae and Ruminococcaceae as well as the fecal bacterial species richness (Chao1) and diversity (Shannon index). Mainly the family Clostridiaceae benefited from the decline in the aforementioned families, being enriched by both dietary treatments. Multigroup data integration using sparse partial least squares-discriminant analysis showed that among the most discriminative operational taxonomic units (OTU) especially two unclassified Clostridiaceae-OTUs, one Prevotella copri-like OTU and one OTU within the vadinCA11 group were associated with calcium and P levels but were negatively linked with complex carbohydrates in feces. Heat-stable toxin A (Sta) of enterotoxigenic E. coli and Stx2e of Shiga-toxin producing E. coli were expressed in feces but were similar among feeding groups. Without modifying the total bacterial gene copies and virulence factor expression of E. coli, both dietary phytase supplementation and LA-treatment of cereals drastically altered the bacterial community composition in pig feces. Results thereby allowed for the characterization of bacterial nutrient dependencies, indicating a link between fecal P availability, complex carbohydrate composition and alterations in the predominant genera.

Project description:The gut-lymph node axis is a critical player in the symbiotic relationship between gut microbiota and the host. However, little is known about the impact of diet-related bacterial shifts in the gut lumen on bacterial translocation into lymph nodes. Here, we (i) characterized changes in the viable microbiota composition along the ileal digesta-mucosa-lymph node axis and (ii) examined the effect of dietary phytase supplementation and lactic acid (LA) soaking of cereals on the bacterial taxonomy along this axis, together with their effect on the mucosal expression of innate immune and barrier function genes in pigs (n?=?8/diet). After 18?days on diets, ileal digesta, mucosa, and ileocecal lymph nodes (ICLNs) were collected for RNA isolation and 16S rRNA-based high-resolution community profiling. Bacterial communities were dominated by Lactobacillaceae and Clostridiaceae, with clearly distinguishable profiles at the three sampling sites. Specific bacterial subsampling was indicated by enrichment of the ICLNs with Lactobacillaceae, Lachnospiraceae, Veillonellaceae, and Methanobacteriaceae and less Clostridiaceae, Pasteurellaceae, Helicobacteraceae, and Enterobacteriaceae compared to that of the mucosa. LA treatment of cereals reduced proteolytic taxa in the lumen, including pathobionts like Helicobacteraceae, Campylobacteraceae, and Fusobacteriaceae When combined, phytase- and LA-treated cereals largely increased species richness, while the single treatments reduced Actinobacteria and Bacteroidetes in ICLNs and increased mucosal MUC2 expression. In contrast, phytase reduced mucosal CDH1 expression, indicating altered barrier function with potential effects on bacterial translocation. Overall, both treatments, although often differently, changed the viable microbiome along the digesta-mucosa-lymph node axis in the ileum, probably due to altered substrate availability and microbial-host interactions.IMPORTANCE A host's diet largely determines the gut microbial composition and therefore may influence bacterial translocation into ICLNs. Due to its importance for cell metabolism, the intestinal phosphorus availability, which was modified here by phytase and LA treatment of cereals, affects the intestinal microbiota. Previous studies mainly focused on bacteria in the lumen. The novelty of this work resides mainly in that we report diet-microbe effects along the digesta-mucosa-ICLN axis and linked those effects to mucosal expression of barrier function genes as crucial components for host health. Lymph nodes can serve as reservoir of pathobionts; therefore, present diet-microbiome-host interactions have implications for food safety.

Project description:This study investigated the effects of supplementing phytase and non-starch polysaccharide-degrading enzymes (NSPases) to corn-soybean meal-based diet on the growth performance, trace element deposition, and intestinal health of growing-finishing pigs. Fifty pigs were randomly assigned into the control (basal diet), phytase (basal diet + 100 g/t of phytase), β-mannanase (basal diet + 40 g/t of β-mannanase), β-glucanase (basal diet + 100 g/t of β-glucanase), and xylanase (basal diet + 100 g/t of xylanase) groups. The results show that the supplementation of phytase and NSPases had no impacts (p > 0.05) on the growth performance of pigs. Compared with the control group, pigs fed with xylanase had higher (p < 0.05) Zn concentrations in the ileum and muscle and those fed with phytase had higher (p < 0.05) Zn concentrations in the ileum. Phytase and xylanase supplementation decreased (p < 0.05) fecal Zn concentrations in pigs compared with the control group (p < 0.05). In addition, phytase, β-mannanase, β-glucanase, and xylanase supplementation up-regulated (p < 0.05) the FPN1 expression, whereas xylanase up-regulated (p < 0.05) the Znt1 expression in the duodenum of pigs compared with the control group. Moreover, phytase, β-glucanase, and xylanase supplementation up-regulated (p < 0.05) the jejunal Znt1 expression compared with the control group. The intestinal morphology results show that the phytase, β-mannanase, and xylanase groups had increased villus heights (VHs), an increased villus height-crypt depth ratio (VH:CD), and decreased crypt depths (CDs) in the duodenum, whereas phytase, β-mannanase, β-glucanase, and xylanase groups had decreased VH and VH:CD, and increased CD in the jejunum compared with the control group (p < 0.05). Pigs fed with exogenous enzymes had decreased bacterial diversity in the cecum. The dietary supplementation of NSPases increased the relative abundance of Firmicutes and decreased spirochaetes (p < 0.05). Compared with the control group, dietary NSPase treatment decreased (p < 0.05) the opportunistic pathogens, such as Treponema_2 and Eubacterium_ruminantium. Moreover, the relative abundances of Lachnospiraceae_XPB1014 and Lachnospiraceae were enriched in the β-glucanase and β-mannanase groups (p < 0.05), respectively. In conclusion, phytase and xylanase supplementation may promote zinc deposition in pigs. Additionally, the supplementation of NSPases may improve the gut health of pigs by modulating the intestinal morphology and microbiota.

Project description:Alpha-ketoglutarate (AKG), a critical molecule in the tricarboxylic acid cycle, is beneficial to intestinal functions. However, its influence on intestinal microbiota and metabolism is not fully understood. We investigated the effects of a low-protein (LP) diet supplemented with AKG on cecal microbial communities and the parameters of microbial metabolism in growing pigs. Twenty-seven young pigs (Large White × Landrace) with an average initial body weight of 11.96 ± 0.18 kg were randomly allotted into three groups (n = 9): a normal protein (NP) diet containing 20% crude protein (CP); LP diet formulated with 17% CP (LP diet); or LP diet supplemented with 10 g kg-1 of AKG (ALP diet). After a 35-day trial period, the digesta of the cecum were collected to analyze the concentrations of ammonia and short-chain fatty acids (SCFAs). We also performed a microbial analysis. Although no significant differences were found in performance among the diet groups, pigs fed the ALP diet had greater average daily gain (ADG) when compared with those in the LP group. Experimental diet did not affect cecal bacterial richness or diversity, as determined by Chao1 and ACE species richness measures and Shannon and Simpson indices, respectively. The predominant phyla Firmicutes, Bacteroidetes, and Proteobacteria increased in relative abundances in the cecum of pigs fed ALP diet. At the genus level, compared to the LP diet, the ALP diet significantly increased the abundances of Lachnospiraceae UCG-005, Lachnospiraceae NK4A136 group, Phascolarctobacterium and Parabacteroides, while decreased Vibrio and Maritalea. Pigs fed the ALP diet increased Oribacterium and Lachnoclostridium when compared with the NP diet. Non-metric multidimensional scaling analysis revealed that the distribution of microbiota at each group was distinctly clustered separately along principal coordinate. In addition, quantitative PCR revealed that the ALP diet was also associated with increases in the amounts of Bacteroides, Bifidobacterium, and Lactobacillus, but a decrease in the level of Escherichia coli. Compared with the NP diet, the ALP diet enhanced the concentrations of valerate and propionate. This ALP diet also increased the concentrations of valerate and isobutyrate when compared with the LP diet. Moreover, the ALP diet was linked with a significant decline in the concentration of ammonia in the cecum. These results indicate that a LP diet supplemented with AKG can alter the balance in microbial communities, increasing the population of SCFA-producing bacteria and the amounts of Bacteroides and Bifidobacterium, while reducing the counts of Escherichia coli and the amount of ammonia in the cecum.

Project description:BACKGROUND: Understanding the taxonomic composition of a sample, whether from patient, food or environment, is important to several types of studies including pathogen diagnostics, epidemiological studies, biodiversity analysis and food quality regulation. With the decreasing costs of sequencing, metagenomic data is quickly becoming the preferred typed of data for such analysis. RESULTS: Rapidly defining the taxonomic composition (both taxonomic profile and relative frequency) in a metagenomic sequence dataset is challenging because the task of mapping millions of sequence reads from a metagenomic study to a non-redundant nucleotide database such as the NCBI non-redundant nucleotide database (nt) is a computationally intensive task. We have developed a robust subsampling-based algorithm implemented in a tool called CensuScope meant to take a 'sneak peak' into the population distribution and estimate taxonomic composition as if a census was taken of the metagenomic landscape. CensuScope is a rapid and accurate metagenome taxonomic profiling tool that randomly extracts a small number of reads (based on user input) and maps them to NCBI's nt database. This process is repeated multiple times to ascertain the taxonomic composition that is found in majority of the iterations, thereby providing a robust estimate of the population and measures of the accuracy for the results. CONCLUSION: CensuScope can be run on a laptop or on a high-performance computer. Based on our analysis we are able to provide some recommendations in terms of the number of sequence reads to analyze and the number of iterations to use. For example, to quantify taxonomic groups present in the sample at a level of 1% or higher a subsampling size of 250 random reads with 50 iterations yields a statistical power of >99%. Windows and UNIX versions of CensuScope are available for download at https://hive.biochemistry.gwu.edu/dna.cgi?cmd=censuscope. CensuScope is also available through the High-performance Integrated Virtual Environment (HIVE) and can be used in conjunction with other HIVE analysis and visualization tools.

Project description:BackgroundStudies of shifts in microbial community composition has many applications. For studies at species or subspecies levels, the 16S amplicon sequencing lacks resolution and is often replaced by full shotgun sequencing. Due to higher costs, this restricts the number of samples sequenced. As an alternative to a full shotgun sequencing we have investigated the use of Reduced Metagenome Sequencing (RMS) to estimate the composition of a microbial community. This involves the use of double-digested restriction-associated DNA sequencing, which means only a smaller fraction of the genomes are sequenced. The read sets obtained by this approach have properties different from both amplicon and shotgun data, and analysis pipelines for both can either not be used at all or not explore the full potential of RMS data.ResultsWe suggest a procedure for analyzing such data, based on fragment clustering and the use of a constrained ordinary least square de-convolution for estimating the relative abundance of all community members. Mock community datasets show the potential to clearly separate strains even when the 16S is 100% identical, and genome-wide differences is < 0.02, indicating RMS has a very high resolution. From a simulation study, we compare RMS to shotgun sequencing and show that we get improved abundance estimates when the community has many very closely related genomes. From a real dataset of infant guts, we show that RMS is capable of detecting a strain diversity gradient for Escherichia coli across time.ConclusionWe find that RMS is a good alternative to either metabarcoding or shotgun sequencing when it comes to resolving microbial communities at the strain level. Like shotgun metagenomics, it requires a good database of reference genomes and is well suited for studies of the human gut or other communities where many reference genomes exist. A data analysis pipeline is offered, as an R package at https://github.com/larssnip/microRMS . Video abstract.

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Dietary Phytase- and Lactic Acid-Treated Cereals Caused Greater Taxonomic Adaptations than Functional Adaptations in the Cecal Metagenome of Growing Pigs.

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