Pectin supplementation accelerates post-antibiotic gut microbiome reconstitution orchestrated with reduced gut redox potential.

Antibiotic-induced gut dysbiosis (AID) presents a big challenge to host health, and the recovery from this dysbiosis is often slow and incomplete. AID is typically characterized by elevation in redox potential, Enterobacteriaceae load, and aerobic metabolism. In our previous study, a pectin-enriched diet was demonstrated to decrease fecal redox potential and modulate the gut microbiome. Therefore, we propose that pectin supplementation may modulate gut redox potential and favor post-antibiotic gut microbiome reconstitution from dysbiosis. In the present study, rats with AIDwere used to investigate the effects of pectin supplementation on post-antibiotic gut microbiome reconstitution from dysbiosis. The results showed that pectin supplementation accelerated post-antibiotic reconstitution of gut microbiome composition and function and led to enhancement of anabolic reductive metabolism and weakening of catabolic oxidative pathways. These results were corroborated by the measurement of redox potential, findings suggesting that pectin favors post-antibiotic recovery from dysbiosis. Pectin-modulated fecal microbiota transplantation accelerated the decrease in antibiotics-elevated redox potential and Enterobacteriaceae load similarly to pectin supplementation. Moreover, both pectin supplementation and Pectin-modulated fecal microbiota transplantation enriched anaerobic members, primarily from Lachnospiraceae orchestration with enhancement of microbial reductive metabolism in post-antibiotic rats. These findings suggested that pectin supplementation accelerated post-antibiotic gut microbiome reconstitution orchestrated with reduced gut redox potential and that the effect of pectin on redox potential was mediated by remodeling of the intestinal microbiota.

Daily fluctuation of Lactobacillus species and their antibiotic resistome in the colon of growing pigs.

There are various types of bacteria inhabiting the intestine that help maintain the balance of the intestinal microbiota. Lactobacillus is one of the important beneficial bacteria and is widely used as a food starter and probiotic. In this study, we investigated the daily fluctuation of the colonic Lactobacillus species and their distribution of antibiotic resistance genes (ARGs) as well as antibiotic susceptibility in pigs. Metagenomic analysis revealed that genus Lactobacillus was one of the most dominant genera in the colon of growing pigs. Rhythmicity analysis revealed that 84 out of 285 Lactobacillus species exhibited rhythmic patterns. Lactobacillus johnsonii and Lactobacillus reuteri were the two most abundant lactobacilli with circadian oscillation, which increased during the day and decreased at night. The profile of the antibiotic resistome was modified over time within 24-h period. Elfamycin resistance genes were the most enriched class found in Lactobacillus. Furthermore, the seven strains of Lactobacillus isolated from the pig intestine mainly exhibited resistance to gentamicin, erythromycin, and lincomycin. The whole genome annotation of four Lactobacillus strains indicated the presence of multiple ARGs, including elfamycin resistance genes, however, the most abundant ARG was optrA in genome of four strains. These results indicate the presence of various Lactobacillus species harboring a large number of ARGs in the swine intestine. This implies that when using animal-derived lactobacilli, it is essential to assess antibiotic resistance to prevent further transmission between animals and the environment.

The Fecal Redox Potential in Healthy and Diarrheal Pigs and Their Correlation with Microbiota.

The redox potential plays a critical role in sustaining the stability of gut microbiota. This study measured the fecal redox potential in healthy and diarrheal pigs using direct and dilution methods and investigated their correlation with microbiota. The results showed that the fluctuations in the redox potential of healthy pig feces were consistent using two different methods and the two methods are equivalent based on an equivalence test. The redox potential was positively correlated with the number of fungi and negatively related to the total bacteria. The relative or absolute abundances of many bacteria at the phyla and genus levels were associated with redox potential. In diarrheal pigs, the potentiometric trends of the two methods demonstrated an opposing pattern and the correlation with total bacteria was reversed. Precipitously elevated redox potential was detected post-diarrhea using dilution methods. The absolute abundance of Escherichia-Shigella and Fuurnierella was positively correlated with redox potential, while both relative and absolute abundances of Limosilactobacillus were positively correlated. These results suggest that both methods are suitable for detecting gut redox potential in healthy pigs, while the dilution method is more suitable for diarrheal pigs. The findings on the correlation of Limosilactobacillus, Prevotella, and Escherichia-Shigella with redox potential offer novel insights for targeted modulation of intestinal health.

Daily fluctuation of colonic microbiome in response to nutrient substrates in a pig model.

Studies on rodents indicate the daily oscillations of the gut microbiota have biological implications for host. However, the responses of fluctuating gut microbes to the dynamic nutrient substrates are not fully clear. In the study, we found that the feed intake, nutrient substrates, microbiota and metabolites in the colon underwent asynchronous oscillation within a day. Short-chain fatty acids (SCFAs) including acetate, propionate, butyrate and valerate peaked during T24 ~ T27 (Timepoint 24, 12:00 pm, T27, 03:00 am) whereas branched SCFAs isobutyrate and isovalerate peaked during T09 ~ T12. Further extended local similarity analysis (eLSA) revealed that the fluctuation of feed intake dynamically correlated with the colonic carbon substrates which further influenced the oscillation of sugar metabolites and acetate, propionate, butyrate and valerate with a certain time shift. The relative abundance of primary degrader Ruminococcaceae taxa was highly related to the dynamics of the carbon substrates whereas the fluctuations of secondary degraders Lactobacillaceae and Streptococcaceae taxa were highly correlated with the sugar metabolites. Meanwhile, colonic nitrogen substrates were correlated with branched amino acids and the branched SCFAs. Furthermore, we validated the evolution of gut microbes under different carbohydrate and protein combinations by using an in vitro fermentation experiment. The study pictured the dynamics of the micro-ecological environment within a day which highlights the implications of the temporal dimension in studies related to the gut microbiota. Feed intake, more precisely substrate intake, is highly correlated with microbial evolution, which makes it possible to develop chronotherapies targeting the gut microbiota through nutrition intervention.

Dynamic analysis of metabolomics reveals the potential associations between colonic peptides and serum appetite-related hormones.

Gut signals, including hormones and metabolites are crucial zeitgebers that regulate the circadian rhythm of host metabolism, but the potential links have been explored more in rodents. Herein, we performed an hour-scale metabolomics analysis of serum and colonic digesta to characterize the circadian rhythmic metabolic patterns using a pig model under ad libitum feeding conditions. Importantly, our findings identified potential associations between colonic and body metabolism, revealing the potential relationships between colonic peptides and host appetite regulation. Concretely, amino acids accounted for the highest proportion in rhythmic serum metabolites, whereas lipids accounted for the highest proportion in rhythmic colonic metabolites. The diurnal difference analysis revealed that the levels of most amino acids and peptides were higher in the light phase, while the levels of most lipids were higher in the dark phase. And more correlations were be checked between serum amino acids, lipids, peptides and colonic metabolites in the light and more correlations were be checked between serum carbohydrates, cofactors and vitamins, energy, nucleotides, xenobiotics and colonic metabolites in the dark. Interestingly, peptides oscillated to a similar extent in serum and colonic digesta. Of note, colonic peptides composed of valine, proline and leucine were checked in positive associations to glucagon-like peptide-1 (GLP-1) in serum. And these peptides were positive with the genera Butyricicoccus, Streptococcus, Clostridioides, Bariatricus and Coriobacteriia_norank, and negative with Prevotella, and showed the potential relationships with colonic microbial biosynthesis of amino acids. Collectively, we mapped the rhythmic profiling on pig serum and colonic metabolites and revealed the relationships between host and gut metabolism. However, the underlying regulatory mechanisms remains to be further investigated.

Short-Term Supplementation of Pectin Alters Substrate Dynamics and Modulates Microbial Carbohydrate Metabolism in the Gut of a Pig Model.

The interaction of pectin and gut microbiota plays an important role in maintaining animal and human health, but this interaction is not fully understood. Here, the impact of pectin supplementation on substrate dynamics and gut microbiota (in the terminal ileum and feces) was integrally investigated in a fistula pig model. Our results showed that a pectin-supplemented diet (PEC) decreased the concentrations of starch, cellulose, and butyrate in feces but not in the terminal ileum. Metagenomic sequencing revealed that PEC had a low impact on the ileal microbiota but significantly increased plant polysaccharide-degrading genera (e.g., Bacteroides, Alistipes, and Treponema) in feces. Additionally, CAZyme profiling indicated that PEC reduced GH68 and GH8 for oligosaccharide degradation in the ileal microbiome, while it enriched GH5, GH57, and GH106 for degradation of carbohydrate substrates in feces. Metabolomic analysis confirmed that PEC increased metabolites involved in carbohydrate metabolism including glucuronate and aconitate. Collectively, pectin could promote complex carbohydrate substrate degradation in the hindgut via modulating the gut microbiota.

Time-restricted feeding affects colonic nutrient substrates and modulates the diurnal fluctuation of microbiota in pigs.

Introduction: Studies demonstrate that time-restricted feeding (TRF) can regulate gut microbiota composition. However, it is unclear whether TRF could affect the gut microbial rhythmicity in growing pigs. Therefore, the present study aimed to explore the effects of TRF on the dynamic fluctuation of the gut microbiota. Methods: A total of 10 healthy growing pigs equipped with T cannula were employed. Pigs were randomly allotted to the free access (FA) and the TRF groups with 5 replicates (1 pig/replicates). Pigs in the FA group were fed free access during the whole experimental period, whereas pigs in the TRF group were fed free access three times per day within limited times (7:00-8:00, 12:00-13:00, 17:00-18:00). The experiment lasted for 15 days, at 06:00 a.m. of the day 16, colonic digesta were collected at a 6-h interval for consecutive 24 h marked as T06 (06:00), T12 (12:00), T18 (18:00), T24 (24:00), T30 (06:00), respectively. Results: Results showed that TRF altered the distribution of feed intake without changing the total feed intake within a day (p = 0.870). TRF decreased the overall concentration of colonic cellulose and altered their oscillating patterns. All alpha-diversity indexes of different time points showed significant differences regardless of feeding pattern with a trough at T18 or T24. TRF shifted the trough of the alpha-diversity index Simpson and Invsimpson. TRF lost the rhythmicity of Prevotellaceae, Ruminococcaceae, Bacteroidales_S24-7_group, and Peptococcaceae and gained the rhythmicity of Pasteurellaceae, Clostridiaceae_1, Veillonellaceae, and Peptostreptococcaceae. Also, TRF altered the interaction pattern by increasing the microbes involved in the co-occurrence network and their crosstalk, especially at T24. Interestingly, the microbial variation at T24 could largely explained by colonic substrates starch (R2 = 0.369; p = 0.001), cellulose (R2 = 0.235; p = 0.009) and NH4-N (R2 = 0.489; p = 0.001). Conclusion: In conclusion, TRF has changed the concentrates of cellulose and the relative abundance of specific microbes and certain microbial metabolites. In addition, TRF has more powerful effects on the fluctuation modes of these nutrient substrates, microbes, and metabolites by shifting their peaks or troughs. This knowledge facilitates the development of precision regulation targeting gut microbial rhythmicity.

The diurnal fluctuation of colonic antibiotic resistome is correlated with nutrient substrates in a pig model.

The increasing prevalence of antimicrobial resistance (AMR) poses a significant threat to public health, and the gut microbiota of livestock (e.g., pigs) are considered a crucial reservoir of antibiotic resistance genes (ARGs), contributing to the long-term persistence of AMR. However, there is still a lack of relevant research on the composition and diurnal fluctuation of ARGs, and their correlation with nutrient substrates in the gut of pigs. To address this knowledge gap, we characterized the antibiotic resistome structure, and circadian oscillations in 45 colonic metagenomically sequenced samples, covering 9-time points within 24 h, from growing pigs. We identified 227 unique types of ARGs, which belonged to 35 drug resistance classes. Tetracycline resistance and antibiotic target protection were the most enriched class and mechanism of drug resistance in colon samples, respectively. The relative abundance of ARGs fluctuated over time within 24 h, with the total abundance peaking at T21 (sampling time at 21:00 p.m.) and the total numbers reaching the peak at T15. A total of 70 core ARGs were identified, which contributed to 99 % of all ARGs. Rhythmicity analysis revealed that 50 out of 227 ARGs and 15 of 49 mobile genetic elements (MGEs) exhibited rhythmic patterns. TetW was the most abundant ARG with circadian rhythm frequently found in Limosilactobacillus reuteri. The concentration of ammonia nitrogen in the colon was significantly correlated with the host genera of rhythmic ARGs. Partial least squares path modeling (PLS-PM) analysis indicated that rhythmic ARGs were significantly correlated with bacterial community, MGEs, and colonic ammonia nitrogen. This study provides new insight into the diurnal fluctuation of ARG profiles in the colon of growing pigs, which was likely driven by the dynamic change of the availability of colonic nutrients substrates.

Reduction of Redox Potential Exerts a Key Role in Modulating Gut Microbial Taxa and Function by Dietary Supplementation of Pectin in a Pig Model.

Pectin exists in a vast range of plants and has a long history of acting as a functional food additive with potential prebiotic effects on intestinal health. However, knowledge of how pectin regulates gut microbial communities is still insufficient and limited. Here, metatranscriptome sequencing revealed that a pectin-enriched diet (PEC) decreased the abundances of fungal keystone taxa (e.g., amino acid-producing Kazachstania spp.) and their genes involved in oxidative phosphorylation, while it increased the abundance of sulfate-reducing Desulfovibrio spp., and methane-producing Methanobrevibacter spp. in colon microbiomes. Furthermore, we first confirmed that PEC decreased fecal redox potential in a fistula pig model, which could be supported by the enrichment of antioxidants (e.g., inosine) in feces. Fecal metagenome analysis disclosed that certain microbial taxa promoted inosine biosynthesis from pectin degradation, including Prevotella, which plays an essential role in pectin biodegradation. Overall, these results demonstrate that pectin decreases the redox potential in pig hindgut to modulate microbial composition and functions, and specific microorganisms generate reducing agents in the course of pectin degradation to decrease redox potential of microbial ecosystem. IMPORTANCE Collective studies indicate that pectin degradation promotes extensive microorganisms that can be involved in pectin degradation directly or indirectly, or benefit from the altered physiological conditions caused by pectin ingestions. Our study focuses on effects of pectin on gut microbial taxa and functions, as well as its interactions with altered environmental features. Our results demonstrate pectin-induced proreducing shifts on colon microbial taxa and functions, and first confirm that pectin decreases hindgut redox potential, which is an important environmental feature that can modulate microbial communities. These results infer that there is bidirectional regulation between microbiota and redox potential during pectin degradation. In general, this investigation proposes new insights into the pectin-modulating gut microbial ecosystem and also provides new perspectives for targeting modulation of gut microbiota.

Metatranscriptomic analysis of colonic microbiota's functional response to different dietary fibers in growing pigs.

BACKGROUND: Dietary fibers are widely considered to be beneficial to health as they produce nutrients through gut microbial fermentation while facilitating weight management and boosting gut health. To date, the gene expression profiles of the carbohydrate active enzymes (CAZymes) that respond to different types of fibers (raw potato starch, RPS; inulin, INU; pectin, PEC) in the gut microbes of pigs are not well understood. Therefore, we investigated the functional response of colonic microbiota to different dietary fibers in pigs through metatranscriptomic analysis. RESULTS: The results showed that the microbial composition and CAZyme structure of the three experimental groups changed significantly compared with the control group (CON). Based on a comparative analysis with the control diet, RPS increased the abundance of Parabacteroides, Ruminococcus, Faecalibacterium and Alloprevotella but decreased Sutterella; INU increased the relative abundance of Fusobacterium and Rhodococcus but decreased Bacillus; and PEC increased the relative abundance of the Streptococcus and Bacteroidetes groups but decreased Clostridium, Clostridioides, Intestinibacter, Gemmiger, Muribaculum and Vibrio. The gene expression of CAZymes GH8, GH14, GH24, GH38, GT14, GT31, GT77 and GT91 downregulated but that of GH77, GH97, GT3, GT10 and GT27 upregulated in the RPS diet group; the gene expression of AA4, AA7, GH14, GH15, GH24, GH26, GH27, GH38, GH101, GT26, GT27 and GT38 downregulated in the INU group; and the gene expression of PL4, AA1, GT32, GH18, GH37, GH101 and GH112 downregulated but that of CE14, AA3, AA12, GH5, GH102 and GH103 upregulated in the PEC group. Compared with the RPS and INU groups, the composition of colonic microbiota in the PEC group exhibited more diverse changes with the variation of CAZymes and Streptococcus as the main contributor to CBM61, which greatly promoted the digestion of pectin. CONCLUSION: The results of this exploratory study provided a comprehensive overview of the effects of different fibers on nutrient digestibility, gut microbiota and CAZymes in pig colon, which will furnish new insights into the impacts of the use of dietary fibers on animal and human health.

Transcriptomic Responses Induced in Muscle and Adipose Tissues of Growing Pigs by Intravenous Infusion of Sodium Butyrate.

Butyrate has a central function in the regulation of energy metabolism as a metabolite of bacterial fermentation. This study evaluated the effects of intravenous sodium butyrate (SB) administration on the transcriptome of muscle and adipose tissue of pigs. Twelve crossbred barrows (Duroc × Landrace × Large White) were fitted with a medical polyethylene cannula via the internal jugular vein and were daily infused with 10 mL SB (200 mmol/L) or the same volume of physiological saline. Muscle transcriptome showed 11 DEGs related to carbohydrate metabolism, 28 DEGs related to lipid metabolism, and 10 DEGs related to amino acid metabolism. Among these, carbohydrate catabolic process-related genes (PPP1R3B, PRPS2, ALDOC), fatty acid synthase (FASN), and lipolysis-related genes (PLIN1) were upregulated, while the carbohydrate biosynthetic process-related genes (PCK1) and most amino acid metabolism-related genes were downregulated. Adipose transcriptome showed 12 DEGs related to carbohydrate metabolism, 27 DEGs related to lipid metabolism, and 10 DEGs related to amino acid metabolism. Among these, carbohydrate metabolism-related genes (IGF1, LEP, SLC2A4) and lipolysis-related genes (LPL) were upregulated, while lipolysis-related genes (ANGPTL4) and most amino acid metabolism-related genes were downregulated. The results suggest that short-term intravenous SB infusion could modulate the muscle and adipose tissue metabolism at the transcriptional level by decreasing amino acid metabolism pathways. Additionally, intravenous SB increased the glucose catabolism in muscle tissue and decreased the glucose utilization in adipose tissue. Intravenous SB increased the fatty acid synthesis, decreased the lipolysis in muscle tissue, and increased the lipolysis in adipose tissue. This suggests that systemic butyrate may display discriminative metabolic regulation in different tissues of barrows.

Swine gut microbiota and its interaction with host nutrient metabolism.

Gut microbiota is generally recognized to play a crucial role in maintaining host health and metabolism. The correlation among gut microbiota, glycolipid metabolism, and metabolic diseases has been well reviewed in humans. However, the interplay between gut microbiota and host metabolism in swine remains incompletely understood. Given the limitation in conducting human experiments and the high similarity between swine and humans in terms of anatomy, physiology, polyphagy, habits, and metabolism and in terms of the composition of gut microbiota, there is a pressing need to summarize the knowledge gained regarding swine gut microbiota, its interplay with host metabolism, and the underlying mechanisms. This review aimed to outline the bidirectional regulation between gut microbiota and nutrient metabolism in swine and to emphasize the action mechanisms underlying the complex microbiome-host crosstalk via the gut microbiota-gut-brain axis. Moreover, it highlights the new advances in knowledge of the diurnal rhythmicity of gut microbiota. A better understanding of these aspects can not only shed light on healthy and efficient pork production but also promote our knowledge on the associations between gut microbiota and the microbiome-host crosstalk mechanism. More importantly, knowledge on microbiota, host health and metabolism facilitates the development of a precise intervention therapy targeting the gut microbiota.

Effects of Early Intervention with Antibiotics and Maternal Fecal Microbiota on Transcriptomic Profiling Ileal Mucusa in Neonatal Pigs.

This study aimed to investigate the effects of early intervention with antibiotics and maternal fecal microbiota on ileal morphology and barrier function, and transcriptomic profiling in neonatal piglets. Piglets in the amoxicillin (AM), fecal microbiota transplantation (FMT), and control (CO) groups were orally administrated with amoxicillin solution (6.94 mg/mL), maternal fecal microbiota suspension [>109 colony forming unit (CFU)/mL], and physiological saline, respectively. Compared with the CO group, early intervention with AM or FMT significantly decreased ileal crypt depth on day 7 and altered gene expression profiles in ileum on days 7 and 21, and especially promoted the expression of chemokines (CCL5, CXCL9, and CXCL11) involved in the toll-like receptor signaling pathway on day 21. FMT changed major immune activities from B cell immunity on day 7 to T cell immunity on day 21 in the ileum. On the other hand, both AM and FMT predominantly downregulated the gene expression of toll-like receptor 4 (TLR4). In summary, both early interventions modulated intestinal barrier function and immune system in the ileum with a low impact on ileal morphology and development.

BRAFV600E mutation analysis in fine-needle aspiration cytology specimens for diagnosis of thyroid nodules: The influence of false-positive and false-negative results.

BACKGROUND: The accurate evaluation of BRAFV600E mutation in preoperative fine-needle aspiration cytology (FNAC) specimens is important for making management decisions in thyroid nodules (TNs). The aim of this study was to assess the false-positive and false-negative BRAFV600E mutations in thyroid FNAC specimens and their influence on diagnosis of TN. METHODS: This prospective study enrolled 292 nodules in 269 patients who underwent BRAFV600E mutation analysis using amplification refractory mutation system-quantitative real-time polymerase chain reaction (ARMS-qPCR) both in FNAC specimens and formalin-fixed, paraffin-embedded (FFPE) tissue samples after surgery. The false-positive and false-negative mutations for BRAFV600E analysis using ARMS-qPCR in FNAC specimens were recorded, with reference to the results of BRAFV600E mutation analysis using ARMS-qPCR in FFPE tissue sample. Diagnostic performances of FNAC, BRAFV600E mutation analysis in FNAC specimens, BRAFV600E mutation analysis in FFPE tissue sample, and the combination of FNAC and BRAFV600E mutation analysis for predicting thyroid malignancy were assessed. RESULTS: The false-positive and false-negative mutations for BRAFV600E analysis using ARMS-qPCR in FNAC specimens were 10.1% (19/189) and 7.1% (7/98), respectively. FNAC combined with preoperative BRAFV600E mutation analysis significantly increased the diagnostic sensitivity from 75.7% to 92.3%, and accuracy from 78.7% to 90.6% in comparison with FNAC alone (both P < .001). No significant differences were found between the combination of FNAC and BRAFV600E mutation analysis in FNAC specimens and the combination of FNAC and BRAFV600E mutation analysis in FFPE tissue sample (sensitivity: 92.3% vs 91.9%; accuracy: 90.6% vs 91.3%; both P > .05). CONCLUSIONS: FNAC combined with preoperative BRAFV600E mutation analysis can significantly increase the diagnostic performance in comparison with FNAC alone. False-positive and false-negative BRAFV600E mutation results are found in preoperative FNAC specimens, whereas it does not affect the overall auxiliary diagnosis of TNs.