Exploring functional genes' correlation with (S)-equol concentration and new daidzein racemase identification.

With its estrogenic activity, (S)-equol plays an important role in maintaining host health and preventing estrogen-related diseases. Exclusive production occurs through the transformation of soy isoflavones by intestinal bacteria, but the reasons for variations in (S)-equol production among different individuals and species remain unclear. Here, fecal samples from humans, pigs, chickens, mice, and rats were used as research objects. The concentrations of (S)-equol, along with the genetic homology and evolutionary relationships of (S)-equol production-related genes [daidzein reductase (DZNR), daidzein racemase (DDRC), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR)], were analyzed. Additionally, in vitro functional verification of the newly identified DDRC gene was conducted. It was found that approximately 40% of human samples contained (S)-equol, whereas 100% of samples from other species contained (S)-equol. However, there were significant variations in (S)-equol content among the different species: rats > pigs > chickens > mice > humans. The distributions of the four genes displayed species-specific patterns. High detection rates across various species were exhibited by DHDR, THDR, and DDRC. In contrast, substantial variations in detection rates among different species and individuals were observed with respect to DZNR. It appears that various types of DZNR may be associated with different concentrations of (S)-equol, which potentially correspond to the regulatory role during (S)-equol synthesis. This enhances our understanding of individual variations in (S)-equol production and their connection with functional genes in vitro. Moreover, the newly identified DDRC exhibits higher potential for (S)-equol synthesis compared to the known DDRC, providing valuable resources for advancing in vitro (S)-equol production. IMPORTANCE: (S)-equol ((S)-EQ) plays a crucial role in maintaining human health, along with its known capacity to prevent and treat various diseases, including cardiovascular diseases, metabolic syndromes, osteoporosis, diabetes, brain-related diseases, high blood pressure, hyperlipidemia, obesity, and inflammation. However, factors affecting individual variations in (S)-EQ production and the underlying regulatory mechanisms remain elusive. This study examines the association between functional genes and (S)-EQ production, highlighting a potential correlation between the DZNR gene and (S)-EQ content. Various types of DZNR may be linked to the regulation of (S)-EQ synthesis. Furthermore, the identification of a new DDRC gene offers promising prospects for enhancing in vitro (S)-EQ production.

Transposon-aided capture (TRACA) of plasmids from the human gut.

The gut microbiota consists of a vast and diverse assemblage of microorganisms that play a pivotal role in maintaining host health. Nevertheless, a significant portion of the human gut microbiota remains uncultivated. Plasmids, a type of MGE, assume a critical function in the biological evolution and adaptation of bacteria to varying environments. To investigate the plasmids present within the gut microbiota community, we used the transposon-aided capture method (TRACA) to explore plasmids derived from the gut microbiota. In this study, fecal samples were collected from two healthy human volunteers and subsequently subjected to the TRACA method for plasmid isolation. Then, the complete sequence of the plasmids was obtained using the genome walking method, and sequence identity was also analyzed. A total of 15 plasmids were isolated. At last, 13 plasmids were successfully sequenced, of which 12 plasmids were highly identical to the plasmids in the National Center for Biotechnology Information (NCBI) database and were all small plasmids. Furthermore, a putative novel plasmid, named pMRPHD, was isolated, which had mobilized elements (oriT and oriV) and a potential type II restriction-modification (R-M) system encoded by DNA cytosine methyltransferase and type II restriction enzyme (Ban I), whose specific functions and applications warrant further exploration.

Genomic Island-Mediated Horizontal Transfer of the Erythromycin Resistance Gene erm(X) among Bifidobacteria.

Antibiotic resistance is a serious medical issue driven by antibiotic misuse. Bifidobacteria may serve as a reservoir for antibiotic resistance genes (ARGs) that have the potential risk of transfer to pathogens. The erythromycin resistance gene erm(X) is an ARG with high abundance in bifidobacteria, especially in Bifidobacterium longum species. However, the characteristics of the spread and integration of the gene erm(X) into the bifidobacteria genome are poorly understood. In this study, 10 tetW-positive bifidobacterial strains and 1 erm(X)-positive bifidobacterial strain were used to investigate the transfer of ARGs. Conjugation assays found that the erm(X) gene could transfer to five other bifidobacterial strains. Dimethyl sulfoxide (DMSO) and vorinostat significantly promoted the transfer of the erm(X) from strain Bifidobacterium catenulatum subsp. kashiwanohense DSM 21854 to Bifidobacterium longum subsp. suis DSM 20211. Whole-genome sequencing and comparative genomic analysis revealed that the erm(X) gene was located on the genomic island BKGI1 and that BKGI1 was conjugally mobile and transferable. To our knowledge, this is the first report that a genomic island-mediated gene erm(X) transfer in bifidobacteria. Additionally, BKGI1 is very unstable in B. catenulatum subsp. kashiwanohense DSM 21854 and transconjugant D2TC and is highly excisable and has an intermediate circular formation. In silico analysis showed that the BKGI1 homologs were also present in other bifidobacterial strains and were especially abundant in B. longum strains. Thus, our results confirmed that genomic island BKGI1 was one of the vehicles for erm(X) spread. These findings suggest that genomic islands play an important role in the dissemination of the gene erm(X) among Bifidobacterium species. IMPORTANCE Bifidobacteria are a very important group of gut microbiota, and the presence of these bacteria has many beneficial effects for the host. Thus, bifidobacteria have attracted growing interest owing to their potential probiotic properties. Bifidobacteria have been widely exploited by the food industry as probiotic microorganisms, and some species have a long history of safe use in food and feed production. However, the presence of antibiotic resistance raises the risk of its application. In this study, we analyzed the transfer of the erythromycin resistance gene erm(X) and revealed that the molecular mechanism behind the spread of the gene erm(X) was mediated by genomic island BKGI1. To the best of our knowledge this is the first report to describe the transfer of the gene erm(X) via genomic islands among bifidobacteria. This may be an important way to disseminate the gene erm(X) among bifidobacteria.

Distribution and Strain Diversity of Immunoregulating Segmented Filamentous Bacteria in Human Intestinal Lavage Samples.

Segmented filamentous bacteria (SFB) are well known for their functions in the immunoregulation of hosts including the promotion of Th17 cell differentiation, B cell maturation, and immune system development. However, most analyses of SFB have focused on animal models, and thus, investigation of SFB prevalence in humans and their roles in human immunoregulation and health is needed. Although little is known overall of SFB prevalence in humans, they are characteristically abundant in animals during weaning. In this study, SFB-like bacteria were detected in ileal lavage samples from human children that were aged between 1 to 17 years old by scanning electron microscopy (SEM) analysis, and their insertion into the mucosa was also observed. In addition, the expression of SFB flagellin at the human bacterial interface was observed by immunohistochemistry (IHC) and western blot. Moreover, two pairs of primers specific for SFB, but targeting different genes, were used to detect SFB in human intestinal lavage samples. These analyses indicated that SFB were present in over 50% of patient ileal samples independent of age. High-throughput gene sequencing indicated that different SFB strains were detected among samples. Between nine and 23 SFB flagellin gene operational taxonomic units were identified. In addition to evaluating the prevalence of SFB in human samples, correlations between SFB presence and chief complaints of clinical symptoms were evaluated, as well as the relationship between SFB and patient serum immunoglobulin concentrations. SFB prevalence was significantly higher in hematochezia patients (68%) than in abdominal pain (56.10%) and diarrhea (43.75%) patients. Furthermore, the concentrations of serum IgA, IgM, and IgE, were similar between SFB-positive and SFB-negative patient groups, although IgG concentrations were significantly higher in the SFB-negative group.

Literature-Based Phenotype Survey and In Silico Genotype Investigation of Antibiotic Resistance in the Genus Bifidobacterium.

Bifidobacteria are typical commensals inhabiting the human intestine and are beneficial to the host because of their probiotic properties. One of the risks concerning probiotics is the potential of introducing antibiotic resistance genes (ARGs) to the host gut pathogens. This study was aimed to depict the general antibiotic resistance characteristics of the genus Bifidobacterium by combining the reported phenotype dataset and in silico genotype prediction. Bifidobacteria were mostly reported to be sensitive to beta-lactams, glycopeptides, chloramphenicol, and rifampicin, but resistant to aminoglycosides, polypeptides, quinolones, and mupirocin. Generally, the resistance phenotypes to erythromycin, tetracycline, fusidic acid, metronidazole, clindamycin, and trimethoprim were variable. Besides cmX and tetQ, characterized in bifidobacterial resident plasmids, 3520 putative ARGs were identified from 831 bifidobacterial genomes through BLASTP search. The identified ARGs matched thirty-eight reference ARGs, four of which seemed to be mutant housekeeping genes. The two high-abundant ARGs, tetW and ermX, were found to have different distribution traits. The predicted ARGs reasonably explained most of the corresponding resistant phenotypes in the published literature.

Effects of bifidobacteria-produced exopolysaccharides on human gut microbiota in vitro.

Exopolysaccharides (EPSs) are carbohydrate polymers that are synthesized and present on the surface of bifidobacteria. Due to their potential applications in diverse sectors, such as food, biotechnology, cosmetics, and medicine, EPSs synthesized by bifidobacteria have recently attracted more attention. EPS production not only has benefits in food and health but also has effects on probiotics in the microbial ecosystem. In this study, we investigated the interaction between bifidobacteria EPSs and human gut microbiota in vitro using thin-layer chromatography, 16S rDNA high-throughput sequencing, and gas chromatography. The results showed that human gut microbiota has the capacity to degrade EPSs, although the degradation rate was approximately 50% after fermenting for 48 h. On the other hand, EPSs regulate the human gut microbiota. Fermented samples in the VI_Bif group clustered together according to the bacterial community compared to the VI_Starch group, in which starch was added as a carbon source. The bifidobacteria EPS promoted the growth of phylum Deinococcus_Thermus, class Deinococci, order Deinococcales, and genus Coprococcus. EPSs also increased the production of propionic acid compared to the starch group. The detection results of Dionex ICS 5000 high-purity capillary ion chromatography system showed that EPSs had absorption peaks of fucose, rhamnose, galactose/acetyl glucosamine, glucose, and ribose, and the molecular proportion of these monosaccharides was approximately 2: 2: 440: 3: 53. The monosaccharide composition of this EPS appears to be more complex than previously reported for bifidobacteria EPS. Additional studies are needed to elucidate its structure and functions.

Host Specificity of Flagellins from Segmented Filamentous Bacteria Affects Their Patterns of Interaction with Mouse Ileal Mucosal Proteins.

Segmented filamentous bacteria (SFB) are known modulators of the mammalian immune system. Currently, the technology for investigating SFB culture in vitro is immature, and as a result, the mechanisms of SFB colonization and immune regulation are not yet fully elucidated. In this study, we investigated the gene diversity and host specificity of SFB flagellin genes. The fliC1 and fliC2 genes are relatively conserved, while the fliC3 and fliC4 genes are more variable, especially at the central and C-terminal regions. Host specificity analysis demonstrated that the fliC1 genes do not cluster together based on the host organism, whereas the fliC3 and fliC4 genes were host specific at the nucleotide and deduced amino acid levels. SFB flagellin protein expression in the ileum mucosa and cecal contents was detected by using fluorescence in situ hybridization (FISH) combined with immunohistochemical (IHC) analysis, immunoblotting, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the purified SFB FliC3 protein originating from both mouse and rat was able to activate Toll-like receptor 5 (TLR5)-linked NF-κB signaling, no host specificity was observed. Interestingly, the patterns of interaction with mouse ileum mucosal proteins were different for mouse FliC3 (mFliC3) and rat FliC3 (rFliC3). Gene Ontology (GO) and KEGG analyses indicated that more adherence-related proteins interacted with mFliC3, while more lysosome- and proteolysis-related proteins interacted with rFliC3. In vitro degradation experiments indicated that the stability of rFliC3 was lower than that of mFliC3 when they were incubated with mouse ileum mucosal proteins. In summary, the gene diversity and host specificity of SFB flagellin genes were investigated, and SFB flagellin expression was detected in gut samples.IMPORTANCE Since SFB genomes contain only one copy of each FliC gene, the diversity of FliC is representative of SFB strain diversity. Currently, little is known regarding the diversity and specificity of members of the group of SFB. The work presented herein demonstrates that select SFB strains, exhibiting unique FliC patterns, are present in a variety of mammalian hosts. SFB fliC genes were found to interact with a number of unique targets, providing further evidence for SFB host selection. Together, this work represents a major advancement in identifying SFB and delineating how members of the group of SFB interact with the host. Future examination of FliC genes will likely enhance our knowledge of intestinal colonization by the gut microbiota.

Fermentation properties of isomaltooligosaccharides are affected by human fecal enterotypes.

Isomaltooligosaccharides (IMOs) are enzymatically synthesized oligosaccharides that have potential prebiotic effects. Five IMO substrates with 2-16° of polymerization (DP) were studied for their fermentation capacities using human microbiomes in an in vitro batch fermentation model. Eleven fecal slurries belonging to three enterotypes, including the Bacteroides-, Prevotella- and Mixed-type, exhibited different degradation rates for long chain IMOs (DP 7 to 16). In contrast, the degradation rates for short chain IMOs (DP 2 to 6) were not affected by enterotypes. Both 16S rRNA gene sequencing and quantitative PCR demonstrated that, after fermentation, the Bifidobacterium growth with IMOs was primarily detected in the Bacteroides- and Mixed-type (non-Prevotella-type), and to a lesser degree in the Prevotella-type. Interestingly, the Prevotella-type microbiome had higher levels of propionic acid and butyric acid production than non-Prevotella-type microbiome after IMOs fermentation. Moreover, principal coordinate analysis (PCoA) of both denaturing gradient gel electrophoresis (DGGE) profiling and 16S rRNA sequencing data demonstrated that the microbiome community compositions were separately clustered based on IMO chain length, suggesting significant impact of DP on the bacterial community structure. The current results clearly demonstrated that the IMO chain length could modulate the structure and composition of the human colonic microbiome. Different responses to short and long chain IMOs were observed from three human enterotypes, indicating that IMOs may be used as therapeutic substrates for directly altering human colonic bacteria.

Equol, a Clinically Important Metabolite, Inhibits the Development and Pathogenicity of Magnaporthe oryzae, the Causal Agent of Rice Blast Disease.

Equol, a metabolite of soybean isoflavone daidzein, has been proven to have various bioactivities related to human health, but little is known on its antifungal activity to plant fungal pathogens. Magnaporthe oryzae is a phytopathogenic fungus that causes rice blast, a devastating disease on rice. Here, we demonstrated that equol influences the development and pathogenicity of M. oryzae. Equol showed a significant inhibition to the mycelial growth, conidial generation and germination, and appressorial formation of M. oryzae. As a result, equol greatly reduced the virulence of M. oryzae on rice and barley leaves. The antifungal activity of equol was also found in several other plant fungal pathogens. These findings expand our knowledge on the bioactivities of equol.

In vitro fermentation of alginate and its derivatives by human gut microbiota.

Alginate (Alg) has a long history as a food ingredient in East Asia. However, the human gut microbes responsible for the degradation of alginate and its derivatives have not been fully understood yet. Here, we report that alginate and the low molecular polymer derivatives of mannuronic acid oligosaccharides (MO) and guluronic acid oligosaccharides (GO) can be completely degraded and utilized at various rates by fecal microbiota obtained from six Chinese individuals. However, the derivative of propylene glycol alginate sodium sulfate (PSS) was not hydrolyzed. The bacteria having a pronounced ability to degrade Alg, MO and GO were isolated from human fecal samples and were identified as Bacteroides ovatus, Bacteroides xylanisolvens, and Bacteroides thetaiotaomicron. Alg, MO and GO can increase the production level of short chain fatty acids (SCFA), but GO generates the highest level of SCFA. Our data suggest that alginate and its derivatives could be degraded by specific bacteria in the human gut, providing the basis for the impacts of alginate and its derivates as special food additives on human health.

Overexpression of CupB5 activates alginate overproduction in Pseudomonas aeruginosa by a novel AlgW-dependent mechanism.

In Pseudomonas aeruginosa, alginate overproduction, also known as mucoidy, is negatively regulated by the transmembrane protein MucA, which sequesters the alternative sigma factor AlgU. MucA is degraded via a proteolysis pathway that frees AlgU from sequestration, activating alginate biosynthesis. Initiation of this pathway normally requires two signals: peptide sequences in unassembled outer-membrane proteins (OMPs) activate the AlgW protease, and unassembled lipopolysaccharides bind periplasmic MucB, releasing MucA and facilitating its proteolysis by activated AlgW. To search for novel alginate regulators, we screened a transposon library in the non-mucoid reference strain PAO1, and identified a mutant that confers mucoidy through overexpression of a protein encoded by the chaperone-usher pathway gene cupB5. CupB5-dependent mucoidy occurs through the AlgU pathway and can be reversed by overexpression of MucA or MucB. In the presence of activating OMP peptides, peptides corresponding to a region of CupB5 needed for mucoidy further stimulated AlgW cleavage of MucA in vitro. Moreover, the CupB5 peptide allowed OMP-activated AlgW cleavage of MucA in the presence of the MucB inhibitor. These results support a novel mechanism for conversion to mucoidy in which the proteolytic activity of AlgW and its ability to compete with MucB for MucA is mediated by independent peptide signals.

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa.

BACKGROUND: Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ(22)) to initiate transcription of the alginate biosynthetic operon. RESULTS: In the current study, we mapped the mucE transcriptional start site, and determined that P(mucE) activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P(mucE) activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. CONCLUSIONS: Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

Whole-Genome Sequencing of Pig Gut Lactobacillus amylovorus CICC 6090.

Lactobacillus amylovorus has been reported to reduce weight and fat content in humans. To identify and understand the mechanism underlying this process, the complete genome of CICC 6090, which was isolated from pig intestines, was sequenced using PromethION and next-generation sequencing.

Whole-genome sequencing of Salmonella pullorum and Sanguinarine-induced mutant.

This work was performed on commercially purchased Salmonella pullorum CVCC519 originally isolated from chicken intestinal content. The Sanguinarine-resistant strain XM3104 was isolated from Sanguinarine-induced CVCC519. To identify possible mechanisms underlying resistance, the complete genomes of CVCC519 and XM3104 were sequenced using PromethION and next-generation sequencing.

Higher-level production of volatile fatty acids in vitro by chicken gut microbiotas than by human gut microbiotas as determined by functional analyses.

The aim of this study was to determine the relationship between the composition and function of gut microbiota. Here, we compared the bacterial compositions and fermentation metabolites of human and chicken gut microbiotas. Results generated by quantitative PCR (qPCR) and 454 pyrosequencing of the 16S rRNA gene V3 region showed the compositions of human and chicken microbiotas to be markedly different, with chicken cecal microbiotas displaying more diversity than human fecal microbiotas. The nutrient requirements of each microbiota growing under batch and chemostat conditions were analyzed. The results showed that chicken cecal microbiotas required simple sugars and peptides to maintain balanced growth in vitro but that human fecal microbiotas preferred polysaccharides and proteins. Chicken microbiotas also produced higher concentrations of volatile fatty acids than did human microbiotas. Our data suggest that the availability of different fermentable substrates in the chicken cecum, which exist due to the unique anatomical structure of the cecum, may provide an environment favorable to the nourishment of microbiotas suited to the production of the higher-energy metabolites required by the bird. Therefore, gut structure, nutrition, immunity, and life-style all contribute to the selection of an exclusive bacterial community that produces types of metabolites beneficial to the host.

Effects of lactobacillus plantarum ZJ316 on pig growth and pork quality.

BACKGROUND: Lactobacillus plantarum is a plant-associated bacterial species but it has also been found in human, mouse and porcine gastrointestinal tracts. It can ferment a broad spectrum of plant carbohydrates; it is tolerant of bile salts and low pH, and it has antagonistic potential against intestinal pathogens. However, experiments reporting the use of L. plantarum as a probiotic are limited. In this study, the effects of L. plantarum ZJ316 isolated from infant fecal samples on pig growth and pork quality were investigated. RESULTS: One hundred and fifty newly weaned pigs were selected randomly and divided into five groups. Group 1 was fed a diet supplemented with the antibiotic mequindox; Groups 2, 3 and 4 were fed a diet supplemented with L. plantarum and no antibiotic; and Group 5 was fed a mixture of mequindox and L. plantarum. After a 60 days initial treatment, samples were collected for evaluation. The results showed that, the L. plantarum ZJ316 has probiotic effects on pig growth and that these effects are dose dependent. The effects of a dose of 1 × 109 CFU/d were more pronounced than those of a dose of 5 × 109 CFU/d or 1 × 1010 CFU/d. In Group 2 (1 × 109 CFU/d), the diarrhea (p = 0.000) and mortality rates (p = 0.448) were lower than in antibiotic-treated pigs (Group 1), and the daily weight gain (p = 0.001) and food conversion ratios were better (p = 0.005). Improved pork quality was associated with Lactobacillus treatment. pH (45 min, p = 0.020), hardness (p = 0.000), stickiness (p = 0.044), chewiness (p = 0.000), gumminess (p = 0.000) and restoring force (p = 0.004) were all significantly improved in Lactobacillus-treated pigs (Group 2). Although we found that L. plantarum exerted probiotic effects on pig growth and pork quality, the mechanisms underlying its action require further study. Polymerase chain reaction-denaturing gradient gel electrophoresis results showed that the gut bacterial communities in Lactobacillus- and antibiotic-treated pigs were very similar and the quantity of L. plantarum ZJ316 was below the detection limits of DGGE-band sequencing. The concentration of short-chain fatty acids in Lactobacillus- and antibiotic-treated fecal samples were not significantly different (p = 0.086). However, the villus height of ilea (p = 0.003), jejuna (p = 0.000) and duodena (p = 0.036) were found to be significantly improved by Lactobacillus treatment. CONCLUSION: L. plantarum ZJ316 was found to have probiotic effects, improving pig growth and pork quality. The probiotic mechanism might not involve L. plantarum colonization and alteration of the gut bacterial community. Rather, it might be related to the inhibition of the growth of opportunistic pathogens and promotion of increased villus height.

[Mining of gene clusters for biosynthesis of secondary metabolites and analysis of genes encoding antibiotic resistance and virulence in 4 644 representative human gut strains].

Genome sequences of 4 644 representative strains from human gut microbiota were analyzed to mine gene clusters for biosynthesis of novel secondary metabolites, as well as genes encoding antibiotic resistance and virulence factors. AntiSMASH analysis showed that more than 60% of the representative strains encoded at least one secondary metabolite gene cluster, and 8 potential novel secondary metabolite gene clusters were identified from 8 unculturable bacteria. The secondary metabolite gene clusters in human intestine are mainly composed of nonribosomal peptide synthetase (NRPS), bacteriocin, arylpolyene, terpene, betalactone and NRPS like gene clusters distributed in Clostridia, Bacilli, Gammaproteobacteria, Bacteroidia, Actinobacteria and Negativicutes. PathoFact analysis showed that genes encoding antibiotic resistance and virulence factors are widely distributed in representative strains, but the frequency encoded by potential pathogens is significantly higher than that of non-potential pathogens. The frequency of genes encoding secretory toxins such as outer membrane protein, PapC N-terminal domain, PapC C-terminal domain, peptidase M16 inactive domain, and non-secretory toxins such as nitroreductase family, AcrB/AcrD/AcrF family, PLD-like domain, Cupin domain, putative hemolysin, S24-like peptidase, phosphotransferase enzyme family, endonuclease/ exonuclease/ phosphatase family, glyoxalase/ bleomycin resistance was high in potential pathogens. This study may facilitate mining new microbial natural products from the intestinal microbiome, understanding the colonization and infection mechanism of intestinal microorganisms, and providing targeted prevention and treatment of intestinal microbial related diseases.

Comparison of tumor-associated and nontumor-associated esophageal mucosa microbiota in patients with esophageal squamous cell carcinoma.

Esophageal microbiota plays important roles in esophageal squamous cell carcinoma (ESCC). The aims of this study were to clarify the changes in the bacterial community during ESCC development and identify latent pathogenic bacteria which may contribute to esophageal carcinogenesis and progression. Fresh tumor and nontumor esophageal mucosal samples were collected from 31 men with ESCC. High-throughput 16s rRNA sequencing was performed, and the operational taxonomic unit data and bacterial classification annotation were obtained and analyzed. The Ace, Chao, Shannon, Simpson indexes, and operational taxonomic unit numbers were higher in nontumor tissues than in tumor tissues, although without statistical significance. There were 4 phyla and 28 genera found to show significant differences between tumor and nontumor samples. The general probiotic Lactobacillus was 1.98-fold higher in nontumor tissues, while the general pathogenic genera Fusobacterium was 4.35-fold higher in tumor tissues. For tumor tissue samples, the genera Treponema and Brevibacillus were significantly higher in N1 and N2 stages, respectively, and Acinetobacter was significantly higher in T3 stage. For nontumor tissues, the genus Fusicatenibacter was significantly higher in T2 stage, and Corynebacterium, Aggregatibacter, Saccharimonadaceae-TM7x, and Cupriavidus were significantly higher in T4 stage. Additionally, bacteria related to nitrotoluene degradation were enriched in nontumor tissues, while bacteria related to base excision repair were enriched in tumor tissues. The relative abundance of several phyla and genera are different between tumor and nontumor tissue samples. The altered bacterial microbiota is correlated with different tumor stages and some microbes may take part in the carcinogenesis and development of ESCC.

Characterization and Identification of a New Daidzein Reductase Involved in (S)-Equol Biosynthesis in Clostridium sp. ZJ6.

(S)-equol (EQ) is an isoflavone with high estrogen-like activity in the human body, and is only produced by some gut bacteria in vivo. It plays an important role in maintaining individual health, however, the dearth of resources associated with (S)-EQ-producing bacteria has seriously restricted the production and application of (S)-EQ. We report here a new functional gene KEC48-07020 (K-07020) that was identified from a chick (S)-EQ-producing bacterium (Clostridium sp. ZJ6, ZJ6). We found that recombinant protein of K-07020 possessed similar function to daidzein reductase (DZNR), which can convert daidzein (DZN) into R/S-dihydrodaidzein (R/S-DHD). Interestingly, K-07020 can reversely convert (R/S)-DHD (DHD oxidase) into DZN even without cofactors under aerobic conditions. Additionally, high concentrations of (S)-EQ can directly promote DHD oxidase but inhibit DZNR activity. Molecular docking and site-directed mutagenesis revealed that the amino acid > Arg75 was the active site of DHD oxidase. Subsequently, an engineered E. coli strain based on K-07020 was constructed and showed higher yield of (S)-EQ than the engineered bacteria from our previous work. Metagenomics analysis and PCR detection surprisingly revealed that K-07020 and related bacteria may be prevalent in the gut of humans and animals. Overall, a new DZNR from ZJ6 was found and identified in this study, and its bidirectional enzyme activities and wide distribution in the gut of humans and animals provide alternative strategies for revealing the individual regulatory mechanisms of (S)-EQ-producing bacteria.

Isomaltooligosaccharides Sustain the Growth of Prevotella Both In Vitro and in Animal Models.

The human digestive tract is colonized by trillions of bacterial cells that play important roles in human health and diseases. It is well known that dietary habits are associated with human microbiota enterotypes. However, the factors that determine the enterotype still remain elusive. In this study, it was first examined, via in vitro batch fermentation, how different carbohydrates affect the Bacteroides and Prevotella enterotypes. Among the 11 substrates (fructo-, galacto-, xylo-, manno-, and isomalto-oligosaccharides [IMO] and lactulose, raffinose, starch, inulin [INU], mannitol, and xylitol) tested, IMO, INU, and starch were found to sustain the growth of Prevotella through batch fermentation. The development of the Prevotella and Bacteroides enterotypes was further simulated in chemostats using fecal samples. IMO coupled with faster dilution rates and lower pH were required to sustain the growth of Prevotella copri in the chemostat based on 16S rRNA gene and metagenomic sequencing. Meanwhile, starch with relatively lower dilution rates and higher pH was required to support the development of the Bacteroides enterotype. Amylo-α-1,6-glucosidase, pectin, and xylan lyases were the carbohydrate-active enzymes associated with the Prevotella enterotype. The Bacteroides enterotype was associated with more diversified carbohydrate-active enzymes. Consistently, since honey contains high isomaltose content, mice fed IMO and honey displayed an increased relative abundance of Prevotella in the colon. In conclusion, both in vitro systems and a mouse model were used to demonstrate that IMO maintains the Prevotella enterotype. This result provides insight into the nutritional requirements underlying gut enterotype formation. IMPORTANCE The Prevotella enterotype type is a human traditional enterotype with high dietary fiber intake, which is related to healthy ageing and Parkinson's disease development. Manipulations of the dwelled gut microbes by dietary isomalto-oligosaccharides efficiently sustained Prevotella type enterotypes, indicating that it can be used in the improvement of elderly health by increasing the gut transit time.