Recombinant Bile Salt Hydrolase Enhances the Inhibition Efficiency of Taurodeoxycholic Acid against Clostridium perfringens Virulence.

Clostridium perfringens is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen C. perfringens. Assays were conducted to evaluate the inhibition of C. perfringens growth, hydrogen sulfide (H2S) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the bsh gene for cloning. The bsh gene from Bifidobacterium longum was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein in E. coli BL21 and Bacillus subtilis 168 (B-sub-BSH), respectively. His-tag-purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and a Western blot (WB) assays. Secretory BSH from B. subtilis was analyzed by a Dot-Blot. B-sub-BSH was evaluated for the inhibition of C. perfringens growth. C. perfringens growth reached 7.8 log10 CFU/mL after 24 h culture. C. perfringens growth was at 8 vs. 7.4, 7.8 vs. 2.6 and 6 vs. 0 log10 CFU/mL in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced C. perfringens H2S production and the virulence gene expression of asrA1, netB, colA, and virT. BSH activity was observed in Lactobacillus johnsonii and B. longum under anaerobe but not L. johnsonii under 10% CO2 air. After the sequence alignment of bsh from ten bacteria, bsh from B. longum was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After the subcloned bsh and amylase signal peptide sequence was inserted into pDR-BSH, B. subtilis was transformed and named B-sub-BSH. The transformation was evaluated using PCR with B. subtilis around 3 kb and B-sub-BSH around 5 kb. Secretory BSH expressed from B-sub-BSH was determined for His-tag using Dot-Blot. Importantly, C. perfringens growth was reduced greater than 59% log10 CFU/mL in the B-sub-BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, TDCA was less potent than DCA against C. perfringens virulence, and recombinant secretory BSH from B-sub-BSH reduced C. perfringens growth, suggesting a new potential intervention against the pathogen-induced chicken NE.

Genetic Dissection of Diverse Seed Coat Patterns in Cowpea through a Comprehensive GWAS Approach.

This study investigates the genetic determinants of seed coat color and pattern variations in cowpea (Vigna unguiculata), employing a genome-wide association approach. Analyzing a mapping panel of 296 cowpea varieties with 110,000 single nucleotide polymorphisms (SNPs), we focused on eight unique coat patterns: (1) Red and (2) Cream seed; (3) White and (4) Brown/Tan seed coat; (5) Pink, (6) Black, (7) Browneye and (8) Red/Brown Holstein. Across six GWAS models (GLM, SRM, MLM, MLMM, FarmCPU from GAPIT3, and TASSEL5), 13 significant SNP markers were identified and led to the discovery of 23 candidate genes. Among these, four specific genes may play a direct role in determining seed coat pigment. These findings lay a foundational basis for future breeding programs aimed at creating cowpea varieties aligned with consumer preferences and market requirements.

Potent Bile Acid Microbial Metabolites Modulate Clostridium perfringens Virulence.

Clostridium perfringens is a versatile pathogen, inducing diseases in the skin, intestine (such as chicken necrotic enteritis (NE)), and other organs. The classical sign of NE is the foul smell gas in the ballooned small intestine. We hypothesized that deoxycholic acid (DCA) reduced NE by inhibiting C. perfringens virulence signaling pathways. To evaluate the hypothesis, C. perfringens strains CP1 and wild-type (WT) HN13 and its mutants were cultured with different bile acids, including DCA and isoallolithocholic acid (isoalloLCA). Growth, hydrogen sulfide (H2S) production, and virulence gene expression were measured. Notably, isoalloLCA was more potent in reducing growth, H2S production, and virulence gene expression in CP1 and WT HN13 compared to DCA, while other bile acids were less potent compared to DCA. Interestingly, there was a slightly different impact between DCA and isoalloLCA on the growth, H2S production, and virulence gene expression in the three HN13 mutants, suggesting possibly different signaling pathways modulated by the two bile acids. In conclusion, DCA and isoalloLCA reduced C. perfringens virulence by transcriptionally modulating the pathogen signaling pathways. The findings could be used to design new strategies to prevent and treat C. perfringens-induced diseases.

Vaccines Using Clostridium perfringens Sporulation Proteins Reduce Necrotic Enteritis in Chickens.

Clostridium perfringens is the prevalent enteric pathogen in humans and animals including chickens, and it remains largely elusive on the mechanism of C. perfringens-induced enteritis because of limited animal models available. In this study, we investigated the role of C. perfringens sporulation proteins as vaccine candidates in chickens to reduce necrotic enteritis (NE). C. perfringens soluble proteins of vegetative cells (CP-super1 and CP-super2) and spores (CP-spor-super1 and CP-spor-super2) were prepared, and cell and chicken experiments were conducted. We found that deoxycholic acid reduced C. perfringens invasion and sporulation using the Eimeria maxima and C. perfringens co-infection necrotic enteritis (NE) model. C. perfringens enterotoxin (CPE) was detected in the CP-spor-super1&2. CP-spor-super1 or 2 induced cell death in mouse epithelial CMT-93 and macrophage Raw 264.7 cells. CP-spor-super1 or 2 also induced inflammatory gene expression and necrosis in the Raw cells. Birds immunized with CP-spor-super1 or 2 were resistant to C. perfringens-induced severe clinical NE on histopathology and body weight gain loss. CP-spor-super1 vaccine reduced NE-induced proinflammatory Ifnγ gene expression as well as C. perfringens luminal colonization and tissue invasion in the small intestine. Together, this study showed that CP-spor-super vaccines reduced NE histopathology and productivity loss.

Triterpenoid CDDO-IM protects against lipopolysaccharide-induced inflammatory response and cytotoxicity in macrophages: The involvement of the NF-κB signaling pathway.

Lipopolysaccharide (LPS), also known as endotoxin, can trigger septic shock, a severe form of inflammation-mediated sepsis with a very high mortality rate. However, the precise mechanisms underlying this endotoxin remain to be defined and detoxification of LPS is yet to be established. Macrophages, a type of immune cells, initiate a key response responsible for the cascade of events leading to the surge in inflammatory cytokines and immunopathology of septic shock. This study was undertaken to determine whether the LPS-induced inflammation in macrophage cells could be ameliorated via CDDO-IM (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline), a novel triterpenoid compound. Data from this study show that gene expression levels of inflammatory cytokine genes such as interleukin-1 beta (IL-1ß), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) were considerably increased by treatment with LPS in macrophages differentiated from ML-1 monocytes. Interestingly, LPS-induced increase in expression of pro-inflammatory cytokine levels is reduced by CDDO-IM. In addition, endogenous upregulation of a series of antioxidant molecules by CDDO-IM provided protection against LPS-induced cytotoxicity in macrophages. LPS-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcriptional activity was also noted to decrease upon treatment with CDDO-IM in macrophages suggesting the involvement of the NF-κB signaling. This study would contribute to improve our understanding of the detoxification of endotoxin LPS by the triterpenoid CDDO-IM.

Microbiota from Specific Pathogen-Free Mice Reduces Campylobacter jejuni Chicken Colonization.

Campylobacter jejuni, a prevalent foodborne bacterial pathogen, is mainly transmitted from poultry with few effective prevention approaches. In this study, we aimed to investigate the role of microbiota on C. jejuni chicken colonization. Microbiota from specific pathogen-free (SPF) mouse stools were collected as SPF-Aerobe and SPF-Anaerobe. Birds were colonized with SPF-Aerobe or SPF-Anaerobe at day 0 and infected with C. jejuni AR101 at day 12. Notably, C. jejuni AR101 colonized at 5.3 and 5.6 log10 C. jejuni CFU/g chicken cecal digesta at days 21 and 28, respectively, while both SPF-Aerobe and SPF-Anaerobe microbiota reduced pathogen colonization. Notably, SPF-Aerobe and SPF-Anaerobe increased cecal phylum Bacteroidetes and reduced phylum Firmicutes compared to those in the nontransplanted birds. Interestingly, microbiota from noninfected chickens, SPF-Aerobe, or SPF-Anaerobe inhibited AR101 in vitro growth, whereas microbiota from infected birds alone failed to reduce pathogen growth. The bacterium Enterobacter102 isolated from infected birds transplanted with SPF-Aerobe inhibited AR101 in vitro growth and reduced pathogen gut colonization in chickens. Together, SPF mouse microbiota was able to colonize chicken gut and reduce C. jejuni chicken colonization. The findings may help the development of effective strategies to reduce C. jejuni chicken contamination and campylobacteriosis.

Natural Compound Resveratrol Attenuates TNF-Alpha-Induced Vascular Dysfunction in Mice and Human Endothelial Cells: The Involvement of the NF-κB Signaling Pathway.

Resveratrol, a natural compound in grapes and red wine, has drawn attention due to potential cardiovascular-related health benefits. However, its effect on vascular inflammation at physiologically achievable concentrations is largely unknown. In this study, resveratrol in concentrations as low as 1 µm suppressed TNF-α-induced monocyte adhesion to human EA.hy926 endothelial cells (ECs), a key event in the initiation and development of atherosclerosis. Low concentrations of resveratrol (0.25-2 µm) also significantly attenuated TNF-α-stimulated mRNA expressions of MCP-1/CCL2 and ICAM-1, which are vital mediators of EC-monocyte adhesion molecules and cytokines for cardiovascular plaque formation. Additionally, resveratrol diminished TNF-α-induced IκB-α degradation and subsequent nuclear translocation of NF-κB p65 in ECs. In the animal study, resveratrol supplementation in diet significantly diminished TNF-α-induced increases in circulating levels of adhesion molecules and cytokines, monocyte adhesion to mouse aortic ECs, F4/80-positive macrophages and VCAM-1 expression in mice aortas and restored the disruption in aortic elastin fiber caused by TNF-α treatment. The animal study also confirmed that resveratrol blocks the activation of NF-κB In Vivo. In conclusion, resveratrol at physiologically achievable concentrations displayed protective effects against TNF-α-induced vascular endothelial inflammation in vitro and In Vivo. The ability of resveratrol in reducing inflammation may be associated with its role as a down-regulator of the NF-κB pathway.

Specific Secondary Bile Acids Control Chicken Necrotic Enteritis.

Necrotic enteritis (NE), mainly induced by the pathogens of Clostridium perfringens and coccidia, causes huge economic losses with limited intervention options in the poultry industry. This study investigated the role of specific bile acids on NE development. Day-old broiler chicks were assigned to six groups: noninfected, NE, and NE with four bile diets of 0.32% chicken bile, 0.15% commercial ox bile, 0.15% lithocholic acid (LCA), or 0.15% deoxycholic acid (DCA). The birds were infected with Eimeria maxima at day 18 and C. perfringens at day 23 and 24. The infected birds developed clinical NE signs. The NE birds suffered severe ileitis with villus blunting, crypt hyperplasia, epithelial line disintegration, and massive immune cell infiltration, while DCA and LCA prevented the ileitis histopathology. NE induced severe body weight gain (BWG) loss, while only DCA prevented NE-induced BWG loss. Notably, DCA reduced the NE-induced inflammatory response and the colonization and invasion of C. perfringens compared to NE birds. Consistently, NE reduced the total bile acids in the ileal digesta, while dietary DCA and commercial bile restored it. Together, this study showed that DCA and LCA reduced NE histopathology, suggesting that secondary bile acids, but not total bile acid levels, play an essential role in controlling the enteritis.

Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion.

Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S. Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S. Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S. Enteritidis. HTC cells were infected with S. Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S. Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S. Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S. Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S. Enteritidis invasion.

Microbiota attenuates chicken transmission-exacerbated campylobacteriosis in Il10-/- mice.

Campylobacter jejuni is a prevalent foodborne pathogen mainly transmitting through poultry. It remains unknown how chicken-transmitted C. jejuni and microbiota impact on human campylobacteriosis. Campylobacter jejuni AR101 (Cj-P0) was introduced to chickens and isolated as passage 1 (Cj-P1). Campylobacter jejuni Cj-P1-DCA-Anaero was isolated from Cj-P0-infected birds transplanted with DCA-modulated anaerobic microbiota. Specific pathogen free Il10-/- mice were gavaged with antibiotic clindamycin and then infected with Cj-P0, Cj-P1, or Cj-P1-DCA-Anaero, respectively. After 8 days post infection, Il10-/- mice infected with Cj-P1 demonstrated severe morbidity and bloody diarrhea and the experiment had to be terminated. Cj-P1 induced more severe histopathology compared to Cj-P0, suggesting that chicken transmission increased C. jejuni virulence. Importantly, mice infected with Cj-P1-DCA-Anaero showed attenuation of intestinal inflammation compared to Cj-P1. At the cellular level, Cj-P1 induced more C. jejuni invasion and neutrophil infiltration into the Il10-/- mouse colon tissue compared to Cj-P0, which was attenuated with Cj-P1-DCA-Anaero. At the molecular level, Cj-P1 induced elevated inflammatory mediator mRNA accumulation of Il17a, Il1ß, and Cxcl1 in the colon compared to Cj-P0, while Cj-P1-DCA-Anaero showed reduction of the inflammatory gene expression. In conclusion, our data suggest that DCA-modulated anaerobes attenuate chicken-transmitted campylobacteriosis in mice and it is important to control the elevation of C. jejuni virulence during chicken transmission process.

Sodium Butyrate Reduces Salmonella Enteritidis Infection of Chicken Enterocytes and Expression of Inflammatory Host Genes in vitro.

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1ß, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

NADPH-quinone oxidoreductase-1 mediates Benzo-[a]-pyrene-1,6-quinone-induced cytotoxicity and reactive oxygen species production in human EA.hy926 endothelial cells.

Numerous studies conducted in the past have reported deaths in the human population due to cardiovascular diseases (CVD) on exposure to air particulate matter (APM). BP-1,6-quinone (BP-1,6-Q) is one of the significant components of APM. However, the mechanism(s) by which it can exert its toxicity in endothelial cells is not yet completely understood. NAD(P)H: quinone oxidoreductase-1 (NQO1) is expressed highly in myocardium and vasculature tissues of the heart and plays a vital role in maintaining vascular homeostasis. This study, demonstrated that BP-1,6-Q diminishes NQO1 enzyme activity in a dose-dependent manner in human EA.hy926 endothelial cells. The decrease in the NQO1 enzyme causes potentiation in BP-1,6-Q-mediated toxicity in EA.hy926 endothelial cells. The enhancement of NQO1 in endothelial cells showed cytoprotection against BP-1,6-Q-induced cellular toxicity, lipid, and protein damage suggesting an essential role of NQO1 in cytoprotection against BP-1,6-Q toxicity. Using various biochemical assays and genetic approaches, results from this study further demonstrated that NQO1 also plays a crucial role in BP-1,6-Q-induced production of reactive oxygen species (ROS). These findings will contribute to elucidating BP-1,6-Q mediated toxicity and its role in the development of atherosclerosis.

Targeting glutathione with the triterpenoid CDDO-Im protects against benzo-a-pyrene-1,6-quinone-induced cytotoxicity in endothelial cells.

Epidemiological studies have exhibited a strong correlation between exposure to air pollution and deaths due to vascular diseases such as atherosclerosis. Benzo-a-pyrene-1,6-quinone (BP-1,6-Q) is one of the components of air pollution. This study was to examine the role of GSH in BP-1,6-Q mediated cytotoxicity in human EA.hy96 endothelial cells and demonstrated that induction of cellular glutathione by a potent triterpenoid, CDDO-Im (1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole), protects cells against BP-1,6-Q induced protein and lipid damage. Incubation of EA.hy926 endothelial cells with BP-1,6-Q caused a significant increase in dose-dependent cytotoxicity as measured by LDH release assay and both apoptotic and necrotic cell deaths as measured by flow cytometric analysis. Incubation of EA.hy926 endothelial cells with BP-1,6-Q also caused a significant decrease in cellular GSH levels. The diminishment of cellular GSH by buthionine sulfoximine (BSO) potentiated BP-1,6-Q-induced toxicity significantly suggesting a critical involvement of GSH in BP-1,6-Q induced cellular toxicity. GSH-induction by CDDO-Im significantly protects cells against BP-1,6-Q induced protein and lipid damage as measured by protein carbonyl (PC) assay and thiobarbituric acid reactive substances (TBARS) assay, respectively. However, the co-treatment of cells with CDDO-Im and BSO reversed the cytoprotective effect of CDDO-Im on BP-1,6-Q-mediated lipid peroxidation and protein oxidation. These results suggest that induction of GSH by CDDO-Im might be the important cellular defense against BP-1,6-Q induced protein and lipid damage. These findings would contribute to better understand the action of BP-1,6-Q and may help to develop novel therapies to protect against BP-1,6-Q-induced atherogenesis.

A secondary bile acid from microbiota metabolism attenuates ileitis and bile acid reduction in subclinical necrotic enteritis in chickens.

BACKGROUND: Clostridium perfringens-induced chicken necrotic enteritis (NE) is responsible for substantial economic losses worldwide annually. Recently, as a result of antibiotic growth promoter prohibition, the prevalence of NE in chickens has reemerged. This study was aimed to reduce NE through titrating dietary deoxycholic acid (DCA) as an effective antimicrobial alternative. MATERIALS AND METHODS: Day-old broiler chicks were assigned to six groups and fed diets supplemented with 0 (basal diet), 0.8, 1.0 and 1.5 g/kg (on top of basal diet) DCA. The birds were challenged with Eimeria maxima (20,000 oocysts/bird) at d 18 and C. perfringens (109 CFU/bird per day) at d 23, 24, and 25 to induce NE. The birds were sacrificed at d 26 when ileal tissue and digesta were collected for analyzing histopathology, mRNA accumulation and C. perfringens colonization by real-time PCR, targeted metabolomics of bile acids, fluorescence in situ hybridization (FISH), or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: At the cellular level, birds infected with E. maxima and C. perfringens developed subclinical NE and showed shortening villi, crypt hyperplasia and immune cell infiltration in ileum. Dietary DCA alleviated the NE-induced ileal inflammation in a dose-dependent manner compared to NE control birds. Consistent with the increased histopathological scores, subclinical NE birds suffered body weight gain reduction compared to the uninfected birds, an effect attenuated with increased doses of dietary DCA. At the molecular level, the highest dose of DCA at 1.5 g/kg reduced C. perfringens luminal colonization compared to NE birds using PCR and FISH. Furthermore, the dietary DCA reduced subclinical NE-induced intestinal inflammatory gene expression and cell apoptosis using PCR and TUNEL assays. Upon further examining ileal bile acid pool through targeted metabolomics, subclinical NE reduced the total bile acid level in ileal digesta compared to uninfected birds. Notably, dietary DCA increased total bile acid and DCA levels in a dose-dependent manner compared to NE birds. CONCLUSION: These results indicate that DCA attenuates NE-induced intestinal inflammation and bile acid reduction and could be an effective antimicrobial alternative against the intestinal disease.

Microbial metabolite deoxycholic acid controls Clostridium perfringens-induced chicken necrotic enteritis through attenuating inflammatory cyclooxygenase signaling.

Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide due to reduced usage of prophylactic antibiotics under consumer preferences and regulatory pressures. The lack of alternative antimicrobial strategies to control this disease is mainly due to limited insight into the relationship between NE pathogenesis, microbiome, and host responses. Here we showed that the microbial metabolic byproduct of secondary bile acid deoxycholic acid (DCA), at as low as 50 µM, inhibited 82.8% of C. perfringens growth in Tryptic Soy Broth (P < 0.05). Sequential Eimeria maxima and C. perfringens challenges significantly induced NE, severe intestinal inflammation, and body weight (BW) loss in broiler chickens. These negative effects were diminished (P < 0.05) by 1.5 g/kg DCA diet. At the cellular level, DCA alleviated NE-associated ileal epithelial death and significantly reduced lamina propria cell apoptosis. Interestingly, DCA reduced C. perfringens invasion into ileum (P < 0.05) without altering the bacterial ileal luminal colonization. Molecular analysis showed that DCA significantly reduced inflammatory mediators of Infγ, Litaf, Il1ß, and Mmp9 mRNA accumulation in ileal tissue. Mechanism studies revealed that C. perfringens induced (P < 0.05) elevated expression of inflammatory mediators of Infγ, Litaf, and Ptgs2 (Cyclooxygenases-2 (COX-2) gene) in chicken splenocytes. Inhibiting the COX signaling by aspirin significantly attenuated INFγ-induced inflammatory response in the splenocytes. Consistent with the in vitro assay, chickens fed 0.12 g/kg aspirin diet protected the birds against NE-induced BW loss, ileal inflammation, and intestinal cell apoptosis. In conclusion, microbial metabolic product DCA prevents NE-induced BW loss and ileal inflammation through attenuating inflammatory response. These novel findings of microbiome protecting birds against NE provide new options on developing next generation antimicrobial alternatives against NE.

Microbial metabolite deoxycholic acid shapes microbiota against Campylobacter jejuni chicken colonization.

Despite reducing the prevalent foodborne pathogen Campylobacter jejuni in chickens decreases campylobacteriosis, few effective approaches are available. The aim of this study was to use microbial metabolic product bile acids to reduce C. jejuni chicken colonization. Broiler chicks were fed with deoxycholic acid (DCA), lithocholic acid (LCA), or ursodeoxycholic acid (UDCA). The birds were also transplanted with DCA modulated anaerobes (DCA-Anaero) or aerobes (DCA-Aero). The birds were infected with human clinical isolate C. jejuni 81-176 or chicken isolate C. jejuni AR101. Notably, C. jejuni 81-176 was readily colonized intestinal tract at d16 and reached an almost plateau at d21. Remarkably, DCA excluded C. jejuni cecal colonization below the limit of detection at 16 and 28 days of age. Neither chicken ages of infection nor LCA or UDCA altered C. jejuni AR101 chicken colonization level, while DCA reduced 91% of the bacterium in chickens at d28. Notably, DCA diet reduced phylum Firmicutes but increased Bacteroidetes compared to infected control birds. Importantly, DCA-Anaero attenuated 93% of C. jejuni colonization at d28 compared to control infected birds. In conclusion, DCA shapes microbiota composition against C. jejuni colonization in chickens, suggesting a bidirectional interaction between microbiota and microbial metabolites.

Human colon mucosal biofilms from healthy or colon cancer hosts are carcinogenic.

Mucus-invasive bacterial biofilms are identified on the colon mucosa of approximately 50% of colorectal cancer (CRC) patients and approximately 13% of healthy subjects. Here, we test the hypothesis that human colon biofilms comprise microbial communities that are carcinogenic in CRC mouse models. Homogenates of human biofilm-positive colon mucosa were prepared from tumor patients (tumor and paired normal tissues from surgical resections) or biofilm-positive biopsies from healthy individuals undergoing screening colonoscopy; homogenates of biofilm-negative colon biopsies from healthy individuals undergoing screening colonoscopy served as controls. After 12 weeks, biofilm-positive, but not biofilm-negative, human colon mucosal homogenates induced colon tumor formation in 3 mouse colon tumor models (germ-free ApcMinΔ850/+;Il10-/- or ApcMinΔ850/+ and specific pathogen-free ApcMinΔ716/+ mice). Remarkably, biofilm-positive communities from healthy colonoscopy biopsies induced colon inflammation and tumors similarly to biofilm-positive tumor tissues. By 1 week, biofilm-positive human tumor homogenates, but not healthy biopsies, displayed consistent bacterial mucus invasion and biofilm formation in mouse colons. 16S rRNA gene sequencing and RNA-Seq analyses identified compositional and functional microbiota differences between mice colonized with biofilm-positive and biofilm-negative communities. These results suggest human colon mucosal biofilms, whether from tumor hosts or healthy individuals undergoing screening colonoscopy, are carcinogenic in murine models of CRC.

Microbial Colonization Coordinates the Pathogenesis of a Klebsiella pneumoniae Infant Isolate.

Enterobacteriaceae are among the first colonizers of neonate intestine. Members of this family, such as Escherichia and Klebsiella, are considered pathobionts and as such are capable of inducing local and systemic disease under specific colonization circumstances. Interplay between developing microbiota and pathogenic function of pathobionts are poorly understood. In this study, we investigate the functional interaction between various colonization patterns on an early colonizer, K. pneumoniae. K. pneumoniae 51-5 was isolated from stool of a healthy, premature infant, and found to contain the genotoxin island pks associated with development of colorectal cancer. Using intestinal epithelial cells, macrophages, and primary splenocytes, we demonstrate K. pneumoniae 51-5 upregulates expression of proinflammatory genes in vitro. Gnotobiotic experiments in Il10-/- mice demonstrate the neonate isolate induces intestinal inflammation in vivo, with increased expression of proinflammatory genes. Regulation of microbiota assembly revealed K. pneumoniae 51-5 accelerates onset of inflammation in Il10-/- mice, most significantly when microbiota is naturally acquired. Furthermore, K. pneumoniae 51-5 induces DNA damage and cell cycle arrest. Interestingly, K. pneumoniae 51-5 induced tumors in ApcMin/+; Il10-/- mice was not significantly affected by absence of colibactin activating enzyme, ClbP. These findings demonstrate pathogenicity of infant K. pneumoniae isolate is sensitive to microbial colonization status.

Microbiome modulates intestinal homeostasis against inflammatory diseases.

Eliminating prophylactic antibiotics in food animal production has exerted pressure on discovering antimicrobial alternatives (e.g. microbiome) to reduce elevated intestinal diseases. Intestinal tract is a complex ecosystem coupling host cells with microbiota. The microbiota and its metabolic activities and products are collectively called microbiome. Intestinal homeostasis is reached through dynamic and delicate crosstalk between host immunity and microbiome. However, this balance can be occasionally broken, which results in intestinal inflammatory diseases such as human Inflammatory Bowel Diseases, chicken necrotic enteritis, and swine postweaning diarrhea. In this review, we introduce the intestinal immune system, intestinal microbiome, and microbiome modulation of inflammation against intestinal diseases. The purpose of this review is to provide updated knowledge on host-microbe interaction and to promote using microbiome as new antimicrobial strategies to reduce intestinal diseases.