Growth performance, organs weight, intestinal histomorphology, and oocyst shedding in broiler chickens offered novel single strain Bacillus subtilis isolated from camel dung and challenged with Eimeria.

We evaluated a single strain Bacillus subtilis BS-9 direct-fed microbial (BSDFM) isolated from camel dung in Eimeria challenged broiler chickens. Seven-hundred d-old Ross 708 male chicks were placed in pens (25 birds/pen) and allocated to 2 treatments (n = 14). From d 0 to 13, control pens received untreated water (-BSDFM), and 2 treated pens received water and 2 mL x 108 colony forming unit/bird/d (+BSDFM); daily water intake (WI) was recorded. On d 9, birds in half (+Eimeria) of pens per treatment received of 1 mL of Eimeria maxima and Eimeria acervulina oocysts orally, and the other half (-Eimeria) sterile saline solution. Birds had ad libitum access to feed and a water line from d 14. Feed intake (FI), body weight (BW) and mortality were recorded for calculating BW gain (BWG) and feed conversion ratio (FCR). On d 14 and 35, samples of birds were necropsied for organ weight and intestinal measurements. Excreta samples were collected from d 14 to 19 for oocyst count. There was no treatment effect (P > 0.05) on growth performance or WI on d 0 to 9. There were interactions between BSDFM and Eimeria on d 19 (P = 0.014) and 29 (P = 0.036) BW with unchallenged +BSDFM birds being heavier than birds in the other treatments. The main effects (P < 0.05) on d 10 to 35 FI, BW, and BWG were such that +BSDFM increased and Eimeria decreased (P < 0.01) these parameters. There was interaction (P = 0.022) between BSDFM and Eimeria on d 10 to 35 FCR such that the FCR of challenged -BSDFM birds was poor than that of unchallenged counterparts, but none differed with +BSDFM birds. There was an interaction (P = 0.039) between BSDFM and Eimeria on d 14 bursa weight with challenged birds exhibiting heavier bursa than unchallenged +BSDFM birds. Eimeria reduced (P = 0.01) and BSDFM (P = 0.002) increased the villi height to crypt depth ratio. Results showed that BSDFM supplementation via water can support the growth performance of broiler chickens challenged with Eimeria and may be a strategy to reduce adverse effects of coccidiosis.

Comparative efficacy of a novel Bacillus subtilis-based probiotic and pharmacological zinc oxide on growth performance and gut responses in nursery pigs.

In this study, we assessed the efficacy of a novel Bacillus subtilis probiotic in improving growth performance and gut responses in comparison to pharmacological zinc oxide (ZnO) in nursery pigs. A total of 96 piglets were randomly assigned to four groups: Negative control (NC), Positive control (PC, 3000 mg Zn /kg feed), B.subtilis low dose (BS9-L, 2 × 107 CFU/pig) and B.subtilis high dose (BS9-H, 2 × 109 CFU/pig). Growth performance, diarrhea rate, gut mucosal gene expression and fecal microbial populations were evaluated. B.subtilis administration did not improve piglet bodyweight. BS9-L showed (P < 0.05) higher average daily gain (ADG) in Period 2 (D14-D28). BS9 groups had (P < 0.001) lower feed conversion ratio (FCR) in Period 2 (D14-D28) and overall. Like the ZnO-group, BS9 groups had lower (P < 0.01) diarrhea rate. A significant reduction (P < 0.05) in fecal E. coli, total coliforms, and an increase in lactic acid bacteria and Bacillus spp. in BS9 groups was observed. BS9 group had reduced (P < 0.05) mRNA levels of intestinal IL-8 and higher levels of MUC-1 and occludin and TJP-1 compared to negative control. These findings suggest that probiotic BS9, may promote growth performance, and ameliorate various indicators of intestinal health in piglets. Hence, it may serve as a prospective alternative to ZnO growth promoter in commercial swine production.

Dispersal of pathogen-associated multispecies biofilm by novel probiotic Bacillus subtilis in a contact-dependent manner.

AIMS: Biofilms are involved in pathogenesis of various bacterial infections. Treatment of biofilm-related bacterial infection remains a major challenge due to the reduced efficacy of antibiotics and associated antibiotic resistance. Given the high prevalence of Enterotoxigenic Escherichia coli (ETEC), Salmonella Typhimurium (S. Typhimurium) and methicillin-resistant Staphylococcus aureus (MRSA)-related infections and associated drug resistance, it is imperative to develop alternative strategies for treatment and prevention. The current study investigated antibiofilm activity of a recently isolated Bacillus subtilis (B. subtilis-9) against these pathogens. METHODS AND RESULTS: Crystal violet staining showed that treatment with B. subtilis-9 significantly reduced biofilm biomass of ETEC (60%-80%), S. Typhimurium (68%-73%) and MRSA (66%-82%). In addition, B. subtilis-9 significantly reduced pre-formed biofilm biomass of ETEC (59%), S. Typhimurium (62%), MRSA (65%) and multispecies (58%). Fluorescence microscopy revealed that B. subtilis-9 treatment significantly reduced the thickness of biofilm and viability of the embedded bacteria. Additionally, B. subtilis-9 significantly reduced planktonic cell growth of ETEC (92%), S. Typhimurium (94%) and MRSA (93%). Interestingly, transwell assay showed that B. subtilis-9 exhibited antibiofilm properties in a cell-to-cell contact-dependent manner and significantly reduced mRNA expression of biofilm-related genes, bssS, luxS and ihfB in ETEC. CONCLUSION: Novel B. subtilis-9 exhibits a strong inhibitory activity against ETEC, S. Typhimurium and MRSA biofilm formation and adhesion to abiotic surfaces. With further investigations, our study could bring forward a novel Bacillus-based probiotic intervention strategy to combat pathogenic biofilms, in clinical and agricultural settings. SIGNIFICANCE AND IMPACT OF THE STUDY: Probiotic bacteria propose a potential alternative in combating biofilm-related infections, however, data on the efficacy and strain selection are limited. Data from this study are critical in further developing Bacillus-based novel probiotic applications that may reduce the use of antibiotics in biofilm-related infections in humans and animals.

A Novel Probiotic Bacillus subtilis Strain Confers Cytoprotection to Host Pig Intestinal Epithelial Cells during Enterotoxic Escherichia coli Infection.

Enteric infections caused by enterotoxic Escherichia coli (ETEC) negatively impact the growth performance of piglets during weaning, resulting in significant economic losses for the producers. With the ban on antibiotic usage in livestock production, probiotics have gained a lot of attention as a potential alternative. However, strain specificity and limited knowledge on the host-specific targets limit their efficacy in preventing ETEC-related postweaning enteric infections. We recently isolated and characterized a novel probiotic Bacillus subtilis bacterium (CP9) that demonstrated antimicrobial activity. Here, we report anti-ETEC properties of CP9 and its impact on metabolic activity of swine intestinal epithelial (IPEC-J2) cells. Our results showed that pre- or coincubation with CP9 protected IPEC-J2 cells from ETEC-induced cytotoxicity. CP9 significantly attenuated ETEC-induced inflammatory response by reducing ETEC-induced nitric oxide production and relative mRNA expression of the Toll-like receptors (TLRs; TLR2, TLR4, and TLR9), proinflammatory tumor necrosis factor alpha, interleukins (ILs; IL-6 and IL-8), augmenting anti-inflammatory granulocyte-macrophage colony-stimulating factor and host defense peptide mucin 1 (MUC1) mRNA levels. We also show that CP9 significantly (P < 0.05) reduced caspase-3 activity, reinstated cell proliferation and increased relative expression of tight junction genes, claudin-1, occludin, and zona occludens-1 in ETEC-infected cells. Finally, metabolomic analysis revealed that CP9 exposure induced metabolic modulation in IPEC J2 cells with the greatest impact seen in alanine, aspartate, and glutamate metabolism; pyrimidine metabolism; nicotinate and nicotinamide metabolism; glutathione metabolism; the citrate cycle (TCA cycle); and arginine and proline metabolism. Our study shows that CP9 incubation attenuated ETEC-induced cytotoxicity in IPEC-J2 cells and offers insight into potential application of this probiotic for ETEC infection control. IMPORTANCE ETEC remains one of the leading causes of postweaning diarrhea and mortality in swine production. Due to the rising concerns with the antibiotic use in livestock, alternative interventions need to be developed. In this study, we analyzed the cytoprotective effect of a novel probiotic strain in combating ETEC infection in swine intestinal cells, along with assessing its mechanism of action. To our knowledge, this is also the first study to analyze the metabolic impact of a probiotic on intestinal cells. Results from this study should provide effective cues in developing a probiotic intervention for ameliorating ETEC infection and improving overall gut health in swine production.

Potential Probiotic Bacillus subtilis Isolated from a Novel Niche Exhibits Broad Range Antibacterial Activity and Causes Virulence and Metabolic Dysregulation in Enterotoxic E. coli.

Microbial life in extreme environments, such as deserts and deep oceans, is thought to have evolved to overcome constraints of nutrient availability, temperature, and suboptimal hygiene environments. Isolation of probiotic bacteria from such niche may provide a competitive edge over traditional probiotics. Here, we tested the survival, safety, and antimicrobial effect of a recently isolated and potential novel strain of Bacillus subtilis (CP9) from desert camel in vitro. Antimicrobial assays were performed via radial diffusion, agar spot, and co-culture assays. Cytotoxic analysis was performed using pig intestinal epithelial cells (IPEC-J2). Real time-PCR was performed for studying the effect on ETEC virulence genes and metabolomic analysis was performed using LC-MS. The results showed that CP9 cells were viable in varied bile salts and in low pH environments. CP9 showed no apparent cytotoxicity in IPEC-J2 cells. CP9 displayed significant bactericidal effect against Enterotoxic E. coli (ETEC), Salmonella Typhimurium, and Methicillin-resistant Staphylococcus aureus (MRSA) in a contact inhibitory fashion. CP9 reduced the expression of ETEC virulent genes during a 5 h co-culture. Additionally, a unique emergent metabolic signature in co-culture samples was observed by LC-MS analysis. Our findings indicate that CP9 exhibits a strong antibacterial property and reveals potential mechanisms behind.

Antiproliferative activity of long chain acylated esters of quercetin-3-O-glucoside in hepatocellular carcinoma HepG2 cells.

Despite their strong role in human health, poor bioavailability of flavonoids limits their biological effects in vivo. Enzymatically catalyzed acylation of fatty acids to flavonoids is one of the approaches of increasing cellular permeability and hence, biological activities. In this study, six long chain fatty acid esters of quercetin-3-O-glucoside (Q3G) acylated enzymatically and were used for determining their antiproliferative action in hepatocellular carcinoma cells (HepG2) in comparison to precursor compounds and two chemotherapy drugs (Sorafenib and Cisplatin). Fatty acid esters of Q3G showed significant inhibition of HepG2 cell proliferation by 85 to 90% after 6 h and 24 h of treatment, respectively. The cell death due to these novel compounds was associated with cell-cycle arrest in S-phase and apoptosis observed by DNA fragmentation, fluorescent microscopy and elevated caspase-3 activity and strong DNA topoisomerase II inhibition. Interestingly, Q3G esters showed significantly low toxicity to normal liver cells than Sorafenib (P < 0.05), a chemotherapy drug for hepatocellular carcinoma. Among all, oleic acid ester of Q3G displayed the greatest antiproliferation action and a high potential as an anti-cancer therapeutic. Overall, the results of the study suggest strong antiproliferative action of these novel food-derived compounds in treatment of cancer.

Flavonoid-enriched apple fraction AF4 induces cell cycle arrest, DNA topoisomerase II inhibition, and apoptosis in human liver cancer HepG2 cells.

Apples are a major source of dietary phytochemicals such as flavonoids in the Western diet. Here we report anticancer properties and possible mechanism of action of apple flavonoid-enriched fraction (AF4) isolated from the peels of Northern Spy apples in human hepatocellular carcinoma cells, HepG2. Treatment with AF4 induced cell growth inhibition in HepG2 cells in time- and dose-dependent manner. Concentration of 50 µg/ml (50 µg total monomeric polyphenols/ml) AF4 was sufficient to induce a significant reduction in cell viability within 6 h of treatment (92%, P < 0.05) but had very low toxicity (minimum 4% to maximum 16%) on primary liver and lung cells, which was significantly lower than currently prescribed chemotherapy drug Sorafenib (minimum 29% to maximum 49%, P < 0.05). AF4 induced apoptosis in HepG2 cells within 6 h of treatment via activation of caspase-3. Cell cycle analysis via flow-cytometer showed that AF4 induced G2/M phase arrest. Further, results showed that AF4 acts as a strong DNA topoisomerase II catalytic inhibitor, which may be a plausible reason to drive the cells to apoptosis. Overall, our data suggests that AF4 possesses a significantly stronger antiproliferative and specific action than Sorafenib in vitro and is a potential natural chemotherapy agent for treatment of liver cancer.

Quercetin-3-O-glucoside induces human DNA topoisomerase II inhibition, cell cycle arrest and apoptosis in hepatocellular carcinoma cells.

BACKGROUND: Dietary flavonoids have been associated with reduced risk of cancer including hepatocellular carcinoma (HCC). Quercetin-3-O-glucoside (Q3G) has been shown to possess anti-proliferative and antioxidant activities. The objectives of this study were to assess the anti-proliferative properties of Q3G in human liver cancer cells (HepG2); assess the cytotoxicity on normal primary cells; and elucidate its possible mechanism of action(s). MATERIALS AND METHODS: Using a dose- and time-dependent study, we evaluated the antiproliferative properties of Q3G in HepG2 cells using MTS cell viability assay and lactate dehydrogenase release assay. To elucidate the mechanism of action, we performed cell-cycle analysis using flow cytometry. Cell death via apoptosis was analyzed by DNA fragmentation assay, caspase-3 induction assay and fluorescence microscopy. DNA topoisomerase II drug screening assay was performed to assess the effect of Q3G on DNA topoisomerase II. RESULTS: Q3G treatment inhibited cell proliferation in a dose- and time-dependent manner in HepG2 cells with the blockade of the cell cycle in the S-phase. Additionally, Q3G exhibited a strong ability to inhibit DNA topoisomerase II. Furthermore, DNA fragmentation and fluorescence microscopy analysis suggested that Q3G induced apoptosis in HepG2 cells with the activation of caspase-3. Interestingly, Q3G exhibited significantly lower toxicity to normal cells (primary human and rat hepatocytes and primary lung cells) than sorafenib (p<0.05), a chemotherapy drug for hepatocellular carcinoma. The results suggest that Q3G is a potential antitumor agent against liver cancer with a possible mechanism of action via cell-cycle arrest and apoptosis. Further research should be performed to confirm these results in vivo.

Conversion of commensal Escherichia coli K-12 to an invasive form via expression of a mutant histone-like protein.

UNLABELLED: The HUα(E38K, V42L) mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397-16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUα(E38K, V42L) was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins Bim(EL) and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUα(E38K, V42L) variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes. IMPORTANCE: Escherichia coli K-12 is well established as a benign laboratory strain and a human intestinal commensal. Recent evidences, however, indicate that the typical noninvasive nature of resident E. coli can be reversed under specific circumstances even in the absence of any major genomic flux. We previously engineered an E. coli strain with a mutant histone-like protein, HU, which exhibited significant changes in nucleoid organization and global transcription. Here we showed that the changes induced by the mutant HU have critical functional consequences: from a strict extracellular existence, the mutant E. coli adopts an almost obligate intracellular lifestyle. The internalized E. coli exhibits many of the prototypical characteristics of traditional intracellular bacteria, like phagosomal escape, intracellular replication, and subversion of host cell apoptosis. We suggest that E. coli K-12 can switch between widely divergent lifestyles in relation to mammalian host cells by reprogramming its cellular transcription program and without gross changes in its genomic content.