Gas Chromatography-Mass Spectrometry Profiling of Volatile Metabolites Produced by Some Bacillus spp. and Evaluation of Their Antibacterial and Antibiotic Activities.

Bacillus species produce different classes of antimicrobial and antioxidant substances: peptides or proteins with different structural compositions and molecular masses and a broad range of volatile organic compounds (VOCs), some of which may serve as biomarkers for microorganism identification. The aim of this study is the identification of biologically active compounds synthesized by five Bacillus species using gas chromatography coupled to mass spectrometry (GC-MS). The current study profoundly enhances the knowledge of antibacterial and antioxidant metabolites ensuring the unambiguous identification of VOCs produced by some Bacillus species, which were isolated from vegetable samples of potato, carrot, and tomato. Phylogenetic and biochemical studies were used to identify the bacterial isolates after culturing. Phylogenetic analysis proved that five bacterial isolates BSS12, BSS13, BSS16, BSS21, and BSS25 showed 99% nucleotide sequence similarities with Bacillus safensis AS-08, Bacillus cereus WAB2133, Bacillus acidiproducens NiuFun, Bacillus toyonesis FORT 102, and Bacillus thuringiensis F3, respectively. The crude extract was prepared from bacterial isolates to assess the antibiotic resistance potency and the antimicrobial potential against various targeted multidrug-resistant strains, including yeast strains such as Candida albicans, Candida krusei, and bacterial strains of Enterococcus hirae, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus group B, Streptococcus mutans, Shigella sonnei, Salmonella enteritidis, Serratia marcescens, Pseudomonas aeruginosa, and Proteus vulgaris. GC-MS analysis of bacterial strains found that VOCs from Bacillus species come in a variety of chemical forms, such as ketones, alcohols, terpenoids, alkenes, etc. Overall, 69 volatile organic compounds were identified from five Bacillus species, and all five were found to share different chemical classes of volatile organic components, which have a variety of pharmacological applications. However, eight antibacterial compounds with different concentrations were commonly found in all five species: acetoin, acetic acid, butanoic acid, 2-methyl-, oxime-, methoxy-phenyl, phenol, 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester, nonanoic acid, and hexadecanoic acid, methyl. The present study has demonstrated that bacterial isolates BSS25, BSS21, and BSS16 display potent inhibitory effects against Candida albicans, while BSS25, BSS21, and BSS13 exhibit the ability to restrain the growth and activity of Candida krusei. Notably, BSS25 and BSS21 are the only isolates that demonstrate substantial inhibitory activity against Klebsiella aerogenes. This disparity in inhibitory effects could be attributed to the higher concentrations of acetoin in BSS25 and BSS21, whereas BSS16 and BSS13 have relatively elevated levels of butanoic acid, 2-methyl-. Certainly, the presence of acetoin and butanoic acid, 2-methyl-, contributes to the enhanced antibacterial potential of these bacterial strains, in conjunction with other organic volatile compounds and peptides, among other factors. The biology and physiology of Bacillus can be better understood using these results, which can also be used to create novel biotechnological procedures and applications. Moreover, because of its exceptional ability to synthesize and produce a variety of different antibacterial compounds, Bacillus species can serve as natural and universal carriers for antibiotic compounds in the form of probiotic cultures and strains to fight different pathogens, including mycobacteria.

Sequential use of nutritional additives in diets for finishing Nellore steers in confinement.

The objective was to evaluate the effect of using prebiotics (Saccharomyces cerevisiae boulardii) or Monensin in the confinement initial phase and replacing monensin with probiotics (Bacillus toyonensis) in the final phase. Forty-eight Nellore steers were used, with an initial mean body weight of 356.2 ± 17.98 kg, distributed in a completely randomized design. Two animals per pen were confined in 80 m2 pens. The experiment was divided into two stages. The first phase lasted from day 1 to the 30th day, during which the animals were divided into two groups of 24 animals each. The treatments were the nutritional additives added to the diet: monensin or prebiotics (Saccharomyces cerevisiae boulardii). In the second phase, each group was subdivided into 12 animals by treatment, which received monensin or probiotics (Bacillus toyonensis). Dry matter intake (DMI), animal performance, and economic evaluation of the use of additives were evaluated. There was no additive effect on DMI, average daily gain, and total weight gain of the animals in the first experimental stage (0-30th day). Likewise, in the second stage (31st-100th day), there was no treatment effect for the variables of intake and performance. There was no effect of the use of different nutritional additives on carcass characteristics. The use of prebiotics sequentially to probiotics promoted gross and net yield that was superior to that of the animals that consumed monensin. Yeasts and bacteria respectively in the first and second phases of confinement can replace monensin in confinement diets.

Toyoncin, a Novel Leaderless Bacteriocin That Is Produced by Bacillus toyonensis XIN-YC13 and Specifically Targets B. cereus and Listeria monocytogenes.

Bacteriocins have attracted increasing interest because of their potential as natural preservatives. Recent studies showed that the Bacillus cereus group is a prominent producer of bacteriocins. Using a laboratory-based screening strategy, we identified a strain in the B. cereus group, Bacillus toyonensis XIN-YC13, with antimicrobial activity against B. cereus. A novel, 70-amino-acid-long leaderless bacteriocin, toyoncin, was purified from the culture supernatant of strain XIN-YC13, and its molecular mass was found to be 7,817.1012 Da. Toyoncin shares no similarity with any other known bacteriocins, and its N-terminal amino acid is formylmethionine rather than methionine. Toyoncin shows good pH and heat stability and exhibits specific antimicrobial activity against two important foodborne pathogens, B. cereus and Listeria monocytogenes. Additionally, toyoncin exerts bactericidal activity and induces cell membrane damage. Toyoncin can also inhibit the outgrowth of B. cereus spores. Preservation assays showed that toyoncin effectively suppressed or eradicated B. cereus and L. monocytogenes in pasteurized skim milk. These results suggest that toyoncin can be used as a new biopreservative against B. cereus and L. monocytogenes in the food industry. IMPORTANCE We identified a novel leaderless bacteriocin, toyoncin, produced by B. toyonensis XIN-YC13. Toyoncin shows good pH and heat stability, and it has specific antimicrobial activity against B. cereus and L. monocytogenes (two important foodborne pathogens), likely by destroying their cell membrane integrity. Toyoncin inhibited the outgrowth of B. cereus spores and effectively inhibited or eliminated B. cereus and L. monocytogenes in a milk model system. These results indicate the potential of toyoncin as a food preservative.

Biohybrid membranes for effective bacterial vehiculation and simultaneous removal of hexavalent chromium (CrVI) and phenol.

The aim of the present study was to obtain an effective vehiculation system in which bacterial agents could maintain viability improving their removal capacity. Herein, we present a novel biohybrid membrane of polymeric nanofibers and free-living bacteria for the simultaneous removal of pollutants. In this system, bacteria are free within the pores between the nanofibers and adsorbed to the surface of the membranes. Association between bacteria and the membranes was performed through a self-formulated medium, and the presence of the bacteria in the polymeric matrix was evidenced through atomic force microscopy (AFM). Biohybrid membranes associated with the remediation agents Bacillus toyonensis SFC 500-1E and Acinetobacter guillouiae SFC 500-1A promoted a reduction of up to 2.5 mg/L of hexavalent chromium and up to 200 mg/L of phenol after 24 h of treatment in synthetic medium containing the contaminants. Similarly, more than 46% of the hexavalent chromium and all of the phenol content were removed after treatment of a tannery effluent with initial concentrations of 7 mg/L of Cr(VI) and 305 mg/L of phenol. Counts of the remediation agents from the membranes were always above 1.107 CFU/g, also in the reutilization assays performed without reinoculation. Biohybrid membranes were hydrolysis-resistant, reusable, and effective in the simultaneous removal of contaminants for more than 5 cycles. Viability of the microorganisms was maintained after long-term storage of the membranes at 4 °C, without the use of microbiological media or the addition of cryoprotectants. Graphical abstract KEY POINTS: • Polymeric membranes were effectively associated with the SFC 500-1 remediation consortium • Biohybrid membranes removed hexavalent chromium and phenol from different matrices • Removal of contaminants was achieved in many successive cycles without reinoculation.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities.

Effects of media components and agricultural by-products on γ-polyglutamic acid production by Bacillus toyonensis As8.

BACKGROUND: Poly-γ­glutamic acid (γ­PGA) provides an environmentally friendly alternative to plastic materials which have widely polluted the environment. OBJECTIVES: The microbial production of γ­PGA, an amino acid biopolymer with glutamic acid subunits, was investigated using renewable agricultural residues in an attempt to find cheaper substitutes for conventional synthetic media components. MATERIAL AND METHODS: Bacteria which produce γ­PGA were isolated through depolymerizing Coix lacryma-jobi, a cellulosic grass, and the effects of various carbon and nitrogen sources, temperature, inoculant load, incubation period, and pH on γ­PGA yield were determined after submerged fermentation. Bacterial growth was measured turbidimetrically at 550 nm. The γ­PGA produced was characterized using Fourier transform infrared (FT-IR) spectroscopy and the polymer shape was determined using scanning electron microscopy (SEM). RESULTS: The best γ­PGA producer was molecularly identified as Bacillus toyonensis As8. The conditions which produced the highest γ­PGA yield were glucose, ammonium sulfate, 25°C, a pH of 5.5, and an incubation period of 48 h. This bacterium yielded the most γ­PGA (26.45 g/L) on cassava peels, while other agro-wastes (corn cob, sorghum leaves, Coix noir leaves, and rice bran) also supported bacterial growth with lower γ­PGA yields than conventional carbon sources. The wrinkled γ­PGA had absorbance peaks of hydroxyl, amide, carbonyl, and amine groups comparable with the ranges of those found in commercial γ­PGA. CONCLUSIONS: The use of agricultural by-products as fermentation substrates increased γ­PGA yield and may therefore be used as substitute components in γ­PGA production.

Genome analysis reveals insights of the endophytic Bacillus toyonensis BAC3151 as a potentially novel agent for biocontrol of plant pathogens.

Diseases caused by phytopathogenic microorganisms account for enormous losses for agribusiness. Although Bacillus species are recognized as being antimicrobial producers and some may provide benefits to plants, the association between Bacillus toyonensis and plants has not been studied. In this study, the whole-genome sequenced endophytic B. toyonensis BAC3151, which has demonstrated antimicrobial activity and quorum sensing inhibition of phytopathogenic bacteria, was investigated for its potential for the production of compounds for biocontrol of plant pathogens. Four whole-genome sequenced B. toyonensis strains shared 3811 protein-coding DNA sequences (CDSs), while strain-specific CDSs, such as biosynthetic gene clusters of antimicrobials, were associated with specific chromosomal regions and mobile genetic elements of the strains. B. toyonensis strains had a higher frequency of putative bacteriocins gene clusters than that of Bacillus species traditionally used for the production of antimicrobials. In addition, gene clusters potentially involved in the production of novel bacteriocins were found in BAC3151, as well as biosynthetic genes of several other compounds, including non-ribosomal peptides, N-acyl homoserine lactonase and chitinases, revealing a genetic repertoire for antimicrobial synthesis greater than that of other Bacillus strains that have demonstrated effective activity against phytopathogens. This study showed for the first time that B. toyonensis has potential to produce various antimicrobials, and the analyses performed indicated that the endophytic strain BAC3151 can be useful for the development of new strategies to control microbial diseases in plants that are responsible for large damages in agricultural crops.

A feed additive containing Bacillus toyonensis (Toyocerin(®) ) protects against enteric pathogens in postweaning piglets.

AIMS: This study evaluated the efficacy of a probiotic containing Bacillus toyonensis spores (Toyocerin(®) ) in postweaning piglets against enteric pathogens. METHODS AND RESULTS: Seven hundred and ninety-two healthy weaning pigs of a commercial farrow-to-finish pig farm were used. The negative control group fed without Toyocerin(®) and two experimental groups fed similar to the negative control group, but supplemented with Toyocerin(®) at t 500 mg kg(-1) diet (Toyocerin 500 group) and 1000 mg kg(-1) diet (Toyocerin 1000 group), respectively. No significant difference (P > 0.05) in morbidity and mortality rate between groups was noticed. The Toyocerin groups showed higher body weight (P < 0.05) and lower feed conversion ratio compared to the negative control group. Diarrhoea score was less in both Toyocerin groups than negative control group (P < 0.05). Moreover, the use of Toyocerin(®) at 1000 mg kg(-1) diet resulted in higher average daily feed intake compared to other groups (P < 0.05), reduction of some enteric pathogens and increase of the number of lactic acid bacteria. CONCLUSIONS: The use of Toyocerin(®) in weaning pigs, especially at 1000 mg kg(-1) diet, improved their health and growth performance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that a feed additive containing B. toyonensis (Toyocerin(®) ) protects against enteric pathogens in postweaning piglets when fed this additive at a proper dose. The use of Toyocerin(®) at 1000 mg kg(-1) diet resulted in higher average daily feed intake, decrease of some enteric pathogens and higher number of lactic acid bacteria. The effect of the probiotic in other age groups remains to be established.

Reduction of Cr(VI) by Bacillus toyonensis LBA36 and its effect on radish seedlings under Cr(VI) stress.

Chromium, being among the most toxic heavy metals, continues to demand immediate attention in the remediation of Cr-contaminated environments. In this study, a strain of LBA36 (Bacillus toyonensis) was isolated from heavy metal contaminated soil in Luanchuan County, Luoyang City, China. The reduction and adsorption rates of LBA36 in 30 mg·L-1 Cr-containing medium were 97.95% and 8.8%, respectively. The reduction mechanism was confirmed by Fourier-transform infrared spectroscopy, and X-ray photoelectron spectroscopy (XPS). Cr(VI) reduction by this strain predominantly occurred outside the cell, with hydroxyl, amide, carboxyl, C-N group, carbonyl, and sulfur carbonyl as the main reaction sites. XPS analysis revealed the presence of Cr2p1/2 and Cr2p3/2. Furthermore, the hydroponic experiment showed that the fresh weight and plant height of radish seedlings increased by 87.87% and 37.07%, respectively, after inoculation with LBA36 strain under 7 mg·L-1 Cr(VI) stress. The levels of chlorophyll, total protein, malondialdehyde, superoxide dismutase and catalase were also affected to different degrees. In conclusion, this study demonstrated the potential of microbial and phytoremediation in the treatment of heavy metal toxicity, and laid the foundation for the development of effective bioremediation methods for Cr(VI) pollution.

The Complete Genome Sequence of Bacillus toyonensis Cbmb3 with Polyvinyl Chloride-Degrading Properties.

The accumulation of high amounts of plastic waste in the environment has raised ecological and health concerns, particularly in croplands, and biological degradation presents a promising approach for the sustainable treatment of this issue. In this study, a polyvinyl chloride (PVC)-degrading bacterium was isolated from farmland soil samples attached to waste plastic, utilizing PVC as the sole carbon source. The circular chromosome of the strain Cbmb3, with a length of 5,768,926 bp, was subsequently sequenced. The average GC content was determined to be 35.45%, and a total of 5835 open reading frames were identified. The strain Cbmb3 was designated as Bacillus toyonensis based on phylogenomic analyses and genomic characteristics. The bioinformatic analysis of the Cbmb3 genome revealed putative genes encoding essential enzymes involved in PVC degradation. Additionally, the potential genomic characteristics associated with phytoprobiotic effects, such as the synthesis of indole acetic acid and secondary metabolite synthesis, were also revealed. Overall, the present study provides the first complete genome of Bacillus toyonensis with PVC-degrading properties, suggesting that Cbmb3 is a potential strain for PVC bioremediation and application.

Application of biohybrid membranes for arsenic and chromium removal and their impact on pollutant accumulation in soybean (Glycine max L.) seedlings.

Chromium and arsenic are among the priority pollutants to be controlled by regulatory and health agencies due to their ability to accumulate in food chains and the harmful effects on health resulting from the ingestion of food contaminated with metals and metalloids. In the present work, four biohybrid membrane systems were developed as alternatives for the removal of these pollutants, three based on polyvinyl alcohol polymeric mesh (PVA, PVA-magnetite, PVA L-cysteine) and one based on polybutylene adipate terephthalate (PBAT), all associated with bioremediation agents. The efficiency of the bioassociation process was assessed through count methods and microscopy. The removal capacity of these systems was evaluated in synthetic liquid medium, both in the absence and in the presence of soybean (Glycine max L.) seedlings. The content of chromium and arsenic was also analyzed in aerial and hypogeous tissues of seedlings grown on contaminated solid substrate. PVA and PVA-magnetite biohybrid membranes showed the highest removal rates, between 57 and 75% of the initial arsenic content and more than 80% of the initial chromium content after 48 h of treatment, when evaluated in synthetic liquid media with initial concentrations of 2.5 ppm of pentavalent arsenic and 5 ppm of hexavalent chromium, both in presence and absence of seedlings. PVA and PBAT promoted a significant reduction of arsenic translocation to the aerial parts, generally edible, of this crop of agronomic interest. The systems tested showed a high potential for biotechnological applications in matrices affected by the presence of arsenic and chromium.

Effect of Bacillus toyonensis BCT-7112T supplementation on growth performance, intestinal morphology, immune-related gene expression, and gut microbiome in Barbary ducks.

This study aimed to investigate the effect of Bacillus toyonensis BCT-7112T supplementation on growth performance, intestinal morphology, immune-related gene expression, and the cecal microbiota of meat ducks. A total of 150 one-day-old male Barbary ducks were divided into 3 groups with 5 replicates (n = 10 ducks per replicate) by completely randomized design and offered diets supplemented with the commercial product Toyocerin (containing 1 × 109B. toyonensis BCT-7112T viable spores/g product) at the levels of 0, 500, or 1,000 mg/kg (0, 500, or 1,000 ppm), respectively, for 8 wk. The results showed that although ducks in the 500 ppm B. toyonensis BCT-7112T group displayed numerically better values (e.g., weight gain and feed conversion ratio) than those in the control group, the growth performance of ducks fed diets supplemented with B. toyonensis BCT-7112T did not differ significantly from that of the control group (P > 0.05). There were no significant differences in the intestinal mucosal morphology of ducks across the experimental groups (P > 0.05). However, ducks in the 500 ppm B. toyonensis BCT-7112T group showed a trend of greater values, for example, villus height per crypt depth of duodenum (P = 0.16) and ileum (P = 0.12) compared with those in the control group. The relative expression of immune-related genes, for example, interferon (IFN) and interleukin-6 (IL-6) in the meat duck spleen was significantly lower in both B. toyonensis BCT-7112T groups at 14 d and 35 d than in the control group (P < 0.05). Beta diversity analysis of the cecal microbiota of ducks in either the 500 ppm or the 1,000 ppm B. toyonensis BCT-7112T group showed to have higher diversity than that in the control group, where at the phylum level, Bacteroidetes was the most abundant, followed by Firmicutes, and at the genus level, Bacteroides, Fusobacterium, and Ruminococcaceae were the top 3 most abundant genera. In conclusion, our study demonstrates that 500 ppm supplementation with B. toyonensis BCT-7112T in duck diets can reduce proinflammatory cytokine gene expression, improve immunological function, and increase the variety of microbial communities in the ceca of meat-type ducks.

Comparable Evaluation of Nutritional Benefits of Lactobacillus plantarum and Bacillus toyonensis Probiotic Supplementation on Growth, Feed Utilization, Health, and Fecal Microbiota in Pre-Weaning Male Calves.

This study was conducted to investigate the impact of probiotic supplementation using Lactobacillus plantarum DSA 20174 and/or Bacillus toyonensis ATCC 55050 on growth performance, blood parameters, hematological measures, and fecal microbiota in pre-weaning Holstein calves. Thirty-two four-day-old male calves with a similar genetic background, weighing an average of 38.27 ± 0.12 kg, were randomly assigned to four groups. The groups consisted of a control group (CON) without supplementation, a group receiving B. toyonensis (BT) at 3 × 109 cfu/calf/day, a group receiving L. plantarum (LP) at 1 × 1010 cfu/calf/day, and a group receiving a combination of LP and BT (LP + BT) at half the dosage for each. The study found that calves supplemented with LP and LP + BT experienced significant improvements in average daily gain and final body weight compared to the control group. The LP + BT group showed the most positive effects on TDMI, starter intake, and CP intake. RBC counts tended to be higher in the probiotic groups, with the LP + BT group having the highest values. The LP + BT group also had higher total protein, albumin, globulin, and hematocrit concentrations. All probiotic groups showed higher serum IgG concentrations. Probiotic supplementation led to increased total bacterial count and decreased levels of E. coli, salmonella, and clostridium. The LP + BT group had a significant decrease in coliform count, while both LP and LP + BT groups had increased Lactobacillus populations. In conclusion, LP + BT probiotic supplement showed the most beneficial effects on growth, feed efficiency, blood constituents, and modulation of fecal microbiota composition.

Identification, Characterization, and Production Optimization of 6-Methoxy-1H-Indole-2-Carboxylic Acid Antifungal Metabolite Produced by Bacillus toyonensis Isolate OQ071612.

Fungal infections currently pose a real threat to human lives. In the current study, soil bacterial isolates were screened for the production of antifungal compounds to combat human fungal pathogens. Notably, the bacterial F1 isolate exhibited antimycotic action towards the Candida albicans ATCC 10231 and Aspergillus niger clinical isolates. By employing phenotypic and molecular techniques, we identified the F1 isolate as the Bacillus toyonensis isolate OQ071612. The purified extract showed stability within a pH range of 6-7 and at temperatures of up to 50 °C. It demonstrated potential antifungal activity in the presence of various surfactants, detergents, and enzymes. The purified extract was identified as 6-methoxy-1H-Indole-2-carboxylic acid using advanced spectroscopic techniques. To optimize the antifungal metabolite production, we utilized response surface methodology (RSM) with a face-centered central composite design, considering nutritional and environmental variables. The optimal conditions were as follows: starch (5 g/L), peptone (5 g/L), agitation rate of 150 rpm, pH 6, and 40 °C temperature. A confirmatory experiment validated the accuracy of the optimization process, resulting in an approximately 3.49-fold increase in production. This is the first documented report on the production and characterization of 6-methoxy-1H-Indole-2-carboxylic acid (MICA) antifungal metabolite from Bacillus toyonensis.

Bacillus toyonensis SAU-19 and SAU-20 Isolated From Ageratina adenophora Alleviates the Intestinal Structure and Integrity Damage Associated With Gut Dysbiosis in Mice Fed High Fat Diet.

This study was performed to identify potential probiotic endophytes from Ageratina adenophora and evaluate their ameliorating effects on gut injury and integrity damage associated with microbiota dysbiosis in mice fed high fat diet. Using morphological and biochemical tests, and 16S rRNA gene sequencing technique, two bacteria endophytes were identified as strains of Bacillus toyonensis and were named Bacillus toyonensis SAU-19 (GenBank No. MW287198) and Bacillus toyonensis SAU-20 (GenBank No. MW287199). Sixty (60) mice were divided into five groups, group 1 was the negative control fed normal diet (NS), group 2 was fed High fat diet (HF), Group 3 was fed High fat diet + 106 Lactobacillus rhamnosus (LGG), group 4 was fed High fat + 106 Bacillus toyonensis SAU-19 and group 5 fed High fat diet + 106 Bacillus toyonensis SAU-20. After 35 days, histological and immunohistochemistry examination were performed in the ileum tissues. Furthermore, DAO and antioxidants activities were measured in serum, mRNA expressions of tight junction proteins (occludin and ZO-1) and inflammation related cytokines (IL-1ß, TFN-α, IL-2, IL-4, and IL-10) in the ileum tissues as well as sIgA levels and total bacteria (Escherichia coli, Salmonella, Staphylococcus, and Lactobacillus) in the small intestine and cecum content. The results showed an increase in the DAO activity, oxidative stress parameter (MDA), pro-inflammation cytokines (IL-1ß, TFN-α, IL-2), reduce immunity (sIgA), and destroyed intestinal structure and integrity (reduce tight junction proteins) in the high fat diet group and this was associated with destruction of the gut microbiota composition (increasing pathogenic bacteria; E. coli, Salmonella, Staphylococcus and reducing beneficial bacteria, Lactobacillus spp.) in mice (P < 0.05). However, the administration of Bacillus toyonensis SAU-19 and SAU-20 reverted these effects. Our findings indicated that, Bacillus toyonensis SAU-19 and SAU-20 isolated from A. adenophora could prevent the excess weight gain from high fat diet feeding, improved antioxidant status and alleviated the intestine integrity damage as well as reduce the population of enteric bacteria such as E. coli, Salmonella, and S. aureus and increasing the population of beneficial bacteria such as Lactobacillus in the gut of mice fed high fat diet, therefore, can serve as a potential probiotics in humans and animals.

Oral Administration of Bacillus toyonensis Strain SAU-20 Improves Insulin Resistance and Ameliorates Hepatic Steatosis in Type 2 Diabetic Mice.

In this study, the ameliorative effects of Bacillus toyonensis-SAU-20 (B. toyo SAU-20), a new probiotic strain isolated and identified by our laboratory from Ageratina adenophora, on the development of insulin resistance and hepatic steatosis in type 2 diabetic (T2DM) mice was investigated. Thirty Specific-pathogen free Kunming (SPFKM) mice were randomly allocated to three groups: control, high fat diet/streptozotocin (HFD/STZ), and HFD/STZ+B. toyo SAU-20 groups with oral administration of B. toyo SAU-20 for 35 days. Biochemistry parameters, glucose tolerance, and insulin resistance were measured in the blood whereas histological analysis, inflammatory cytokines and lipogenic genes in the liver tissues. The results showed that, the levels of serum glucose, lipid profile, mRNA expression of lipogenic related genes and pro-inflammatory cytokines were significantly increased in T2DM mice. However, after B. toyo SAU-20 administration, the elevation of these parameters was significantly suppressed (P<0.05). In addition, the feeding of B. toyo SAU-20 significantly improved the morphological changes of the liver with significant alleviation of dyslipidemia, oxidative stress status and inflammation (P<0.05) indicating the ameliorating effect of B. toyo SAU-20 in hepatic steatosis in T2DM. Therefore, we concluded that, B. toyo SAU-20 alleviated insulin resistance and hepatic steatosis by improving the lipid profiles, antioxidant status and downregulating lipogenic genes as well as pro-inflammation cytokines expression.

Genomic characterisation of multidrug-resistant Bacillus toyonensis strain 4HC1 isolated from marine plastic in Norway.

OBJECTIVES: Bacillus toyonensis is widespread in nature. Multidrug-resistant B. toyonensis strain 4HC1 was isolated from polyethylene submerged in the water column near a beach in Øygarden, Norway. We analysed the whole genome sequence of strain 4HC1 in order to understand the genetic basis of the observed phenotypic antibiotic resistance. METHODS: Whole-genome sequencing of B. toyonensis strain 4HC1 was performed on Illumina MiSeq platform using 2 × 300 bp chemistry. The genome sequence was assembled using SPAdes v.3.13.0 and was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). RESULTS: The draft genome of strain 4HC1 is 6 156 259 bp (133 contigs) in size with a GC content of 34.95%. The genome comprises 6089 protein-coding genes, 86 tRNAs and 24 rRNAs. Strain 4HC1 is resistant to cefotaxime, trimethoprim and ampicillin and carries various antibiotic resistance genes (ARGs), including several ß-lactamases, aminoglycoside 6-adenylyltransferase, a TetM family tetracycline resistance gene, two different tetracycline efflux pumps, and a bleomycin resistance gene. Several virulence genes including genes involved in immune evasion, iron acquisition and toxins were also detected in strain 4HC1. CONCLUSION: The draft genome sequence of B. toyonensis strain 4HC1 released here shows the presence of various ARGs and virulence genes in a multidrug-resistant strain isolated from marine plastic.

Improving the bioremediation technology of contaminated wastewater using biosurfactants produced by novel bacillus isolates.

Biosurfactants have many advantages outside chemical one, led for application it through different sectors. So, the present study aimed for improving the bioremediation technology of contaminated wastewater using biosurfactants produced by novel bacillus isolates. In this regard, Bacillus thuringiensis and Bacillus toyonensis strains were obtained as most producing isolates of highly active biosurfactants. The optimized conditions for high biosurfactants yield production were established. Also, the stability of the produced biosurfactants at various conditions, pH, temperature and salinity was studied. The biosurfactant has been reported up to 120 °C, pH 12 and 10% of NaCl. The identified biosurfactants, decanoic acid and oleamide were applied for wastewater remediation from oil residues and pathogens contamination. The biosurfactant was had high antibacterial activity compared with references antimicrobial drugs, as well as it is enhanced bioremediation technology for petroleum oil residues contaminating sites. Thus, we can say, these biosurfactants could achieve the objectives of sustainable development.

Characterization of a Deep Sea Bacillus toyonensis Isolate: Genomic and Pathogenic Features.

Bacillus toyonensis is a group of Gram-positive bacteria belonging to the Bacillus cereus group and used in some cases as probiotics or biocontrol agents. To our knowledge, B. toyonensis from the deep sea (depth >1,000 m) has not been documented. Here, we report the isolation and characterization of a B. toyonensis strain, P18, from a deep sea hydrothermal field. P18 is aerobic, motile, and able to grow at low temperatures (4°C) and high concentrations of NaCl (8%). P18 possesses a circular chromosome of 5,250,895 bp and a plasmid of 536,892 bp, which encode 5,380 and 523 genes, respectively. Of these genes, 2,229 encode hypothetical proteins that could not be annotated based on the COG database. Comparative genomic analysis showed that P18 is most closely related to the type strain of B. toyonensis, BCT-7112T. Compared to BCT-7112T, P18 contains 1,401 unique genes, 441 of which were classified into 20 COG functional categories, and the remaining 960 genes could not be annotated. A total of 319 putative virulence genes were identified in P18, including toxin-related genes, and 24 of these genes are absent in BCT-7112T. P18 exerted strong cytopathic effects on fish and mammalian cells that led to rapid cell death. When inoculated via injection into fish and mice, P18 rapidly disseminated in host tissues and induced acute infection and mortality. Histopathology revealed varying degrees of tissue lesions in the infected animals. Furthermore, P18 could survive in fish and mouse sera and possessed hemolytic activity. Taken together, these results provide the first evidence that virulent B. toyonensis exists in deep sea environments.

Bacillus toyonensis SAU-19 Ameliorates Hepatic Insulin Resistance in High-Fat Diet/Streptozocin-Induced Diabetic Mice.

Insulin resistance (IR) is a hallmark of type 2 diabetes mellitus (T2DM). This study was performed to investigate the antidiabetic effect of Bacillus toyonensis SAU-19 and its possible mechanisms of action in mice with type 2 diabetes mellitus (T2DM). Thirty SPFKM mice were randomly assigned to three groups: control, diabetic model, and diabetes + Bacillus toyonensis SAU-19 group. After 35 days, blood was collected for biochemical analysis and liver tissue samples for histopathological analysis using H&E staining, qPCR, and ELISA. The results showed that the administration of B. toyonensis SAU-19 significantly improved the blood glucose, hepatic insulin resistance, and morphological changes of the liver characterized by significant improvement of dyslipidemia, glycogen synthesis, and antioxidant status (p < 0.05), indicating the strains' ameliorating effects on hepatic insulin resistance in T2DM. In conclusion, the probiotic strain (B. toyonensis SAU-19) inhibits T2DM by reducing insulin resistance, improving antioxidant status, and downregulating genes related to glucose synthesis; hence, it may be used in treating diabetes and other metabolic disorders. This study provides the basis for further studies into the molecular mechanisms of B. toyonensis SAU-19 in treating T2DM.