An Investigation of Quorum Sensing Inhibitors against Bacillus cereus in The Endophytic Fungus Pithomyces sacchari of the Laurencia sp.

Bacillus cereus, a common food-borne pathogen, forms biofilms and generates virulence factors through a quorum sensing (QS) mechanism. In this study, six compounds (dankasterone A, demethylincisterol A3, zinnimidine, cyclo-(L-Val-L-Pro), cyclo-(L-Ile-L-Pro), and cyclo-(L-Leu-L-Pro)) were isolated from the endophytic fungus Pithomyces sacchari of the Laurencia sp. in the South China Sea. Among them, demethylincisterol A3, a sterol derivative, exhibited strong QS inhibitory activity against B. cereus. The QS inhibitory activity of demethylincisterol A3 was evaluated through experiments. The minimum inhibitory concentration (MIC) of demethylincisterol A3 against B. cereus was 6.25 µg/mL. At sub-MIC concentrations, it significantly decreased biofilm formation, hindered mobility, and diminished the production of protease and hemolysin activity. Moreover, RT-qPCR results demonstrated that demethylincisterol A3 markedly inhibited the expression of QS-related genes (plcR and papR) in B. cereus. The exposure to demethylincisterol A3 resulted in the downregulation of genes (comER, tasA, rpoN, sinR, codY, nheA, hblD, and cytK) associated with biofilm formation, mobility, and virulence factors. Hence, demethylincisterol A3 is a potentially effective compound in the pipeline of innovative antimicrobial therapies.

Impact of Bacillus cereus SPB-10 on Growth Promotion of Wheat (Triticum aestivum L.) Under Arsenic-Contaminated Soil.

This study investigates the impact of bacteria on arsenic reduction in wheat plants, highlighting the potential of microbe-based eco-friendly strategies for plant growth. In the present study, bacterial isolate SPB-10 was survived at high concentration against both form of arsenic (As3+ and As5+). SPB-10 produced 5.2 g/L and 11.3 g/L of exo-polysaccharide at 20 ppm of As3+ and As5+, respectively, whereas qualitative examination revealed the highest siderophores ability. Other PGP attributes such as IAA production were recorded 52.12 mg/L and 95.82 mg/L, phosphate solubilization was 90.23 mg/L and 129 mg/L at 20 ppm of As3+ and As5+, respectively. Significant amount of CAT, APX, and Proline was also observed at 20 ppm of As3+ and As5+ in SPB-10. Isolate SPB-10 was molecularly identified as Bacillus cereus through 16S rRNA sequencing. After 42 days, wheat plants inoculated with SPB-10 had a 25% increase in shoot length and dry weight, and 26% rise in chlorophyll-a pigment under As5+ supplemented T4 treatment than control. Reducing sugar content was increased by 24% in T6-treated plants compared to control. Additionally, SPB-10 enhanced the content of essential nutrients (NPK), CAT, and APX in plant's-leaf under both As3+ and As5+ stressed conditions after 42 days. The study found that arsenic uptake in plant roots and shoots decreased in SPB-10-inoculated plants, with the maximum reduction observed in As5+ treated plants. Bio-concentration factor-BCF was reduced by 90.89% in SPB-10-inoculated treatment T4 after 42 days. This suggests that Bacillus cereus-SPB-10 may be beneficial for plant growth in arsenic-contaminated soil.

Synthesis, structure, properties and antimicrobial activity of para trifluoromethyl phenylboronic derivatives.

The [2-formyl-4-(trifluoromethyl)phenyl]boronic acid as well as its benzoxaborole and bis(benzoxaborole) derivatives were obtained and their properties studied. The 2-formyl compound displays an unusual structure in the crystalline state, with a significant twist of the boronic group, whereas in DMSO solution it tautomerizes with formation of a cyclic isomer. All the studied compounds exhibit relatively high acidity as well as a reasonable antimicrobial activity. Docking studies showed interactions of all the investigated compounds with the binding pocket of Candida albicans LeuRS. High activity against Bacillus cereus was determined for the 2-formyl compound as well as for the novel bis(benzoxaborole), whereas the studied benzoxaborole shows high antifungal action with MIC values equal to 7.8and 3.9 µg/mL against C. albicans and A. niger respectively. None of the studied compounds exhibits reasonable activity against E. coli.

Effect of chlorhexidine gluconate on mechanical and anti-microbial properties of thermoplastic cassava starch.

Antimicrobial thermoplastic starch (TPS) was developed using cassava starch, glycerol, and chlorhexidine gluconate (CHG) blend. CHG was added at concentrations of 1%, 5%, 10%, and 20% (wt./wt.) as an antimicrobial additive. The tensile strength and hardness of the blended samples increased with the chlorhexidine gluconate content, especially for 1% CHG. wt./wt. (12.6 MPa and 94, respectively). The TPS/CHG20 blend exhibited a large phase of CHG and recrystallization of TPS. The water solubility decreased with the addition of CHG. Nuclear magnetic resonance data confirmed a reaction between the hydroxyl groups of TPS and the amino groups of CHG. The TPS/CHG20% exhibited an inhibition zone for three bacterial types (Staphylococcus aureus, Escherichia coli, and Bacillus cereus) and three fungal types (Rhizopus oligosporus, Aspergillus oryzae, and Candida albicans). CHG acted simultaneously as a chain extender and an antimicrobial additive for TPS, improving its tensile strength, hardness, and anti-microbial properties.

Heterologous expression of a natural product biosynthetic gene cluster from Cordyceps militaris.

Cordyceps is a genus of ascomycete fungi widely used in old Chinese medicine, and many investigations have focus on uncovering their biological activities. Until now, only a few compounds have been identified from Cordyceps, mainly due to their poor yield. So as to make full use of Cordyceps, we used the strategy of genome mining and heterologous expression to discover natural products (NPs) from Cordyceps militaris. Analysis of the genome sequence of Cordyceps militaris CM01 showed the presence of a cryptic gene cluster encoding a highly-reducing polyketide synthetase (HR-PKS), enoyl-reductase (ER) and cytochrome P450. Heterologous expression in Aspergillus nidulans enabled the identification of two new polyketides, cordypyrone A and B. Their structures were determined by 1D and 2D NMR techniques. They showed only modest activities against pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Mycobacteria tuberculosis and Bacillus cereus.

NMR Hydrophilic Metabolomic Analysis of Bacterial Resistance Pathways Using Multivalent Antimicrobials with Challenged and Unchallenged Wild Type and Mutated Gram-Positive Bacteria.

Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both Gram-positive and Gram-negative bacteria. Proton Nuclear Magnetic Resonance (1H NMR) metabolomics is an important method for studying resistance development in bacteria, since this is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. In this project, the metabolic differences between wild type Bacillus cereus (B. cereus) samples and B. cereus that was mutated through 33 growth cycles in a nonlethal dose of a multivalent antimicrobial agent were identified. For additional comparison, samples for analysis of the wild type and mutated strains of B. cereus were prepared in both challenged and unchallenged conditions. A C16-DABCO (1,4-diazabicyclo-2,2,2-octane) and mannose functionalized poly(amidoamine) dendrimer (DABCOMD) were used as the multivalent quaternary ammonium antimicrobial for this hydrophilic metabolic analysis. Overall, the study reported here indicates that B. cereus likely change their peptidoglycan layer to protect themselves from the highly positively charged DABCOMD. This membrane fortification most likely leads to the slow growth curve of the mutated, and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for the membrane fortifications to occur as well as to the decreased diffusion of nutrients across the mutated membrane.

Antibacterial Properties of Melanoidins Produced from Various Combinations of Maillard Reaction against Pathogenic Bacteria.

Novel melanoidins are produced by the Maillard reaction. Here, melanoidins with high antibacterial activity were tested by examining various combinations of reducing sugars and amino acids as reaction substrates. Twenty-two types of melanoidins were examined by combining two reducing sugars (glucose and xylose) and eleven l-isomers of amino acids (alanine, arginine, glutamine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine) to confirm the effects of these melanoidins on the growth of Listeria monocytogenes at 25°C. The melanoidins produced from the combination of d-xylose with either l-phenylalanine (Xyl-Phe) or l-proline (Xyl-Pro), for which absorbance at 420 nm was 3.5 ± 0.2, completely inhibited the growth of L. monocytogenes at 25°C for 48 h. Both of the melanoidins exhibited growth inhibition of L. monocytogenes which was equivalent to the effect of nisin (350 IU/mL). The antimicrobial spectrum of both melanoidins was also investigated for 10 different species of bacteria, including both Gram-positive and Gram-negative species. While Xyl-Phe-based melanoidin successfully inhibited the growth of Bacillus cereus and Brevibacillus brevis, Xyl-Pro-based melanoidin inhibited the growth of Salmonella enterica Typhimurium. However, no clear trend in the antimicrobial spectrum of the melanoidins against different bacterial species was observed. The findings in the present study suggest that melanoidins generated from xylose with phenylalanine and/or proline could be used as potential novel alternative food preservatives derived from food ingredients to control pathogenic bacteria. IMPORTANCE Although the antimicrobial effect of melanoidins has been reported in some foods, there have been few comprehensive investigations on the antimicrobial activity of combinations of reaction substrates of the Maillard reaction. The present study comprehensively investigated the potential of various combinations of reducing sugars and amino acids. Because the melanoidins examined in this study were produced simply by heating in an autoclave at 121°C for 60 min, the targeted melanoidins can be easily produced. The melanoidins produced from combinations of xylose with either phenylalanine or proline exhibited a wide spectrum of antibiotic effects against various pathogens, including Listeria monocytogenes, Bacillus cereus, and Salmonella enterica Typhimurium. Since the antibacterial effect of the melanoidins on L. monocytogenes was equivalent to that of a nisin solution (350 IU/mL), we might expect a practical application of melanoidins as novel food preservatives.

Antibacterial and antioxidant activities of endophytic fungi and nettle (Urtica dioica L.) leaves as their host.

Nettle (Urtica dioica L), as a plant rich in biologically active compounds, is one of the most important plants used in herbal medicine. Studies have shown that this plant has antioxidant, antiplatelet, hypoglycemic and hypocholesterolemia effects. In this study, we characterized three Alternaria endophytic fungi isolated from their host U. dioica. We hypothesized that these endophytic fungi can produce new bioactive metabolites, which may possess the bioactive property with potential application in the medical and pharmaceutical industries. The antibacterial activity was evaluated against reference and isolated strains, including Methicillin-Resistant Staphylococcus aureus. A wide range of antimicrobial activities similar to those measured in nettle leaves was detected especially for Alternaria sorghi. Furthermore, the highest antioxidant activity detected with DPPH free radical scavenging was measured for A. sorghi and nettle leaves ethyl acetate extracts. In addition, whereas catalase activity was similar in the three isolated fungi and nettle leaves, total thiol content and superoxide dismutase activity were significantly higher in leaves. A. sorghi showed the best activities compared to other isolated fungi. The characterization and further production of bioactive compounds produced by this endophyte should be investigated to fight bacteria and especially those that develop drug multi-resistance.

Hazelnut Shells as Source of Active Ingredients: Extracts Preparation and Characterization.

Hazelnut shells represent a waste material (about 42% of the total biomass) deriving from hazelnut harvest. These are mainly used as a heating source; however, they represent an interesting source of polyphenols useful in health field. The impact on phenolic profile and concentrations of hazelnut shell extracts obtained by three extraction methods (maceration, ultrasonic bath, and high-power ultrasonic), as well as temperature, extraction time, and preventive maceration, was studied. The prepared extracts were characterized in terms of chemical composition, antioxidant and antimicrobial activities. Eighteen different phenolic compounds were identified and quantified by chemical analysis and gallic acid was the most abundant in all the extracts analyzed. Other relevant compounds were chlorogenic acid, protocatechuic acid and catechin. Preventive maceration had a positive effect on the extraction of different types of compounds regardless of the method performed. Application of the high-power ultrasonic method had different effects, either positive or negative, depending on the type of compound and extraction time. All the prepared extracts showed antioxidant activity especially those prepared by maceration, and many of them were able to inhibit the growth of both B. subtilis and B. cereus.

Synthesis, antimicrobial activity and cytotoxicity of triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl nucleoside analogues.

Four new triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl nucleoside analogues were synthesized by coupling with 8-bromoctyl- or 10- bromdecyltriphenylphosphonium bromide and evaluated for the in vitro antibacterial activity against S. aureus, B. cereus, E. faecalis, two MRSA strains isolated from patients and resistant to fluoroquinolone antibiotic ciprofloxacin and ß-lactam antibiotic amoxicillin, E. coli, antifungal activity against T. mentagrophytes C. albicans and cytotoxicity against human cancer cell lines M-HeLa, MCF-7, A549, HuTu-80, PC3, PANC-1 and normal cell line Wi-38. In these compounds a TPP cation was attached via an octyl or a decyl linker to the N 3 atom of the heterocycle moiety (thymine, 6-methyluracil, quinazoline-2,4-dione) which was bonded with 2',3',5'-tri- O - acetyl-greek beta-d-ribofuranose residue by the (1,2,3-triazol-4-il)methyl bridge. All synthesized compounds showed high antibacterial activity against S. aureus within the range of MIC values 1.2-4.3 greek muM, and three of them appeared to be bactericidal with respect to tis bacterium at MBC values 4.1-4.3 greek muM. Two lead compounds showed both high antibacterial activity against the MRSA strains resistant to Ciprofloxacin and Amoxicillin within the range of MIC values 1.0-4.3 greek muM and high cytotoxicity against human cancer cell lines HuTu-80 and MCF-7 within the range of IC50 values 6.4-10.2 greek muM. This is one of the few examples when phosphonium salts exhibited both antibacterial activity and cytotoxicity against human cancer cell lines. According to the results obtained the bactericidal effect of the lead compounds, unlike classical surfactants, was not caused by a violation of the integrity of the cytoplasmic membrane of bacteria and their cytotoxic activity is most likely associated both with the induction of apoptosis along the mitochondrial pathway and the arrest of the cell cycle in the G0/G1 phase.

Impact of Phytochemicals on Viability and Cereulide Toxin Synthesis in Bacillus cereus Revealed by a Novel High-Throughput Method, Coupling an AlamarBlue-Based Assay with UPLC-MS/MS.

Due to its food-poisoning potential, Bacillus cereus has attracted the attention of the food industry. The cereulide-toxin-producing subgroup is of particular concern, as cereulide toxin is implicated in broadscale food-borne outbreaks and occasionally causes fatalities. The health risks associated with long-term cereulide exposure at low doses remain largely unexplored. Natural substances, such as plant-based secondary metabolites, are widely known for their effective antibacterial potential, which makes them promising as ingredients in food and also as a surrogate for antibiotics. In this work, we tested a range of structurally related phytochemicals, including benzene derivatives, monoterpenes, hydroxycinnamic acid derivatives and vitamins, for their inhibitory effects on the growth of B. cereus and the production of cereulide toxin. For this purpose, we developed a high-throughput, small-scale method which allowed us to analyze B. cereus survival and cereulide production simultaneously in one workflow by coupling an AlamarBlue-based viability assay with ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS). This combinatory method allowed us to identify not only phytochemicals with high antibacterial potential, but also ones specifically eradicating cereulide biosynthesis already at very low concentrations, such as gingerol and curcumin.

Tyrosine-Templated Dual-Component Silver Nanomaterials Exhibit Photoluminescence and Versatile Antimicrobial Properties through ROS Generation.

The role of small molecules in the preparation of metal nanomaterials generates considerable interest in the fields from materials science to interdisciplinary sciences. In this study, a small amino acid, l-tyrosine (Tyr), has been used as a ligand precursor for the preparation of silver nanomaterials (AgNMs) comprising a dual system: smaller silver nanoclusters (responsible exclusively for the photophysical properties) and larger silver nanoparticles (responsible exclusively for the antimicrobial properties). The luminescent properties of this AgNM system substantiate the role played by Tyr as a capping and a reducing agent outside the protein environment. An interesting feature of this report is the promising antimicrobial properties of the AgNMs against Saccharomyces cerevisiae, Candida albicans, Escherichia coli, and Bacillus cereus cell lines. The importance of this work is that this investigation demonstrates the combating ability of our AgNM system against pathogenic strains (C. albicans and B. cereus) as well. Moreover, the mechanistic aspects of the antimicrobial activity of the AgNMs were elucidated using various methods, such as propidium iodide staining, monitoring reactive oxygen species generation, leakage of proteins, DNA cleavage, etc. We propose that AgNM-mediated cytotoxicity in S. cerevisiae stems from the generation of singlet oxygen (1O2) species that create oxidative stress, disrupting the cell membrane and thereby resulting in leakage of proteins from the cells. This study can pave the way toward elucidating the role of a small molecule, Tyr, in the formation of NMs and describes the use of new NMs in potential antimicrobial applications.

Talinum paniculatum (Jacq.) Gaertn. leaves - source of nutrients, antioxidant and antibacterial potentials.

BACKGROUND: The diet of most of the population is limited to a reduced number of plants, even in areas that have a varied and extensive diversity, such as Brazil. Unconventional Food Plants (UFPs) are plants considered exotic, native, and wild that grow naturally and can be used as food. Among these is Talinum paniculatum (Jacq.) Gaertn., which is widespread throughout Brazil and can be a potential source of nutrients. Due to the potential of utilization of UFPs in human food and the lack of studies regarding the composition of T. paniculatum, this study aimed to assess the nutritional value of T. paniculatum leaves, their antioxidant capacity, and their antimicrobial activity for possible use in food. METHODS: The characterization of the leaves of T. paniculatum was carried out through analyses of proximal composition, color, ascorbic acid, mineral profile, and antinutritional factors showing the presence of condensed and hydrolysable tannins and nitrates in low concentrations. Solvents of water, ethanol, ethanol/water, methanol, methanol/water, methanol/acetic acid and acetone/water/acetic acid were used to evaluate the extraction yield of phenolic compounds, antioxidant capacity, and antibacterial activity of the extracts. RESULTS: High contents of protein (18.61 g 100 g-1), insoluble dietary fiber (34.75 g 100 g-1), ascorbic acid (81.03 mg 100 g-1), magnesium, potassium, and calcium (649.600, 411.520 and 228.117 mg 100 g-1, respectively) were observed. Extraction using the mixture of solvents of methanol/acetic acid showed the highest yield of phenolic compounds (432.73 mg EAG 100 g-1) and antioxidant capacity using the DPPH assay (3144.92 mg 100 g-1). Bacillus cereus growth was inhibited by the T. paniculatum extracts. CONCLUSIONS: T. paniculatum leaves are a source of nutrients and their extracts have antioxidant and antibacterial potentials which can be used as supplements in food to improve one's health.

Immobilization and Release Studies of Triazole Derivatives from Grafted Copolymer Based on Gellan-Carrying Betaine Units.

New polymer-bioactive compound systems were obtained by immobilization of triazole derivatives onto grafted copolymers and grafted copolymers carrying betaine units based on gellan and N-vinylimidazole. For preparation of bioactive compound, two new types of heterocyclic thio-derivatives with different substituents were combined in a single molecule to increase the selectivity of the biological action. The 5-aryl-amino-1,3,4 thiadiazole and 5-mercapto-1,2,4-triazole derivatives, each containing 2-mercapto-benzoxazole nucleus, were prepared by an intramolecular cyclization of thiosemicarbazides-1,4 disubstituted in acidic and basic medium. The structures of the new bioactive compounds were confirmed by elemental and spectral analysis (FT-IR and 1H-NMR). The antimicrobial activity of 1,3,4 thiadiazoles and 1,2,4 triazoles was tested on gram-positive and gram-negative bacteria. The triazole compound was chosen to be immobilized onto polymeric particles by adsorption. The Langmuir, Freundlich, and Dubinin-Radushkevich adsorption isotherm were used to describe the adsorption equilibrium. Also, the pseudo-first and pseudo-second models were used to elucidate the adsorption mechanism of triazole onto grafted copolymer based on N-vinylimidazole and gellan (PG copolymer) and grafted copolymers carrying betaine units (PGB1 copolymer). In vitro release studies have shown that the release mechanism of triazole from PG and PGB1 copolymers is characteristic of an anomalous transport mechanism.

Preparation and characterization of 2-hydroxyethyl starch microparticles for co-delivery of multiple bioactive agents.

The present study reports the generation of 2-hydroxyethyl starch microparticles for co-delivery and controlled release of multiple agents. The obtained microparticles are characterized by using Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray diffraction analysis, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. By using ofloxacin and ketoprofen as drug models, the release sustainability of the microparticles is examined at pH 1.2, 5.4, and 6.8 at 37 °C, with Fickian diffusion being found to be the major mechanism controlling the kinetics of drug release. Upon being loaded with the drug models, the microparticles show high efficiency in acting against Escherichia coli and Bacillus cereus. The results suggest that our reported microparticles warrant further development for applications in which co-administration of multiple bioactive agents is required.

Characterization of the antibacterial activity from ethanolic extracts of the botanical, Larrea tridentata.

BACKGROUND: ß-lactam antibiotics are a class of broad-spectrum antibiotics consisting of all antibiotic agents that contain a ß-lactam ring in their molecular structures. ß-lactam antibiotics are only known to be isolated from fungi (e.g. Acremonium chrysogenum, Penicillium chrysogenum and Aspergillus nidulans) and bacteria (e.g. Streptomyces clavuligerus). We have shown that botanical extracts prepared from Larrea tridentata have strong antimicrobial activity against several bacteria, including members of Staphylococcus and Streptococcus genera. METHODS: Through resistance studies, inhibitor assays, and ELISA testing, we demonstrated L. tridentata extracts may contain a ß-lactam type antibiotic activity. RESULTS: Based on the estimated ß-lactam concentration within the extract, the antimicrobial activity of the L. tridentata extract was approximately 2000-8000-fold greater against Staphylococcus as compared to other ß-lactams, penicillin or ampicillin. In the L. tridentata extract, this increased activity was found to be associated with the likely presence of a cofactor leading to increased potentiation of the ß-lactam activity. This potentiation activity was also observed to enhance the activity of exogenously added natural penicillin antibiotics. CONCLUSIONS: Although constituents were not isolated in this study, the results obtained strongly support the presence of ß-lactam type antibiotic activity and antibiotic potentiation activity present in ethanolic extracts prepared from L. tridentata.

Implication of a Key Region of Six Bacillus cereus Genes Involved in Siroheme Synthesis, Nitrite Reductase Production and Iron Cluster Repair in the Bacterial Response to Nitric Oxide Stress.

Bacterial response to nitric oxide (NO) is of major importance for bacterial survival. NO stress is a main actor of the eukaryotic immune response and several pathogenic bacteria have developed means for detoxification and repair of the damages caused by NO. However, bacterial mechanisms of NO resistance by Gram-positive bacteria are poorly described. In the opportunistic foodborne pathogen Bacillus cereus, genome sequence analyses did not identify homologs to known NO reductases and transcriptional regulators, such as NsrR, which orchestrate the response to NO of other pathogenic or non-pathogenic bacteria. Using a transcriptomic approach, we investigated the adaptation of B. cereus to NO stress. A cluster of 6 genes was identified to be strongly up-regulated in the early phase of the response. This cluster contains an iron-sulfur cluster repair enzyme, a nitrite reductase and three enzymes involved in siroheme biosynthesis. The expression pattern and close genetic localization suggest a functional link between these genes, which may play a pivotal role in the resistance of B. cereus to NO stress during infection.

Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes.

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

Aminoglycosides Antagonize Bacteriophage Proliferation, Attenuating Phage Suppression of Bacterial Growth, Biofilm Formation, and Antibiotic Resistance.

The common cooccurrence of antibiotics and phages in both natural and engineered environments underscores the need to understand their interactions and implications for bacterial control and antibiotic resistance propagation. Here, aminoglycoside antibiotics that inhibit protein synthesis (e.g., kanamycin and neomycin) impeded the replication of coliphage T3 and Bacillus phage BSP, reducing their infection efficiency and mitigating their hindrance of bacterial growth, biofilm formation, and tolerance to antibiotics. For example, treatment with phage T3 reduced subsequent biofilm formation by Escherichia coli liquid cultures to 53% ± 5% of that of the no-phage control, but a smaller reduction of biofilm formation (89% ± 10%) was observed for combined exposure to phage T3 and kanamycin. Despite sharing a similar mode of action with aminoglycosides (i.e., inhibiting protein synthesis) and antagonizing phage replication, albeit to a lesser degree, tetracyclines did not inhibit bacterial control by phages. Phage T3 combined with tetracycline showed higher suppression of biofilm formation than when combined with aminoglycosides (25% ± 6% of the no-phage control). The addition of phage T3 to E. coli suspensions with tetracycline also suppressed the development of tolerance to tetracycline. However, this suppression of antibiotic tolerance development disappeared when tetracycline was replaced with 3 mg/liter kanamycin, corroborating the greater antagonism with aminoglycosides. Overall, this study highlights this overlooked antagonistic effect on phage proliferation, which may attenuate phage suppression of bacterial growth, biofilm formation, antibiotic tolerance, and maintenance of antibiotic resistance genes. IMPORTANCE The coexistence of residual antibiotics and phages is common in many environments, which underscores the need to understand their interactive effects on bacteria and the implications for antibiotic resistance propagation. Here, aminoglycosides acting as bacterial protein synthesis inhibitors impeded the replication of various phages. This alleviated the suppressive effects of phages against bacterial growth and biofilm formation and diminished bacterial fitness costs that suppress the emergence of tolerance to antibiotics. We show that changes in bacteria caused by environmentally relevant concentrations of sublethal antibiotics can affect phage-host dynamics that are commonly overlooked in vitro but can result in unexpected environmental consequences.

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.