Integrated Genomic and Metabolomic Analysis Illuminates Key Secreted Metabolites Produced by the Novel Endophyte Bacillus halotolerans Cal.l.30 Involved in Diverse Biological Control Activities.

The endophytic strain Cal.l.30, isolated from the medicinal plant Calendula officinalis, was selected among seven Bacillus strains with plant growth promoting activity and strong biological potential against the postharvest fungal pathogen Botrytis cinerea. Treatment by inoculating Cal.l.30 bacterial cell culture or cell free supernatant on harvested grapes and cherry tomato fruits, significantly reduced gray mold disease severity index and disease incidence. Based on 16S rRNA sequence analysis and whole genome phylogeny, Cal.l.30 was identified as Bacillus halotolerans. Genome mining revealed that B. halotolerans Cal.l.30 is endowed with a diverse arsenal of secondary metabolite biosynthetic gene clusters (SM-BGCs) responsible for metabolite production with antimicrobial properties. A sub-set of the identified SM-BGCs (mojavensin A, 'bacillunoic acid') appears to be the result of recent horizontal gene transfer events. Its genome was also mined for CAZymes associated with antifungal activity. Further UHPLC-HRMS analysis indicated that Cal.l.30 synthesizes and secretes secondary metabolites with antimicrobial activity, including the lipopeptides, fengycin, surfactin and mojavensin A, bacillaene isoforms, L-dihydroanticapsin and bacillibactin. Other compounds with known antimicrobial activity were also detected, such as azelaic acid, 15- hydroxypentadecanoid acid and 2-hydroxyphenylacetic acid. The genomic and metabolomic features of the B. halotolerans Cal.l.30 provided new perspectives on the exploitation of novel Bacillus sp. as a biocontrol agent.

Genomic and Metabolomic Insights into Secondary Metabolites of the Novel Bacillus halotolerans Hil4, an Endophyte with Promising Antagonistic Activity against Gray Mold and Plant Growth Promoting Potential.

The endophytic bacterial strain Hil4 was isolated from leaves of the medicinal plant Hypericum hircinum. It exhibited antifungal activity against Botrytis cinerea and a plethora of plant growth promoting traits in vitro. Whole genome sequencing revealed that it belongs to Bacillus halotolerans and possesses numerous secondary metabolite biosynthetic gene clusters and genes involved in plant growth promotion, colonization, and plant defense elicitation. The Mojavensin cluster was present in the genome, making this strain novel among plant-associated B. halotolerans strains. Extracts of secreted agar-diffusible compounds from single culture secretome extracts and dual cultures with B. cinerea were bioactive and had the same antifungal pattern on TLC plates after bioautography. UHPLC-HRMS analysis of the single culture secretome extract putatively annotated the consecutively produced antimicrobial substances and ISR elicitors. The isolate also proved efficient in minimizing the severity of gray mold post-harvest disease on table grape berries, as well as cherry tomatoes. Finally, it positively influenced the growth of Arabidopsis thaliana Col-0 and Solanum lycopersicum var. Chondrokatsari Messinias after seed biopriming in vitro. Overall, these results indicate that the B. halotolerans strain Hil4 is a promising novel plant growth promoting and biocontrol agent, and can be used in future research for the development of biostimulants and/or biological control agents.

Reverse micelles as nanocarriers of nisin against foodborne pathogens.

Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively designed. Structural characterization of the RMs was assessed using Electron Paramagnetic Resonance Spectroscopy and Small Angle X-ray Scattering in the presence and absence of nisin. No conformational changes occurred in the presence of nisin for the nanocarriers. To assess efficacy of the loaded systems, their antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes was tested in lettuce leaves and minced meat, respectively. Antimicrobial activity was evident in both cases. Interestingly, a synergistic antimicrobial effect was observed in lettuce leaves and to a lesser extent in minced meat between nisin and some of the nanocarriers' constituents (probably ethanol). Our findings suggest complex interactions that take place when RMs are applied in different food matrices.

Microemulsions as Potential Carriers of Nisin: Effect of Composition on Structure and Efficacy.

Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers in order to ensure its effectiveness as a biopreservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil, and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by electrical conductivity measurements, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles' size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased, whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the well diffusion assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations, but no significant difference was observed between the two used vegetable oils (ROO and SO).

Effects of local application of simvastatin on bone regeneration in femoral bone defects in rabbit.

Simvastatin (SIM), which is widely used in hyperlipidemia treatment, has also attracted attention due to its anabolic effects on bones. This study is designed to investigate the effectiveness of 2 mg SIM combined with 3 different carriers as delivery systems. Bone defects were surgically created in the femoral bones of 14 New Zealand white rabbits. The carriers used were the inorganic bovine bone graft (BOS), the hydroxyapatite combined with calcium sulfate (HACS), and the collagen sponge (COS). The bone defects were divided for each time period into 7 groups, as follows: passive control group (CONT), active control groups (BOS), (HACS) and (COS) (no simvastatin), and groups (BOS + SIM), (HACS + SIM) (carrier and simvastatin combination). Animal were sacrificed at 4 and 8 weeks postoperatively, and bone defects areas were prepared for histological examination and histomorphometric evaluation. Analysis of variance demonstrated statistically significant differences between groups depending on the carrier used. At 4 weeks, the BOS + SIM group presented higher rates of new bone formation, whereas at 8 weeks more new bone formation was noted for the HACS + SIM group. This study suggests that local application of simvastatin, combined with an appropriate carrier, can promote new bone formation.

Feed supplementation of Lactobacillus plantarum PCA 236 modulates gut microbiota and milk fatty acid composition in dairy goats--a preliminary study.

This study aimed to evaluate the potential of a promising Lactobacillus plantarum isolate (PCA 236) from cheese as a probiotic feed supplement in lactating goats. The ability of L. plantarum to survive transit through the goat gastrointestinal tract and to modulate selected constituents of the gut microbiota composition, monitored at faecal level was assessed. In addition, L. plantarum effects on plasma immunoglobulins and antioxidant capacity of the animals as well as on the milk fatty acid composition were determined. For the purpose of the experiment a field study was designed, involving 24 dairy goats of the Damascus breed, kept in a sheep and goat dairy farm. The goats were divided in terms of body weight in two treatments of 12 goats each, namely: control (CON) without addition of L. plantarum and probiotic (PRO) treatment with in feed administration of L. plantarum so that the goats would intake 12 log CFU/day. The experiment lasted 5 weeks and at weekly time intervals individual faecal, blood and milk samples were collected and analysed. All faecal samples were examined for the presence of L. plantarum PCA 236. In addition, the culturable population levels of mesophilic aerobes, coliforms lactic acid bacteria (LAB), Streptococcus, Enterococcus, mesophilic anaerobes, Clostridium and Bacteroides in faeces were also determined by enumeration on specific culture media. In parallel, plasma IgA, IgM and IgG and antioxidant capacity of plasma and milk were determined. No adverse effects were observed in the animals receiving the lactobacillus during the experiment. Lactobacillus plantarum PCA 236 was recovered in the faeces of all animals in the PRO treatment. In addition, PRO treatment resulted in a significant (P