Search Results (160)

Fusarium wilt disease, caused by Fusarium oxysporum f. sp. cucumerinum (FOC), leads to widespread yield losses and quality deterioration in cucumber. Endophytes, as environmentally friendly control agents that enhance pathogen resistance in their host plants, may mitigate these problems. In this study, we isolated 14 endophytic bacteria from invasive Ambrosia artemisiifolia and screened the strain Bacillus subtilis TCX1, which exhibited significant antagonistic activity against FOC (inhibitory rate of 86.0%). TCX1 killed Fusarium oxysporum by being highly likely to produce lipopeptide and producing wall hydrolytic enzymes including protease, cellulase, and β-glucanase, thereby inhibiting mycelial growth and spore germination and causing peroxidation of FOC’s cytoplasmic membrane. In addition to its direct effects, TCX1 exerts indirect effects by inducing cucumber resistance to FOC. When cucumber seedlings were inoculated with TCX1, antioxidant enzymes related to disease resistance, including Superoxide dismutase (SOD), Peroxidase (POD), Polyphenol oxidase (PPO) and Phenylalanine ammonialyase (PAL) in cucumber, were significantly increased. The marker genes involved in induced systemic resistance and the salicylic acid signaling pathway, such as npr1, pr1a, pr2, pr9, lox1, and ctr1, were also dramatically upregulated, indicating these pathways played an important role in improving cucumber resistance. Notably, TCX1 can also promote cucumber growth through producing indole-3-acetic acid, solubilizing phosphate, and secreting siderophores. Given that TCX1 has dual functions as both a biological control agent and a biofertilizer, it offers an effective strategy for managing cucumber seedling blight while enhancing plant productivity. Full article

The COVID-19 pandemic exposed the urgent need for innovative tools to strengthen pandemic preparedness and health defense, especially in low- and middle-income countries (LMICs). While vaccination has been the cornerstone of the defense strategy against many infectious agents, there is a critical gap in vaccine equity, ensuring it is accessible to all, especially among the most vulnerable populations. The conventional vaccine delivery platforms, through parenteral administration, face notable limitations, including reliance on trained personnel, sterile conditions, and cold chain logistics. The parenteral vaccines often fail to induce robust mucosal immunity, which is critical for preventing infections at mucosal surfaces, the primary entry point for many pathogens. Bacillus subtilis, a Gram-positive, spore-forming bacterium, has emerged as a promising platform for mucosal vaccine delivery owing to its Generally Recognized as Safe (GRAS) status. Its robust spores are highly resilient to harsh environmental conditions, which may eliminate the need for cold chain storage and further facilitate distribution in LMICs. This review explores the potential of B. subtilis as a next-generation vaccine delivery platform, focusing on its unique characteristics, mechanisms of action, and applications in addressing global health challenges. This review also examines existing research demonstrating the safety, immunogenicity, and efficacy of B. subtilis spore-based vaccines while identifying limitations and future directions for optimization as a scalable and adaptable solution for resilient health defense, particularly in LMICs. Full article

Background/Objectives: The ongoing evolution of SARS-CoV-2 has highlighted the limitations of parenteral vaccines in preventing viral transmission, largely due to their failure to elicit robust mucosal immunity. Methods: Here, we evaluated an intranasal (IN) vaccine formulation consisting of recombinant receptor-binding domain (RBD) adsorbed onto human probiotic Bacillus subtilis DG101 spores. Results: In BALB/c mice, IN spore-RBD immunization induced strong systemic and mucosal humoral responses, including elevated specific IgG, IgM, and IgA levels in serum, bronchoalveolar lavage fluid (BALF), nasal-associated lymphoid tissue (NALT), and saliva. It further promoted mucosal B cell and T cell memory, along with a Th1/Tc1-skewed T cell response, characterized by increased IFN-γ-expressing CD4⁺ and CD8⁺ T cells in the lungs. Conclusions: All in all, these findings highlight the potential of intranasal vaccines adjuvanted with probiotic B. subtilis spores in inducing sterilizing immunity and limiting SARS-CoV-2 transmission. Full article

Powdery mildew caused by Sphaerotheca fusca poses a significant threat to cucumber (Cucumis sativus L.) production worldwide, underscoring the need for sustainable disease management strategies. This study investigates the potential of L-lysine, abundantly produced by Bacillus subtilis M 320 (BSM320), to prime systemic acquired resistance (SAR) pathways in cucumber plants. Liquid chromatography–mass spectrometry analysis identified L-lysine as the primary bioactive metabolite in the BSM320 culture filtrate. Foliar application of purified L-lysine significantly reduced powdery mildew symptoms, lowering disease severity by up to 92% at concentrations ≥ 2500 mg/L. However, in vitro spore germination assays indicated that L-lysine did not exhibit direct antifungal activity, indicating that its protective effect is likely mediated through the activation of plant immune responses. Quantitative reverse transcription PCR revealed marked upregulation of key defense-related genes encoding pathogenesis-related proteins 1 and 3, lipoxygenase 1 and 23, WRKY transcription factor 20, and L-type lectin receptor kinase 6.1 within 24 h of treatment. Concurrently, salicylic acid (SA) levels increased threefold in lysine-treated plants, confirming the induction of an SA-dependent SAR pathway. These findings highlight L-lysine as a sustainable, residue-free priming agent capable of enhancing broad-spectrum plant immunity, offering a promising approach for amino acid-based crop protection. Full article

Medium-high-hydrostatic-pressure (MHHP) treatment can induce the spore to germinate via activating the germination receptor, subsequently resulting in the loss of the heat resistance of the spore and finally killing the germinated spore, although the ungerminated spore, even after MHHP treatment, can survive. This study aims to clarify the pasteurization effect of the combination of MHHP treatment with post-/pre-heating treatment on Bacillus subtilis spores suspended in soy milk as a food model. Regarding the results, the D value, as a known heat resistance indicator of the MHHP-treated spore, decreased in comparison with the untreated spore. However, the activation energies required for killing both the untreated and the MHHP-treated spores were equivalent, which indicated that the heat conductivity of the ungerminated spores might be increased by MHHP treatment. When the spore was subjected to pre-heating treatment and subsequently to MHHP treatment, the pasteurization effect of MHHP treatment differed with the pre-heating temperature. Pre-heating treatment at 80 °C could promote pasteurization, while that at 90–100 °C could suppress it, which might be caused by the heat activation/inactivation of germination receptors. From these results, the presence of post-/pre-heat treatment could be an important factor for the pasteurization of B. subtilis spores via MHHP treatment. Full article

This study evaluates various strains of soil bacterial for use in the development of new biopreparations. Mesophilic spore-forming bacteria were isolated from cultivated soil and analysed for their enzymatic activity, ability to decompose crop residues, and antagonistic properties towards selected phytopathogens. Notably, this is the first cytotoxicity assessment of soil bacterial metabolites on Spodoptera frugiperda Sf-9 (fall armyworm). Bacillus subtilis, Bacillus licheniformis, Bacillus velezensis, Paenibacillus amylolyticus, and Prestia megaterium demonstrated the highest hydrolytic potential for the degradation of post-harvest residues from maize, winter barley, and triticale. They exhibited antimicrobial activity against at least three of the tested phytopathogens and demonstrated the ability to solubilize phosphorus. Metabolites of B. licheniformis (IC50 = 8.3 mg/mL) and B. subtilis (IC50 = 144.9 mg/mL) were the most cytotoxic against Sf-9. We recommend the use of the tested strains in industrial practice as biocontrol agents, plant growth biostimulants, crop residue decomposition stimulants, and bioinsecticides. Future studies should focus on assessing the efficacy of using these strains under conditions simulating the target use, such as plant microcosms and greenhouses and the impact of these strains on the abundance and biodiversity of native soil microbiota. This research can serve as a model procedure for screening other strains of bacteria for agricultural purposes. Full article

Background/Objectives: The emergence of multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) as a leading cause of fatal hospital-acquired infections underscores the urgent need for effective vaccines. While oral vaccines using live Bacillus subtilis spores expressing A. baumannii TonB-dependent receptor (TBDR) show promise, biosafety concerns regarding recombinant spore persistence necessitate alternative strategies. Here, we evaluated chemically inactivated B. subtilis spores displaying TBDR as a safer yet immunogenic vaccine candidate. Methods: Recombinant spores were inactivated using iron-ethanol sporicidal solution and administered to BALB/c mice (8–12 weeks old) to assess safety and immunogenicity. Toxicity was evaluated through clinical monitoring, serum biochemistry, and histopathology. Immune responses were characterized by T/B cell activation, IgG/IgA titers, and mucosal sIgA levels. Protective efficacy was determined by challenging immunized mice with MDR A. baumannii Ab35 and quantifying bacterial loads and examining tissue pathology. Results: The inactivated spores exhibited an excellent safety profile, with no adverse effects on clinical parameters, organ function, or tissue integrity. Immunization induced robust systemic and mucosal immunity, evidenced by elevated CD4+/CD8+ T cells, B cells, and antigen-specific IgG/IgA in serum and mucosal secretions. Following the challenge, vaccinated mice showed significantly reduced pulmonary bacterial burdens (>90% reduction), and preserved lung and spleen architecture compared to controls, which developed severe inflammation and tissue damage. Conclusions: These findings demonstrate that inactivated B. subtilis spores expressing TBDR are a safe, orally administrable vaccine platform that elicits protective immunity against MDR A. baumannii. By addressing biosafety concerns associated with live spores while maintaining efficacy, this approach represents a critical advance toward preventing high-risk nosocomial infections. Full article

The rapid expansion of aquaculture is vital for global food security, yet it faces persistent threats from disease outbreaks, vaccine inefficacy, and antibiotic overuse, all of which undermine sustainability. Conventional vaccines often fail to induce robust mucosal immunity, spurring interest in probiotics as adjuvants to enhance immunogenicity. Probiotics such as Bacillus subtilis and Lactobacillus casei modulate fish microbiomes, fortify mucosal barriers, and activate innate immune responses via mechanisms including Toll-like receptor signaling and cytokine production. These actions prime the host environment for prolonged adaptive immunity, improving antigen uptake and pathogen clearance. Experimental advances—such as Bacillus subtilis-engineered spores increasing survival rates to 86% in Vibrio anguillarum-challenged European seabass—demonstrate the potential of this synergy. Innovations in delivery systems, including chitosan–alginate microcapsules and synbiotic formulations, further address oral vaccine degradation, enhancing practicality. Probiotics also suppress pathogens while enriching beneficial gut taxa, amplifying mucosal IgA and systemic IgM responses. However, challenges such as strain-specific variability, environmental dependencies, and unresolved ecological risks persist. Optimizing host-specific probiotics and advancing multi-omics research is critical to unlocking this synergy fully. Integrating probiotic mechanisms with vaccine design offers a pathway toward antibiotic-free aquaculture, aligning with One Health principles. Realizing this vision demands interdisciplinary collaboration to standardize protocols, validate field efficacy, and align policies with ecological sustainability. Probiotic–vaccine strategies represent not merely a scientific advance but an essential evolution for resilient, ecologically balanced aquaculture systems. Full article

Probiotics play a pivotal role in animal production by promoting growth, enhancing gut health, and modulating immune responses. Bacillus subtilis, a widely utilized probiotic, forms robust spores that exhibit exceptional resistance, making it ideal for feed applications. While B. subtilis spores have been shown to stimulate innate immune signaling, the specific contributions of spore coat proteins to immune modulation remain poorly characterized. In this study, we investigated the immunostimulatory effects of spores deficient in six key coat proteins: SpoIVA, SafA, CotE, CotX, CotZ, and CgeA. These proteins are essential for the assembly and structural integrity of the spore’s multi-layered coat, and are involved in recruiting other coat components. Deletion of these genes result in defects in spore coat architecture, potentially altering spore–host interactions. Using porcine alveolar macrophages (MΦ3D4/2), we assessed cytokine responses to each mutant strain. Our findings demonstrate that the absence of specific structural proteins significantly impacts immune activation, particularly through Toll-like receptor pathways. This work provides novel insights into the immunomodulatory functions of spore coat proteins and lays the foundation for the rational design of next-generation B. subtilis-based probiotics with enhanced immunological properties for agricultural applications. Full article

This study evaluated the antimicrobial potential of a Bacillus subtilis spore-based probiotic cocktail to reduce foodborne pathogens in both nutrient-rich laboratory media and a complex food matrix (hummus). Three common foodborne pathogens—Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Typhimurium—were cultured individually in full-strength, half-strength, and quarter-strength tryptic soy broth (TSB) with or without the probiotic spores (~7 log CFU/mL). Additionally, a commercial hummus formulation was inoculated with L. monocytogenes (~3 log CFU/g) and B. subtilis spores (~7 log CFU/g) and stored at 30 °C to simulate temperature abuse. In TSB, E. coli and Salmonella grew to ~8.2 log CFU/mL in full-strength media, with no significant inhibition by the probiotics. However, L. monocytogenes showed substantial suppression: in nutrient-limited TSB, viable counts dropped below the detection limit of 1.48 log CFU/mL by 24 h in the presence of probiotics. In hummus, L. monocytogenes grew to an average of 8.22 log CFU/g in the absence of probiotics but remained significantly lower at an average of 5.03 log CFU/g when co-inoculated with B. subtilis (p < 0.05). Germination of probiotic spores was confirmed within 6 h under all conditions. These findings suggest that B. subtilis spores selectively inhibit Listeria, particularly under nutrient stress or abuse conditions. While the probiotic had limited impact on Gram-negative pathogens, its application may serve as a clean-label strategy for suppressing L. monocytogenes in ready-to-eat (RTE) foods. This dual-model approach provides insights into both mechanistic activity and practical limitations of spore probiotics in complex food matrices. Full article

Background/Objectives: Mucosal vaccines are rare but commercially desirable because of their real and theoretical biological advantages. Spores and vegetative forms from Bacillus have been used as probiotics due to their stability under various environmental conditions, including heat, gastric acidity, and moisture. Preclinical studies have shown that Bacillus subtilis (B. subtilis) spores can serve as effective mucosal adjuvants. Our study aimed to evaluate B. subtilis spores as a mucosal adjuvant. Methods and Results: We demonstrate in rodents that the fusion protein (F) from respiratory syncytial virus (RSV), when combined with either heat-inactivated or live B. subtilis spores, elicits robust IgG binding and neutralizes antibody titers following both systemic and intranasal administration in mice. The spores facilitate TH-1 and local IgA responses, which could enhance antiviral protection. However, this vaccine failed to elicit measurable antibodies when immunized using a strict intranasal administration method in cotton rats. Conclusions: Our findings illustrate the differing immune responses between the two rodent species, highlighting the need for the careful consideration of validated methods when evaluating intranasal vaccines in preclinical studies. Full article

During Bacillus subtilis spore germination/outgrowth, the rehydration of the spore core and activation of aerobic metabolism can generate reactive oxygen species (ROS)-promoted DNA lesions that are repaired via the base excision repair pathway (BER). Accordingly, spores deficient in the AP-endonucleases (APEs) Nfo and ExoA exhibit a delayed outgrowth that is suppressed following disruption of the checkpoint protein DisA. Here, we report that DisA-independent DNA damage checkpoints operate during B. subtilis spore outgrowth. Consistent with this notion, spores lacking Nfo, ExoA, and Nth, which functions as an APE, did not suppress delayed outgrowth following disA disruption. Furthermore, in reference to the ∆nfo ∆exoA ∆nth spores, spores deficient for these APEs and DisA displayed a significantly higher number of oxidative genetic lesions and failed to properly segregate its chromosome during the first round of replication in the outgrowth stage. Finally, we found that DisA promotes low-fidelity repair and replication events, as revealed by DNA-alkaline gel electrophoresis (AGE) as well as spontaneous and H₂O₂-promoted Rif^R mutagenesis. Overall, our results unveil the existence of DisA-independent DNA damage checkpoint(s) that are activated by genomic lesions of an oxidative nature during spore germination and outgrowth, ensuring a proper transition to vegetative growth. Full article

In the food industry, food spoilage caused by spores is a pressing scientific challenge that needs to be addressed urgently, and spore germination is a key approach to solving this problem. Studies have shown that peptidoglycan-induced spore germination represents a novel mechanism of action, which can bind to the PASTA domain of the serine/threonine kinase PrkC. However, the signaling mechanism of peptidoglycan-induced spore germination remains unclear. This study focuses on Bacillus subtilis, using pull-down experiments to screen for proteins interacting with PrkC. There are 80 interaction proteins of PrkC that were identified in the spore. GO analysis reveals that PrkC-interacting proteins in the spore are mainly involved in metabolic processes, cell part and catalysis. KEGG results indicate that PrkC-interacting proteins in the spore are mainly involved in RNA degradation, quorum sensing, oxidative phosphorylation, etc. Additionally, proteins are categorized into six groups by function based on events that may be associated with post-germination triggered by peptidoglycan-induced activation of the PrkC signaling pathway, including “stimulate translation initiation” and “ATP synthesis and energy metabolism”. The experimental results provide a theoretical basis for further elucidating the signaling mechanism of PrkC, revealing the signaling pathway of peptidoglycan-induced spore germination, and identifying targeted inducers and repressors of spore germination. Full article

Bacillus subtilis NCD-2 demonstrates exceptional biocontrol potential against cotton Verticillium wilt. While previous studies have established its direct antifungal activity (e.g., inhibiting Verticillium dahliae mycelial growth and spore germination), our work reveals a novel mechanism: NCD-2 primes systemic resistance in cotton by activating plant immune-signaling pathways. Firstly, transcriptional profiling uncovered that NCD-2 triggers a defense response in roots analogous to V. dahliae infection, allowing cotton to maintain a more balanced state when confronted with pathogen attacks. Meanwhile, the mutant strains ∆fen and ∆srf—defective in lipopeptide synthesis—also improved cotton resistance to Verticillium wilt by activating partially identical defense pathways in cotton roots. Furthermore, the application of lipopeptide compounds derived from NCD-2, particularly surfactin and fengycin, could enhance host resistance to V. dahliae. Using an RT-qPCR approach, we found that numerous resistance-related genes were induced by these lipopeptide compounds. The up-regulation of SA/JA pathway markers (e.g., NPR1, ICS1, COI1, and LOX1) revealed NCD-2’s activation of plant immune signaling. Using virus-induced gene silencing (VIGS), we conclusively linked SA and JA signaling to NCD-2-induced defense priming. Silencing either pathway abolished resistance, highlighting their indispensable coordination. By bridging mechanistic insights and agricultural applicability, our work positions NCD-2 as a sustainable alternative to conventional fungicides, addressing both crop productivity and environmental health. Full article

Microbial-induced carbonate precipitation technology allows concrete to detect and diagnose cracks autonomously. However, the concrete’s compact structure and alkaline environment necessitate the adoption of a proper carrier material to safeguard microorganisms. In this study, various bacterial strains, including Bacillus subtilis, Bacillus sphaericus, and Bacillus megaterium, were immobilized in lightweight expanded clay aggregates (LECA) to investigate their effect on the self-healing performance, mechanical strength, and freeze–thaw durability. Self-healing concrete specimens were prepared using immobilized LECA, directly added bacterial spores, polyvinyl acetate (PVA) fibers, and air-entraining admixture (AEA). The pre-cracked prisms were monitored for 224 days to assess self-healing efficiency through ultrasonic pulse velocity (UPV) and surface crack analysis methods. A compressive strength restoration test was conducted by pre-loading the cube specimens with 60% of the failure load and re-testing them after 28 days for strength regain. Additionally, X-ray diffraction and scanning electron microscopy (SEM) were conducted to analyze the precipitate material. The findings revealed that self-healing efficiency improved with the biomineralization activity over the healing period demonstrated by the bacterial strains. Compression and flexural strengths decreased for the bacterial specimens attributed to porous LECA. However, restoration in compression strength and freeze–thaw durability significantly improved for the bacterial mixes compared to control and reference mixes. XRD and SEM analyses confirmed the formation of calcite as a self-healing precipitate. Overall, results indicated the superior performance of Bacillus megaterium followed by Bacillus sphaericus and Bacillus subtilis. The findings of the current study provide important insights for the construction industry, showcasing the potential of bacteria to mitigate the degradation of concrete structures and advocating for a sustainable solution that reduces reliance on manual repairs, especially in inaccessible areas of the structures. Full article