[Deficiency in N-acetylglucosamine transport affects the sporulation process and increases the hemolytic activity of the S-layer protein in Lysinibacillus sphaericus ASB13052]. / La deficiencia en el transporte de N-acetilglucosamina en Lysinibacillus sphaericus ASB13052 afecta el proceso de esporulación e incrementa la actividad hemolítica de su S-layer.

Lysinibacillus sphaericus is a bacterium that, along with Bacillus thuringiensis var. israelensis, is considered the best biological insecticide for controlling mosquito larvae and an eco-friendly alternative to chemical insecticides. It depends on peptidic molecules such as N-acetylglucosamine to obtain carbon sources and possesses a phosphotransferase system (PTS) for their incorporation. Some strains carry S-layer proteins, whose involvement in metal retention and larvicidal activity against disease-carrying mosquitoes has been demonstrated. Alterations in the amino sugar incorporation system could affect the protein profile and functionality. Strain ASB13052 and the isogenic mutant in the ptsH gene, which is predominant in the PTS signaling pathway, were used in this study. For the first time, the presence of N-glycosylated S-layer proteins was confirmed in both strains, with a variation in their molecular weight pattern depending on the growth phase. In the exponential phase, an S-layer protein greater than 130 kDa was found in the ptsH mutant, which was absent in the wild-type strain. The mutant strain exhibited altered and incomplete low quality sporulation processes. Hemolysis analysis, associated with larvicidal activity, showed that the ptsH mutant has higher lytic efficiency, correlating with the high molecular weight protein. The results allow us to propose the potential effects that arise as a result of the absence of amino sugar transport on hemolytic activity, S-layer isoforms, and the role of N-acetylglucosamine in larvicidal activity.

The Resistance of Bacillus Spores: Implications for the Strain-Specific Response to High-Performance Disinfectants.

Bacterial spores in materials and equipment pose significant biosecurity risks, making effective disinfection crucial. This study evaluated Ortho-phthalaldehyde (OPA) and a quaternary ammonia-glutaraldehyde solution (AG) for inactivating spores of Bacillus thuringiensis (BT), B. cereus (BC), and two strains of B. velezensis (BV1 and BV2). Spores of BV1 and BT were treated with 22.5 mg/m3 OPA by dry fumigation or 1 mg/mL AG by spray for 20 min, according to the manufacturer's recommendation. As no sporicidal effect was observed, OPA was tested at 112.5 mg/m3 for 40 min, showing effectiveness for BT but not for BV1. Minimum bactericidal concentration (MBC) tests revealed higher MBC values for glutaraldehyde, prompting an overnight test with 112.5 mg/m3 OPA by dry fumigation and 50 mg/mL AG by spray, using formaldehyde as a control. AG reduced all Bacillus strains, but with limited sporicidal effect. OPA was sporicidal for BT and BV1 but not for BC and BV2, indicating a strain-dependent effect. Formaldehyde performed better overall but did not completely inactivate BV2 spores. Our findings suggest that OPA and AG have potential as formaldehyde replacements in wet disinfection procedures.

Identification of pathways to high-level vancomycin resistance in Clostridioides difficile that incur high fitness costs in key pathogenicity traits.

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.

Is there a role for intestinal sporobiota in the antimicrobial resistance crisis?

Antimicrobial resistance (AMR) is a complex issue requiring specific, multi-sectoral measures to slow its spread. When people are exposed to antimicrobial agents, it can cause resistant bacteria to increase. This means that the use, misuse, and excessive use of antimicrobial agents exert selective pressure on bacteria, which can lead to the development of "silent" reservoirs of antimicrobial resistance genes. These genes can later be mobilized into pathogenic bacteria and contribute to the spread of AMR. Many socioeconomic and environmental factors influence the transmission and dissemination of resistance genes, such as the quality of healthcare systems, water sanitation, hygiene infrastructure, and pollution. The sporobiota is an essential part of the gut microbiota that plays a role in maintaining gut homeostasis. However, because spores are highly transmissible and can spread easily, they can be a vector for AMR. The sporobiota resistome, particularly the mobile resistome, is important for tracking, managing, and limiting the spread of antimicrobial resistance genes among pathogenic and commensal bacterial species.

Inhibition of Monilinia fructicola sporulation and pathogenicity through eucalyptol-mediated targeting of MfCat2 by Streptomyces lincolnensis strain JCP1-7.

Peach brown rot, attributed to Monilinia fructicola, presents a significant threat to postharvest peach cultivation, causing losses of up to 80%. With an increasing number of countries, spearheaded by the European Union, imposing bans on chemical agents in fruit production, there is a growing interest in mining highly active antibacterial compounds from biological control strains for postharvest disease management. In this study, we highlight the unique ability of Streptomyces lincolnensis strain JCP1-7 to inhibit M. fructicola sporulation, despite its limited antimicrobial efficacy. Through GC-MS analysis, eucalyptol was identified as the key compound. Fumigation of diseased fruits with eucalyptol at a concentration of 0.0335 µg cm-3 demonstrated an in vivo inhibition rate against M. fructicola of 93.13%, completely suppressing spore formation. Transcriptome analysis revealed the impact of eucalyptol on multiple pathogenesis-related pathways, particularly through the inhibition of catalase 2 (Cat2) expression. Experiments with a MfCat2 knockout strain (ΔMfCat2) showed reduced pathogenicity and sensitivity to JCP1-7 and eucalyptol, suggesting MfCat2 as a potential target of JCP1-7 and eucalyptol against M. fructicola. Our findings elucidate that eucalyptol produced by S. lincolnensis JCP1-7 inhibits M. fructicola sporulation by regulating MfCat2, thereby effectively reducing postharvest peach brown rot occurrence. The use of fumigation of eucalyptol offers insights into peach brown rot management on a large scale, thus making a significant contribution to agricultural research.

Decontamination Effect of Hypochlorous Acid Dry Mist on Selected Bacteria, Viruses, Spores, and Fungi as Well as on Components of Electronic Systems.

This publication presents the effect of hypochlorous acid dry mist as a disinfectant on selected bacteria, viruses, spores, and fungi as well as on portable Microlife OXY 300 finger pulse oximeters and electronic systems of Raspberry Pi Zero microcomputers. The impact of hypochlorous acid on microbiological agents was assessed at concentrations of 300, 500, and 2000 ppm of HClO according to PN-EN 17272 (Variant I). Studies of the impact of hypochlorous acid fog on electronic components were carried out in an aerosol chamber at concentrations of 500 ppm and 2000 ppm according to two models consisting of 30 (Variant II) and 90 fogging cycles (Variant III). Each cycle included the process of generating a dry mist of hypochlorous acid (25 mL/m3), decontamination of the test elements, as well as cleaning the chamber of the disinfectant agent. The exposure of the materials examined on hypochlorous acid dry mist in all variants resulted in a decrease in the number of viruses, bacteria, spores, and fungi tested. In addition, the research showed that in the variants of hypochlorous acid fogging cycles analyzed, no changes in performance parameters and no penetration of dry fog of hypochlorous acid into the interior of the tested medical devices and electronic systems were observed.

Inhibition and Mechanism of Citral on Bacillus cereus Vegetative Cells, Spores, and Biofilms.

Bacillus cereus causes food poisoning by producing toxins that cause diarrhea and vomiting and, in severe cases, endocarditis, meningitis, and other diseases. It also tends to form biofilms and spores that lead to contamination of the food production environment. Citral is a potent natural antibacterial agent, but its antibacterial activity against B. cereus has not been extensively studied. In this study, we first determined the minimum inhibitory concentrations and minimum bactericidal concentrations, growth curves, killing effect in different media, membrane potential, intracellular adenosine triphosphate (ATP), reactive oxygen species levels, and morphology of vegetative cells, followed by germination rate, morphology, germination state of spores, and finally biofilm clearance effect. The results showed that the minimum inhibitory concentrations and minimum bactericidal concentrations of citral against bacteria ranged from 100 to 800 µg/mL. The lag phase of bacteria was effectively prolonged by citral, and the growth rate of bacteria was slowed down. Bacteria in Luria-Bertani broth were reduced to below the detection limit by citral at 800 µg/mL within 0.5 h. Bacteria in rice were reduced to 3 log CFU/g by citral at 4000 µg/mL within 0.5 h. After treatment with citral, intracellular ATP concentration was reduced, membrane potential was altered, intracellular reactive oxygen species concentration was increased, and normal cell morphology was altered. After treatment with citral at 400 µg/mL, spore germination rate was reduced to 16.71%, spore morphology was affected, and spore germination state was altered. It also had a good effect on biofilm removal. The present study showed that citral had good bacteriostatic activity against B. cereus vegetative cells and its spores and also had a good clearance effect on its biofilm. Citral has the potential to be used as a bacteriostatic substance for the control of B. cereus in food industry production.

Enhancing UV radiation protection of Bacillus thuringiensis formulations using sulfur quantum dots: synthesis and efficacy evaluation.

Bacillus thuringiensis (Bt) is a widely used microbial insecticide, but its effectiveness is limited due to the degradation of Bt spores and crystals under UV radiation from sunlight. The objective of this study was to develop a novel Bt formulation with improved UV protection by utilizing sulfur quantum dots (SQDs) as stabilizing agents in a Pickering emulsion. The SQDs were comprehensively characterized using FTIR, XRD, TEM, HRTEM, UV, and fluorescence analyses, which confirmed the formation of well-dispersed, spherical SQDs. The microcapsule formulation with SQDs demonstrated superior UV stability, as it maintained 57.77% spore viability after 96 h of UV exposure, in comparison to 33.74% and 31.25% for the SQDs formulation (non-microcapsules) and unprotected Bt formulations (free spore, as a control), respectively. Furthermore, the microcapsule formulation exhibited higher insecticidal activity, resulting in a larval mortality of 71.22%, as opposed to 42.34% and 38.42% for the other formulations. These findings emphasize the effectiveness of microcapsule formulation with SQDs in safeguarding Bt spores and crystals against UV radiation, thereby enhancing their practical application in pest control. This approach presents a promising strategy for the development of biopesticides that are more resilient and have a longer shelf life.

Study on the inactivation effect and mechanism of EGCG disinfectant on Bacillus subtilis.

The widespread use of chlorine-based disinfectants in drinking water treatment has led to the proliferation of chlorine-resistant bacteria and the risk of disinfection byproducts (DBPs), posing a serious threat to public health. This study aims to explore the effectiveness and potential applications of epigallocatechin gallate (EGCG) against chlorine-resistant Bacillus and its spores in water, providing new insights for the control of chlorine-resistant bacteria and improving the biological stability of distribution systems. The inactivation effects of EGCG on Bacillus subtilis (B. subtilis) and its spores were investigated using transmission electron microscopy, ATP measurement, and transcriptome sequencing analysis to determine changes in surface structure, energy metabolism, and gene expression levels, thereby elucidating the inactivation mechanism. The results demonstrate the potential application of EGCG in continuously inhibiting chlorine-resistant B. subtilis in water, effectively improving the biological stability of the distribution system. However, EGCG is not suitable for treating raw water with high spore content and is more suitable as a supplementary disinfectant for processes with strong spore removal capabilities, such as ozone, ultraviolet, or ultrafiltration. EGCG exhibits a disruptive effect on the morphological structure and energy metabolism of B. subtilis and suppresses the synthesis of substances, energy metabolism, and normal operation of the antioxidant system by inhibiting the expression of multiple genes, thereby achieving the inactivation of B. subtilis.

Inactivation mechanism of phenyllactic acid against Bacillus cereus spores and its application in milk beverage.

Phenyllactic acid (PLA) as a natural phenolic acid exhibits antibacterial activity against non-spore-forming bacteria, while the inhibitory effect against bacterial spore remained unknown. Herein, this study investigated the inactivation effect of PLA against Bacillus cereus spores. The results revealed that the minimum inhibitory concentration of PLA was 1.25 mg/mL. PLA inhibited the outgrowth of germinated spores into vegetative cells rather than germination of spores. PLA disrupted the spore coat, and damaged the permeability and integrity of inner membrane. Moreover, PLA disturbed the establishment of membrane potential due to the inhibition of oxidative metabolism. SEM observations further visualized the morphological changes and structural disruption caused by PLA. Besides, PLA caused the degradation of DNA of germinated spores. Finally, PLA was applied in milk beverage, and showed promising inhibitory effect against B. cereus spores. This finding could provide scientific basis for the application of PLA against spore-forming bacteria in food industry.

Assessing the stability and sporicidal efficacy of oxidizing disinfectants.

BACKGROUND: The role of the healthcare environment in the transmission of clinical pathogens is well established. EN 17126:2018 was developed to address the need for regulated sporicidal product testing and includes a realistic medical soil to enable validation of products that claim combined cleaning and disinfection efficacy. AIM: To investigate the chemical stability and sporicidal efficacy of oxidizing disinfectant products in the presence of simulated clean and medical dirty conditions. METHODS: Disinfectant stability and sporicidal efficacy were evaluated in like-for-like ratios of soil:product. Disinfectants were exposed to simulated test soils and free chlorine, chlorine dioxide or peracetic acid concentrations were measured using standard colorimetric methods. Efficacy of disinfectants against C. difficile R027 endospores was assessed as per EN 17126:2018. Comparisons of performance between clean and medical dirty conditions were performed using one-way analysis of variance. Correlation analysis was performed using Pearson product-moment correlation. FINDINGS: Performance of chlorine-releasing agents (sodium dichloroisocyanurate, chlorine dioxide and hypochlorous acid) was concentration dependent, with 1000 ppm chlorine showing reduced stability and efficacy in dirty conditions. By contrast, peracetic acid product demonstrated stability and consistently achieved efficacy in dirty conditions. CONCLUSION: These results have implications for clinical practice, as ineffective environmental decontamination may increase the risk of transmission of pathogens that can cause healthcare-associated infections.

Single-cell analysis reveals microbial spore responses to sodium hypochlorite.

Pollution from toxic spores has caused us a lot of problems because spores are extremely resistant and can survive most disinfectants. Therefore, the detection of spore response to disinfectant is of great significance for the development of effective decontamination strategies. In this work, we investigated the effect of 0.5% sodium hypochlorite on the molecular and morphological properties of single spores of Bacillus subtilis using single-cell techniques. Laser tweezers Raman spectroscopy showed that sodium hypochlorite resulted in Ca2+-dipicolinic acid release and nucleic acid denaturation. Atomic force microscopy showed that the surface of treated spores changed from rough to smooth, protein shells were degraded at 10 min, and the permeability barrier was destroyed at 15 min. The spore volume decreased gradually over time. Live-cell imaging showed that the germination and growth rates decreased with increasing treatment time. These results provide new insight into the response of spores to sodium hypochlorite.

Emulsified and Nanoemulsified Essential Oils in the Control of Clostridium botulinum and Clostridium sporogenes in Mortadella.

Clostridium botulinum is a foodborne pathogen responsible for severe neuroparalytic disease associated with the ingestion of pre-formed toxin in food, with processed meats and canned foods being the most affected. Control of this pathogen in meat products is carried out using the preservative sodium nitrite (NaNO2), which in food, under certain conditions, such as thermal processing and storage, can form carcinogenic compounds. Therefore, the objective was to use nanoemulsified essential oils (EOs) as natural antimicrobial agents, with the aim of reducing the dose of NaNO2 applied in mortadella. The antimicrobial activity of nanoemulsions prepared with mixtures of EOs of garlic, clove, pink pepper, and black pepper was evaluated on endospores and vegetative cells of C. botulinum and Clostridium sporogenes (surrogate model) inoculated in mortadella prepared with 50 parts per million NaNO2. The effects on the technological (pH, water activity, and color) and sensory characteristics of the product were also evaluated. The combinations of EOs and their nanoemulsions showed sporicidal effects on the endospores of both tested microorganisms, with no counts observed from the 10th day of analysis. Furthermore, bacteriostatic effects on the studied microorganisms were observed. Regarding the technological and sensorial characteristics of the product, the addition of the combined EOs had a negative impact on the color of the mortadella and on the flavor/aroma. Despite the strong commercial appeal of adding natural preservatives to foods, the effects on flavor and color must be considered. Given the importance of controlling C. botulinum in this type of product, as well as the reduction in the amount of NaNO2 used, this combination of EOs represents a promising antimicrobial alternative to this preservative, encouraging further research in this direction.

Review of techniques for the in-situ sterilization of soil contaminated with Bacillus anthracis spores or other pathogens.

This review summarizes the literature on efficacy of techniques to sterilize soil. Soil may need to be sterilized if contaminated with pathogens such as Bacillus anthracis. Sterilizing soil in-situ minimizes spread of the bio-contaminant. Soil is difficult to sterilize, with efficacy generally diminishing with depth. Methyl bromide, formaldehyde, and glutaraldehyde are the only soil treatment options that have been demonstrated at full-scale to effectively inactivate Bacillus spores. Soil sterilization modalities with high efficacy at bench-scale include wet and dry heat, metam sodium, chlorine dioxide gas, and activated sodium persulfate. Simple oxidants such as chlorine bleach are ineffective in sterilizing soil.

Antibacterial and Sporicidal Activity Evaluation of Theaflavin-3,3'-digallate.

Theaflavin-3,3'-digallate (TFDG), a polyphenol derived from the leaves of Camellia sinensis, is known to have many health benefits. In this study, the antibacterial effect of TFDG against nine bacteria and the sporicidal activities on spore-forming Bacillus spp. have been investigated. Microplate assay, colony-forming unit, BacTiter-GloTM, and Live/Dead Assays showed that 250 µg/mL TFDG was able to inhibit bacterial growth up to 99.97%, while 625 µg/mL TFDG was able to inhibit up to 99.92% of the spores from germinating after a one-hour treatment. Binding analysis revealed the favorable binding affinity of two germination-associated proteins, GPR and Lgt (GerF), to TFDG, ranging from -7.6 to -10.3 kcal/mol. Semi-quantitative RT-PCR showed that TFDG treatment lowered the expression of gpr, ranging from 0.20 to 0.39 compared to the control in both Bacillus spp. The results suggest that TFDG not only inhibits the growth of vegetative cells but also prevents the germination of bacterial spores. This report indicates that TFDG is a promising broad-spectrum antibacterial and anti-spore agent against Gram-positive, Gram-negative, acid-fast bacteria, and endospores. The potential anti-germination mechanism has also been elucidated.

Recent Advances in the Application of the Antimicrobial Peptide Nisin in the Inactivation of Spore-Forming Bacteria in Foods.

Conventional thermal and chemical treatments used in food preservation have come under scrutiny by consumers who demand minimally processed foods free from chemical agents but microbiologically safe. As a result, antimicrobial peptides (AMPs) such as bacteriocins and nisin that are ribosomally synthesised by bacteria, more prominently by the lactic acid bacteria (LAB) have appeared as a potent alternative due to their multiple biological activities. They represent a powerful strategy to prevent the development of spore-forming microorganisms in foods. Unlike thermal methods, they are natural without an adverse impact on food organoleptic and nutritional attributes. AMPs such as nisin and bacteriocins are generally effective in eliminating the vegetative forms of spore-forming bacteria compared to the more resilient spore forms. However, in combination with other non-thermal treatments, such as high pressure, supercritical carbon dioxide, electric pulses, a synergistic effect with AMPs such as nisin exists and has been proven to be effective in the inactivation of microbial spores through the disruption of the spore structure and prevention of spore outgrowth. The control of microbial spores in foods is essential in maintaining food safety and extension of shelf-life. Thus, exploration of the mechanisms of action of AMPs such as nisin is critical for their design and effective application in the food industry. This review harmonises information on the mechanisms of bacteria inactivation from published literature and the utilisation of AMPs in the control of microbial spores in food. It highlights future perspectives in research and application in food processing.

Inhibition of spores to prevent the recurrence of Clostridioides difficile infection - A possibility or an improbability?

Clostridioides difficile is one of the most common nosocomial gastrointestinal pathogens, and recurrence is a problematic issue because approximately 20-30% of patients experience at least one episode of recurrence, even after treatment with a therapeutic drug of choice for C. difficile infection (CDI), such as vancomycin. CDI recurrence has a multifactorial complex mechanism, in which gut microbiota disruption coincident with viable C. difficile spores, is considered the most important factor. The effectiveness of an anti-C. difficile antimicrobial agent against CDI cannot guarantee its inhibitory effect on C. difficile spores and vice versa. However, an antimicrobial agent, such as fidaxomicin, which has a good inhibitory effect on both C. difficile vegetative cells and spores is assumed to not only treat CDI but also prevent its recurrence. Prolonged adherence to the exosporium has been proposed as a possible mechanism of inhibiting spores, and as a result, redesigning anti-C. difficile antimicrobial agents with the ability to adhere to the exosporium may provide another pathway for the development of anti-C. difficile spore agents. For example, vancomycin lacks an inhibitory effect against C. difficile spores, but a vancomycin-loaded spore-targeting iron oxide nanoparticle that selectively binds to C. difficile spores has been developed to successfully delay spore germination. Some new antimicrobial agents in phase II clinical trials, including cadazolid and ridinilazole, have shown exceptional anti-C. difficile and spore-inhibiting effects that can be expected to not only treat CDI but also prevent its recurrence in the future.