Bacillus Spores Research Articles

Identification of Clinical Isolates of the Bacillus cereus Group and Their Characterization by Mass Spectrometry and Electron Microscopy

Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

Semi-automated water sampling module for repeated sampling and concentration of Bacillus cereus group spores

Monitoring the presence of pathogenic Bacillus spores is important for industrial applications, as well as necessary for ensuring human health. Bacillus thuringiensis is used as a biopesticide against several insect pests. Bacillus cereus spores are a significant cause of food poisoning, and Bacillus anthracis is a recognized biosecurity threat. Laboratory-based methods, such as polymerase chain reaction, enzyme-linked immunosorbent assay, or matrix-assisted laser desorption ionization spectroscopy provide sensitive detection of bacteria and spores, but the application of those methods for quasi-continuous environmental monitoring presents a significant challenge requiring frequent human intervention. To address this challenge, we developed a workstation for quasi-autonomous monitoring of water reservoirs for the presence of bacteria and spores, and designed and validated the functionality of a microprocessor-controlled module capable of repetitive collection and pre-concentration of spores in liquid samples tested with fiberglass (FG), polyether sulfone and polyvinylidene fluoride filters. The best results were obtained with FG filters delivering a 20× concentration of B. thuringiensis and B. cereus spores from saline suspensions. The successful 20× pre-concentration of Bacillus spores demonstrated with FG filters could be repeated up to 3 times when bleach decontamination is applied between filtrations. Taken together, our results demonstrate an attractive instrument suitable for semi-automated, quasi-continuous sampling and pre-processing of water samples for biosensing of bacterial spores originating from a complex environment.

Study of the composition of the residual microflora of milk after pasteurization

The article presents the results of studies of the composition of the residual microflora of pasteurized milk, depending on the bacterial landscape and the initial contamination of raw milk. The thermal stability of test cultures of microorganisms that significantly affect the quality and storage capacity of fermented dairy products has been studied. To study the composition of the residual microflora of milk after pasteurization, sterile milk was infected with test cultures of microorganisms at doses from 101 CFU/cm3 to 107 CFU/cm3. After infection, the milk was pasteurized at temperatures of (72 ± 1) °C and (80 ± 1) °C with a holding time of 10–20 seconds. The detection and enumeration of microorganisms was carried out by standardized microbiological methods. Microorganisms were identified by visual assessment of dominant colonies and cell morphology in micropreparations. The thermal stability of microorganisms important for dairy products, in particular cheeses, the source of which is raw milk, has been studied. It has been established that of the coccal forms, the greatest risks are associated with enterococci. Escherichia coli at infection doses above 106 CFU/cm3 partially retains viability both at low-temperature and at high-temperature pasteurization. Pasteurization temperatures do not have a lethal effect on spore bacilli, their number in pasteurized milk does not decrease, regardless of the initial dose of infection. Low-temperature pasteurization activates the process of clostridial spore germination. The ability to reactivate cells after thermal shock was observed in Escherichia coli, Staphylococcus aureus, Pseudomonas, and mold fungi. Thus, the residual microflora of milk subjected to low-temperature pasteurization is represented by enterococci, thermophilic streptococci, micrococci, staphylococci, asporogenous bacilli and spore bacteria. The above microorganisms constitute the residual microflora of pasteurized milk and are involved in the maturation of cheeses, determining their quality and safety, [as well as] affecting the storage capacity of the finished product.

Potential antifungal activity of essential oil from Cymbopogon citratus (DC) Stapf and its performances as biostabilization of tiger nut milk (Cyperus exculentus)

Tiger nut milk is not heated during processing and is usually contaminated with spoilage microorganisms due to its high starch content. The present study aims to preserve tiger nut milk using essential oil from Cymbopogon citratus as an antimicrobial agent. The antifungal activity was evaluated by agar diffusion and micro-dilution methods. Storage tests were performed at 4°C by incorporating tiger nut milk with different concentrations of EO (i.e. 0.05; 0.10; 0.15; 0.20; 0.25; 0.30; 0.35; 0.40; 0.45 and 0.50 (% v/v)), after which the physicochemical and hygienic characteristics were assessed. The EO showed significant (p ˂ 0.05) inhibitory activity against A. niger (19.89 mm), C. albicans (26.30 mm), C. kefir (26.45 mm), C. tropicalis (27.31 mm), R. nigricans (27.45 mm) and S. cerevisiae KVL013 (25.52 mm) with MICs 15.62 (A. niger), 0.98 (C. albicans), 1.63 (C. kefir), 1.95 (C. tropicalis), 7.81 (R. nigricans) and 0.98 (S. cerevisiae KVL013). The results of the shelf-life monitoring showed that the milk samples were free of total and thermotolerant coliforms, Bacillus spores and sulphite-reducing anaerobes spores. At a concentration of 0.40 to 0.50% of EO and after 21 days, flora microbial load decreased in unpasteurized milk from 103 to 102 (CFU/mL) and yeasts/moulds decreased from 102 to 101 (CFU/mL), whereas in pasteurized milk, flora decreased from 103 to 10 (CFU/mL), and yeasts/moulds decreased from 102 to less than 101 (CFU/mL). This shows that the tiger nut milk remained stable and its hygienic quality complied with the standards set for spoilage flora and spores for 21 days at 4°C under refrigeration. The results obtained indicated that EO of C. citratus has strong antimicrobial activity on spoilage flora and the stabilisation concentration can be modulated for sensory acceptability.

Accurate identification of living Bacillus spores using laser tweezers Raman spectroscopy and deep learning

Accurately, rapidly, and noninvasively identifying Bacillus spores can greatly contribute to controlling a plenty of infectious diseases. Laser tweezers Raman spectroscopy (LTRS) has confirmed to be a powerful tool for studying Bacillus spores at a single cell level. In this study, we constructed a single-cell Raman spectra dataset of living Bacillus spores and utilized deep learning approach to accurately, nondestructively identify Bacillus spores. The trained convolutional neural network (CNN) could efficiently extract tiny Raman spectra features of five spore species, and provide a prediction accuracy of specie identification as high as 100 %. Moreover, the spectral feature differences in three Raman bands at 660, 826, and 1017 cm−1 were confirmed to mostly contribute to producing such high prediction accuracy. In addition, optimal CNN model was employed to monitor and identify sporulation process at different metabolic phases in one growth cycle. The obtained average prediction accuracy of metabolic phase identification was approximately 88 %. It can be foreseen that, LTRS combined with CNN approach have great potential for accurately identifying spore species and metabolic phases at a single cell level, and can be gradually extended to perform identification for many unculturable bacteria growing in soil, water, and food.

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) invitro and onto rDNA invivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

Decolorization of Textile Dye by Spore Surface Displayed Small Laccase for the Enhanced Thermal Stability and Robust Repeated Reaction.

In this study, we tried to decolorize synthetic dyes using small laccase (SLAC) from Streptomyces coelicolor, which is resistant to pH, temperature change, and traditional inhibitors for the actual industrial applications using spore surface display system. We inserted SLAC-His6 tag at the C-terminal of CotE anchoring motif. The proper surface expression of CotE-SLAC fusion protein on the surface of Bacillus subtilis spore was verified with flow cytometry using FITC labeled anti-His6 tag antibody. After 6 h of reaction, more than 90% of Indigo carmine was decomposed using recombinant SLAC displaying Bacillus spore, whereas less than 10% of Indigo carmine was decomposed with wild type spore. Over 70% of laccase activity was retained with recombinant SLAC displaying spore, which was heat-treated for 3 h at 90°C. For eight rounds of repeated decomposition of Indigo carmine, no significant decrease of enzymatic activity was observed. This showed the robust characteristics of spore display format for repeated and harsh condition reactions.

Alginate films as carriers of probiotic bacteria and Pickering emulsion

Bioactive edible films have been proposed from a calcium-crosslinked alginate matrix added with probiotic bacteria (Bacillus coagulans, as the bioactive component) and/or bacterial cellulose nanocrystal (BCNC)-stabilized palm olein Pickering emulsion (as a hydrophobizing component). The emulsion, however, instead of increasing the surface hydrophobicity and the barrier to water vapor, decreased both, due to discontinuities on the film structure and possibly the BCNC layer around the droplets minimizing the surface exposure of the hydrophobic phase (and its effects on film hydrophobicity). The probiotic bacteria, on the other hand, besides increasing the elastic modulus (which was ascribed to electrostatic interactions between teichoic acid and calcium), also enhanced both surface hydrophobicity and barrier to water vapor, probably due to the hydrophobicity of Bacillus spores. The probiotics exhibited high viability (> 8 log cfu.g−1, on a dry film basis) during film drying, storage, and digestion in a simulated gastrointestinal tract.

CotG Mediates Spore Surface Permeability in Bacillus subtilis.

Proteins and glycoproteins that form the surface layers of the Bacillus spore assemble into semipermeable arrays that surround and protect the spore cytoplasm. Such layers, acting like molecular sieves, exclude large molecules but allow small nutrients (germinants) to penetrate. We report that CotG, a modular and abundant component of the Bacillus subtilis spore coat, controls spore permeability through its central region, formed by positively charged tandem repeats. These repeats act as spacers between the N and C termini of the protein, which are responsible for the interaction of CotG with at least one other coat protein. The deletion but not the replacement of the central repeats with differently charged repeats affects the spore resistance to lysozyme and the efficiency of germination-probably by reducing the coat permeability to external molecules. The presence of central repeats is a common feature of the CotG-like proteins present in most Bacillus species, and such a wide distribution of this protein family is suggestive of a relevant role for the structure and function of the Bacillus spore. IMPORTANCE Bacterial spores are quiescent cells extremely resistant to a variety of unphysiological conditions, including the presence of lytic enzymes. Such resistance is also due to the limited permeability of the spore surface, which does not allow lytic enzymes to reach the spore interior. This article proposes that the spore permeability in B. subtilis is mediated by CotG, a modular protein formed by a central region of repeats of positively charged amino acid acting as a "spacer" between the N and C termini. These, in turn, interact with other coat proteins, generating a protein layer whose permeability to external molecules is controlled by the distance between the N and C termini of CotG. This working model is most likely expandable to most sporeformers of the Bacillus genus, since they all have CotG-like proteins, not homologous to CotG of B. subtilis but similarly characterized by central repeats.

Direct Observation of the Sporicidal Action of Hydrogen Peroxide on Bacillus atrophaeus Spores By Optical Trapping Raman Microscopy

Hydrogen peroxide (H2O2), a strong oxidizer, is frequently used for chemical sterilization in aseptic food processing. Due to their inherent robustness, bacterial spores, i.e. spores of Bacillus atrophaeus, are commonly employed to evaluate the efficacy of the sterilization procedure [1]. Despite its widespread use, however, the details of the sporicidal action of H2O2 are not yet fully understood, with various mechanisms being discussed in the literature [2,3]. Here, we employ optical trapping Raman microscopy to elucidate the sporicidal mechanism of H2O2. Optical trapping Raman microscopy allows us to isolate, investigate, and manipulate individual spores from a sample (Fig. 1A). Combining optical microscopy with the real-time, label-free chemical information obtained from Raman spectroscopy directly reveals the sequence of steps that lead to oxidative spore death after H2O2 exposure (Fig. 1B).We find that spore degradation begins with the fast, cooperative release of dipicolinic acid (DPA), a key spore biomarker and a major component of spore’s core. This indicates a breach of the inner membrane surrounding the core. The onset of DPA release depends on the concentration of H2O2. DPA outflux is accompanied by the formation of oxidation products (mostly carbonyl compounds) and oxidation continues after exchange of DPA with the surrounding medium is complete. The remaining spore shell then slowly disintegrates and continues to shrink on a timescale of hundreds of seconds. Simultaneous observation of spore morphology and size by optical microscopy and particle tracking corroborates this three-step mechanism.The presented assay allows on-line monitoring of spore composition and morphology, which directly reveals the mechanism underlying the sporicidal action of H2O2. In addition, trapped spores can be exposed to different chemical or physical stimuli (temperature, light, chemical composition of the surrounding medium, etc.) to investigate a variety of industrially relevant sterilization conditions. Jildeh, Z. B., Wagner, P. H. & Schöning, M. J. Sterilization of Objects, Products, and Packaging Surfaces and Their Characterization in Different Fields of Industry: The Status in 2020. physica status solidi (a) 218, 2000732 (2021). Setlow, P. & Christie, G. What’s new and notable in bacterial spore killing. World J Microbiol Biotechnol 37, 144 (2021). Setlow, P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101, 514-525 (2006). Figure 1

The Role of Probiotic Bacillus Spores and Amino Acids with Immunoglobulins on a Rat Enteropathy Model.

Non-steroidal anti-inflammatory drugs (NSAIDs) are some of the most widely used drugs due to their anti-inflammatory, analgesic and antipyretic pharmacological effects. Gastrointestinal side effects are some of the most severe and frequent side effects of NSAIDs. These depend on the balance of the gut microbiome, the abundance of Gram-negative bacteria, and the amount of lipopolysaccharide released. Therefore, restoring or improving gut bacteria balance with probiotic supplements could prove to be an adjuvant therapy against mild NSAID-induced enteropathy. Twenty-five Wistar albino male rats were divided into five groups. The negative control group was administered carboxymethylcellulose and the positive control group diclofenac (DIC), 8 mg/kg for 7 days, which represented the enteropathy model. Treatment groups consisted of a combination of pro-biotic spores (MSB), amino acids and immunoglobulins supplement (MM), which were also administered for 7 days. We analyzed hepatic injury markers (AST, ALT) and creatinine, and inflammatory markers, IL-6, TNF-α, PGE2, iNOS, as well as total antioxidant capacity. The results obtained in the present study suggest that the modulation of the intestinal microbiota by administration of probiotics (Bacillus spores), alone or in combination with immunoglobulins and amino acids, represents an attractive therapy for the prevention of NSAID-induced enteropathy.

Autoclaved Diet with Inactivated Spores of Bacillus spp. Decreased Reproductive Performance of Muc2-/- and Muc2+/- Mice.

Simple SummaryWithin barrier facilities for the housing of laboratory animals, the sterilization of feed, bedding, and cages is used to reduce contact with bacteria. However, in nature, animals come into contact with a lot of bacteria. We investigated the effect of an autoclaved diet on the reproductive performance of Muc2−/− mice. Muc2−/− mice develop intestinal barrier defects and are sensitive to changes of the gut microbiota. We have shown that the autoclaved diet negatively affects the reproductive performance of Muc2−/− females and their healthy Muc2+/− siblings. Thus, the autoclaved diet led to earlier rectal prolapse of Muc2−/− females combined with intestinal inflammation, compared to mice fed with the non-autoclaved diet. We hypothesize that this effect is due to the reduction of the diet nutritional value and inactivation of Bacillus spp. spores in the autoclaved diet. Within barrier facilities, autoclaved diet and bedding are used for husbandry of laboratory rodents. Bacillus spp. are ubiquitous in nature and some of them are known as probiotics. Inactivation of the Bacillus spores and reduction of the diet nutritional value due to autoclavation could be especially critical for immunodeficient mice. We studied the effect of the autoclaved and non-autoclaved diets on the reproductive performance and the age of prolapse manifestation in Muc2−/− mice with impaired gut barrier function and, therefore, sensitive to change of microbiota. We found that the non-autoclaved diet led to enhancement of the fertility index of Muc2−/− and Muc2+/− female mice. The non-autoclaved diet affected the prolapse of Muc2−/− mice that occurred later in comparison with females eating the autoclaved diet. We showed that Bacillus spp. was present in the non-autoclaved diet and feces of mice on the non-autoclaved diet. Bacterial strains of the non-autoclaved diet and feces belonged to B. amyloliquefaciens, B. thuringiensis, B. subtilis, Lysinibacillus macrolides, B. cereus, and other representatives of Bacillus spp. Moreover, autoclavation of the diet affected on the percent of the blood and spleen immune cells, the bacterial composition of the intestine, and increased the level of methionine in the thigh muscle of mice. Enhanced reproductive performance and delayed prolapse manifestation in Muc2−/− mice could be due to improved digestion, as Bacillus spp. from diet and feces had enzymatic activity.

The Genus Iodidimonas: From Its Discovery to Potential Applications.

The genus Iodidimonas was recently proposed in the class Alphaproteobacteria. Iodidimonas strains are aerobic, mesophilic, neutrophilic, moderately halophilic, and chemo-organotrophic. They were first discovered in natural gas brine water containing a very high level of iodide (I−). They exhibited a unique phenotypic feature of iodide oxidation to form molecular iodine (I2). Iodidimonas was also enriched and isolated from surface seawater supplemented with iodide, and it is clearer now that their common habitats are those enriched with iodide. In such environments, Iodidimonas species seem to attack microbial competitors with the toxic form I2 to occupy their ecological niche. The iodide-oxidizing enzyme (IOX) purified from the Iodidimonas sp. strain Q-1 exhibited high catalytic efficiency for iodide and consisted of at least two proteins IoxA and IoxC. IoxA is a putative multicopper oxidase with four conserved copper-binding regions but is phylogenetically distinct from other bacterial multicopper oxidases. The IOX/iodide system could be used as a novel enzyme-based antimicrobial system which can efficiently kill Bacillus spores. Furthermore, the IOX/iodide system can be applied to the decolorization of recalcitrant dyes, where iodide may function as a novel inorganic natural redox mediator.

Autoclaved diet with inactivated spores of Bacillus spp. decreased reproductive performance of Muc2-/- and Muc2+/- mice

Autoclaved diet with inactivated spores of Bacillus spp. decreased reproductive performance of Muc2-/- and Muc2+/- mice

Nasal-spraying Bacillus spores as an effective symptomatic treatment for children with acute respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a leading cause of Acute Respiratory Tract Infections (ARTIs) in young children. However, there is currently no vaccine or treatment available for children. Here, we demonstrated that nasal-spraying probiotics containing 5 billion of Bacillus spores (LiveSpo Navax) is an effective symptomatic treatment in a 6-day randomized controlled clinical study for RSV-infected children (n = 40–46/group). Navax treatment resulted in 1-day faster recovery-time and 10–50% better efficacy in relieving ARTI symptoms. At day 3, RSV load and level of pro-inflammatory cytokines in nasopharyngeal samples was reduced by 630 folds and 2.7–12.7 folds respectively. This showed 53-fold and 1.8–3.6-fold more effective than those in the control-standard of care-group. In summary, nasal-spraying Bacillus spores can rapidly and effectively relieve symptoms of RSV-induced ARTIs while exhibit strong impacts in reducing viral load and inflammation. Our nasal-spraying probiotics may provide a basis for simple-to-use, low-cost, and effective treatment against viral infection in general.

The Novel Monooxygenase Gene dipD in the dip Gene Cluster of Alcaligenes faecalis JQ135 Is Essential for the Initial Catabolism of Dipicolinic Acid.

Dipicolinic acid (DPA), an essential pyridine derivative biosynthesized in Bacillus spores, constitutes a major proportion of global biomass carbon pool. Alcaligenes faecalis strain JQ135 could catabolize DPA through the "3HDPA (3-hydroxydipicolinic acid) pathway." However, the genes involved in this 3HDPA pathway are still unknown. In this study, a dip gene cluster responsible for DPA degradation was cloned from strain JQ135. The expression of dip genes was induced by DPA and negatively regulated by DipR. A novel monooxygenase gene, dipD, was crucial for the initial hydroxylation of DPA into 3HDPA and proposed to encode the key catalytic component of the multicomponent DPA monooxygenase. The heme binding protein gene dipF, ferredoxin reductase gene dipG, and ferredoxin genes dipJ/dipK/dipL were also involved in the DPA hydroxylation and proposed to encode other components of the multicomponent DPA monooxygenase. The 18O2 stable isotope labeling experiments confirmed that the oxygen atom in the hydroxyl group of 3HDPA came from dioxygen molecule rather than water. The protein sequence of DipD exhibits no significant sequence similarities with known oxygenases, suggesting that DipD was a new member of oxygenase family. Moreover, bioinformatic survey suggested that the dip gene cluster was widely distributed in many Alpha-, Beta-, and Gammaproteobacteria, including soil bacteria, aquatic bacteria, and pathogens. This study provides new molecular insights into the catabolism of DPA in bacteria. IMPORTANCE Dipicolinic acid (DPA) is a natural pyridine derivative that serves as an essential component of the Bacillus spore. DPA accounts for 5 to 15% of the dry weight of spores. Due to the huge number of spores in the environment, DPA is also considered to be an important component of the global biomass carbon pool. DPA could be decomposed by microorganisms and enter the global carbon cycling; however, the underlying molecular mechanisms are rarely studied. In this study, a DPA catabolic gene cluster (dip) was cloned and found to be widespread in Alpha-, Beta-, and Gammaproteobacteria. The genes responsible for the initial hydroxylation of DPA to 3-hydroxyl-dipicolinic acid were investigated in Alcaligenes faecalis strain JQ135. The present study opens a door to elucidate the mechanism of DPA degradation and its possible role in DPA-based carbon biotransformation on earth.

Wet foam flow: A suitable method for improving surface hygiene in the food industry

In the food industry, the cleaning of contaminated surfaces requires new strategies to be adopted which can provide greater cleaning efficiency with minimal energy and water consumption. The use of wet foams was proposed to clean stainless steel surfaces contaminated by droplets containing Bacillus spores. Methods such as polarography, conductimetry and foam structure identification were used. Foam flow conditions with varying wall shear stresses and bubble sizes were observed to measure their impacts on surface contaminant removal kinetics. Compared to conventional cleaning-in-place method, foam flow more effectively removed both hydrophilic and hydrophobic spores. The combination of high shear stress and small bubble sizes (<0.2 mm) showed promise for improving the cleaning efficiency of the foam. Mechanisms such as fluctuation in local stresses, or in the liquid film thickness between the bubbles and the steel wall induced by bubble passage, foam imbibition and liquid film drainage were then investigated.

Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores.

Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40-72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20min) and lysozyme (100μg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.

The sporicidal activity of a disinfectant with peracetic acid against the spores of Bacillus subtilis and Bacillus cereus according to the european standard PN-EN 17126: 2019-01.

The assessment of the sporicidal effectiveness of disinfectants is important from the point of view of the prevention of nosocomial infections and spore contamination of clinical samples, medical equipment and materials used in patient care. The rods of Bacillus spp. cause infections of the digestive system, bloodstream and, less often, respiratory tract. Cases were diagnosed in immunocompromised patients, malignant neoplasms and in neonatal wards. The source of the infection was hospital linen, reusable towels, catheters or milk from the human milk bank. Determination of the minimal sporicidal parameters of a disinfectant containing peracetic acid. The sporicidal activity of a disinfectant containing peracetic acid against Bacillus subtilis and Bacillus cereus spore suspensions was tested in a defined concentration range during a contact time of 15 minutes, in the presence of various interfering substances (clean and dirty conditions) according to the European Standard PN-EN 17126: 2019-01. The disinfecting preparation containing peracetic acid showed sporicidal activity against Bacillus subtilis at a concentration of 1% for 15 minutes under clean and dirty conditions and at concentrations of 0.5%, 1.00% and 1.25% against the Bacillus cereus spores during the same contact time but only under dirty conditions. The preparation showed no sporicidal activity against Bacillus cereus at concentrations of 2%, 3%, 4% and 5% during a contact time of 15 minutes under both dirty and clean conditions. In areas where there is a risk of infecting a patient or contaminating clinical specimens, materials and equipment with spores of Bacillus spp., it is necessary to use disinfectants with sporicidal activity confirmed according to the PN-EN 17126: 2019-01 standard. The sporicidal activity of disinfectants containing peracetic acid may depend on the method of preparing the solutions, their concentration, pH, temperature and the contamination degree of the disinfected surface.

Structure of deposits formed by drying of droplets contaminated with Bacillus spores determines their resistance to rinsing and cleaning

The formation of deposits by evaporation of droplets contaminated by Bacillus spores was investigated, focusing on the role of spore and material properties. Droplets containing hydrophilic to hydrophobic spores were deposited on materials (stainless steels, polypropylene, glass). The presence of spores within the droplets, as well as material hydrophobicity plays a major role in the kinetics of the droplet shape during drying while the pattern of the dried deposit was affected by both material and spore properties. The resistance to detachment of the different deposits was then investigated. After a single rinsing procedure, a very high resistance to detachment of adherent spores was observed (up to 90% of residual spores). The hydrophilic Bs PY79 spores were the least resistant to removal, while the differences between materials were not pronounced. The ease of cleaning of the dried deposits was much greater. The least resistant spores were still the Bs PY79 ones, while the detachment was 3–100 times more effective on glass than on other materials. These results highlight the predominant role of hydrophilic/hydrophobic properties of particles and materials on the structure of deposits and their further resistance to rinsing and cleaning procedures.