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Clostridioides difficile detection in community settings is time-intensive, resulting in delays in diagnosing and quarantining infected individuals. However, with the advent of semi-automated devices and improved algorithms in recent decades, the ability to discern CDI infection from asymptomatic carriage has significantly improved. This, in turn, has led to efficiently regulated monitoring systems, further reducing endemic risk, with recent concerns regarding a possible surge in hospital-acquired Clostridioides difficile infections post-COVID failing to materialize. This review highlights established and emerging technologies used to detect community-acquired Clostridioides difficile in research and clinical settings. [ABSTRACT FROM AUTHOR]

Gold nanoparticles (AuNPs) have a wide range of applications due to their optoelectronic properties. Particularly, biological synthesis of AuNPs highlights due to the advantages related to the environmental care and the stabilization provided by capping biomolecules during biosynthesis. The antimicrobial activity of AuNPs was reported, however, their activity against resistant structures such as bacterial endospores have not been evaluated. This work shows the extracellular biosynthesis of AuNPs using Epicoccum nigrum (Au1-8) throughout an ecofriendly, simple, and efficient method. Physicochemical characterization revealed that stable quasi-spherical AuNPs in a range size of 2 to 30 nm, with crystal structure and metallic Au0 composition were obtained. The AuNPs sporicidal activity against Bacillus subtilis endospores was evaluated by its exposure to 300 mg/L AuNPs during 48 h, which produced a decrease of 90.6% of the endospore germination. The analysis of the endospore-AuNP interaction by scanning electron microscopy (SEM) and infrared (IR) spectroscopy revealed injury of the endospore coat and lysis. These results are promising for the advance in the development of low-aggressive agents used for environmental decontamination regimens against pathogenic bacterial spores, or even as an alternative for the treatment of illnesses produced by endospore-former causative agents of food spoilage/poisoning and human diseases.

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements. [ABSTRACT FROM AUTHOR]

Clostridioides difficile detection in community settings is time-intensive, resulting in delays in diagnosing and quarantining infected individuals. However, with the advent of semi-automated devices and improved algorithms in recent decades, the ability to discern CDI infection from asymptomatic carriage has significantly improved. This, in turn, has led to efficiently regulated monitoring systems, further reducing endemic risk, with recent concerns regarding a possible surge in hospital-acquired Clostridioides difficile infections post-COVID failing to materialize. This review highlights established and emerging technologies used to detect community-acquired Clostridioides difficile in research and clinical settings.

Plant-parasitic nematodes are important economic pests of a range of tropical crops. Strategies for managing these pests have relied on a range of approaches, including crop rotation, the utilization of genetic resistance, cultural techniques, and since the 1950’s the use of nematicides. Although nematicides have been hugely successful in controlling nematodes, their toxicity to humans, domestic animals, beneficial organisms, and the environment has raised concerns regarding their use. Alternatives are therefore being sought. The Pasteuria group of bacteria that form endospores has generated much interest among companies wanting to develop microbial biocontrol products. A major challenge in developing these bacteria as biocontrol agents is their host-specificity; one population of the bacterium can attach to and infect one population of plantparasitic nematode but not another of the same species. Here we will review the mechanism by which infection is initiated with the adhesion of endospores to the nematode cuticle. To understand the genetics of the molecular processes between Pasteuria endospores and the nematode cuticle, the review focuses on the nature of the bacterial adhesins and how they interact with the nematode cuticle receptors by exploiting new insights gained from studies of bacterial infections of Carnorhabditis elegans. A new Velcro-like multiple adhesin model is proposed in which the cuticle surface coat, which has an important role in endospore adhesion, is a complex extracellular matrix containing glycans originating in seam cells. The genes associated with these seam cells appear to have a dual role by retaining some characteristics of stem cells. [ABSTRACT FROM AUTHOR]

Plant-parasitic nematodes are important economic pests of a range of tropical crops. Strategies for managing these pests have relied on a range of approaches, including crop rotation, the utilization of genetic resistance, cultural techniques, and since the 1950’s the use of nematicides. Although nematicides have been hugely successful in controlling nematodes, their toxicity to humans, domestic animals, beneficial organisms, and the environment has raised concerns regarding their use. Alternatives are therefore being sought. The Pasteuria group of bacteria that form endospores has generated much interest among companies wanting to develop microbial biocontrol products. A major challenge in developing these bacteria as biocontrol agents is their host-specificity; one population of the bacterium can attach to and infect one population of plant-parasitic nematode but not another of the same species. Here we will review the mechanism by which infection is initiated with the adhesion of endospores to the nematode cuticle. To understand the genetics of the molecular processes between Pasteuria endospores and the nematode cuticle, the review focuses on the nature of the bacterial adhesins and how they interact with the nematode cuticle receptors by exploiting new insights gained from studies of bacterial infections of Carnorhabditis elegans. A new Velcro-like multiple adhesin model is proposed in which the cuticle surface coat, which has an important role in endospore adhesion, is a complex extracellular matrix containing glycans originating in seam cells. The genes associated with these seam cells appear to have a dual role by retaining some characteristics of stem cells.

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements.

The disinfection of wastewater is a treatment that allows for its reuse. However, not all pathogenic microorganisms or their resistant structures, e.g., endospores, cysts, or oocysts, are eliminated in conventional treatments. This work compared the removal efficacy of sodium hypochlorite (NaClO) and peracetic acid (PAA) on three strains of bacteria isolated from the tertiary effluent of a wastewater treatment plant. The results of the inactivation kinetics showed that complete inactivation of S. pasteuri was achieved with both NaClO (>12 log, t = 5–10 min, 15–30 mg/L) and PAA (~9 log, t = 20–30 min, 15 mg/L). Likewise, with higher concentration of both disinfectants, the inactivation time decreased. K. pneumoniae showed greater resistance to PAA (3 log, t = 30 min) than to NaClO (8 log, t = 15 min). B. subtilis endospores showed resistance to NaClO (3 log, t = 60–100 min, 15 mg/L); however, PAA was more effective (~4 log, t = 45–100 min), with 15 mg/L regarding these latter four kinetics. The inactivation curves of these strains showed concave and linear tendencies with tail effects, fitting the Weibull and Geeraerd models. Both the inactivation kinetics and the models established for vegetative cells and endospores in this study are conclusive to understand the differences between these biological forms and, consequently, their ability to survive disinfection. [ABSTRACT FROM AUTHOR]

Butyric acid producing clostridia (BAPC) cause the so-called late-blowing defect, a serious quality problem in semi-hard and hard cheeses. Late-blown cheeses are characterized by undesired slits and cracks, irregular eyes, and off-flavors due to excessive amounts of gas and organic acids produced by clostridia. Clostridial transfer to raw milk can occur during milking through dirty teats. Therefore, teat cleaning before milking is a key factor in preventing clostridial contamination of the milk. However, different cleaning methods are used, and little information is available on the efficacy of routine teat cleaning in reducing clostridial endospores. The main objectives of this study were to assess the extent of udder contamination with BAPC spores and to investigate the efficacy of routine teat cleaning on BAPC spore counts in milk. In a longitudinal study, eight dairy farms were visited during five sampling events. Clostridial spore counts were quantified from teat skin before and after routine teat cleaning, in pooled quarter milk samples from individual cows, and in bulk tank milk samples using a most probable number method. In addition, farm management data were collected periodically through a survey, and average cow cleanliness was assessed by a veterinarian. On average, teat cleaning resulted in a 0.6 log unit reduction in BAPC spores on teat skin, and a strong positive correlation was found between BAPC spore concentrations on teat skin after cleaning and in pooled quarter milk samples. Seasonal variations and the potential influence of differences in farm management were also noted. Interestingly, average cow cleanliness correlated strongly with BAPC spore levels in milk, suggesting the potential for a quick and rough estimation method of clostridial contamination that could be implemented by farmers. [ABSTRACT FROM AUTHOR]

Bacillus subtilis (Bs) is a bacterium that benefits plants and is used in the production of bio-fungicides. The cultivation of Bs is a crucial step in bio-control preparation production, as it greatly impacts the quality and price of the final product. In a series of shake flask experiments, we investigated the economically feasible broth composition for spore production of Bacillus subtilis MSCL 897, a Latvian soil isolate. Our study investigated the impact of utilizing legume-based flours (such as broad bean, grey pea, and soybean) as the primary nitrogen source, along with sugar-beet molasses, sucrose, or glucose as the carbon source, and yeast extract, peptone, and corn-steep liquor as growth factor additives. Additionally, we examined the effect of using (NH4)2HPO4 or urea as supplementary nitrogen sources, as well as previously established media formulations, on spore yield. Our results showed that a culture medium composed of broad bean flour (10 g/L) and molasses (10 g/L) led to spore productivity of 1.35 ± 0.47 × 108 CFU/mL at 48 h. By enriching the culture medium base constituents with a minor (0.5–1.0 g/L) yeast extract or corn-steep liquor additive, a notable increase in spore productivity was observed, with values of 2.00 ± 0.28 × 108 and 2.34 ± 0.18 × 108 CFU/mL at 48 h, respectively, and sporulation efficiency > 80–90%. As a result, we achieved a high spore yield of the Bacillus subtilis MSCL 897 strain, demonstrating the competitiveness of our approach, which relied on a low-cost medium made mainly from locally available and renewable raw materials. [ABSTRACT FROM AUTHOR]

Understanding interactions of bacteria with fiber-based packaging materials is fundamental for appropriate food packaging. We propose a laboratory model to evaluate microbial growth and survival in liquid media solely consisting of packaging materials with different fiber types. We evaluated food contaminating species (Escherichia coli, Staphylococcus aureus, Bacillus cereus), two packaging material isolates and bacterial endospores for their growth abilities. Growth capacities differed substantially between the samples as well as between bacterial strains. Growth and survival were strongest for the packaging material entirely made of recycled fibers (secondary food packaging) with up to 10.8 log10 CFU/ml for the packaging isolates. Among the food contaminating species, B. cereus and E. coli could grow in the sample of entirely recycled fibers with maxima of 6.1 log10 and 8.6 log10 CFU/mL, respectively. Escherichia coli was the only species that was able to grow in bleached fresh fibers up to 7.0 log10 CFU/mL. Staphylococcus aureus perished in all samples and was undetectable after 1–6 days after inoculation, depending on the sample. The packaging material strains were isolated from recycled fibers and could grow only in samples containing recycled fibers, indicating an adaption to this environment. Spores germinated only in the completely recycled sample. Additionally, microbial digestion of cellulose and xylan might not be a crucial factor for growth. This is the first study describing bacterial growth in food packaging materials itself and proposing functionalization strategies toward active food packaging through pH-lowering. [ABSTRACT FROM AUTHOR]

Archaeological studies need to use laboratory techniques, including analytical methods like Atomic Absorption Spectrometry, gas chromatography - mass spectrometry, and high-performance liquid chromatography, as well as genetic methods to resolve and verify scientific hypotheses. However, additional tools are still needed in the case of the cultural practices and traditions of ancient societies. Archaeological examinations of cultural practices have made significant progress in recent years, but additional tools are still needed to fully understand the complexity and diversity of these practices. In this work, we demonstrate how the genotyping of soil bacteria that can produce endospores is a potentially additional method for discovering past funeral rituals in various human populations who used food during their ceremonies. Endospores were isolated from soil samples taken from inside earthenware cup and pot-type vessels from a burial ground identified with the Tarnobrzeg Lusatian culture (SE Poland). The detected species of spore-forming bacteria strains were mostly environmental (originating from soil and / or water). However, the presence of some of the taxa i.e. (Peanibacillus, Bacillus) may provide a valuable source of archaeological information. We found that a combination of molecular and microbiological analysis can support archaeological studies of burial grounds and - in particular - individual graves, especially when they are characterized by a complete lack of bones. [ABSTRACT FROM AUTHOR]

Antibody detection is the critical first step for tracking the spread of many diseases including COVID-19. Lateral flow immunoassay (LFIA) is the most commonly used method for rapid antibody detection because it is easy-to-use and inexpensive. However, LFIA has limited sensitivity when gold nanoparticles (AuNPs) are used as the signals. In this study, the endospores of Bacillus subtilis were used in combination with AuNP in a LFIA to detect antibodies. The endospores serve as a signal amplifier. The detection limit was about 10−8 M for anti-beta galactosidase antibody detection whereas the detection limit of conventional LFIA is about 10−6 M. Furthermore, the proposed methods have no additional user steps compared with the traditional LFIA. This method, therefore, improved the sensitivity 100-fold without compromising any advantages of LFIA. We believe that the proposed method will be useful for detection of antibodies against HIV, Zika virus, SARS-CoV-2, and so on. [Display omitted] • An anti-β galactosidase antibody was detected using endospores as signal amplifier in a lateral flow immunoassay (LFIA). • Compared with conventional LFIA, the LOD of the proposed method using gold nanoparticles is 100 times better. • The proposed method takes 10–20 min to produce results and is user-friendly. • The proposed method improves the sensitivity of LFIA for antibody detection which is useful for disease diagnosis. [ABSTRACT FROM AUTHOR]

Rhinosporidiosis is a chronic granulomatous fungal infection caused by Rhinosporidium seeberi. Usual site of infection is the nasal mucosa & nasopharynx. Male urethra is extremely rare site involving this disease. Here we are reporting a rare case where rhinosporidiosis presented as a prolapsing mass from urethra during voiding.

ABSTRACT 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.

Honey is a value-added product rich in several types of phenolic compounds, enzymes, and sugars recently explored in biomedical and food applications. Nevertheless, even though it has a low water activity (aW ≈ 0.65) that hinders the development of pathogenic and spoilage microorganisms, it is still prone to contamination by pathogenic microorganisms (vegetative and spores) and may constitute harm to special groups, particularly by immunosuppressed people and pregnant women. Thus, an efficient processing methodology needs to be followed to ensure microbial safety while avoiding 5-hydroxymethylfurfural (HMF) formation and browning reactions, with a consequent loss of biological value. In this paper, both thermal (pressure-assisted thermal processing, PATP) and nonthermal high-pressure processing (HPP), and another pressure-based methodology (hyperbaric storage, HS) were used to ascertain their potential to inactivate Bacillus subtilis endospores in honey and to study the influence of aW on the inactivation on this endospore. The results showed that PATP at 600 MPa/15 min/75 °C of diluted honey (52.9 °Brix) with increased aW (0.85 compared to ≈0.55, the usual honey aW) allowed for inactivating of at least 4.0 log units of B. subtilis spores (to below detection limits), while HS and HPP caused neither the germination nor inactivated spores (i.e., there was neither a loss of endospore resistance after heat shock nor endospore inactivation as a consequence of the storage methodology). PATP of undiluted honey even at harsh processing conditions (600 MPa/15 min/85 °C) did not impact the spore load. The results for diluted honey open the possibility of its decontamination by spores' inactivation for medical and pharmaceutical applications. [ABSTRACT FROM AUTHOR]

Microbially mediated processes in a given habitat tend to be catalyzed by abundant populations that are ecologically adapted to exploit specific environmental characteristics. Typically, metabolic activities of rare populations are limited but may be stimulated in response to acute environmental stressors. Community responses to sudden changes in temperature and pressure can include suppression and activation of different populations, but these dynamics remain poorly understood. The permanently cold ocean floor hosts countless low-abundance microbes including endospores of thermophilic bacteria. Incubating sediments at high temperature resuscitates viable spores, causing the proliferation of bacterial populations. This presents a tractable system for investigating changes in a microbiome's community structure in response to dramatic environmental perturbations. Incubating permanently cold Arctic fjord sediments at 50°C for 216 h with and without volatile fatty acid amendment provoked major changes in community structure. Germination of thermophilic spores from the sediment rare biosphere was tracked using mass spectrometry-based metabolomics, radiotracer-based sulfate reduction rate measurements, and high-throughput 16S rRNA gene sequencing. Comparing community similarity at different intervals of the incubations showed distinct temporal shifts in microbial populations, depending on organic substrate amendment. Metabolite patterns indicated that amino acids and other sediment-derived organics were decomposed by fermentative Clostridia within the first 12-48 h. This fueled early and late phases of exponential increases in sulfate reduction, highlighting the cross-feeding of volatile fatty acids as electron donors for different sulfate-reducing Desulfotomaculia populations. The succession of germinated endospores triggered by sudden exposure to high temperature and controlled by nutrient availability offers a model for understanding the ecological response of dormant microbial communities following major environmental perturbations. [ABSTRACT FROM AUTHOR]

Aims: The worldwide spread of the coronavirus SARS‐CoV‐2 has highlighted the need for fast and simple disinfection processes, amongst others for ambulance cars on site. To overcome current drawbacks regarding room disinfection, the use of cold atmospheric plasma in remote operation represents a promising alternative for the disinfection of larger volumes. In this study, a compact plasma system was evaluated regarding its disinfection efficiency inside an ambulance car. Methods and Results: The developed plasma device is based on a dielectric barrier discharge (DBD) and operates with ambient air as process gas. The humidified afterglow from the plasma nozzle was introduced into an ambulance car with a volume of approximately 10 m3 while Bacillus atrophaeus endospores, Staphylococcus aureus or Phi 6 bacteriophages dried on different surfaces (PET‐films, glass slides or aluminum foil) were exposed to the reactive gas inside the ambulance vehicle at eight different positions. Reductions of spores by more than 4 orders of magnitude were found on all surfaces and positions within 2 h. Due to their higher susceptibility, Phi 6 bacteriophages and S. aureus counts were reduced by at least 4 orders of magnitude within 30 min on all surfaces. Conclusion: The results show that different microorganisms dried on variable surfaces can be inactivated by several orders of magnitude inside an ambulance by plasma gas from of a compact DBD plasma nozzle. Significance and Impact of the Study: Plasma gas generated on site by a DBD plasma nozzle proved to be highly efficient for the disinfection of the interior of an ambulance car. Compact plasma systems could be a viable alternative for the disinfection of vehicles or rooms. [ABSTRACT FROM AUTHOR]

We present the genomic sequences of 10 spore-forming bacteria from the Bacillaceae family isolated from fecal samples of mice residing in the Tisch Family Biblical Zoo, Jerusalem. These isolates suggest Bacillus bacteria are a native component of rodent gut flora, facilitating further research into gut colonization and microbiome diversity., Competing Interests: The authors declare no conflict of interest.

Denitrification is a key metabolic process in the global nitrogen cycle and is performed by taxonomically diverse microorganisms. Despite the widespread importance of this metabolism, challenges remain in identifying denitrifying populations and predicting their metabolic end-products based on their genotype. Here, genome-resolved metagenomics was used to explore the denitrification genotype of Bacillota enriched in nitrate-amended high temperature incubations with confirmed N 2 O and N 2 production. A set of 12 hidden Markov models (HMMs) was created to target the diversity of denitrification genes in members of the phylum Bacillota . Genomic potential for complete denitrification was found in five metagenome-assembled genomes from nitrate-amended enrichments, including two novel members of the Brevibacillaceae family. Genomes of complete denitrifiers encode N 2 O reductase gene clusters with clade II-type nosZ and often include multiple variants of the nitric oxide reductase gene . The HMM set applied to all genomes of Bacillota from the Genome Taxonomy Database identified 17 genera inferred to contain complete denitrifiers based on their gene content. Among complete denitrifiers it was common for three distinct nitric oxide reductases to be present (qNOR, bNOR, and sNOR) that may reflect the metabolic adaptability of Bacillota in environments with variable redox conditions., Competing Interests: CRJH and MF have patented oil sands emissions reduction technology that depends on biogas production by thermophilic bacteria, related to the presented research. EB, JC and WDLR declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Microbially mediated processes in a given habitat tend to be catalyzed by abundant populations that are ecologically adapted to exploit specific environmental characteristics. Typically, metabolic activities of rare populations are limited but may be stimulated in response to acute environmental stressors. Community responses to sudden changes in temperature and pressure can include suppression and activation of different populations, but these dynamics remain poorly understood. The permanently cold ocean floor hosts countless low-abundance microbes including endospores of thermophilic bacteria. Incubating sediments at high temperature resuscitates viable spores, causing the proliferation of bacterial populations. This presents a tractable system for investigating changes in a microbiome's community structure in response to dramatic environmental perturbations. Incubating permanently cold Arctic fjord sediments at 50°C for 216 h with and without volatile fatty acid amendment provoked major changes in community structure. Germination of thermophilic spores from the sediment rare biosphere was tracked using mass spectrometry-based metabolomics, radiotracer-based sulfate reduction rate measurements, and high-throughput 16S rRNA gene sequencing. Comparing community similarity at different intervals of the incubations showed distinct temporal shifts in microbial populations, depending on organic substrate amendment. Metabolite patterns indicated that amino acids and other sediment-derived organics were decomposed by fermentative Clostridia within the first 12–48 h. This fueled early and late phases of exponential increases in sulfate reduction, highlighting the cross-feeding of volatile fatty acids as electron donors for different sulfate-reducing Desulfotomaculia populations. The succession of germinated endospores triggered by sudden exposure to high temperature and controlled by nutrient availability offers a model for understanding the ecological response of dormant microbial communities following major environmental perturbations.

The disinfection of wastewater is a treatment that allows for its reuse. However, not all pathogenic microorganisms or their resistant structures, e.g., endospores, cysts, or oocysts, are eliminated in conventional treatments. This work compared the removal efficacy of sodium hypochlorite (NaClO) and peracetic acid (PAA) on three strains of bacteria isolated from the tertiary effluent of a wastewater treatment plant. The results of the inactivation kinetics showed that complete inactivation of S. pasteuri was achieved with both NaClO (>12 log, t = 5–10 min, 15–30 mg/L) and PAA (~9 log, t = 20–30 min, 15 mg/L). Likewise, with higher concentration of both disinfectants, the inactivation time decreased. K. pneumoniae showed greater resistance to PAA (3 log, t = 30 min) than to NaClO (8 log, t = 15 min). B. subtilis endospores showed resistance to NaClO (3 log, t = 60–100 min, 15 mg/L); however, PAA was more effective (~4 log, t = 45–100 min), with 15 mg/L regarding these latter four kinetics. The inactivation curves of these strains showed concave and linear tendencies with tail effects, fitting the Weibull and Geeraerd models. Both the inactivation kinetics and the models established for vegetative cells and endospores in this study are conclusive to understand the differences between these biological forms and, consequently, their ability to survive disinfection.

Butyric acid producing clostridia (BAPC) cause the so-called late-blowing defect, a serious quality problem in semi-hard and hard cheeses. Late-blown cheeses are characterized by undesired slits and cracks, irregular eyes, and off-flavors due to excessive amounts of gas and organic acids produced by clostridia. Clostridial transfer to raw milk can occur during milking through dirty teats. Therefore, teat cleaning before milking is a key factor in preventing clostridial contamination of the milk. However, different cleaning methods are used, and little information is available on the efficacy of routine teat cleaning in reducing clostridial endospores. The main objectives of this study were to assess the extent of udder contamination with BAPC spores and to investigate the efficacy of routine teat cleaning on BAPC spore counts in milk. In a longitudinal study, eight dairy farms were visited during five sampling events. Clostridial spore counts were quantified from teat skin before and after routine teat cleaning, in pooled quarter milk samples from individual cows, and in bulk tank milk samples using a most probable number method. In addition, farm management data were collected periodically through a survey, and average cow cleanliness was assessed by a veterinarian. On average, teat cleaning resulted in a 0.6 log unit reduction in BAPC spores on teat skin, and a strong positive correlation was found between BAPC spore concentrations on teat skin after cleaning and in pooled quarter milk samples. Seasonal variations and the potential influence of differences in farm management were also noted. Interestingly, average cow cleanliness correlated strongly with BAPC spore levels in milk, suggesting the potential for a quick and rough estimation method of clostridial contamination that could be implemented by farmers.

The ever-increasing complexity in critical spacecraft hardware and materials has led to the development of new microbial reduction procedures as well as to changes in established processes such as heat microbial reduction (HMR). In the space biology field of Planetary Protection, 500°C for 0.5 s is the current HMR recommendation to reduce microorganisms from flight hardware. However, more studies are needed to effectively determine the microbial reduction capability of high-temperature (more than 200°C), short-duration (under 30 s) heat exposures. One of the many recent microbial reduction bioengineering research avenues harnesses electromagnetic energy for microbial reduction, with previous investigations demonstrating that infrared heaters are capable of the short temperature ramp time required for rapid heating investigations above 200°C. Therefore, this study employed a 6 kW infrared heater to determine the survivability of heat resistant Bacillus canaveralius 29669 to high-temperature, short-duration infrared temperatures. While B. canaveralius 29669 spores can survive microbial heat reduction processes above 200°C, we found evidence suggesting that the 500°C for 0.5 s temperature sterilization specification for Planetary Protection should be updated. This research presents spore survival data and a corresponding model pointing to a re-evaluation of the recommended HMR exposure of 500°C for 0.5 s, while simultaneously meeting requirements on the forward biological contamination of solar system bodies and opening up design possibilities for future spacecraft hardware. [ABSTRACT FROM AUTHOR]

The inactivation processes of coliform bacteria (total and fecal) and sulphito-reducing Clostridium bacteria (vegetative species and spores) in water maturation lagoon of a low-cost nature-based wastewater treatment plant using constructed wetlands and through processes of photolysis in a pilot photoreactor have been comparatively studied. The different inactivation mechanisms by photolysis of these bacteria have been studied following the criteria of different statistical and kinetic models. Clostridium disinfection treatments fit models in which two types of bacteria populations coexist, one sensitive (vegetative species) and the other (spores) resistant to the treatment, the sensitive one (94%) with an inactivation rate of k = 0.24 ± 0.07 min−1 and the resistant one (6%) with k = 0.11 ± 0.05 min−1. Total coliform photolytic disinfection also shows two populations with different physiological state. The time required to reduce the first logarithmic decimal cycle of the different types of bacteria (physiological states) are δ1 = 4.2 ± 0.9 and δ2 = 8.3 ± 1.1 min, respectively. For fecal coliform photolytic disinfection, only bacteria population, with k = 1.15 ± 0.19 min−1, is found. The results obtained confirm the photolytic disinfection processes and maturation lagoon are effective systems for Clostridia bacteria removal after water treatment by nature-based systems. Total removal of coliform bacteria is not achieved by maturation lagoons, but their reduction is significant using low doses of cumulative radiation. [ABSTRACT FROM AUTHOR]

The ever-increasing complexity in critical spacecraft hardware and materials has led to the development of new microbial reduction procedures as well as to changes in established processes such as heat microbial reduction (HMR). In the space biology field of Planetary Protection, 500°C for 0.5 s is the current HMR recommendation to reduce microorganisms from flight hardware. However, more studies are needed to effectively determine the microbial reduction capability of high-temperature (more than 200°C), short-duration (under 30 s) heat exposures. One of the many recent microbial reduction bioengineering research avenues harnesses electromagnetic energy for microbial reduction, with previous investigations demonstrating that infrared heaters are capable of the short temperature ramp time required for rapid heating investigations above 200°C. Therefore, this study employed a 6 kW infrared heater to determine the survivability of heat resistant Bacillus canaveralius 29669 to high-temperature, short-duration infrared temperatures. While B. canaveralius 29669 spores can survive microbial heat reduction processes above 200°C, we found evidence suggesting that the 500°C for 0.5 s temperature sterilization specification for Planetary Protection should be updated. This research presents spore survival data and a corresponding model pointing to a re-evaluation of the recommended HMR exposure of 500°C for 0.5 s, while simultaneously meeting requirements on the forward biological contamination of solar system bodies and opening up design possibilities for future spacecraft hardware.

Bacillus subtilis (Bs) is a bacterium that benefits plants and is used in the production of bio-fungicides. The cultivation of Bs is a crucial step in bio-control preparation production, as it greatly impacts the quality and price of the final product. In a series of shake flask experiments, we investigated the economically feasible broth composition for spore production of Bacillus subtilis MSCL 897, a Latvian soil isolate. Our study investigated the impact of utilizing legume-based flours (such as broad bean, grey pea, and soybean) as the primary nitrogen source, along with sugar-beet molasses, sucrose, or glucose as the carbon source, and yeast extract, peptone, and corn-steep liquor as growth factor additives. Additionally, we examined the effect of using (NH4)2HPO4 or urea as supplementary nitrogen sources, as well as previously established media formulations, on spore yield. Our results showed that a culture medium composed of broad bean flour (10 g/L) and molasses (10 g/L) led to spore productivity of 1.35 ± 0.47 × 108 CFU/mL at 48 h. By enriching the culture medium base constituents with a minor (0.5–1.0 g/L) yeast extract or corn-steep liquor additive, a notable increase in spore productivity was observed, with values of 2.00 ± 0.28 × 108 and 2.34 ± 0.18 × 108 CFU/mL at 48 h, respectively, and sporulation efficiency > 80–90%. As a result, we achieved a high spore yield of the Bacillus subtilis MSCL 897 strain, demonstrating the competitiveness of our approach, which relied on a low-cost medium made mainly from locally available and renewable raw materials.

Analysis of the dipicolinic acid (DPA) released from Clostridium botulinum spores during thermal processing is crucial to obtaining a mechanistic understanding of the factors involved in spore heat resistance and related food safety applications. Here, we developed a novel mixed-mode liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for detection of the DPA released from C.botulinum type A, nonproteolytic types B and F strains, and nonpathogenic surrogate Clostridium sporogenes PA3679 spores. DPA was retained on a mixed-mode C18/anion exchange column and was detected using electrospray ionization (ESI) positive mode within a 4-min analysis time. The intraday and interday precision (%CV) was 1.94–3.46% and 4.04–8.28%, respectively. Matrix effects were minimal across proteolytic type A Giorgio-A, nonproteolytic types QC-B and 202-F, and C. sporogenes PA3679 spore suspensions (90.1–114% of spiked DPA concentrations). DPA recovery in carrot juice and beef broth ranged from 105 to 118%, indicating limited matrix effects of these food products. Experiments that assessed the DPA released from Giorgio-A spores over the course of a 5-min thermal treatment at 108 °C found a significant correlation (R = 0.907; P < 0.05) between the log reduction of spores and amount of DPA released. This mixed-mode LC–MS/MS method provides a means for rapid detection of DPA released from C.botulinum spores during thermal processing and has the potential to be used for experiments in the field of food safety that assess the thermal resistance characteristics of various C. botulinum spore types. [ABSTRACT FROM AUTHOR]

Clostridium butyricum DKU-11, a bacterium has been isolated from the stools of breastfed infants near Cheonan-Asan city, Republic of Korea, and has a genome sequence of 4,630,814 bp. The GC content is 28.7% and a total of 4,137 coding sequences in two contigs., Competing Interests: The author declares no conflict of interest.

The genome of Paenibacillus phoenicis, a spore-forming bacterium isolated from the spacecraft assembly facility of the Phoenix mission, was generated via hybrid assembly by merging short and long reads. Examining this genome may shed light on strategies to minimize the risk of contaminating extraterrestrial environments with Earth-based microorganisms., Competing Interests: The authors declare no conflict of interest.

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 (NaNO 2 ), 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 NaNO 2 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 NaNO 2 . 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 NaNO 2 used, this combination of EOs represents a promising antimicrobial alternative to this preservative, encouraging further research in this direction.

In original experiments, Bak et al. (Wind-Driven Saltation: An Overlooked Challenge for Life on Mars. Astrobiology 2019;19(4):497–505) suggest a new mechanism for the destruction of spores on Mars: abrasion by wind-driven saltation. Bak et al. found that the tumbling of spores on grain surfaces (simulating saltation) was, by far, most lethal at the outset of their experiments. They suggest that it may be sharp edges of the freshly crushed basalt particles used in their experiments that destroy the spores and that these edges abrade away over the course of each experiment. But prior Mars analogue experiments, observations of particles from terrestrial deserts, and imaging from Mars landers suggest that most martian dust has been rounded by billions of years of aeolian processes. If so, saltation on Mars is more likely well simulated by the later stages of the Bak et al. experiments, reducing implied lethality by orders of magnitude. Experiments could test this by beginning with particles that had been already abraded. Even assuming the highest lethality found in their experiments, saltation "hop" distances on Mars suggest that abrasion would not prevent ∼1% of released spores from remaining viable while traveling hundreds or even thousands of kilometers. [ABSTRACT FROM AUTHOR]

Aim: The survival of Clostridioides difficile (previously Clostridium difficile) vegetative cells and endospores was compared at different levels of indigenous microflora using autoclaved and unautoclaved dairy composts with different moisture contents (MCs). Methods and Results: Both types of composts adjusted to 20, 30 and 40% MCs were inoculated with a suspension of C. difficile that contained both vegetative cells (c. 5–6 log CFU per gram) and endospores (c. 5·0 CFU per gram), and then stored aerobically inside a humidity‐controlled chamber at room temperature 22·5 ± 0·8°C for 1 year. The level of indigenous microflora was very stable during the storage after day 7 in both types of compost. The greatest reductions of C. difficile vegetative cell counts occurred during the first 24 h of storage in autoclaved and unautoclaved composts, which had 4·7 and 5·5 log CFU per gram with 20% MC, 1·8 and 2·1 log CFU per gram with 30% MC, and 2·3 and 1·3 log CFU per gram with 40% MC, respectively. Both MC and the duration of storage have significant (P < 0·05) effects on the survival of vegetative cells for first 120 days of storage. The slow inactivation of C. difficile vegetative cells at higher MCs during aerobic storage was confirmed by exponentially decaying modelling data during the early stage of aerobic exposure. The reduction of endospore counts (<1·0 log CFU per gram) during the storage for both types of compost at all MCs was not significant (P > 0·05) except for the autoclaved compost with 30% MC. Conclusion: The highly resistant C. difficile endospores to the unfavourable environmental conditions survived for more than a year while vegetative cells died off exponentially upon the initial aerobic exposure. Significance and Impact of the Study: The long‐term survival of C. difficile endospores in contaminated compost may transmit the pathogen to fresh produce, animals or water in pre‐harvest conditions. [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to evaluate the antimicrobial effect of the essential oils of cinnamon, cardamom, clove, oregano, and thyme and their synergism on vegetative cells and endospores of Clostridium perfringens type A inoculated in meat sausage (mortadella), as well as the influence of blends on the color, and lipid oxidation through the determination of thiobarbituric acid reactive substances (TBARS index). The anticlostridial action of the oil blends was established. The two added oil blends (Treat. 1: oregano, clove, and thyme; Treat. 2: oregano, clove, and cinnamon) in combination with reduced nitrite content (75 ppm) promoted a lower growth of C. perfringens in mortadella stored at 15 °C for 21 days in comparison to treatments containing only 75 ppm of nitrite. The essential oil blends showed antioxidant action and did not alter food color, thus possessing potential application as a preservative for the meat products industry.

The efficiency of plasmas sources for the decontamination of heat-sensitive devices has been proven for more than 20 years, but commercial plasma-based sterilizers still have a narrow range of applications. This can be partially explained by difficulties to determine reliable bio-indicators and standardized microbiological test procedures required by industrial uses. In this paper, we examine the influence of environmental factors on the inactivation rate of microorganisms deposited on surfaces and treated by plasma sources. In addition, we present a literature review showing that several in-discharge and afterglow plasma sterilizers offer shorter treatment times than conventional low-temperature sterilizers to reduce the concentration of endospores on contaminated surfaces by 6-log. Finally we make a few recommendations for future plasma decontamination standards.

The toxin-producing bacterium Bacillus cereus is an important and neglected human pathogen and a common cause of food poisoning. Several toxins have been implicated in disease, including the pore-forming toxins hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE). Recent work revealed that HBL binds to the mammalian surface receptors LITAF and CDIP1 and that both HBL and NHE induce potassium efflux and activate the NLRP3 inflammasome, leading to pyroptosis. These mammalian receptors, in part, contribute to inflammation and pathology. Other putative virulence factors of B. cereus include cytotoxin K, cereulide, metalloproteases, sphingomyelinase, and phospholipases. In this review, we highlight the latest progress in our understanding of B. cereus biology, epidemiology, and pathogenesis, and discuss potential new directions for research in this field. Bacillus cereus is an important human pathogen, and new findings have expanded our understanding of how this bacterium causes disease. B. cereus Hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE) induce membrane pore formation, leading to activation of the NLRP3 inflammasome, systemic inflammation, and death. Lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (LITAF) and cell death-inducing P53 target 1 (CDIP1) are bona fide mammalian surface receptors of HBL. These newly identified toxin receptors and the NLRP3 inflammasome represent unique targets for potential future therapies against severe B. cereus infections. [ABSTRACT FROM AUTHOR]

Abstract Background Southern root-knot nematode Meloidogyne incognita (Kofoid and White, 1919), Chitwood, 1949 is a key pest of agricultural crops. Pasteuria penetrans is a hyperparasitic bacterium capable of suppressing the nematode reproduction, and represents a typical coevolved pathogen-hyperparasite system. Attachment of Pasteuria endospores to the cuticle of second-stage nematode juveniles is the first and pivotal step in the bacterial infection. RNA-Seq was used to understand the early transcriptional response of the root-knot nematode at 8 h post Pasteuria endospore attachment. Results A total of 52,485 transcripts were assembled from the high quality (HQ) reads, out of which 582 transcripts were found differentially expressed in the Pasteuria endospore encumbered J2 s, of which 229 were up-regulated and 353 were down-regulated. Pasteuria infection caused a suppression of the protein synthesis machinery of the nematode. Several of the differentially expressed transcripts were putatively involved in nematode innate immunity, signaling, stress responses, endospore attachment process and post-attachment behavioral modification of the juveniles. The expression profiles of fifteen selected transcripts were validated to be true by the qRT PCR. RNAi based silencing of transcripts coding for fructose bisphosphate aldolase and glucosyl transferase caused a reduction in endospore attachment as compared to the controls, whereas, silencing of aspartic protease and ubiquitin coding transcripts resulted in higher incidence of endospore attachment on the nematode cuticle. Conclusions Here we provide evidence of an early transcriptional response by the nematode upon infection by Pasteuria prior to root invasion. We found that adhesion of Pasteuria endospores to the cuticle induced a down-regulated protein response in the nematode. In addition, we show that fructose bisphosphate aldolase, glucosyl transferase, aspartic protease and ubiquitin coding transcripts are involved in modulating the endospore attachment on the nematode cuticle. Our results add new and significant information to the existing knowledge on early molecular interaction between M. incognita and P. penetrans.

Endospore formation is used by members of the phylum Firmicutes to withstand extreme environmental conditions. Several recent studies have proposed endospore formation in species outside Firmicutes, particularly in Rhodobacter johrii and Serratia marcescens, members of the phylum Proteobacteria.Inthisstudy, we aimed to investigate endospore formation in these two species by using advanced imaging and analytical approaches. Examination of the phase-bright structures observed in R. johrii and S. marcescens using cryo-electron tomography failed to identify endospores or stages of endospore formation. We determined that the phase-bright objects in R. johrii cells were triacylglycerol storage granules and those in S. marcescens were aggregates of cellular debris. In addition, R. johrii and S. marcescens containing phase-bright objects do not possess phenotypic and genetic features of endospores, including enhanced resistance to heat, presence of dipicolinic acid, or the presence of many of the genes associated with endospore formation. Our results support the hypothesis that endospore formation is restricted to the phylum Firmicutes. [ABSTRACT FROM AUTHOR]

The human gut microbiome is a diverse and complex ecosystem that plays a critical role in health and disease. The composition of the gut microbiome has been well studied across all stages of life. In recent years, studies have investigated the production of endospores by specific members of the gut microbiome. An endospore is a tough, dormant structure formed by members of the Firmicutes phylum, which allows for greater resistance to otherwise inhospitable conditions. This innate resistance has consequences for human health and disease, as well as in biotechnology. In particular, the formation of endospores is strongly linked to antibiotic resistance and the spread of antibiotic resistance genes, also known as the resistome. The term sporobiota has been used to define the spore-forming cohort of a microbial community. In this review, we present an overview of the current knowledge of the sporobiota in the human gut. We discuss the development of the sporobiota in the infant gut and the perinatal factors that may have an effect on vertical transmission from mother to infant. Finally, we examine the sporobiota of critically important food sources for the developing infant, breast milk and powdered infant formula.

The bacterium Bacillus subtilis has long been an important subject for basic studies. However, this organism has also had industrial applications due to its easy genetic manipulation, favorable culturing characteristics for large-scale fermentation, superior capacity for protein secretion, and generally recognized as safe (GRAS) status. In addition, as the metabolically dormant form of B. subtilis, its spores have attracted great interest due to their extreme resistance to many environmental stresses, which makes spores a novel platform for a variety of applications. In this review, we summarize both conventional and emerging applications of B. subtilis spores, with a focus on how their unique characteristics have led to innovative applications in many areas of technology, including generation of stable and recyclable enzymes, synthetic biology, drug delivery, and material sciences. Ultimately, this review hopes to inspire the scientific community to leverage interdisciplinary approaches using spores to address global concerns about food shortages, environmental protection, and health care. [ABSTRACT FROM AUTHOR]

Background: Clostridioides difficile is transmitted through endospores. Most disinfection procedures for these structures deploy high concentrations of chlorine-derived compounds such as sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC). However, these substances are linked to undesirable public health and environmental issues.Aim: To compare the efficacy of NaCl-derived electrochemically activated solution (ECAS, 0.18% w/v NaOCl, pH=9.6-10.3), commercial bleach (5000 ppm, 2.83% w/v NaOCl, pH=5.6) and NaDCC (1000 ppm, pH=6.8) to inactivate C. difficile endospores on surfaces using a standard quantitative test (EPA MO-21-03).Findings: Ten representative reference and field strains from multi-locus sequence typing Clades 1-5 were assayed (N=10). Irrespective of the phylogenetic background of the strains, ECAS showed comparable or better log reduction values [mean=3.22, 95% confidence interval (CI) 0.40-5.56] than bleach (mean=2.74, 95% CI 0.12-5.50) and NaDCC (mean=2.02, 95% CI 0.10-5.12). Cyclic voltammetry measurements revealed similar electrochemical behaviours and open-circuit potentials for ECAS and NaOCl. Congruently, similar morphologies for spores treated with these two compounds were observed by transmission electron microscopy. A factorial design demonstrated that exposure time, but not activation time, influenced the efficacy of ECAS.Conclusions: ECAS and NaOC were found to have functional equivalence and may have a common mechanism of action. [ABSTRACT FROM AUTHOR]