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To investigate the heating sterilization indicator bacteria used in the production of "Hyuganatsu" straight juice stored in paper containers using the aseptic method, we examined the degree of contamination by various bacterial spores in the untreated raw material, "Hyuganatsu" juice. In a total of 11 samples, 51 bacterial strains were isolated by aerobic culture, 12 strains were isolated by anaerobic culture, and thermophilic acidophilic bacteria (TAB) were not detected. Among the isolated strains, Paenibacillus spp. were detected most frequently, followed by Bacillus spp., Paraclostridium spp., and Clostridium spp., but these bacteria could not grow in "Hyuganatsu" straight juice (pH 3.1), so there was no need to evaluate them as sterilization indicator bacteria. TAB was not detected in this experiment, but considering past failures and the characteristics of this species, it is necessary to consider TAB as sterilization indicator bacteria. Therefore, in the future, we will conduct a growth test in "Hyuganatsu" straight juice using TAB, and if growth occurs, we will consider it necessary to evaluate TAB as sterilization indicator bacteria by setting the appropriate heating sterilization conditions. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacterial spores are metabolically dormant, resistant to microbicides, and vectors of food spoilage and diseases, while germinated spores are easy to kill. Consequently, understanding germination mechanisms may facilitate the development of “germinate-to-eradicate” strategies. Spores germinate in response to many compounds (called germinants). They can also retain the memory of a germinant exposure, such that a second exposure triggers more efficient germination, but how is not clear. A recent high-profile paper [Science (2022) 378:43] suggested that increasing spore electrochemical potential is how memory is “stored” based on measurements of Bacillus subtilis spores’ accumulation of the dye thioflavin-T after germinant exposure. Indeed, we found that wild-type spores of three Bacillus and one Clostridioides species all exhibited this early thioflavin-T accumulation during nutrient pulses. However, our data indicate that inferring spores’ electrochemical potential from thioflavin-T accumulation is problematic. We found that B. subtilis spores lacking their proteinaceous coat exhibited memory but did not accumulate thioflavin-T prior to germinant addition or during nutrient pulses. Furthermore, wild-type Bacillus spores germinating with dodecylamine, which also elicits memory, showed no thioflavin-T accumulation. Finally, we found that thioflavin-T accumulation by a germinating spore is outside the spore core at early stages but inside the spore core as germination proceeds. These findings suggest that thioflavin-T accumulation during the early stages of germination is due to its binding to one or more protein in the spore coat rather than to changes in spores’ electrochemical potential; thus, thioflavin-T is not a potentiometric dye for the study of spore memory of germinant pulses. IMPORTANCE Bacillus and Clostridium spores cause food spoilage and disease because of spores’ dormancy and resistance to microbicides. However, when spores “come back to life” in germination, their resistance properties are lost. Thus, understanding the mechanisms of spore germination could facilitate the development of “germinate to eradicate” strategies. One germination feature is the memory of a pulsed germinant stimulus leading to greater germination following a second pulse. Recent observations of increases in spore binding of the potentiometric dye thioflavin-T early in their germination of spores led to the suggestion that increasing electrochemical potential is how spores “remember” germinant pulses. However, new work finds no increased thioflavin-T binding in the physiological germination of Coatless spores or of intact spores germinating with dodecylamine, even though spore memory is seen in both cases. Thus, using thioflavin-T uptake by germinating spores to assess the involvement of electrochemical potential in memory of germinant exposure, as suggested recently, is questionable.

Aims To assess low concentration hydrogen peroxide (LCHP) (H2O2) vapor dispersed with a home humidifier for its ability to decontaminate vehicle interiors contaminated with Bacillus anthracis surrogate Bacillus atrophaeus spores. Methods and results Efficacy of a vaporized 3% H2O2 solution was evaluated for liquid volumes, on/off vehicle heating, ventilation, and air conditioning (HVAC) system operations, and temperatures that ranged from 5 to 27°C. Survival of the spores was assessed by quantification of remaining viable spores with efficacy quantified in terms of mean log10 reduction. Decontamination efficacy after the 6-day dwell time increased when the 3% H2O2 liquid volume was doubled, increasing from 4-of-10 to 10-of-10 nondetects (zero colonies counted using standard dilution and filter plating) inside the vehicle cabin. Recirculating cabin air through the HVAC system during decontamination decreased efficacy to 6-of-10 non-detects. While no 6-log10 reduction in viable spores was observed on the cabin filter with the cabin filter kept in place, a 6-log10 reduction was achieved after its removal and placement in the cabin during treatment. Conclusions Results from this study allow for informed decisions on the use of LCHP vapor as an effective decontamination approach for vehicle interiors. [ABSTRACT FROM AUTHOR]

ABSTRACT There is growing evidence that shows Clostridium (Clostridioides) difficile is a pathogen of One Health importance with a complex dissemination pathway involving animals, humans, and the environment. Thus, environmental discharge and agricultural recycling of human and animal waste have been suspected as factors behind the dissemination of Clostridium difficile in the community. Here, the presence of C. difficile in 12 wastewater treatment plants (WWTPs) in Western Australia was investigated. Overall, C. difficile was found in 90.5% (114/126) of raw sewage influent, 48.1% (50/104) of treated effluent, 40% (2/5) of reclaimed irrigation water, 100% (38/38) of untreated biosolids, 95.2% (20/21) of anaerobically digested biosolids, and 72.7% (8/11) of lime-amended biosolids. Over half of the isolates (55.3% [157/284]) were toxigenic, and 97 C. difficile ribotypes (RTs) were identified, with RT014/020 the most common (14.8% [42/284]). Thirteen C. difficile isolates with the toxin gene profile A+ B+ CDT+ (positive for genes coding for toxins A and B and the binary C. difficile transferase toxin [CDT]) were found, including the hypervirulent RT078 strain. Resistance to the antimicrobials fidaxomicin, vancomycin, metronidazole, rifaximin, amoxicillin-clavulanate, meropenem, and moxifloxacin was uncommon; however, resistance to clindamycin, erythromycin, and tetracycline was relatively frequent at 56.7% (161/284), 14.4% (41/284), and 13.7% (39/284), respectively. This study revealed that toxigenic C. difficile was commonly encountered in WWTPs and being released into the environment. This raises concern about the possible spillover of C. difficile into animal and/or human populations via land receiving the treated waste. In Western Australia, stringent measures are in place to mitigate the health and environmental risk of recycling human waste; however, further studies are needed to elucidate the public health significance of C. difficile surviving the treatment processes at WWTPs. IMPORTANCE Clostridium difficile infection (CDI) is a leading cause of antimicrobial-associated diarrhea in health care facilities. Extended hospital stays and recurrences increase the cost of treatment and morbidity and mortality. Community-associated CDI (CA-CDI) cases, with no history of antimicrobial use or exposure to health care settings, are increasing. The isolation of clinically important C. difficile strains from animals, rivers, soil, meat, vegetables, compost, treated wastewater, and biosolids has been reported. The objective of this study was to characterize C. difficile in wastewater treatment plants (WWTPs) in Australia. We found that C. difficile can survive the treatment processes of WWTPs, and toxigenic C. difficile was being released into the environment, becoming a potential source/reservoir for CA-CDI.

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency. Bacillus subtilis spores were pressurized for 50 s – 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions. The ∼7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43–52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions. The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality. • Bacterial spores superdormant (SD) to very high pressure (vHP) were characterized. • Flow cytometry is usable for SD spores after ≤1 min at vHP and post-HP incubation. • The vHP (550 MPa, 60 °C) SD spores had a normal dipicolinic acid content. • Superdormancy (resistance to vHP) was lost over time and not genetically inherited. • Superdormancy to vHP was unlikely caused by permanent cellular properties. [ABSTRACT FROM AUTHOR]

The study explored the effects of electric field strength, temperature, and frequency on the inactivation of Clostridium sporogenes PA3679 spores (a surrogate of Clostridium botulinum) within buffer. The experiments were conducted at three different field strengths (30, 40, and 50 V/cm), temperatures (110, 120, and 130 °C), and frequencies (60, 1000, and 5000 Hz). Ohmic heating resulted in significantly greater reduction of C. sporogenes compared to conventional heating. Electric field strength significantly influenced spore inactivation. It was found that lower frequencies led to quicker rates of inactivation. Given that high field strengths have dual benefits of increasing both heating and spore kill rate, the possibility exists to process foods to sterility without a hold. This is expected to result in improved product quality, reduced process times and increased efficiency. • Ohmic heating results in higher bacterial spore inactivation than conventional heating. • This study also confirms the existence of non-thermal effects of ohmic heating. • Higher field strength offered significantly higher inactivation of spores. • 60Hz and 5000 Hz offered highest and lowest inactivation of Clostridium sporogenes. [ABSTRACT FROM AUTHOR]

Pulsed electric field (PEF) treatment, an alternative to thermal processing in the food industry, is insufficient to inactivate bacterial spores. Although spores that have been treated in this manner remain alive, specific understanding of their physiological properties is limited. The purpose of this study is to describe the morphology, viability, and germination behavior of Bacillus atrophaeus spores treated with PEF. Our findings indicate that nonlethal PEF treatment results in spore deformation, dipicolinic acid (DPA) leakage, and a shorter and more uniform germination lag time (Tlag), but that there is no change in release duration (ΔTrelease), germination ratio, or viability. Based on our findings, we conclude that an intact morphologic state and DPA content are not prerequisites for germination and full viability and that, in contrast to nutrient-induced germination in which initially slowly released DPA triggers subsequent germination events, leaked DPA during PEF treatment does not. Spores that have been subjected to this procedure remain dormant and preserve their full germinability. We found that PEF-treated spores respond to germinants more quickly and with less heterogeneity, possibly because the tiny cracks formed on the spore surface facilitate the germinants’ access to the germination receptors situated on the spore’s inner membrane. The consensus view that nonlethal PEF has less impact on spores that are still capable of forming CFUs under proper conditions is one-sided. This research advances our understanding of how spores behave following nonlethal PEF treatment and gives information on the topics of nosocomial sterilization, food safety, and public health. [ABSTRACT FROM AUTHOR]

Abstract Bio-concrete using bacterially produced calcium carbonate can repair microcracks but is still relatively expensive due to the addition of bacteria, nutrients, and calcium sources. Agricultural by-products and oyster shells were used to produce economical bio-concrete. Sesame meal was the optimal agricultural by-product for low-cost spore production of the alkaliphilic Bacillus miscanthi strain AK13. Transcriptomic dataset was utilized to compare the gene expressions of AK13 strain under neutral and alkaline conditions, which suggested that NaCl and riboflavin could be chosen as growth-promoting factors at alkaline pH. The optimal levels of sesame meal, NaCl, and riboflavin were induced with the central composite design to create an economical medium, in which AK13 strain formed more spores with less price than in commercial sporulation medium. Calcium nitrate obtained from nitric acid treatment of oyster shell powder increased the initial compressive strength of cement mortar. Non-ureolytic calcium carbonate precipitation by AK13 using oyster shell-derived calcium ions was verified by energy-dispersive X-ray spectroscopy and X-ray diffraction analysis. Stereomicroscope and field emission scanning electron microscopy confirmed that oyster shell-derived calcium ions, along with soybean meal-solution, increased the bacterial survival and calcium carbonate precipitation inside mortar cracks. These data suggest the possibility of commercializing bacterial self-healing concrete with economical substitutes for culture medium, growth nutrient, and calcium sources.

Little is known about final spores components when bacteria undergo sporulation under different nutrient conditions. Different degrees of resistance and germination rates were observed in the three types of spores of Lysinibacillus boronitolerans YS11 (SD, Spores formed in Difco sporulation medium™; SC and SF, Spores formed in an agricultural byproduct medium with 10 mM CaCl2 and with 10 mM FeSO4, respectively). Stronger UV resistance was recorded for SF with 1.8–2.3-fold greater survival than SC and SD under UV treatment. The three spore types showed similar heat resistances at 80°C, but survival rates of SC and SD were much higher (∼1,000 times) than those of SF at 90°C. However, germination capacity of SF was 20% higher than those of SD and SC on Luria-Bertani agar plates for 24 h. SF germinated more rapidly in a liquid medium with high NaCl concentrations than SC and SD, but became slower under alkaline conditions. Raman spectroscopy was used to analyze the heterogeneities in the three types of vegetative cells and their spores under different nutritional conditions. Exponentially grown-each vegetative cells had different overall Raman peak values. Raman peaks of SC, SD, and SF also showed differences in adenine and amide III compositions and nucleic acid contents. Our data along with Raman spectroscopy provided the evidence that spores formed under under different growth conditions possess very different cellular components, which affected their survival and germination rates. [ABSTRACT FROM AUTHOR]

Aim: The aim of the present study was to examine the vaccine immune response in ewes supplemented with Bacillus toyonensis BCT‐7112T during a period of 5‐day supplementation before vaccination against a recombinant Clostridium perfringens epsilon toxin (rETX). Methods and Results: Ewes were vaccinated with 200 µg of rETX adjuvanted with 10% aluminium hydroxide. The treat group was orally supplemented with B. toyonensis BCT‐7112T (3 × 108 viable spores) for 5 days prior to the first and second vaccination. Ewes supplemented with B. toyonensis BCT‐7112T showed higher neutralizing antibody titres than the non‐supplemented ewes (P < 0·05), with an increase in serum levels for total IgG anti‐rETX by 3·2‐fold (P < 0·0001), and for both IgG isotypes IgG1 and IgG2 by 2·1‐fold and 2·3‐fold (P < 0·01), respectively, compared with the control group. The peripheral blood mononuclear cells of ewes in the supplemented group had a higher (P < 0·05) cytokine mRNA transcription levels for IL‐2 (6·4‐fold increase), IFN‐γ (2·9‐fold increase) and transcription factor Bcl6 (2·3‐fold increase) compared with the control group. Conclusion: We conclude that a 5 days of supplementation with B. toyonensis BCT‐7112T prior vaccination is sufficient to significantly improve the humoral immune response of ewes against C. perfringens recombinant ETX vaccine. Significance and Impact of the study: These findings open a new perspective in the utilization of B. toyonensis BCT‐7112T as an immunomodulator since a 5 days period of probiotic supplementation is sufficient to improve the vaccine immune response. [ABSTRACT FROM AUTHOR]

Bio-concrete using bacterially produced calcium carbonate can repair microcracks but is still relatively expensive due to the addition of bacteria, nutrients, and calcium sources. Agricultural by-products and oyster shells were used to produce economical bio-concrete. Sesame meal was the optimal agricultural by-product for low-cost spore production of the alkaliphilic Bacillus miscanthi strain AK13. Transcriptomic dataset was utilized to compare the gene expressions of AK13 strain under neutral and alkaline conditions, which suggested that NaCl and riboflavin could be chosen as growth-promoting factors at alkaline pH. The optimal levels of sesame meal, NaCl, and riboflavin were induced with the central composite design to create an economical medium, in which AK13 strain formed more spores with less price than in commercial sporulation medium. Calcium nitrate obtained from nitric acid treatment of oyster shell powder increased the initial compressive strength of cement mortar. Non-ureolytic calcium carbonate precipitation by AK13 using oyster shell-derived calcium ions was verified by energy-dispersive X-ray spectroscopy and X-ray diffraction analysis. Stereomicroscope and field emission scanning electron microscopy confirmed that oyster shell-derived calcium ions, along with soybean meal-solution, increased the bacterial survival and calcium carbonate precipitation inside mortar cracks. These data suggest the possibility of commercializing bacterial self-healing concrete with economical substitutes for culture medium, growth nutrient, and calcium sources. [ABSTRACT FROM AUTHOR]

The urgency for energy efficient, responsive architectures has propelled smart material development to the forefront of scientific and architectural research. This paper explores biological, physical, and morphological factors influencing the programming of a novel microbial-based smart hybrid material which is responsive to changes in environmental humidity. Hygromorphs respond passively, without energy input, by expanding in high humidity and contracting in low humidity. Bacillus subtilis develops environmentally robust, hygromorphic spores which may be harnessed within a bilayer to generate a deflection response with potential for programmability. The bacterial spore-based hygromorph biocomposites (HBCs) were developed and aggregated to enable them to open and close apertures and demonstrate programmable responses to changes in environmental humidity. This study spans many fields including microbiology, materials science, design, fabrication and architectural technology, working at multiple scales from single cells to 'bench-top' prototype.Exploration of biological factors at cellular and ultracellular levels enabled optimisation of growth and sporulation conditions to biologically preprogramme optimum spore hygromorphic response and yield. Material explorations revealed physical factors influencing biomechanics, preprogramming shape and response complexity through fabrication and inert substrate interactions, to produce a palette of HBCs. Morphological aggregation was designed to harness and scale-up the HBC palette into programmable humidity responsive aperture openings. This culminated in pilot performance testing of a humidity-responsive ventilation panel fabricated with aggregated Bacillus HBCs as a bench-top prototype and suggests potential for this novel biotechnology to be further developed., (Creative Commons Attribution license.)

Isostatic high pressure (HP) of 150 MPa can trigger the germination of bacterial spores, making them lose their extreme resistance to stress factors, and increasing their susceptibility to milder inactivation strategies. However, germination response of spores within a population is very heterogeneous, and tools are needed to study this heterogeneity. Here, classical methods were combined with more recent and powerful techniques such as flow cytometry (FCM) and fluorescence activated cell sorting (FACS) to investigate spore germination behavior under HP. Bacillus subtilis spores were treated with HP at 150 MPa and 37°C, stained with SYTO16 and PI, and analyzed via FCM. Four sub-populations were detected. These sub-populations were for the first time isolated on single cell level using FACS and characterized in terms of their heat resistance (80°C, 10 min) and cultivability in a nutrient-rich environment. The four isolated sub-populations were found to include (1) heat-resistant and mostly cultivable superdormant spores, i.e., spores that remained dormant after this specific HP treatment, (2) heat-sensitive and cultivable germinated spores, (3) heat-sensitive and partially-cultivable germinated spores, and (4) membrane-compromised cells with barely detectable cultivability. Of particular interest was the physiological state of the third sub-population, which was previously referred to as “unknown”. Moreover, the kinetic transitions between different physiological states were characterized. After less than 10 min of HP treatment, the majority of spores germinated and ended up in a sublethally damaged stage. HP treatment at 150 MPa and 37°C did not cause inactivation of all geminated spores, suggesting that subsequent inactivation strategies such as mild heat inactivation or other inactivation techniques are necessary to control spores in food. This study validated FCM as a powerful technique to investigate the heterogeneous behavior of spores under HP, and provided a pipeline using FACS for isolation of different sub-populations and subsequent characterization to understand their physiological states.

Abstract Background and aims: Drinking water pollution is a serious threat for public water supply. Water disinfection has a very important role in the reduction of pathogenic microorganism. Water electrodisinfection is an efficient and cost-effective method in water disinfection. The aim of this study was the survey of inactivation in two types bacteria (as index and resistant) by electrochemical process in the presence of supporting electrolytes. Materials and methods:Inactivation of E.Coli and Bacillus subtilis spore was studied at current intensity of 500mA, with each of the supporting electrolytes including CuI, NaBr and KCl. The type of electrodes was steel and the distance between electrodes was 2cm. Furthermore, the tests carried out in monopolar mode Fe-Fe, neutral pH and experimental temperature (21o c). The number of bacteria, amount of supporting electrolyte, voltage, and electrochemical action time were investigated. Results:Adding the supporting electrolytes to the electrochemical disinfection process decreased bacterial resistan Copper iodide (CuI) on 2 mg/l concentration showed more efficiency in bacterial inactivation. Conclusion:Supporting electrolyte CuI with concentration of 2 mg/l both enhances the electrochemical process performance due to an increase in ionic transmission rate and voltage current. Also, this decreases electrochemical inactivation time and increases bactericidal effect.

Isostatic high pressure (HP) of 150 MPa can trigger the germination of bacterial spores, making them lose their extreme resistance to stress factors, and increasing their susceptibility to milder inactivation strategies. However, germination response of spores within a population is very heterogeneous, and tools are needed to study this heterogeneity. Here, classical methods were combined with more recent and powerful techniques such as flow cytometry (FCM) and fluorescence activated cell sorting (FACS) to investigate spore germination behavior under HP. Bacillus subtilis spores were treated with HP at 150 MPa and 37°C, stained with SYTO16 and PI, and analyzed via FCM. Four sub-populations were detected. These sub-populations were for the first time isolated on single cell level using FACS and characterized in terms of their heat resistance (80°C, 10 min) and cultivability in a nutrient-rich environment. The four isolated sub-populations were found to include (1) heat-resistant and mostly cultivable superdormant spores, i.e., spores that remained dormant after this specific HP treatment, (2) heat-sensitive and cultivable germinated spores, (3) heat-sensitive and partially-cultivable germinated spores, and (4) membrane-compromised cells with barely detectable cultivability. Of particular interest was the physiological state of the third sub-population, which was previously referred to as "unknown". Moreover, the kinetic transitions between different physiological states were characterized. After less than 10 min of HP treatment, the majority of spores germinated and ended up in a sublethally damaged stage. HP treatment at 150 MPa and 37°C did not cause inactivation of all geminated spores, suggesting that subsequent inactivation strategies such as mild heat inactivation or other inactivation techniques are necessary to control spores in food. This study validated FCM as a powerful technique to investigate the heterogeneous behavior of spores under HP, and provided a pipeline using FACS for isolation of different sub-populations and subsequent characterization to understand their physiological states. [ABSTRACT FROM AUTHOR]

Bacterial spore control strategies based on the germination-inactivation principle can lower the thermal load needed to inactivate bacterial spores and thus preserve food quality better. However, the success of this strategy highly depends on the germination of spores, and a subpopulation of spores that fail to germinate or germinate extremely slowly hinders the application of this strategy. This subpopulation of spores is termed ‘superdormant (SD) spores.’ Depending on the source of the germination stimulus, SD spores are categorized as nutrient-SD spores, Ca2+-dipicolinic acid SD spores, dodecylamine-SD spores, and high pressure SD spores. In recent decades, research has been done to isolate these different groups of SD spores and unravel the cause of their germination deficiency as well as their germination capacities. This review summarizes the challenges caused by SD spores, their isolation and characterization, the underlying mechanisms of their germination deficiency, and the future research directions needed to tackle this topic in further depth.

Low energy electron beam (LEEB) treatment is an emerging non-thermal technology that performs surface decontamination with a minimal influence on food quality. Bacterial spore resistance toward LEEB treatment and its influencing factors were investigated in this study. Spores from Geobacillus and Bacillus species were treated with a lab-scale LEEB at energy levels of 80 and 200 keV. The spore resistances were expressed as D-values (the radiation dose required for one log10 reduction at a given energy level) calculated from the linear regression of log10 reduction against absorbed dose of the sample. The results revealed that the spore inactivation efficiency by LEEB is comparable to that of other ionizing radiations and that the inactivation curves are mostly log10-linear at the investigated dose range (3.8 – 8.2 kGy at 80 keV; 6.0 – 9.8 kGy at 200 keV). The D-values obtained from the wildtype strains varied from 2.2 – 3.0 kGy at 80 keV, and from 2.2 – 3.1 kGy at 200 keV. Bacillus subtilis mutant spores lacking α/β-type small, acid-soluble spore proteins showed decreased D-values (1.3 kGy at 80 and 200 keV), indicating that spore DNA is one of the targets for LEEB spore inactivation. The results revealed that bacterial species, sporulation conditions and the treatment dose influence the spore LEEB inactivation. This finding indicates that for the application of this emerging technology, special attention should be paid to the choice of biological indicator, physiological state of the indicator and the processing settings. High spore inactivation efficiency supports the application of LEEB for the purpose of food surface decontamination. With its environmental, logistical, and economic advantages, LEEB can be a relevant technology for surface decontamination to deliver safe, minimally processed and additive-free food products.

Summary: High‐pressure freezing limits the size of biological samples, because only small samples can be frozen without ice damage. Additionally, these samples must fit into the dimensions of the sample holder provided by the high‐pressure freezer. We explored the potential of a 10 μm thin polyester filter membrane (PE‐filter) as a versatile sample substrate for high‐pressure freezing. Planktonic bacteria, bacterial spores and suspended eukaryotic cells could be concentrated on the PE‐filter, whereas biofilm, bacterial microcolonies and HeLa cells were able to grow directly on the PE‐filter. These microorganism‐loaded PE‐filters were used for high‐pressure freezing, freeze‐substitution and plastic embedding in Epon or Lowicryl. Embedded filters were cross‐sectioned so that the interface between microorganism and substrate as well as the overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa‐cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism‐loaded PE‐filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze‐substituted PE‐filters samples with scanning electron microscopy, demonstrates the versatility of the PE‐filter as a sample substrate for a wide array of microorganisms. Lay Description: In order to investigate biological samples in the transmission electron microscope it is imperative to remove all their water content, or the specimens will be destroyed by boiling in the high vacuum of the microscope. In order to avoid dramatic morphology‐changes due to drying artefacts or the impact of chemical stabilisers, high‐pressure freezing (HPF) was developed. This protocol allows freezing biological samples in an instant (within a few milliseconds) down to –196°C while applying high pressure at the same time so that the specimen retains all its water in a solidified noncrystalline form. However, the formation of morphology‐destroying ice crystals is only avoided, if the cooling of the sample is faster than the ice crystal formation, which is only possible with very thin samples (up to a maximum of 200 μm in optimal cases). High‐pressure freezing is regarded as the gold‐standard for sample preparation of cells, tissues and small organisms. However, all of these samples must fit into the dimensions of the specific sample holder of the high‐pressure freezer and their transfer into the high‐pressure freezing machine must be achieved without significant impact on sample physiology. Additionally, it may also necessary to concentrate and immobilise a biological specimen before they can be placed in the HPF sample holder. Although a few number of strategies and sample substrates have been used for different types of biological samples, we explored the potential of a 10 μm thin polyester filter membrane (PE‐filter) as a versatile sample substrate for HPF. In culture medium suspended bacteria, suspended bacterial spores and in medium suspended higher cells could be concentrated on the PE‐filter, whereas bacterial biofilm or bacterial microcolonies from an agar plate, and surface‐adhering higher cells were able to grow directly on the PE‐filter. These microorganism‐loaded PE‐filters could be directly used for high‐pressure freezing, and were finally embedded in a plastic resin like Epon or Lowicryl. Embedded filters were cross‐sectioned so that the interface between microorganism and substrate or overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa‐cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism‐loaded PE‐filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze‐substituted PE‐filters samples with scanning‐electron microscopy, demonstrates the versatility of the PE‐filter as a sample substrate for a wide array of microorganisms. [ABSTRACT FROM AUTHOR]

Bacterial spores displaying heterologous proteins have been proposed as a safe and efficient method for delivery of antigens and enzymes to animal mucosal surfaces. Initial studies have been performed using Bacillus subtilis spores, but other spore forming organisms have also been considered. B. megaterium spores have been shown capable of displaying large amounts of a model heterologous protein ( Discosoma red fluorescent protein mRFP) that in part crossed the exosporium to localize in the space between the outer coat layer and the exosporium. Here, B. megaterium spores have been used to adsorb Bcp1 (bacterioferritin comigratory protein 1), a peroxiredoxin of the archaeon Sulfolobus solfataricus, known to have an antioxidant activity. The spores were highly efficient in adsorbing the heterologous enzyme which, once adsorbed, retained its activity. The adsorbed Bcp1 localized beneath the exosporium, filling the space between the outer coat and the exosporium. This unusual localization contributed to the stability of the enzyme-spore interaction and to the protection of the adsorbed enzyme in simulated intestinal or gastric conditions. [ABSTRACT FROM AUTHOR]

Low energy electron beam (LEEB) treatment is an emerging non-thermal technology that performs surface decontamination with a minimal influence on food quality. Bacterial spore resistance toward LEEB treatment and its influencing factors were investigated in this study. Spores from Geobacillus and Bacillus species were treated with a lab-scale LEEB at energy levels of 80 and 200 keV. The spore resistances were expressed as D -values (the radiation dose required for one log10 reduction at a given energy level) calculated from the linear regression of log10 reduction against absorbed dose of the sample. The results revealed that the spore inactivation efficiency by LEEB is comparable to that of other ionizing radiations and that the inactivation curves are mostly log10-linear at the investigated dose range (3.8 – 8.2 kGy at 80 keV; 6.0 – 9.8 kGy at 200 keV). The D -values obtained from the wildtype strains varied from 2.2 – 3.0 kGy at 80 keV, and from 2.2 – 3.1 kGy at 200 keV. Bacillus subtilis mutant spores lacking α/β-type small, acid-soluble spore proteins showed decreased D -values (1.3 kGy at 80 and 200 keV), indicating that spore DNA is one of the targets for LEEB spore inactivation. The results revealed that bacterial species, sporulation conditions and the treatment dose influence the spore LEEB inactivation. This finding indicates that for the application of this emerging technology, special attention should be paid to the choice of biological indicator, physiological state of the indicator and the processing settings. High spore inactivation efficiency supports the application of LEEB for the purpose of food surface decontamination. With its environmental, logistical, and economic advantages, LEEB can be a relevant technology for surface decontamination to deliver safe, minimally processed and additive-free food products. [ABSTRACT FROM AUTHOR]

Importance: Bacillus and Clostridium spores cause food spoilage and disease because of spores' dormancy and resistance to microbicides. However, when spores "come back to life" in germination, their resistance properties are lost. Thus, understanding the mechanisms of spore germination could facilitate the development of "germinate to eradicate" strategies. One germination feature is the memory of a pulsed germinant stimulus leading to greater germination following a second pulse. Recent observations of increases in spore binding of the potentiometric dye thioflavin-T early in their germination of spores led to the suggestion that increasing electrochemical potential is how spores "remember" germinant pulses. However, new work finds no increased thioflavin-T binding in the physiological germination of Coatless spores or of intact spores germinating with dodecylamine, even though spore memory is seen in both cases. Thus, using thioflavin-T uptake by germinating spores to assess the involvement of electrochemical potential in memory of germinant exposure, as suggested recently, is questionable., Competing Interests: The authors declare no conflict of interest.

Abstract: High‐pressure thermal sterilization (HPTS) and ultra‐high‐pressure homogenization (UHPH) are two emerging sterilization techniques that have not been implemented in the food industry yet. The two technologies apply different acting principles as HPTS uses isostatic pressure in combination with heat whereas UHPH uses dynamic pressure in combination with shear stress, cavitation, impingement, and heat. Both technologies offer significant benefits in terms of spore inactivation in food production with reduced thermal intensity and minimized effects on sensory and nutritional profiles. These benefits have resulted in relevant research efforts on both technologies over the past few decades. This state of the art of the discontinuous HPTS‐based and the continuous UHPH‐based sterilization concepts are assessed within this review. Further, various basic principles and promising future preservation applications of HPTS and UHPH for food processing, that are also applicable in the pharmaceutical, biochemical, and biotechnological sectors, are summarized. In addition, the applications and limitations of these technologies in terms of optimizations needed to overcome the identified challenges are emphasized. [ABSTRACT FROM AUTHOR]

Since the 2001 anthrax attacks, researchers have focused on the development of a bacterial spore biomarker detector with rapid response time and high sensitivity and selectivity. Hence, a ratiometric fluorescent nanosensor based on lanthanide-functionalized attapulgite (Atta) was designed and prepared using dye-doped Atta as an internal reference. Atta, which is famous because of Maya Blue, is a hydrated magnesium aluminum silicate present in nature, with pronounced adsorption and a large specific surface area rich in silicon hydroxyl. The adsorption of dye molecules benefits the formation of an internal reference with orange fluorescence. Meanwhile, silicon hydroxyl would be available for terbium (Tb 3+ ) complex grafting, which favors the sensitization of the bacterial spore biomarker. With increasing dipicolinic acid (DPA) concentrations, energy transfer from DPA to Tb 3+ gradually enhanced, thereby resulting in strong and predominant green fluorescence. The ratiometric fluorescence intensity is highly sensitive. It has a limit of detection of 9.8 nM, which is much lower than the infectious dosage of Bacillus anthracis spores (60 μM) for humans. The rapid response time (10s), high selectivity, and reliable and practical performances in actual applications make this ratiometric fluorescent nanosensor useful in the detection of biomarkers for various bacterial spores. Moreover, the reported construction of visual ratiometric detection system follows the sustainable development idea of ‘‘from nature, for nature, into the nature’’ and promotes the use of naturally renewable resources to produce promising and eco-friendly functional materials for various applications. [ABSTRACT FROM AUTHOR]

Since bacterial spores, such as Bacillus anthracis spores, are extremely hazardous to human beings and animals, efforts have focused on the development of bacterial spore detector with rapid response and high selectivity and sensitivity. Therefore, we reported a facile one-step chelating-reagent-assisted hydrothermal synthesis of lanthanide-doped fluorapatite (FA) nanoprobes for detecting the biomarker of bacterial spores. In FA synthesis, ethylenediaminetetraacetic acid (EDTA) can serve not only as a shape controller and a stabilizer but also as a chelating reagent for lanthanide ions. After the fabrication, terbium (or europium) ions were not only embedded into lattice models of FA nanocrystals, but also chelated by EDTA on the surface of the FA nanocrystals, which can be available for detecting the biomarker (i.e., dipicolinic acid) of bacterial spores. The obtained FA: Ln–EDTA nanoprobes can provide reference fluorescence which play crucial roles in the calibration and correction of concentration. The fluorescence intensities of FA: Tb–EDTA or FA: Eu–EDTA from Tb 3+ or Eu 3+ emission were highly sensitive and increased gradually with increasing DPA concentrations. The limit of detection of FA: Tb–EDTA was 8.2 nM, which is lower than that of FA: Eu–EDTA (20.9 nM). It can be attributed to the fact that DPA can achieve a more effective energy transfer to Tb 3+ than to Eu 3+ . Additionally, the nanoprobes were successfully applied for the determination of DPA in fetal calf serum samples. Given their rapid response and high sensitivity and selectivity, these ratiometric fluorescent nanoprobes are promising tools for the detection of bacterial spores. [ABSTRACT FROM AUTHOR]

Methods for microbial inactivation are important in the food industry; however, conventional external heating (CH) reduces food quality. Accordingly, the nonthermal effects of ohmic heating (OH) on Bacillus subtilis spores in a sodium chloride aqueous solution at 101°C (i.e., the boiling point), as well as the effects of electric field intensity and frequency during OH, were investigated. Survival kinetics were compared between OH and external CH. The inactivation effect on B. subtilis was greater for all electric field conditions (5, 10, and 20 V/cm) than for CH. In particular, 20 V/cm showed a significantly higher inactivation effect ( P < 0.05) on B. subtilis than those of CH at 8, 10, 12, 14, and 16 min. The survival data were fitted to various primary kinetic models. In the Weibull model and the log-linear model, there were significant differences ( P < 0.05) in the rate parameters δ and kmax between OH at 20 V/cm and CH. However, there were no significant differences ( P > 0.05) in survival kinetics between 20, 40, and 60 kHz; B. subtilis spores were inactivated more efficiently as the frequency increased. B. subtilis spores were almost completely inactivated at 14 to 16 min for the 60-kHz treatment, but spores were still alive at 20 and 40 kHz for the same treatment times. These results demonstrated that OH inactivates B. subtilis spores more effectively than CH. OH conditions with high electric field intensities and high frequencies resulted in efficient B. subtilis spore inactivation. [ABSTRACT FROM AUTHOR]

There is growing evidence that shows Clostridium ( Clostridioides ) difficile is a pathogen of One Health importance with a complex dissemination pathway involving animals, humans, and the environment. Thus, environmental discharge and agricultural recycling of human and animal waste have been suspected as factors behind the dissemination of Clostridium difficile in the community. Here, the presence of C. difficile in 12 wastewater treatment plants (WWTPs) in Western Australia was investigated. Overall, C. difficile was found in 90.5% (114/126) of raw sewage influent, 48.1% (50/104) of treated effluent, 40% (2/5) of reclaimed irrigation water, 100% (38/38) of untreated biosolids, 95.2% (20/21) of anaerobically digested biosolids, and 72.7% (8/11) of lime-amended biosolids. Over half of the isolates (55.3% [157/284]) were toxigenic, and 97 C. difficile ribotypes (RTs) were identified, with RT014/020 the most common (14.8% [42/284]). Thirteen C. difficile isolates with the toxin gene profile A + B + CDT + (positive for genes coding for toxins A and B and the binary C. difficile transferase toxin [CDT]) were found, including the hypervirulent RT078 strain. Resistance to the antimicrobials fidaxomicin, vancomycin, metronidazole, rifaximin, amoxicillin-clavulanate, meropenem, and moxifloxacin was uncommon; however, resistance to clindamycin, erythromycin, and tetracycline was relatively frequent at 56.7% (161/284), 14.4% (41/284), and 13.7% (39/284), respectively. This study revealed that toxigenic C. difficile was commonly encountered in WWTPs and being released into the environment. This raises concern about the possible spillover of C. difficile into animal and/or human populations via land receiving the treated waste. In Western Australia, stringent measures are in place to mitigate the health and environmental risk of recycling human waste; however, further studies are needed to elucidate the public health significance of C. difficile surviving the treatment processes at WWTPs. IMPORTANCE Clostridium difficile infection (CDI) is a leading cause of antimicrobial-associated diarrhea in health care facilities. Extended hospital stays and recurrences increase the cost of treatment and morbidity and mortality. Community-associated CDI (CA-CDI) cases, with no history of antimicrobial use or exposure to health care settings, are increasing. The isolation of clinically important C. difficile strains from animals, rivers, soil, meat, vegetables, compost, treated wastewater, and biosolids has been reported. The objective of this study was to characterize C. difficile in wastewater treatment plants (WWTPs) in Australia. We found that C. difficile can survive the treatment processes of WWTPs, and toxigenic C. difficile was being released into the environment, becoming a potential source/reservoir for CA-CDI.

[Display omitted] • UVc effect explored on spore metabolites: DPA and its salt CaDPA. • Photoproducts of DPA characterized fluorescence using emission. • Spectroscopy and DPA assay demonstrated the instability of photoproducts. • New insights into photoproducts production by mass spectrometry. • Novel microscopy methodology implemented for photoproducts vizualization. Bacterial spores can cause significant problems such as food poisoning (like neurotoxin or emetic toxin) or serious illnesses (like anthrax or botulism). This dormant form of bacteria, made of several layers of barriers which provide extreme resistance to many abiotic stresses (radiation, temperature, pressure, etc.), are difficult to investigate in situ. To better understand the biological and chemical mechanisms involved and specific to spores resistance, the acquisition of environmental parameters is necessary. For that purpose, our research has been focused on the detection and analysis of a unique spore component, dipicolinic acid (DPA), used as the main in situ metabolite for sporulating bacteria detection. In its native form, DPA is only weakly fluorescent but after Ultraviolet irradiation at the wavelength of 254 nm (UVc), DPA photoproducts (DPAp) exhibit a remarkable fluorescence signal. These photoproducts are rarely identified and part of this study gives new insights offered by mass spectrometry (MS) in the determination of DPA photoproducts. Thanks to DPA assay techniques and fluorescence spectrometry, we highlighted the instability of photoproducts and introduced new assumptions on the effects of UVc on DPA. Studies in spectroscopy and microscopy allowed us to better understand these native probes in bacterial spores and will allow the implementation of a new method for studying the physico-chemical parameters of spore resistance. [ABSTRACT FROM AUTHOR]

Ultra high pressure homogenization (UHPH) opens up new areas for dynamic high pressure assisted thermal sterilization of liquids. Bacillus amyloliquefaciens spores are resistant to high isostatic pressure and temperature and were suggested as potential surrogate for high pressure thermal sterilization validation. B. amyloliquefaciens spores suspended in PBS buffer (0.01 M, pH 7.0), low fat milk (1.5%, pH 6.7) and whole milk (3.5%, pH 6.7) at initial concentration of ~106 CFU/mL were subjected to UHPH treatments at 200, 300 and 350 MPa with an inlet temperature at ~80 °C. Thermal inactivation kinetics of B. amyloliquefaciens spores in PBS and milk were assessed with thin wall glass capillaries and modeled using mechanistic linear first order and Weibull models. The residence time during UHPH treatments was estimated to determine the contribution of temperature to spore inactivation by UHPH. No sublethal injury was detected after UHPH treatments using sodium chloride as selective component in the nutrient agar medium. The inactivation profiles of spores in PBS buffer and milk were compared and fat provided no clear protective effect for spores against treatments. Treatment at 200 MPa with valve temperatures lower than 125 °C caused no reduction of spores. A reduction of 3.5 log10 CFU/mL of B. amyloliquefaciens spores was achieved by treatment at 350 MPa with a valve temperature higher than 150 °C. The modeled thermal inactivation and observed inactivation during UHPH treatments suggest that temperature could be the main lethal effect driving inactivation.

Background: Bacterial spores have been proposed as vehicles to display heterologous proteins for the development of mucosal vaccines, biocatalysts, bioremediation and diagnostic tools. Two approaches have been developed to display proteins on the spore surface: a recombinant approach, based on the construction of gene fusions between DNA molecules coding for a spore surface protein (carrier) and for the heterologous protein to be displayed (passenger); and a non-recombinant approach based on spore adsorption, a spontaneous interaction between negatively charged, hydrophobic spores and purified proteins. The molecular details of spore adsorption have not been fully clarified yet. Results: We used the monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp. and Bacillus subtilis spores of a wild type and an isogenic mutant strain lacking the CotH protein to clarify the adsorption process. Mutant spores, characterized by a strongly altered coat, were more efficient than wild type spores in adsorbing mRFP but the interaction was less stable and mRFP could be in part released by raising the pH of the spore suspension. A collection of isogenic strains carrying GFP fused to proteins restricted in different compartments of the B. subtilis spore was used to localize adsorbed mRFP molecules. In wild type spores mRFP infiltrated through crust and outer coat, localized in the inner coat and was not surface exposed. In mutant spores mRFP was present in all surface layers, inner, outer coat and crust and was exposed on the spore surface. Conclusions: Our results indicate that different spores can be selected for different applications. Wild type spores are preferable when a very tight protein-spore interaction is needed, for example to develop reusable biocatalysts or bioremediation systems for field applications. cotH mutant spores are instead preferable when the heterologous protein has to be displayed on the spore surface or has to be released, as could be the case in mucosal delivery systems for antigens and drugs, respectively. [ABSTRACT FROM AUTHOR]

Clostridium species and Bacillus spp. are spore-forming bacteria that cause hospital infections. The spores from these bacteria are transmitted from patient to patient via healthcare workers' hands. Although alcohol-based hand rubbing is an important hand hygiene practice, it is ineffective against bacterial spores. Therefore, healthcare workers should wash their hands with soap when they are contaminated with spores. However, the extent of health care worker hand contamination remains unclear. The aim of this study is to determine the level of bacterial spore contamination on healthcare workers' hands. The hands of 71 healthcare workers were evaluated for bacterial spore contamination. Spores attached to subject's hands were quantitatively examined after 9 working hours. The relationship between bacterial spore contamination and hand hygiene behaviors was also analyzed. Bacterial spores were detected on the hands of 54 subjects (76.1%). The mean number of spores detected was 468.3 CFU/hand (maximum: 3300 CFU/hand). Thirty-seven (52.1%) and 36 (50.7%) subjects were contaminated with Bacillus subtilis and Bacillus cereus , respectively. Nineteen subjects (26.8%) were contaminated with both Bacillus species. Clostridium difficile was detected on only one subject's hands. There was a significant negative correlation between the hand contamination level and the frequency of handwashing ( r = −0.44, P < 0.01) and a significant positive correlation between the hand contamination level and the elapsed time since last handwashing ( r = 0.34, P < 0.01). Healthcare workers' hands may be frequently contaminated with bacterial spores due to insufficient handwashing during daily patient care. [ABSTRACT FROM AUTHOR]

Ultra-high-pressure homogenization (UHPH) offers new opportunities for continuous high-pressure-assisted thermal sterilization of liquids and production of stable emulsions. As relevant indicator, Bacillus amyloliquefaciens spores were suspended in a coarse oil-in-water (o/w) emulsion (pH 7.6) with 10 % soybean oil and 4 % soy protein isolate at the initial concentration of ~10 CFU/mL. The o/w emulsion was subjected to UHPH treatments at 200, 300 and 350 MPa with the inlet temperature at ~80 °C to inactivate the respective bacterial spores and produce fine emulsion simultaneously. After treatment at 350 MPa with the valve temperature higher than 150 °C, a reduction up to 4 log CFU/mL of bacterial spores was obtained, and the emulsion was characterized by smaller particle size and narrow particle size distributions, with the D[3, 4] of 0.210 μm and the span of 0.746. No spore germination was found after any of these UHPH treatments. The microstructure of the emulsion was observed under confocal laser scanning microscopy with fluorescein isothiocyanate and Nile red to stain proteins and fats, respectively. UHPH showed potential to combine sterilization and emulsification into one single process. [ABSTRACT FROM AUTHOR]

In this study, we develop a characterization of bacterial spore resistance to NIR pulsed light under modalities traditionally used in multiphoton microscopy. Energy dose and laser power are both key parameters in spore and bacterial cell inactivation. Surprisingly, spores and vegetative cells seem to show a similar sensitivity to pulsed NIR, spores being only 2-fold more resistant than their vegetative counterparts. This work enables us to eliminate certain hypotheses concerning the main driver of spore inactivation processes. Our findings suggest that damage leading to inactivation is mainly caused by photochemical reactions characterized by multiple possible pathways, including DNA damage or oxidation processes. • Pulsed Near Infrared Radiation can lead to bacterial spore inactivation. • Unusual close inactivation rate are observed between bacterial spore and vegetative cells. • Photochemicals damage are proposed as the main driver of spore inactivation. [ABSTRACT FROM AUTHOR]

Bacterial spores have a strong resistance to both chemical and physical hurdles and create a risk for food industry which has been tackled by applying high thermal intensity treatments to sterilize food. These strong thermal treatments lead to reduction of the organoleptic and nutritional properties of food and alternative are actively searched for. Innovative hurdles offer an alternative to inactivate bacterial spores. In particular, recent technological developments have enabled a new generation of high pressure homogenizer working at pressures up to 400 MPa and thus opening new opportunities for high pressure sterilization of foods. In this short review, we summarize the work conducted on (ultra)-high pressure homogenization (U)HPH to inactivate endospores in model and food systems. Specific attention is given to process parameters (pressure, inlet and valve temperatures). This review gathers the current state of the art and underlines the potential of UHPH sterilization of pumpable foods while highlighting the needs for future work.

This study aimed to clarify how the phenolic monoterpene carvacrol and its structural isomer thymol both as essential oil components (EOCs) inhibit the germination of Bacillus subtilis spore. Germination was evaluated by the OD 600 reduction rate in a growth medium and phosphate buffer containing either l-alanine (l-Ala) system or l-asparagine, d-glucose, d-fructose plus KCl (AGFK) system. The germination of the wild-type spores in the Trypticase Soy broth (TSB) was found to be greatly inhibited by thymol than by carvacrol. Such a difference in the germination inhibition was confirmed by the dipicolinic acid (DPA) release from germinating spores in the AGFK buffer system, but not in the l-Ala system. Similar to the wild-type spores, no difference in the inhibitory activity between the EOCs was also indicated with the gerB, gerK-deletion mutant spores in the l-Ala buffer system and the above substantial difference was also done with the gerA-deleted mutant spores in the AGFK. Fructose was found to release spores from the EOC inhibition and inversely even stimulated. Increased concentrations of glucose and fructose partially suppressed the germination inhibition by carvacrol. The results obtained should contribute to the elucidation of the control effects of these EOCs on bacterial spores in foods.

This paper presents a novel technique for designing a microwave power delivery system targeted at compact and portable microwave-assisted diagnostic healthcare applications to help tackle the growing problem of anti-microbial resistance. The arrangement comprises a purpose-built cylindrical cavity resonator within which, the bacterial samples are exposed, driven by a high-efficiency 10-W GaN amplifier, critically coupled via a simple, adjustable internal loop antenna. The experimental work considers the exposure of different sample volumes of water and the change in the natural impedance environment of the cavity that results. For the first time, it is shown that impedance variation cannot only be accommodated, but can actually be exploited, allowing “continuous,” high-efficiency performance to be achieved while processing a wide range of sample volumes. Specifically, using only transistor package parasitics, the impedance of the cavity itself together with a single-series transmission line allows a continuous class-F ^-1 mode loading condition to be realized. Measured results show more than 70% average drain efficiency, above 40-dBm average output power, and more than 10-dB gain over the cavity's operational bandwidth. [ABSTRACT FROM PUBLISHER]

Fatty acid esters are food additives with strong antibacterial activity against spore-forming bacteria. The antibacterial activity of monoglycerol fatty acid esters (MGs) and sucrose fatty acid esters (SEs) with various fatty acid chain lengths was systematically investigated on four typical Bacillus species: B. cereus, B. subtilis, B. megaterium, and B. coagulans. Monoglycerol monolaurate (MG12) and monoglycerol monomyristate (MG14) showed relatively strong bactericidal effects on vegetative cells of these species among the MGs tested at both pH 6.0 and pH 8.0. Different SEs showed bactericidal effects on different species at pH 6.0, while the SEs had antibacterial effects only on B. coagulans at pH 8.0. SEs showed antibacterial effects on vegetative cells of these species, as was the case with MGs. In addition, it was found that MGs and SEs showed strong antibacterial effects on both B. cereus and B. subtilis in the logarithmic phase of growth, while the antibacterial activities of MGs persisted longer than those of SEs. [ABSTRACT FROM AUTHOR]

A new generation of high pressure homogenizers reaching up to 400 MPa offers opportunities for spore inactivation and high pressure sterilization of foods. A Stansted Fluid Power ultra-high pressure homogenization (UHPH) unit was tested to inactivate bacterial spores in a model buffer system. Bacillus subtilis PS832 and Geobacillus stearothermophilus ATCC7953 spores’ thermal resistances were assessed (D, z-value and E a ). The pressure and valve temperature were monitored during UHPH. Residence times under pressure and high temperature were estimated and enabled comparison with thermal inactivation, indicating the estimated thermal contribution to inactivation. Spore germination was also assessed but no germination was observed. Up to five log 10 B. subtilis and two log 10 G. stearothermophilus spores were inactivated by the harshest treatments (> 300 MPa –T valve > 145 °C for ~ 0.24 s). The inactivation profiles were similar to the predicted thermal inactivation suggesting that the valve temperature might be a dominant parameter leading to bacterial spore inactivation. Industrial relevance This work showed the UHPH potential for spore inactivation and the need of highly thermoresistant surrogates for process validation, for instance, G. stearothermophilus ATCC7953 spores. Both these findings are highly relevant for industrial application as, to date, no surrogate for this process was suggested while the first patents for the technology transfer to industry are being issued. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial spores are a major concern for food safety due to their high resistance to conventional preservation hurdles. Innovative hurdles can trigger bacterial spore germination or inactivate them. In this work, Geobacillus stearothermophilus spore high pressure (HP) germination and inactivation mechanisms were investigated by in situ infrared spectroscopy (FT-IR) and fluorometry. G. stearothermophilus spores' inner membrane (IM) was stained with Laurdan fluorescent dye. Time-dependent FT-IR and fluorescence spectra were recorded in situ under pressure at different temperatures. The Laurdan spectrum is affected by the lipid packing and level of hydration, and provided information on the IM state through the Laurdan generalized polarization. Changes in the –CH2 and –CH3 asymmetric stretching bands, characteristic of lipids, and in the amide I′ band region, characteristic of proteins' secondary structure elements, enabled evaluation of the impact of HP on endospores lipid and protein structures. These studies were complemented by ex situ analyses (plate counts and microscopy). The methods applied showed high potential to identify germination mechanisms, particularly associated to the IM. Germination up to 3 log10 was achieved at 200 MPa and 55 °C. A molecular-level understanding of these mechanisms is important for the development and validation of multi-hurdle approaches to achieve commercial sterility. [Copyright &y& Elsevier]

This review briefly describes the concerns of nanobiotechnology in the design and development of novel vaccines using the most known nanocarriers, including nature-made nanocarriers (such as bacterial spores, virus-like particles, exosomes, and bacteriophages), man-made nanocarriers (such as Proteosomes, liposomes, virosomes, SuperFluids, and nanobeads), and their applications in therapeutic and protective immunization, as well as their advantages and disadvantages. Here, we focus on the development of nano-based vaccines as “nanovaccines” for inducing immune systems, and the foreseeable promises and problems when compared with existing vaccines. Also, we review a potential nano-hazard for vaccines, so-called nanobacterial contamination. [ABSTRACT FROM AUTHOR]

Abstract: Pulsed electric field (PEF) treatment, an alternative to thermal processing in the food industry, is insufficient to inactivate bacterial spores. Although spores that have been treated in this manner remain alive, specific understanding of their physiological properties is limited. The purpose of this study is to describe the morphology, viability, and germination behavior of Bacillus atrophaeus spores treated with PEF. Our findings indicate that nonlethal PEF treatment results in spore deformation, dipicolinic acid (DPA) leakage, and a shorter and more uniform germination lag time (\(\def\upalpha{\unicode[Times]{x3B1}}\)\(\def\upbeta{\unicode[Times]{x3B2}}\)\(\def\upgamma{\unicode[Times]{x3B3}}\)\(\def\updelta{\unicode[Times]{x3B4}}\)\(\def\upvarepsilon{\unicode[Times]{x3B5}}\)\(\def\upzeta{\unicode[Times]{x3B6}}\)\(\def\upeta{\unicode[Times]{x3B7}}\)\(\def\uptheta{\unicode[Times]{x3B8}}\)\(\def\upiota{\unicode[Times]{x3B9}}\)\(\def\upkappa{\unicode[Times]{x3BA}}\)\(\def\uplambda{\unicode[Times]{x3BB}}\)\(\def\upmu{\unicode[Times]{x3BC}}\)\(\def\upnu{\unicode[Times]{x3BD}}\)\(\def\upxi{\unicode[Times]{x3BE}}\)\(\def\upomicron{\unicode[Times]{x3BF}}\)\(\def\uppi{\unicode[Times]{x3C0}}\)\(\def\uprho{\unicode[Times]{x3C1}}\)\(\def\upsigma{\unicode[Times]{x3C3}}\)\(\def\uptau{\unicode[Times]{x3C4}}\)\(\def\upupsilon{\unicode[Times]{x3C5}}\)\(\def\upphi{\unicode[Times]{x3C6}}\)\(\def\upchi{\unicode[Times]{x3C7}}\)\(\def\uppsy{\unicode[Times]{x3C8}}\)\(\def\upomega{\unicode[Times]{x3C9}}\)\(\def\bialpha{\boldsymbol{\alpha}}\)\(\def\bibeta{\boldsymbol{\beta}}\)\(\def\bigamma{\boldsymbol{\gamma}}\)\(\def\bidelta{\boldsymbol{\delta}}\)\(\def\bivarepsilon{\boldsymbol{\varepsilon}}\)\(\def\bizeta{\boldsymbol{\zeta}}\)\(\def\bieta{\boldsymbol{\eta}}\)\(\def\bitheta{\boldsymbol{\theta}}\)\(\def\biiota{\\boldsymbol{\iota}}\)\(\def\bikappa{\boldsymbol{\kappa}}\)\(\def\bilambda{\boldsymbol{\lambda}}\)\(\def\\bimu{\boldsymbol{\mu}}\)\(\def\binu{\boldsymbol{\nu}}\)\(\def\bixi{\boldsymbol{\xi}}\)\(\def\biomicron{\boldsymbol{\micron}}\)\(\def\bipi{\boldsymbol{\pi}}\)\(\def\birho{\boldsymbol{\rho}}\)\(\def\bisigma{\boldsymbol{\sigma}}\)\(\def\bitau{\boldsymbol{\\tau}}\)\(\def\biupsilon{\boldsymbol{\upsilon}}\)\(\def\biphi{\boldsymbol{\phi}}\)\(\def\bichi{\boldsymbol{\chi}}\)\(\def\bipsy{\boldsymbol{\psy}}\)\(\def\biomega{\boldsymbol{\omega}}\)\(\def\bupalpha{\bf{\alpha}}\)\(\def\bupbeta{\bf{\beta}}\)\(\def\bupgamma{\bf{\gamma}}\)\(\def\bupdelta{\bf{\delta}}\)\(\def\bupvarepsilon{\bf{\varepsilon}}\)\(\def\bupzeta{\bf{\zeta}}\)\(\def\bupeta{\bf{\eta}}\)\(\def\buptheta{\bf{\theta}}\)\(\def\bupiota{\bf{\iota}}\)\(\def\bupkappa{\bf{\kappa}}\)\(\def\\buplambda{\bf{\lambda}}\)\(\def\bupmu{\bf{\mu}}\)\(\def\bupnu{\bf{\nu}}\)\(\def\bupxi{\bf{\xi}}\)\(\def\bupomicron{\bf{\micron}}\)\(\def\buppi{\bf{\pi}}\)\(\def\buprho{\bf{\rho}}\)\(\def\bupsigma{\bf{\sigma}}\)\(\def\buptau{\bf{\tau}}\)\(\def\bupupsilon{\bf{\upsilon}}\)\(\def\bupphi{\bf{\phi}}\)\(\def\bupchi{\bf{\chi}}\)\(\def\buppsy{\bf{\psy}}\)\(\def\bupomega{\bf{\omega}}\)\(\def\bGamma{\bf{\Gamma}}\)\(\def\bDelta{\bf{\Delta}}\)\(\def\bTheta{\bf{\Theta}}\)\(\def\bLambda{\bf{\Lambda}}\)\(\def\bXi{\bf{\Xi}}\)\(\def\bPi{\bf{\Pi}}\)\(\def\bSigma{\bf{\Sigma}}\)\(\def\bPhi{\bf{\Phi}}\)\(\def\bPsi{\bf{\Psi}}\)\(\def\bOmega{\bf{\Omega}}\)\({T&#95;{{\rm{lag}}}}\)), but that there is no change in release duration (\(\Delta {T&#95;{{\rm{release}}}}\)), germination ratio, or viability. Based on our findings, we conclude that an intact morphologic state and DPA content are not prerequisites for germination and full viability and that, in contrast to nutrient-induced germination in which initially slowly released DPA triggers subsequent germination events, leaked DPA during PEF treatment does not. Spores that have been subjected to this procedure remain dormant and preserve their full germinability. We found that PEF-treated spores respond to germinants more quickly and with less heterogeneity, possibly because the tiny cracks formed on the spore surface facilitate the germinants' access to the germination receptors situated on the spore's inner membrane. The consensus view that nonlethal PEF has less impact on spores that are still capable of forming CFUs under proper conditions is one-sided. This research advances our understanding of how spores behave following nonlethal PEF treatment and gives information on the topics of nosocomial sterilization, food safety, and public health., (Copyright ©, International Association for Food Protection.)

Background: Modern microbiology laboratories are designed to protect workers and the environment from microbial aerosols produced during microbiological procedures and accidents. However, there is only limited data available on the aerosols generated from common microbiology procedures., Methods: A series of common microbiological procedures were undertaken with high concentration spore suspensions while air samplers were operated to sample the aerosols generated. Surface contamination from droplets was visualized using sodium fluorescein within the suspension. A total of 36 procedures were studied using different sample volumes (0.1-10 mL) and two spore suspension titers (10 7 and 10 9 colony forming units [cfu]/mL)., Results: The aerosol concentrations generated varied from 0 to 13,000 cfu/m 3 . There was evidence to suggest that titer, volume, and poor use of equipment were significant factors in increased aerosol generation from some of the procedures. A risk assessment undertaken using the data showed that any aerosol generated from these processes would be contained within a correctly operating biological safety cabinet. Therefore, with these procedures, the operator and the environment would not require any additional protective measures such as respiratory protective equipment or a negative pressure laboratory to prevent aerosol exposure or release., Conclusions: Aerosol generation from common laboratory processes can be minimized by reducing sample volumes and concentrations if possible. Training laboratory staff in good microbiological techniques would further mitigate aerosols generated from common laboratory processes., Competing Interests: No competing financial interests exist., (© Thomas Pottage et al. 2022; Published by Mary Ann Liebert, Inc.)

This review focuses on the antibacterial activities of visible light-responsive titanium dioxide (TiO) photocatalysts. These photocatalysts have a range of applications including disinfection, air and water cleaning, deodorization, and pollution and environmental control. Titanium dioxide is a chemically stable and inert material, and can continuously exert antimicrobial effects when illuminated. The energy source could be solar light; therefore, TiO photocatalysts are also useful in remote areas where electricity is insufficient. However, because of its large band gap for excitation, only biohazardous ultraviolet (UV) light irradiation can excite TiO, which limits its application in the living environment. To extend its application, impurity doping, through metal coating and controlled calcination, has successfully modified the substrates of TiO to expand its absorption wavelengths to the visible light region. Previous studies have investigated the antibacterial abilities of visible light-responsive photocatalysts using the model bacteria Escherichia coli and human pathogens. The modified TiO photocatalysts significantly reduced the numbers of surviving bacterial cells in response to visible light illumination. They also significantly reduced the activity of bacterial endospores; reducing their toxicity while retaining their germinating abilities. It is suggested that the photocatalytic killing mechanism initially damages the surfaces weak points of the bacterial cells, before totally breakage of the cell membranes. The internal bacterial components then leak from the cells through the damaged sites. Finally, the photocatalytic reaction oxidizes the cell debris. In summary, visible light-responsive TiO photocatalysts are more convenient than the traditional UV light-responsive TiO photocatalysts because they do not require harmful UV light irradiation to function. These photocatalysts, thus, provide a promising and feasible approach for disinfection of pathogenic bacteria; facilitating the prevention of infectious diseases. [ABSTRACT FROM AUTHOR]

Bc1245 is a monocistronic chromosomal gene of Bacillus cereus ATCC 14579 encoding a putative protein of 143 amino acids identified in this study to have a spore-related function in B. cereus. Bc1245 is highly conserved in the genome of members of the B. cereus group, indicating an important function of the gene in this group of bacteria. Quantitative PCR revealed that bc1245 is transcribed late in sporulation (upon formation of phase-bright spores) and at the same time as the mother cell-specific transcription factor σK. The σK regulon includes structural components of the spore (such as coat proteins), and it is therefore plausible that bc1245 might encode a structural outer spore protein. This was confirmed by detection of BC1245 in exosporium extracts from B. cereus by immunoblotting against BC1245 antiserum. [ABSTRACT FROM AUTHOR]

Abstract: Spores of Geobacillus stearothermophilus and Bacillus subtilis adhering to the surface of stainless steel (SS) and polypropylene (PP) were subjected to rinsing in water or NaOH solution to study their removability. The spores were made adherent through deposition from suspension under humid and dry conditions. The humid condition kept the surface wet throughout the deposition, whereas the dry condition made them dry up finally. Experimental results demonstrated that drying significantly enhanced both quantity and strength of the spore adhesion for both types of surface. No removal of the spores adhering to surfaces exposed to dry environment was observed through water rinsing with shear force application. Even with 0.2% NaOH, they were hardly removed irrespective of temperature. Although 1% NaOH removed or inactivated most of spores on SS surface, it left considerable viable spores on PP surface. Thus enhanced alkali tolerance was indicated for spores adhering to surfaces, especially made of PP, exposed to dry environment. [Copyright &y& Elsevier]

The article discusses a study which examines the enzymatic properties of YvdO protein. Findings of the study reveal that the enzyme was specified under the middle-chain fatty acids with estimated optimum potential of hydrogen (pH) of 7.5. It indicates that a free myristic acid was determined during its reaction with 1-myristoly-2-lyso-sn-glycero-3-phosphocholine. It concludes that YvdO is a novel thermostable lipolytic enzyme which has the capacity to hydrolyze lysophospholipids.

Abstract: Both atmospheric pressure plasma and photo-catalyst metal oxide titanium dioxide (TiO2) are well known for their microorganism inactivation and chemical material decomposition abilities. In this work, radio-frequency atmospheric pressure plasma and TiO2 are used together to inactivate Bacillus subtilis spores that have a very high degree of environmental resistance to ultra-violet (UV) photons and heat. The combinational use of the plasma and TiO2 demonstrates an enhanced performance of B. subtilis spore inactivation by showing a decrease in the decimal reduction time of as large as 40% compared with the use of plasma alone. A significant increase of hydroxyl (OH) and excited oxygen atomic emission line (O I) intensities in the presence of TiO2 suggests that the atmospheric pressure plasma assisted by TiO2 is very effective at generating reactive oxygen radicals, which is known to be a dominant factor in bacterial spore inactivation. Possible TiO2 activation mechanism by the plasma is investigated. [Copyright &y& Elsevier]

Abstract: Germination of Clostridium perfringens is known to be triggered by nutrients such as l-alanine and inosine, and facilitated by CO2, however the role of CO2 has not been fully understood. During the studies of the germination-specific protease GSP, we found that CO2 could be replaced by bicarbonate or weakly acidic pH (pH 6.0–6.5). We also found that the spores obtained from the C. perfringens S40 overproducing GSP could germinate without CO2. Moreover, the spores could germinate in the absence of nutrients, when the spores were incubated with bicarbonate or under weakly acidic pH. GSP, which might consist of three homologous proteases, CspA, CspB, and CspC, is one of the key enzymes involved in the spore germination, and converts the pre-mature form of the spore cortex-lytic enzyme, SleC, to the mature form. Maturation of SleC in the spores obtained from the mother strain of C. perfringens S40 requires nutrients plus bicarbonate or weakly acidic pH. In contrast, mature SleC was found in the spores obtained from the cells overpoducing GSP, when the spores were treated by nutrients, bicarbonate or weakly acidic pH. Each nutrients, bicarbonate and weakly acidic pH can trigger the germination of the spores obtained from C. perfringens cells overproducing GSP. [Copyright &y& Elsevier]

The advantage of using high pressure (HP) cycling treatment compared with continuous HP treatment was investigated for the inactivation of bacterial spores. The effects of parameters such as pulse number, pressure level, treatment temperature, compression and decompression rates, and time between pulses were evaluated. For this purpose, Bacillus subtilis and B. cereus spores (108 and 106 CFU/mL respectively) were suspended in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution, tryptone salt (TS) buffer solution, or infant milk and treated by HP cycling at 300–400 MPa, at 38–60 °C, for 1–5 pulses. Pressure cycling reduced the number of viable spores by 1.8 and 5.9 log respectively for B. subtilis and B. cereus species. Continuous HP treatments were performed at the same pressure and temperature for similar treatment durations. Our results showed that the spore inactivation ratio was correlated with the cumulative exposure time to pressure rather than to effects of the cycling process. Greater spore inactivation caused by HP cycling was observed only when faster compression and decompression rates were applied, probably due to adiabatic heating. A three-step kinetic model was developed, which seemed to support our hypothesis regarding the mechanisms of inactivation by pressure cycling and continuous HP treatments. The resistance of bacterial spores to HP limits the industrial applications to refrigerated food products. In this study, we investigated the use of pressure cycling as a means to improve spore baroinactivation at moderate temperatures (T < 60 °C). We showed that cycling pressure does not significantly increase bacterial spore inactivation in comparable treatment duration, but certainly increases material fatigue in HP vessels. Thus, under moderate temperature, cycling pressure treatment is not industrially relevant. • The highest inactivation was 1.8 and 5.9 log for B. subtilis and B. cereus spores respectively. • Spore inactivation was mainly related to the pressure holding time and not to the cycling effect. • A three-step kinetic model with a good fit was developped for spore inactivation by pressure cycling. • Higher inactivation with slow compression kinetic due to holding time under pressure during compression time [ABSTRACT FROM AUTHOR]

The article analyzes cortex degradation products to understand better the hydrolytic process of Clostridium perfringens S40 spore peptidoglycan during germination. According to the authors, SleC is most likely a bifunctional enzyme with lytic transglycosylase and N-acetylmuramoyl-L-alanine amidase activities. They add that during germination of Clostridium perfringens spores, SleC causes merely small and local changes in the cortex structure, which are needed before SleM can function.

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release duringl-alanine induced germination via terbium ion (Tb3+)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295±19 spores mL−1 and 369±36 spores mL−1, respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27±2 spores mL−1. The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 × 103±6.7 × 102 cells mL−1. Culturing attempts yielded 2 CFU mL−1 (4°C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays. [ABSTRACT FROM AUTHOR]