Heat Resistance Of Spores Research Articles

Variation in heat resistance and biofilm formation of Bacillus cereus spores in various fermented soybean foods

This study investigated the heat resistance of Bacillus cereus spores (as well as spores in intact biofilm) in two types of Korean fermented soybean foods and presumed the potential key parameters (physicochemical and nutritional properties) associated with their heat resistance. For example, the D100°C-values of B. cereus ATCC 10987 and CH3 spores with strong heat resistance and prolific biofilm-forming capability were compared in various Jjigae-type (Cheonggukjang jjigae, Doenjang jjigae, and Gochujang jjigae) and Jang-type (Cheonggukjang, Doenjang, and Gochujang) foods commonly consumed in Korea. The D100°C-values of planktonic spores were significantly different depending on the type of food, that is, Jang and Jjigae. Compared with Jjigae-type foods, a higher heat resistance of B. cereus spores was found in Jang-type foods (particularly Doenjang and Gochujang) with low water activity and high salinity. In Jjigae-type foods, spore heat resistance showed a positive correlation with the pH of Jjigaes, indicating that an acidic environment weakens the spores. A negative correlation between the total fat content and spore heat resistance was found in Jjigae-type foods but not in Jang-type foods. Meanwhile, regarding the heat resistance of B. cereus spores in intact biofilm, the D100°C-values were significantly higher (up to 6.5-fold) than those of planktonic spores in all Jjigae-type foods. The slightly acidic pH and amount of carbohydrates are likely related to the large formation of extracellular polymeric substances and strong heat resistance of B. cereus spores in biofilm. This study may provide a comprehensive understanding of the relationship between the key parameters of foods and heat resistance of B. cereus spores with or without intact biofilm and methods to control their risks in different types of fermented soybean foods.

Bacillus cereus in rice: A review on food poisoning, antimicrobial resistance, and control measures.

Rice is often associated with Bacillus cereus (B. cereus) food poisoning. This review aims to explore the food poisoning activity, antimicrobial resistance, and control measures of B. cereus in rice from 1974 to October 2023. We searched for eligible studies from the PubMed database based on explicit criteria following the PRISMA checklist. A total of 117 articles were collected, and the final analysis included 29 studies. Quality appraisal was performed using AMSTAR 2, SANRA 2, and Critical Appraisal Tool standards. B. cereus can grow and multiply in food to cause emetic vomiting or diarrheal syndrome. The primary etiology of B. cereus contamination is improper food handling and storage temperature during the cooking, cooling, and reheating stages of rice. The alarming rise of antimicrobial resistance in B. cereus to beta-lactam antibiotics necessitates alternatives from natural antimicrobial preservatives such as carvacrol, chitosan, or trans-cinnamaldehyde to prevent microbial infestation and toxin production. Implementing food safety strategies tailored to specific food settings, such as restaurants and factorymanufactured ready-to-eat rice, is critical for preventing food contamination by B. cereus. Given the heat-resistant spores and intoxication properties of B. cereus, it is important to develop effective interventions and hygienic protocols from farm to fork.

Role of Dipicolinic Acid in Heat Resistance of Spores of Clostridium botulinum and Clostridium sporogenes PA3679 by Thermal and Pressure-assisted Thermal Processing

Dipicolinic acid (DPA) is a major constituent of spores and reportedly provides protection against inactivation by various thermal processes; however, the relationship between DPA and resistance towards pressure-assisted thermal processing is not well understood. Thermal and pressure-assisted thermal inactivation studies of Clostridium botulinum nonproteolytic strains QC-B and 610-F, proteolytic strain Giorgio-A, and thermal surrogate Clostridium sporogenes PA3679 spores suspended in ACES buffer (0.05 M, pH 7.0) were performed to determine if a relationship exists between DPA release and log reduction of spores. Thermal inactivation at 80, 83, and 87 °C for nonproteolytic strains and 101, 105, and 108 °C for the proteolytic strain and thermal surrogate were conducted. Pressure-assisted thermal inactivation for nonproteolytic strains at 83 °C/600 MPa and for the proteolytic strain and thermal surrogate at 105 °C/600 MPa were performed. Surviving spores were enumerated by 5-tube MPN method for log reductions and analyzed for released DPA by liquid chromatography-tandem mass spectrometry. The correlation between MPN log reductions, released DPA, and D-values were calculated. A positive correlation between released DPA and log reduction of spores was observed for QC-B and 610-F at 80 and 83 °C (r = 0.6073 − 0.7755; P < 0.01). At 87 °C, a positive correlation was detected for 610-F (r = 0.4242, P < 0.05) and no correlation was observed for QC-B (r = 0.1641; P > 0.05). A strong, positive correlation (r = 0.8359 − 0.9284; P < 0.05) between released DPA and log reduction of spores was observed for Giorgio-A at 101, 105, and 108 °C, and a strong, positive correlation (r = 0.8402; P < 0.05) was observed for PA3679 at 101 °C. A positive correlation (r = 0.5646 − 0.6724; P < 0.01) was observed for QC-B, 610-F, and Giorgio-A after pressure-assisted thermal treatment. No correlation (r = 02494; P > 0.05) was found for PA3679 after pressure-assisted thermal treatment. These results suggest a correlation exists between DPA release and heat resistance; however, the level of correlation varied between strains and temperatures. The findings from this research may aid in the development of spore inactivation strategies targeting the thermal resistance profiles of various strains of C. botulinum spores.

Screening and characterization of lactic acid bacteria with antibacterial effect against heat-resistant spore outgrowth of Bacillus sporothermodurans

This study is the first to screen of lactic acid bacteria strains with antimicrobial effect against spore outgrowth of Bacillus sporothermodurans and characterize their technological properties. Eighty strains were isolated from Tunisia raw milk but only 8 isolates were selected based on their antagonistic activity against germinated spores of B. sporothermodurans and nine pathogenic bacteria. Phenotypic and genotypic characterization allowed us to distinguish three different species, namely Lactiplantibacillus plantarum, Levilactobacillus brevis and Lactococcus lactis. The lactic acid strains able to produce protease and lipase enzymes. The important antagonistic activity was observed in presence of L. lactis TLAB6 strain which produced a bacteriocin and lactic acid. The bacteriocin produced by the L. lactis TLAB6 is heat stable and resistant to trypsin. The results suggest that the selected LAB with antibacterial activity against germinated spore and technological properties may be used to preserve the food and formulate of bioactive functional foods.

Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

A rare blood transfusion complication: Bacillus thermoamylovorans bacteremia and diffuse pustular rash

Bacillus thermoamylovorans is a species of bacillus that causes structural defects in milk by contaminating milk with its heat-resistant spores. We report a case of diffuse pustular rash on the extremities and bacillus thermoamylovorans bacteremia in blood culture after erythrocyte suspension replacement. On admission, his acute phase reactants were elevated and he had a generalized rash. After appropriate antibiotherapy, his lesions regressed almost completely. There is no case report of blood transfusion-associated bacillus thermoamylovorans bacteremia in the literature. This case is presented to contribute to the literature to show that Bacillus thermoamylovorans bacteremia may play a role in the etiology of pustular rash and infectious reactions associated with blood transfusion.

Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

Oil addition increases the heat resistance of Clostridium sporogenes spores in braised sauce beef: Perspectives from spore surface characteristics and microstructure

During thermal processing of braised sauce beef, the lipid content of circularly used sauce increased accordingly because of lipid migration from beef to sauce, which may impact the bacterial heat resistance in the products. This study aims to characterize the heat resistance of Clostridium sporogenes spores in braised sauce beef, and investigate the effects of oil on the spore surface characteristics and microstructure. The results indicated that the heat resistance of C. sporogenes spores in beef was significantly higher than that in sauce. Oil addition remarkably enhanced the spore heat resistance in sauce, with D95°C value three times more than that without oil added, and even higher than that in beef. The results of spore surface characteristics indicated that oil addition led to an increase of hydrophobicity and a decrease of zeta potential, which ultimately increased spore heat resistance. Microstructure analysis indicated that exosporium maintenance and cortex expansion induced by oil addition might contribute to the increase of spore heat resistance. This study has sufficiently verified the importance of oil content on the heat resistance of C. sporogenes spores, which should be taken into consideration when developing thermal processes for controlling the spores in food matrices.

In silico analysis of Bacillus stearothermophilus as a bioindicator for sterilizationco analysis of Bacillus stearothermophilus as a bioindicator for sterilization

To kill every kind of viable bacterial cells, sterilization in food and pharmaceutical industries is essential. In this study, bio indicators are used to validate accurate sterility. Bacillus stearothermophilus is used as a reference standard heat stable bio indicator. A 16S ribosomal RNA ribotyping sequence of Bacillus stearothermophilus is compared with different bacterial bioindicators including Bacillus pumilus, Bacillus atrophaeus, Clostridium sporogenes, and Brevundimonas diminuta. Results showed that Bacillus pumilus has the highest percentage (90.88%) similarity with the Geobacillus stearothermophilus followed by the Bacillus atrophaeus (90.84%), and Clostridium sporogenes (85.35%). The Brevundimonas diminuta was the least similar organism having a percentage similarity of 81.71% with the Geobacillus stearothermophilus. Bacterial spores are used as biological indicators for sterilization because they exhibit resistance to chemical and physical sterilization techniques. The protein common in all bacteria under study is Spo0A that make the bacteria heat resistant. The eradication of all these kind of heat resistant spores needs a high temperature which will also kill other viable cells. Keywords: Bacillus stearothermophilus; Bacillus pumilus; Bacillus atrophaeus; Clostridium sporogenes and Brevundimonas diminuta; Spo0A protein.

Effects of spore purity on the wet heat resistance of Clostridium perfringens, Bacillus cereus and Bacillus subtilis spores

Heat resistance of spores of Clostridium perfringens 8238 (Hobbs Serotype 2), Bacillus cereus NCTC 11143 (4810/72), and Bacillus subtilis PS533, an isogenic derivative of strain PS832 (a 168 strain) was determined in ground beef at 95 °C. Spore purification was by centrifugation and washing with sterile distilled water (dH2O), followed by sonication and then Histodenz centrifugation for B. subtilis and C. perfringens, and centrifugation and washing with sterile dH2O followed by Histodenz centrifugation for B. cereus. Bags containing inoculated beef samples were submerged in a temperature-controlled water bath and held at 95 °C for predetermined lengths of time. Surviving spore populations were enumerated by plating on mannitol egg yolk polymyxin agar (MYP) agar plates for B. cereus and B. subtilis, and on tryptose-sulfite-cycloserine agar (TSC) agar plates for C. perfringens. Survivor curves were fitted to linear, linear with tail, and Weibull models using the USDA Integrated Pathogen Modeling Program (IPMP) 2013 software. The Weibull model provided a relatively better fit to the data since the root mean square error (RMSE), mean square error (MSE), sum of squared errors (SSE), and Akaike information criterion (AIC) values were lower than the values obtained using the linear or the linear with tail models. Additionally, the Weibull model accurately predicted the observed D-values at 95 °C for the three spore-formers since the accuracy factor (Af) values ranged from 1.03 to 1.08 and the bias factor (Bf) values were either 1.00 or 1.01. Times at 95 °C to achieve a 3-log reduction decreased from 206 min for C. perfringens spores purified with water washes alone to 191 min with water washes followed by sonication and Histodenz centrifugation, from 7.9 min for B. cereus spores purified with water washes alone to 1.4 min with water washes followed by Histodenz centrifugation, and from 20.6 min for B. subtilis spores purified with water washes alone to 6.7 min for water washes followed by sonication and Histodenz centrifugation. Thermal-death-time values reported in this study will assist food processors to design thermal processes to guard against bacterial spores in cooked foods. In addition, clearly spore purity is an additional factor in spore wet heat resistance, although the cause of this effect is not clear.

Compatible solutes determine the heat resistance of conidia

BackgroundAsexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle.ResultsIn this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance.ConclusionsHeat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

Levels and types of microbial contaminants in different plant-based ingredients used in dairy alternatives

In this study levels and types of microbial contaminants were investigated in 88 different plant-based ingredients including many that are used to manufacture dairy alternatives. Studied ingredients encompassed samples of pulses (pea, faba bean, chickpea, and mung bean), cereals/pseudocereals (oat, rice, amaranth and quinoa) and drupes (coconut, almond and cashew). The microbial analysis included: i) total viable count (TVC), ii) total aerobic mesophilic spore count (TMS), iii) heat resistant aerobic thermophilic spore count (HRTS), iv) anaerobic sulfite reducing Clostridium spore count (SRCS), and v) Bacillus cereus spore count (BCES). Microorganisms isolated from the counting plates with the highest sample dilutions were identified using 16S rRNA and MALDI-TOF MS analyses.Many of the investigated ingredients showed a high proportion of spores as part of their total aerobic mesophilic counts. In 63 % of the samples, the difference between TVC and TMS counts was 1 Log10 unit or less. This was particularly the case for the majority of pea isolates and concentrates, faba bean isolates, oat kernels and flakes, and for single samples of chickpea isolate, almond, amaranth, rice, quinoa, and coconut flours. Concentrations of TVC ranged between <1.0 and 5.3 Log10 CFU/g in different samples, and TMS varied between <1.0 and 4.1 Log10 CFU/g. Levels of HTRS, BCES and SRCS were generally low, typically around or below the LOD of 1.0 Log10 CFU/g.In total, 845 individual bacterial colonies were isolated belonging to 33 different genera. Bacillus licheniformis and B. cereus group strains were most frequently detected among Bacillus isolates, and these species originated primarily from pea and oat samples. Geobacillus stearothermophilus was the main species encountered as part of the HRTS. Among the Clostridium isolates, Clostridum sporogenes/tepidum were predominant species, which were mostly found in pea and almond samples. Strains with potential to cause foodborne infection or intoxication were typed using the PCR-based method for toxin genes detection. In the B. cereus group, 9 % of isolates contained the ces gene, 28 % contained hbl, 42 % cytK, and 69 % were positive for the nhe gene. Absence of the boNT-A and -B genes was confirmed for all isolated C. sporogenes/tepidum strains. Nearly all (98 %) B. licheniformis isolates were positive for the lchAA gene.Insight into the occurrence of microbial contaminants in plant-based ingredients, combined with knowledge of their key inactivation and growth characteristics, can be used for the microbial risk assessment and effective design of plant-based food processing conditions and formulations to ensure food safety and prevent spoilage.

Phylogenetic characterization and biodiversity of spore-forming bacteria isolated from Brazilian UHT milk.

Among the milk contaminating microorganisms, those which are able to form heat-resistant spores are concerning, especially for dairy companies that use ultra-high temperature (UHT) technology. These spores, throughout storage, can germinate and produce hydrolytic enzymes that compromise the quality of the final product. This study evaluated 184 UHT milk samples from different batches collected from seven Brazilian dairy companies with a possible microbial contamination problem. The bacteria were isolated, phenotypically characterized, clustered by REP-PCR, and identified through 16S rDNA sequencing. The presence of Bacillus sporothermodurans was verified using biochemical tests (Gram staining, catalase and oxidase test, glucose fermentation, esculin hydrolysis, nitrate reduction, and urease test). According to these tests, none of the isolates presented typical characteristics of B. sporothermodurans. In sequence, the isolates, that presented rod-shapes, were submitted to molecular analyses in order to determine the microbial biodiversity existing among them. The isolates obtained were grouped into 16 clusters, four of which were composed of only one individual. A phylogenetic tree was constructed using the sequences obtained from the 16S rDNA sequencing and some reference strains of species close to those found using BLAST search in the NCBI nucleotide database. Through this tree, it was possible to verify the division of the isolates into two large groups, the Bacillus subtilis and the Bacillus cereus groups. Furthermore, most isolates are phylogenetically closely related, which makes it even more difficult to identify them at the species level. In conclusion, it was possible to assess, in general, the groups of sporulated contaminants in Brazilian UHT milk produced in the regions evaluated. In addition, it was also possible to determine the biodiversity of spore-forming bacteria found in UHT milk samples, thus opening up a range of possible research topics regarding the effects of the presence of these microorganisms on milk quality.

Effect of spo0A, sigE, sigG, and sigK disruption on butanol production and spore formation in Clostridium saccharoperbutylacetonicum strain N1-4 (ATCC13564)

Clostridium saccharoperbutylacetonicum strain N1-4 (ATCC13564) is a butanol-producing strain suitable for application to butanol production from cellulosic materials by co-culture with cellulolytic and thermophilic species, such as Hungateiclostridium thermocellum (synonym: Clostridium thermocellum). The optimal temperature for butanol production by strain N1-4 is 30°C, and the strain is sensitive to a high culture temperature of 37°C. Given that spore formation is observed at high frequency when strain N1-4 is cultivated at 37°C, we assumed in a previous study that the initiation of sporulation is related to a decrease in butanol production. Therefore, to investigate the relationship between butanol production and spore formation, we generated strain N1-4 isolates in which genes related to spore formation were disrupted. The sporulation-related gene disruptants of spo0A, sigE, sigG, and sigK lost the ability to produce heat-resistant spores, irrespective of the culture temperature. Among the gene disruptants produced, only the spo0A disruptant lost butanol-producing ability when cultivated at 30°C. Interestingly, the sigE disruptant maintained butanol productivity similar to that observed at 30°C, even when cultivated at 37°C. In addition, the sigE disruptant successfully produced butanol from Avicel cellulose by co-culture with H.thermocellum at a fermentation temperature of 37°C.

High yield of heat-resistant spores of Bacillus megaterium in bioreactors

Microbial inoculants have been proposed as a sustainable and cost-effective alternative to conventional agricultural practices. However, the effectiveness of these bio-based products can be negatively impacted by reduced cell viability during product development, storage, and commercialization. To address these limitations, it is critical to identify and optimize the cultivation conditions that can lead to the production of highly resistant spores for use as inoculants. In this study, optimal cultivation conditions for obtaining Bacillus megaterium B119 spores in bioreactors were reached, with increases in the sporulation efficiency and heat resistance. The best nutrient medium and optimal temperature and initial pH conditions were obtained on shake flask experiments and subsequently validated in two bench-scale bioreactor models (stirred and aerated tank and the bubble column). The optimized temperature and initial pH were found to be 37 °C and 5.6. The highest concentration of heat-resistant Bacillus spores of 1.31·1012 CFU/mL was obtained in the bubble column bioreactor with a sporulation efficiency of 100.0%, a heat resistance of 78.4%, and an energetic efficiency on spores production of 4.83·1010 CFU/J. The production of highly resistant spores can significantly improve the survival rate of microorganisms during inoculant production and application, thereby enhancing their effectiveness in the field.

Clostridium perfringens-Opportunistic Foodborne Pathogen, Its Diversity and Epidemiological Significance.

The C. perfringens species is associated with various environments, such as soils, sewage, and food. However, it is also a component of the gastrointestinal (GI) microflora (i.e., microbiota) of sick and healthy humans and animals. C. perfringens is linked with different systemic and enteric diseases in livestock and humans, such as gas gangrene, food poisoning, non-foodborne diarrhoea, and enterocolitis. The strains of this opportunistic pathogen are known to secrete over 20 identified toxins that are considered its principal virulence factors. C. perfringens belongs to the anaerobic bacteria community but can also survive in the presence of oxygen. The short time between generations, the multi-production capability of toxins and heat-resistant spores, the location of many virulence genes on mobile genetic elements, and the inhabitance of this opportunistic pathogen in different ecological niches make C. perfringens a very important microorganism for public health protection. The epidemiological evidence for the association of these strains with C. perfringens-meditated food poisoning and some cases of non-foodborne diseases is very clear and well-documented. However, the genetic diversity and physiology of C. perfringens should still be studied in order to confirm the importance of suspected novel virulence traits. A very significant problem is the growing antibiotic resistance of C. perfringens strains. The aim of this review is to show the current basic information about the toxins, epidemiology, and genetic and molecular diversity of this opportunistic pathogen.

Are Bacillus thuringiensis strains like any other Bacillus cereus strains? Phenotypic-based tools to locate Bacillus thuringiensis in the diversity of the Bacillus cereus sensu lato group

Some Bacillus thuringiensis (Bt) strains are used as pesticide agent. This species belongs to Bacillus cereus (Bc) group which contains many species with a high phenotypic diversity, and could be pathogenic like B. cereus. The aim of this study was to characterize the phenotype of 90 strains belonging to Bc group, half of which were Bt. Knowing that Bt strains belong to different phylogenetic Bc groups, do Bt strains have the same phenotype than other Bc group strains?Five phenotypic parameters were estimated for 90 strains in the Bc group, of which 43 were Bt strains: minimal, maximal and optimal growth temperature, cytotoxicity on Caco-2 cells, heat resistance of spores. The dataset was processed by principal component analysis, showing that 53% of the variance of the profiles corresponded to factors linked to growth, heat resistance and cytotoxicity. The phenotype followed the phylogenetic groups based on panC. Bt strains showed similar behavior to other strains in the Bc group, in our experimental conditions. Commercial bio-insecticide strains were mesophilic with low heat resistance.

Bacillus subtilis, a Swiss Army Knife in Science and Biotechnology.

Next to Escherichia coli, Bacillus subtilis is the most studied and best understood organism that also serves as a model for many important pathogens. Due to its ability to form heat-resistant spores that can germinate even after very long periods of time, B. subtilis has attracted much scientific interest. Another feature of B. subtilis is its genetic competence, a developmental state in which B. subtilis actively takes up exogenous DNA. This makes B. subtilis amenable to genetic manipulation and investigation. The bacterium was one of the first with a fully sequenced genome, and it has been subject to a wide variety of genome- and proteome-wide studies that give important insights into many aspects of the biology of B. subtilis. Due to its ability to secrete large amounts of proteins and to produce a wide range of commercially interesting compounds, B. subtilis has become a major workhorse in biotechnology. Here, we review the development of important aspects of the research on B. subtilis with a specific focus on its cell biology and biotechnological and practical applications from vitamin production to concrete healing. The intriguing complexity of the developmental programs of B. subtilis, paired with the availability of sophisticated tools for genetic manipulation, positions it at the leading edge for discovering new biological concepts and deepening our understanding of the organization of bacterial cells.

Identification and characterization of new proteins crucial for bacterial spore resistance and germination.

2Duf, named after the presence of a transmembrane (TM) Duf421 domain and a small Duf1657 domain in its sequence, is likely located in the inner membrane (IM) of spores in some Bacillus species carrying a transposon with an operon termed spoVA 2mob. These spores are known for their extreme resistance to wet heat, and 2Duf is believed to be the primary contributor to this trait. In this study, we found that the absence of YetF or YdfS, both Duf421 domain-containing proteins and found only in wild-type (wt) B. subtilis spores with YetF more abundant, leads to decreased resistance to wet heat and agents that can damage spore core components. The IM phospholipid compositions and core water and calcium-dipicolinic acid levels of YetF-deficient spores are similar to those of wt spores, but the deficiency could be restored by ectopic insertion of yetF, and overexpression of YetF increased wt spore resistance to wet heat. In addition, yetF and ydfS spores have decreased germination rates as individuals and populations with germinant receptor-dependent germinants and increased sensitivity to wet heat during germination, potentially due to damage to IM proteins. These data are consistent with a model in which YetF, YdfS and their homologs modify IM structure to reduce IM permeability and stabilize IM proteins against wet heat damage. Multiple yetF homologs are also present in other spore forming Bacilli and Clostridia, and even some asporogenous Firmicutes, but fewer in asporogenous species. The crystal structure of a YetF tetramer lacking the TM helices has been reported and features two distinct globular subdomains in each monomer. Sequence alignment and structure prediction suggest this fold is likely shared by other Duf421-containing proteins, including 2Duf. We have also identified naturally occurring 2duf homologs in some Bacilli and Clostridia species and in wt Bacillus cereus spores, but not in wt B. subtilis. Notably, the genomic organization around the 2duf gene in most of these species is similar to that in spoVA 2mob, suggesting that one of these species was the source of the genes on this operon in the extremely wet heat resistant spore formers.

Quantitative microbial spoilage risk assessment of plant-based milk alternatives by Geobacillus stearothermophilus in Europe

Geobacillus stearothermophilus is one of the predominant spoilers of UHT-treated food products, due to its extremely heat-resistant spores. However, the surviving spores should be exposed to temperature higher than their minimum growth temperature for a certain time to germinate and grow to spoilage levels. Considering the projected temperature increase due to climate change, the events of non-sterility during distribution and transportation are expected to escalate. Hence, the aim of this study was to build a quantitative microbial spoilage risk assessment (QMRSA) model to quantify the risk of spoilage of plant-based milk alternatives within Europe. The model consists of four main steps: 1. Initial contamination of raw materials 2. Heat inactivation of spores during UHT treatment 3. Partitioning 4. Germination and outgrowth of spores during distribution and storage. The risk of spoilage was defined as the probability of G. stearothermophilus to reach its maximum concentration (Nmax = 107.5 CFU/mL) at the time of consumption. The assessment was performed for North (Poland) and South (Greece) Europe, and the risk of spoilage was estimated for the current climatic conditions and a climate change scenario. Based on the results, the risk of spoilage was negligible for the North European region, while the risk of spoilage in South Europe was 6.2 × 10−3 95% CI (2.3 × 10−3;1.1 × 10−2) under the current climatic conditions. The risk of spoilage was increased for both tested countries under climate change scenario; from zero to 1.0 × 10−4 in North Europe, risk multiplied 2 or 3 in South Europe depending on air conditioning implementation at consumer’s place. Therefore, the heat treatment intensity and the use of insulated trucks during distribution were investigated as mitigation strategies and led to significant reduction of the risk. Overall, the QMRSA model developed in this study can support risk management decisions of these products by quantify the potential risk under current climatic conditions and climate change scenarios.