Comparing resistance of bacterial spores and fungal conidia to pulsed light and UVC radiation at a wavelength of 254 nm.

Pulsed light (PL) inactivates microorganisms by UV-rich, high-irradiance and short time pulses (250 µs) of white light with wavelengths from 200 nm to 1100 nm. PL is applied for disinfection of food packaging material and food-contact equipment. Spores of seven Bacillus ssp. strains and one Geobacillus stearothermophilus strain and conidia of filamentous fungi (One strain of Aspergillus brasiliensis, A. carbonarius and Penicillium rubens) were submitted to PL (fluence from 0.23 J/cm2 to 4.0 J/cm2) and UVC (at λ = 254 nm; fluence from 0.01 J/cm2 to 3.0 J/cm2). One PL flash at 3 J/cm2 allowed at least 3 log-reduction of all tested microorganisms. The emetic B. cereus strain F4810/72 was the most resistant of the tested spore-forming bacteria. The PL fluence to 3 log-reduction (F3 PL) of its spores suspended in water was 2.9 J/cm2 and F3 UVC was 0.21 J/cm2, higher than F3 PL and F3 UVC of spores of B. pumilus SAFR-032 2.0 J/cm2 and 0.15 J/cm2, respectively), yet reported as a highly UV-resistant spore-forming bacterium. PL and UVC sensitivity of bacterial spores was correlated. Aspergillus spp. conidia suspended in water were poorly sensitive to PL. In contrast, PL inactivated Aspergillus spp. conidia spread on a dry surface more efficiently than UVC. The F2 PL of A. brasiliensis DSM1988 was 0.39 J/cm2 and F2 UVC was 0.83 J/cm2. The resistance of spore-forming bacteria to PL could be reasonably predicted from the knowledge of their UVC resistance. In contrast, the sensitivity of fungal conidia to PL must be specifically explored.

Counts of mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic sporeforming bacteria and persistence of Bacillus cereus spores throughout cocoa powder processing chain.

Sporeforming bacteria are a concern in some food raw materials, such as cocoa powder. Samples (n = 618) were collected on two farms and at several stages during cocoa powder manufacture in three commercial processing lines to determine the impact of each stage on bacterial spore populations. Mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic, and Bacillus cereus spore populations were enumerated in all the samples. Genetic diversity in B. cereus strains (n = 110) isolated from the samples was examined by M13 sequence-based PCR typing, partial sequencing of the panC gene, and the presence/absence of ces and cspA genes. The counts of different groups of sporeforming bacteria varied amongst farms and processing lines. For example, the counts of mesophilic aerobic spore-forming (MAS) populations of cocoa bean fermentation were lower than 1 log spore/g in Farm 1 but higher than 4 log spore/g in Farm 2. B. cereus isolated from cocoa powder was also recovered from cocoa beans, nibs, and samples after roasting, refining, and pressing, which indicated that B. cereus spores persist throughout cocoa processing. Phylogenetic group IV was the most frequent (73%), along with processing. Strains from phylogenetic group III (14 %) did not show the ces gene's presence.

Proteome of spores from biological indicators in sterilization processes: Bacillus pumilus and Bacillus atrophaeus.

Bacillus atrophaeus and Bacillus pumilus spores are widely used as biological indicators to assess the effectiveness of decontamination procedures. Spores are intricate, multi-layered cellular structures primarily composed of proteins, which significantly contribute to their extreme resistance. Therefore, conducting a comprehensive proteome analysis of spores is crucial to identify the specific proteins conferring spore resistance. Here, we employed a high-throughput shotgun proteomic approach to compare the spore proteomes of B. atrophaeus DSM675 and B. pumilus DSM492, identifying 1312 and 1264 proteins, respectively. While the overall number of proteins found in both strains is roughly equivalent, a closer examination of a subset of 54 spore-specific proteins revealed noteworthy distinctions. Among these 54 proteins, 23 were exclusively detected in one strain, while others were shared between both. Notably, of the 31 proteins detected in both strains, 10 exhibited differential abundance levels, including key coat layer morphogenetic proteins. The exploration of these 54 proteins, considering their presence, absence, and differential abundance, provides a unique molecular signature that may elucidate the differences in sensitivity/resistance profiles between the two strains.

Comparison of pesticide residue and specific nutrient levels in peeled and unpeeled apples.

BACKGROUND: Studies have shown that the consumption of apples has a beneficial effect on cardiovascular diseases and some cancers, largely as a result of their micronutrient and phytoconstituent contents. Apple peel not only contains more polyphenols than the flesh, but also is likely to contain pesticide residues. The present study aimed to compare the contents of certain micronutrients and residual pesticide levels in peeled and unpeeled apples. RESULTS: Peeled apples contained fewer pesticide residues at lower concentrations than unpeeled apples. However, whether samples were peeled or not, the exposure values for pesticide residues in apples never exceeded the acceptable daily intake (ADI), but ranged between 0.04% and 2.10% of the ADI in adults for food intake estimated at the 95th percentile (277 g per person per day). Determination of polyphenol, fibre, magnesium and vitamin C levels showed that the nutritional differences observed between peeled and unpeeled apples were marginal. CONCLUSION: The consumption of apples, such as the apples tested in the present study, results in an exposure to pesticides that is low for unpeeled apples, and lower for peeled apples. Moreover, there was no significant loss of nutritional value from eating peeled apples based on the nutrients investigated. © 2022 Society of Chemical Industry.

Bacillus cereus cshA Is Expressed during the Lag Phase of Growth and Serves as a Potential Marker of Early Adaptation to Low Temperature and pH.

Bacterial adaptation is characterized by a lag phase during which cells do not multiply or modify their physiology to cope with the constraints of their environment. Our aim was to determine a sequence of events during the lag phase of growth at low temperature and pH for three Bacillus cereus strains. The onsets of expression of two genes, one of which is essential for stress adaptation (cshA, coding for a RNA helicase) and one of which is involved in the transition between lag phase and exponential phase (abrB, coding for a transition regulator), were determined using fluorescent transcriptional reporter systems. Regardless of the stressing conditions and the tested strains, the cshA promoter was active very early, while the biomass increased and always did so before the first cell division. At 12°C and pH 7.0, the onset of cshA promoter activity occurred at between 3 h and 7 h, while the bacterial counts started to increase at between 12 h and 13 h. At pH 5.0 and at 20°C or 30°C, the onset of cshA promoter activity occurred before 1 h and earlier than at pH 7.0. In contrast, the onset of abrB promoter activity depended on the strain and the stressing conditions. In the ATCC 14579 strain, the onset of abrB promoter activity always started at between 30 min and 3 h, before biomass increased and cell division occurred. For the other strains, it took place along with the first cell division at 12°C but did so much later during growth under the other tested conditions.IMPORTANCE The spore-forming bacterium B. cereus is a major cause of foodborne outbreaks in Europe. Some B. cereus strains can grow at low temperatures and low pH in many processed foods. Modeling of the bacterial lag time is hampered by a lack of knowledge of the timing of events occurring during this phase. In this context, the identification of lag phase markers, not currently available, could be a real advance for the better prediction of lag time duration. Currently, no molecular markers of this phase are available. By determining that cshA was always expressed early during the lag phase, we provide a molecular marker of the early adaptation process of B. cereus cells when exposed to low temperature and pH.

The fate of Bacillus cereus and Geobacillus stearothermophilus during alkalization of cocoa as affected by alkali concentration and use of pre-roasted nibs.

Alkalization is a step of cocoa processing and consists of the use of alkali and high temperature to improve the sensorial and technological qualities of cocoa. Intense food processing can select spores, which can compromise safety and quality of the final product. Thus, the aim of this study was to evaluate the fate of B. cereus and G. stearothermophilus spores during the alkalization of pre-roasted (Pr) nibs (held at 120 °C) and unroasted (Ur) nibs (held at 90 °C) using potassium carbonate (0, 2, 4 and 6% w/w). In all conditions, log-linear inactivation kinetics with a tail was observed. The inactivation rate (kmax) for B. cereus varied from 0.065 to 1.67 min-1, whereas the kmax for G. stearothermophilus varied from 0.012 to 0.063 min-1. For both microorganisms, the lowest kmax values were observed during Ur nibs alkalization. The carbonate concentration increase promoted kmax values reduction. The highest tail values were observed for G. stearothermophilus in Ur nibs alkalization, reaching 3.04 log spores/g. Tail formation and low kmax values indicated that cocoa alkalization does not cause significant reductions on bacterial spore population. Therefore, the microbiological control should be primarily ensured by the raw material quality and by avoiding recontamination in the cocoa chain.

Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores.

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2 and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.

Genotypic and phenotypic characterization of foodborne Geobacillus stearothermophilus.

Geobacillus stearothermophilus is the main thermophilic spore former involved in flat sour spoilage of canned foods. Three typing methods were tested and applied to differentiate strains at intra-species level: panC sequence analysis, REP-PCR and M13-PCR. panC gene was highly conserved within the studied strains, suggesting a low intra-specific diversity. This was supported by REP-PCR primary assays and M13-PCR results. M13-PCR profile analysis succeeded in differentiating six closely related groups (at 79% threshold similarity) among 127 strains from a range of spoiled canned food products and from different canneries. Phenotypic traits were investigated among 20 selected strains representing groups and origins. Ranges of growth under different temperatures (from 40 °C to 70 °C), pH (from 5.0 to 6.5), NaCl concentrations (from 1 to 5%) and sporulation conditions poorly differed between strains, but wet heat resistance of spores showed a 20-fold variation between strains. Furthermore, in this study, strains that belonged to the same M13-PCR genetic group did not share phenotypic characteristics or common origin. The work emphasizes a low diversity within the G. stearothermophilus species but data from this study may contribute to a better control of G. stearothermophilus spoilage in canned food.

Variation of cardinal growth parameters and growth limits according to phylogenetic affiliation in the Bacillus cereus Group. Consequences for risk assessment.

The growth rates of strains covering the seven major phylogenetic groups of Bacillus cereus sensu lato (as defined by Guinebretiere et al., 2008) at a range of temperature (7 °C-55 °C), pH (4.6-7.5) and a(w) (0.929-0.996, with 0.5%-10% NaCl as humectant) were determined. Growth rates were fitted by non-linear regression to determine the cardinal parameters T(min), T(opt), T(max), pH(min), pH(opt), a(wmin) and µ(opt). We showed that cardinal parameters reflected the differences in the temperature adaptation observed between B. cereus phylogenetic groups I to VII. The ability of growing at low pH (up to 4.3) or low a(w) (from a(w) 0.929 and up to 10% NaCl) varied among strains. The strains of groups III and VII, the most tolerant to heat, were also the most adapted to high NaCl (all strains growing at 8% NaCl) and the ones of groups I and VI the least adapted (no growth at 7% NaCl). All strains of groups II and VII were able to grow at pH 4.6, and only a few strains of group VI. Phenotypic differences between the two psychrotrophic groups II and VI were revealed by contrasted acid and salt tolerance. The cardinal values determined in this work were validated by comparing with cardinal parameters of a panel of strains published elsewhere and with predictions of growth in a range of foods.

Inferring an augmented Bayesian network to confront a complex quantitative microbial risk assessment model with durability studies: application to Bacillus cereus on a courgette purée production chain.

The Monte Carlo (MC) simulation approach is traditionally used in food safety risk assessment to study quantitative microbial risk assessment (QMRA) models. When experimental data are available, performing Bayesian inference is a good alternative approach that allows backward calculation in a stochastic QMRA model to update the experts' knowledge about the microbial dynamics of a given food-borne pathogen. In this article, we propose a complex example where Bayesian inference is applied to a high-dimensional second-order QMRA model. The case study is a farm-to-fork QMRA model considering genetic diversity of Bacillus cereus in a cooked, pasteurized, and chilled courgette purée. Experimental data are Bacillus cereus concentrations measured in packages of courgette purées stored at different time-temperature profiles after pasteurization. To perform a Bayesian inference, we first built an augmented Bayesian network by linking a second-order QMRA model to the available contamination data. We then ran a Markov chain Monte Carlo (MCMC) algorithm to update all the unknown concentrations and unknown quantities of the augmented model. About 25% of the prior beliefs are strongly updated, leading to a reduction in uncertainty. Some updates interestingly question the QMRA model.

A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus.

The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.

Influence of anaerobiosis and low temperature on Bacillus cereus growth, metabolism, and membrane properties.

The impact of simultaneous anaerobiosis and low temperature on growth parameters, metabolism, and membrane properties of Bacillus cereus ATCC 14579 was studied. No growth was observed under anaerobiosis at 12°C. In bioreactors, growth rates and biomass production were drastically reduced by simultaneous anaerobiosis and low temperature (15°C). The two conditions had a synergistic effect on biomass reduction. In anaerobic cultures, fermentative metabolism was modified by low temperature, with a marked reduction in ethanol production leading to a lower ability to produce NAD(+). Anaerobiosis reduced unsaturated fatty acids at both low optimal temperatures. In addition, simultaneous anaerobiosis and low temperatures markedly reduced levels of branched-chain fatty acids compared to all other conditions (accounting for 33% of total fatty acids against more 71% for low-temperature aerobiosis, optimal-temperature aerobiosis, and optimal-temperature anaerobiosis). This corresponded to high-melting-temperature lipids and to low-fluidity membranes, as indicated by differential scanning calorimetry, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, and infrared spectroscopy. This is in contrast to requirements for cold adaptation. A link between modification in the synthesis of metabolites of fermentative metabolism and the reduction of branched-chain fatty acids at low temperature under anaerobiosis, through a modification of the oxidizing capacity, is assumed. This link may partly explain the impact of low temperature and anaerobiosis on membrane properties and growth performance.

Modeling heat resistance of Bacillus weihenstephanensis and Bacillus licheniformis spores as function of sporulation temperature and pH.

Although sporulation environmental factors are known to impact on Bacillus spore heat resistance, they are not integrated into predictive models used to calculate the efficiency of heating processes. This work reports the influence of temperature and pH encountered during sporulation on heat resistance of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 spores. A decrease in heat resistance (δ) was observed for spores produced either at low temperature, at high temperature or at acidic pH. Sporulation temperature and pH maximizing the spore heat resistance were identified. Heat sensitivity (z) was not modified whatever the sporulation environmental factors were. A resistance secondary model inspired by the Rosso model was proposed. Sporulation temperatures and pHs minimizing or maximizing the spore heat resistance (T(min(R)), T(opt(R)), T(max(R)), pH(min(R)) and pH(opt(R))) were estimated. The goodness of the model fit was assessed for both studied strains and literature data. The estimation of the sporulation temperature and pH maximizing the spore heat resistance is of great interest to produce spores assessing the spore inactivation in the heating processes applied by the food industry.

Sporulation boundaries and spore formation kinetics of Bacillus spp. as a function of temperature, pH and a(w).

Sporulation niches in the food chain are considered as a source of hazard and are not clearly identified. Determining the sporulation environmental boundaries could contribute to identify potential sporulation niches. Spore formation was determined in a Sporulation Mineral Buffer. The effect of incubation temperature, pH and water activity on time to one spore per mL, maximum sporulation rate and final spore concentration was investigated for a Bacillus weihenstephanensis and a Bacillus licheniformis strain. Sporulation boundaries of B. weihenstephanensis and of B. licheniformis were similar to, or included within, the range of temperatures, pH and water activities supporting growth. For instance, sporulation boundaries of B. weihenstephanensis were evaluated at 5°C, 35°C, pH 5.2 and a(w) 0.960 while growth boundaries were observed at 5°C, 37°C, pH 4.9 and a(w) 0.950. Optimum spore formation was determined at 30°C pH 7.2 for B. weihenstephanensis and at 45°C pH 7.2 for B. licheniformis. Lower temperatures and pH delayed the sporulation process. For instance, the time to one spore per mL was tenfold longer when sporulation occurred at 10°C and 20°C, for each strain respectively, than at optimum sporulation temperature. The relative effect of temperature and pH on sporulation rates and on growth rates is similar. This work suggests that the influence of environmental factors on the quantitative changes in sporulation boundaries and rates was similar to their influence on changes in growth rate.

Clostridium botulinum type C, D, C/D, and D/C: An update.

Clostridium botulinum is the main causative agent of botulism, a neurological disease encountered in humans as well as animals. Nine types of botulinum neurotoxins (BoNTs) have been described so far. Amongst these "toxinotypes," the A, the B and E are the most frequently encountered in humans while the C, D, C/D and D/C are mostly affecting domestic and wild birds as well as cattle. In France for instance, many cases and outbreaks are reported in these animal species every year. However, underestimation is very likely at least for avifauna species where the detection of dead animals can be challenging. Knowledge about BoNTs C, D, C/D, and D/C and the diseases they cause in animals and humans is still scarce and unclear. Specifically, the potential role of animal botulism outbreaks in cattle and poultry as a source of human illness needs to be further assessed. In this narrative review, we present the current knowledge about toxinotypes C, D, C/D, and D/C in cattle and poultry with, amongst various other aspects, their epidemiological cycles. We also discuss the zoonotic potential of these toxinotypes and some possible ways of risk mitigation. An adapted and effective management of botulism outbreaks in livestock also requires a better understanding of these less common and known toxinotypes.

Role of the five RNA helicases in the adaptive response of Bacillus cereus ATCC 14579 cells to temperature, pH, and oxidative stresses.

In this study, growth rates and lag times of the five RNA helicase-deleted mutants of Bacillus cereus ATCC 14579 were compared to those of the wild-type strain under thermal, oxidative, and pH stresses. Deletion of cshD and cshE had no impact under any of the tested conditions. Deletion of cshA, cshB, and cshC abolished growth at 12°C, confirming previous results. In addition, we found that each RNA helicase had a role in a specific temperature range: deletion of cshA reduced growth at all the tested temperatures up to 45°C, deletion of cshB had impact below 30°C and over 37°C, and deletion of cshC led mainly to a cold-sensitive phenotype. Under oxidative conditions, deletion of cshB and cshC reduced growth rate and increased lag time, while deletion of cshA increased lag time only with H(2)O(2) and reduced growth rate at a high diamide concentration. Growth of the ΔcshA strain was affected at a basic pH independently of the temperature, while these conditions had a limited effect on ΔcshB and ΔcshC strain growth. The RNA helicases CshA, CshB, and CshC could participate in a general adaptation pathway to stressful conditions, with a stronger impact at low temperature and a wider role of CshA.

Origin of bacterial spores contaminating foods.

Bacterial spores (=endospores) are common contaminants in foods. Sources of contamination in the food chain may include soil, faeces, animal feeds and food ingredients and processing chain themselves. Sporulation may occur in very diverse environments. The environment of sporulation has a strong influence on spore properties relevant for food quality and safety.

Spores of Bacillus cereus strain KBAB4 produced at 10 °C and 30 °C display variations in their properties.

Spores of the psychrotrophic Bacillus cereus KBAB4 strain were produced at 10 °C and 30 °C in fermentors. Spores produced at 30 °C were more resistant to wet heat at 85 °C, 1% glutaraldehyde, 5% hydrogen peroxide, 1M NaOH and pulsed light at fluences between 0.5 and 1.75 Jcm(-2) and to a lesser extent to monochromatic UV-C at 254 nm. No difference in resistance to 0.25 mM formaldehyde, 1M nitrous acid and 0.025 gl(-1) calcium hypochlorite was observed. Spores produced at 10 °C germinated more efficiently with 10 mM and 100 mM l-alanine than spores produced at 30 °C, while no difference in germination was observed with inosine. Dipicolinic acid (DPA) content in the spore was significantly higher for spores prepared at 30 °C. Composition of certain fatty acids varied significantly between spores produced at 10 °C and 30 °C.

Short-Chain and Unsaturated Fatty Acids Increase Sequentially From the Lag Phase During Cold Growth of Bacillus cereus.

Fatty acids of two mesophilic and one psychrotrophic strains of the foodborne pathogen Bacillus cereus were analyzed by gas chromatography coupled to mass spectrometry during growth at cold (10 and 12°C) vs. optimal (30°C) temperatures and during the whole growth process (6-7 sampling times) from lag to stationary phase. In all these strains, a sequential change of fatty acids during cold growth was observed. Fatty acids were modified as soon as the end of lag, with an increase of the short-chain fatty acids (less than 15 carbons), particularly i13. These short-chain fatty acids then reached a maximum at the beginning of growth and eventually decreased to their initial level, suggesting their importance as a rapid cold adaptation mechanism for B. cereus. In a second step, an increase in Δ5,10 di-saturated fatty acids and in monounsaturated fatty acids in Δ5 position, at the expense of unsaturation in Δ10, started during exponential phase and continued until the end of stationary phase, suggesting a role in growth consolidation and survival at cold temperatures. Among these unsaturated fatty acids, those produced by unsaturation of n16 increased in the three strains, whereas other unsaturated fatty acids increased in some strains only. This study highlights the importance of kinetic analysis of fatty acids during cold adaptation.

The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus.

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of BacillaceaeIMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.