Whole-cell biocatalytic of Bacillus cereus WZZ006 strain to synthesis of indoxacarb intermediate: (S)-5-Chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester.

In this study, a newly isolated strain screened from the indoxacarb-rich agricultural soils, Bacillus cereus WZZ006, has a high stereoselectivity to racemic substrate 5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester. (S)-5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester was obtained by bio-enzymatic resolution. After the 36-hour hydrolysis in 50-mM racemic substrate under the optimized reaction conditions, the e.e.s was up to 93.0% and the conversion was nearly 53.0% with the E being 35.0. Therefore, B cereus WZZ006 performed high-level ability to produce (S)-5-chloro-1-oxo-2,3-dihydro-2-hydroxy-1H-indene-2-carboxylic acid methyl ester. This study demonstrates a new biocatalytic process route for preparing the indoxacarb chiral intermediates and provides a theoretical basis for the application of new insecticides in agricultural production.

Proteins Encoded by the gerP Operon Are Localized to the Inner Coat in Bacillus cereus Spores and Are Dependent on GerPA and SafA for Assembly.

The germination of Bacillus spores is triggered by certain amino acids and sugar molecules which permeate the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here, we use the ellipsoid localization microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex-lytic enzymes within the inner coat. We also reveal that the GerPA protein alone can localize in the absence of all other GerP proteins and that it has an essential role for the localization of all other GerP proteins within the spore. Its essential role is also demonstrated to be dependent on SafA, but not CotE, for localization, which is consistent with an inner coat location. GerP-null spores are shown also to have reduced permeability to fluorescently labeled dextran molecules compared to wild-type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability.IMPORTANCE The bacterial spore coat comprises a multilayered proteinaceous structure that influences the distribution, survival, and germination properties of spores in the environment. The results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ellipsoid localization microscopy (ELM) image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, health care, and environmental sectors.

Bacillus cereus bacteremia with central nervous system involvement: A neuropathological study.

Bacillus cereus is a widely-distributed, gram-positive or variable, rod-shaped bacterium frequently considered a contaminant in clinical specimens. It is recognized as a potential pathogen inducing self-limiting emetic or diarrheal food poisoning or localized infection in immunocompetent patients. True B. cereus bacteremia is uncommon and mainly observed in fragile patients, notably in immunocompromised individuals. We report clinical, radiological, and pathological findings of a 64-year-old patient with a history of acute myeloid leukemia who initially presented a fever while neutropenic after the induction of a second cycle of chemotherapy. He developed B. cereus bacteremia with invasive infection and a fatal outcome. The clinical and radiological data of this case are compared to a previously-published series of 21 patients from our institution with B. cereus bacteremia. This study highlights the clinical challenge to diagnose B. cereus and the importance of the delay between the detection of B. cereus and the initiation of an effective targeted antibiotic therapy. This case presented an aggressive evolution with multiple necrotic and hemorrhagic foci in the brain. Upon histological examination, B. cereus virulence was notably reflected by the dissection of blood vessel walls by the bacilli and luminal occlusion, a pattern that has not been yet reported.
.

Bacterial Whole Cell Typing by Mass Spectra Pattern Matching with Bootstrapping Assessment.

Bacterial typing is of great importance in clinical diagnosis, environmental monitoring, food safety analysis, and biological research. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is now widely used to analyze bacterial samples. Identification of bacteria at the species level can be realized by matching the mass spectra of samples against a library of mass spectra of known bacteria. Nevertheless, in order to reasonably type bacteria, identification accuracy should be further improved. Herein, we propose a new framework to the identification and assessment for MALDI-MS based bacterial analysis. Our approach combines new measures for spectra similarity and a novel bootstrapping assessment. We tested our approach on a general data set containing the mass spectra of 1741 strains of bacteria and another challenging data set containing 250 strains, including 40 strains in the Bacillus cereus group that were previously claimed to be impossible to resolve by MALDI-MS. With the bootstrapping assessment, we achieved much more reliable predictions at both the genus and species level, and enabled to resolve the Bacillus cereus group. To the best of the authors' knowledge, our method is the first to provide a statistical assessment to MALDI-MS based bacterial typing that could lead to more reliable bacterial typing.

Contrasting evolutionary patterns of spore coat proteins in two Bacillus species groups are linked to a difference in cellular structure.

BACKGROUND: The Bacillus subtilis-group and the Bacillus cereus-group are two well-studied groups of species in the genus Bacillus. Bacteria in this genus can produce a highly resistant cell type, the spore, which is encased in a complex protective protein shell called the coat. Spores in the B. cereus-group contain an additional outer layer, the exosporium, which encircles the coat. The coat in B. subtilis spores possesses inner and outer layers. The aim of this study is to investigate whether differences in the spore structures influenced the divergence of the coat protein genes during the evolution of these two Bacillus species groups. RESULTS: We designed and implemented a computational framework to compare the evolutionary histories of coat proteins. We curated a list of B. subtilis coat proteins and identified their orthologs in 11 Bacillus species based on phylogenetic congruence. Phylogenetic profiles of these coat proteins show that they can be divided into conserved and labile ones. Coat proteins comprising the B. subtilis inner coat are significantly more conserved than those comprising the outer coat. We then performed genome-wide comparisons of the nonsynonymous/synonymous substitution rate ratio, dN/dS, and found contrasting patterns: Coat proteins have significantly higher dN/dS in the B. subtilis-group genomes, but not in the B. cereus-group genomes. We further corroborated this contrast by examining changes of dN/dS within gene trees, and found that some coat protein gene trees have significantly different dN/dS between the B subtilis-clade and the B. cereus-clade. CONCLUSIONS: Coat proteins in the B. subtilis- and B. cereus-group species are under contrasting selective pressures. We speculate that the absence of the exosporium in the B. subtilis spore coat effectively lifted a structural constraint that has led to relaxed negative selection pressure on the outer coat.

Cinnamon oil nanoemulsion formulation by ultrasonic emulsification: investigation of its bactericidal activity.

Cinnamon oil (extracted from Cinnamomum zeylanicum) nanoemulsion was formulated using Tween 80 and water by ultrasonic emulsification. Process of nanoemulsion formulation was optimized for parameters such as surfactant concentration, oil-surfactant mixing ratio and emulsification time. Surfactant concentration was found to be inversely related to droplet size and directly related to stability. Increase in emulsification time resulted in decrease in droplet diameter. Stable cinnamon oil formulation (CF3) having droplet diameter of 65 nm was formulated after sonication for 30 min. Formulated nanoemulsion was evaluated for bactericidal efficacy against Bacillus cereus. Time and concentration dependent killing of B. cereus cells was observed upon treatment with nanoemulsion. Even at a higher dilution of CF3, significant reduction in bacterial population was observed. Alteration in membrane permeability of interacted samples was suggested by quantifying the release of UV absorbing materials. Bacterial staining with acridine orange/ethidium bromide supported kinetics of killing data and also substantiated the above findings of alteration in membrane permeability. FTIR illustrated disappearance of peak corresponding phosphate vibration at 1078 cm(-1) and 536 cm(-1), and peak associated with vibration of acyl chains of lipid at 2852 cm(-1) was shifted to 2854 cm(-1) which suggested deformation of membrane phospholipids in nanoemulsion treated cells. SEM observations demonstrated membrane distortion leading to cell lysis. These results propose the potential use of cinnamon oil nanoemulsion for preservation of minimally processed food.

Low temperatures and fermentative metabolism limit peptidoglycan digestion of Bacillus cereus. Impact on colony forming unit counts.

The impact of fermentative metabolism at low temperature on cell division of Bacillus cereus was studied. Fermentation at 37 °C had no influence on the division of bacteria. Aerobic cultures at 15 °C produced larger cells than at 37 °C, but cell division was normal. In fermentative cultures at 15 °C, no increase in CFU ml(-1) was observed. However, A(600) increased, due to formation of long filaments. Transmission electronic microscopy and light microscopy with fluorescent staining showed several nucleic acid entities separated by a hydrophobic membrane, indicating that each filament contained several individual cells attached by peptidoglycan. When left in air at room temperature, one filament gave several daughter cells, this means that one CFU formed by one filament may represent a greater contamination potential than one CFU formed by a single cell. Division was observed in cultures at 15 °C with anaerobic respiration in the presence of nitrates. Possible filamentous growth must thus be taken into account to avoid underestimating B. cereus growth in vacuum or modified atmosphere packaged foods stored at low temperature.

A novel structured bioreactor for solid-state fermentation.

A novel patented solid-state bioreactor (251 L) with honeycomb loading device was designed and its performance was tested. First, this apparatus gave a 66.87 % of calculated loading coefficient (volume ratio), which was almost twofold compared with conventional fermenters. Next, considering the crucial effect of heat transfer on bed loading and microbial growth, the performance was validated by temperature variance during fermentation and spore viability of Bacillus cereus DM423. Air pressure pulsation or external water jacket was used to control temperature; the maximal temperature variation was 7.7 versus 19.8 °C, respectively during fermentation. The difference was mainly due to the continuous gas phase characterized by solid-state fermentation (SSF). The average living spores of (1.50 ± 0.07) × 10(11) cfu/g at 40 h obtained from the device was higher than (0.70 ± 0.03) × 10(11) cfu/g from flask at 48 h. The results indicated that this new loading bioreactor with air pressure pulsation could be a good prospect for industrialization of SSF employing bacterial cultures.

A multivalent probe for AI-2 quorum-sensing receptors.

Multivalency is a common principle in the recognition of cellular receptors, and multivalent agonists and antagonists have played a major role in understanding mammalian cell receptor biology. The study of bacterial cell receptors using similar approaches, however, has lagged behind. Herein we describe our efforts toward the development of a dendrimer-based multivalent probe for studying AI-2 quorum-sensing receptors. From these studies, we have discovered a chemical probe specific for Lsr-type AI-2 quorum-sensing receptors with the potential for enabling the identification of new bacterial species that utilize AI-2 as a quorum-sensing signaling molecule.

Monitoring the wet-heat inactivation dynamics of single spores of Bacillus species by using Raman tweezers, differential interference contrast microscopy, and nucleic acid dye fluorescence microscopy.

Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca(2+) with dipicolinic acid (CaDPA) was released rapidly at a highly variable time T(lag), the levels of spore nucleic acids remained nearly unchanged, and the T(lag) times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ~50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before T(lag) and reached maximum at a time slightly later than T(release). However, the fluorescence intensities of wet-heat-inactivated spores were ~15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment.

Global gene expression profile for swarming Bacillus cereus bacteria.

Bacillus cereus can use swarming to move over and colonize solid surfaces in different environments. This kind of motility is a collective behavior accompanied by the production of long and hyperflagellate swarm cells. In this study, the genome-wide transcriptional response of B. cereus ATCC 14579 during swarming was analyzed. Swarming was shown to trigger the differential expression (>2-fold change) of 118 genes. Downregulated genes included those required for basic cellular metabolism. In accordance with the hyperflagellate phenotype of the swarm cell, genes encoding flagellin were overexpressed. Some genes associated with K(+) transport, phBC6A51 phage genes, and the binding component of the enterotoxin hemolysin BL (HBL) were also induced. Quantitative reverse transcription-PCR (qRT-PCR) experiments indicated an almost 2-fold upregulation of the entire hbl operon during swarming. Finally, BC1435 and BC1436, orthologs of liaI-liaH that are known to be involved in the resistance of Bacillus subtilis to daptomycin, were upregulated under swarming conditions. Accordingly, phenotypic assays showed reduced susceptibility of swarming B. cereus cells to daptomycin, and P(spac)-induced hyper-expression of these genes in liquid medium highlighted the role of BC1435 and BC1436 in the response of B. cereus to daptomycin.

Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel.

Bacillus cereus spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of B. cereus ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of B. cereus ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in B. anthracis.

The potential of flow cytometry in the study of Bacillus cereus.

Flow cytometry (FCM) is a rapid method allowing the acquisition of multiparametric data from thousands of individual cells within a sample. As well as measuring the intrinsic light scattering properties of cells, a plethora of fluorescent dyes may be employed to yield information on macromolecule content, surface antigens present or physiological status. Despite FCM's indispensability within other fields e.g. immunology, it is underutilized within microbiological research. In this review, a strong case is presented for the potential of FCM in the study of Gram-positive spore-former, Bacillus cereus. Previous reports where FCM was successfully used in the study of B. cereus are reviewed along with relevant studies involving other members of the genus. Under headings reflecting common research themes associated with B. cereus, specific instances where FCM has generated novel data, providing a unique insight into the organism, are discussed. Further applications are posited, based on the authors' own research with FCM and B. cereus and work extant in the broader field of microbial cytometry. The authors conclude that, while the expense of equipment and reagents is an undeniable disadvantage, FCM is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not possible with any other technique.

Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells.

Bacteria are able to cope with the challenges of a sudden increase in salinity by activating adaptation mechanisms. In this study, exponentially growing cells of the pathogen Bacillus cereus ATCC 14579 were exposed to both mild (2.5% [wt/vol] NaCl) and severe (5% [wt/vol] NaCl) salt stress conditions. B. cereus continued to grow at a slightly reduced growth rate when it was shifted to mild salt stress conditions. Exposure to severe salt stress resulted in a lag period, and after 60 min growth had resumed, with cells displaying a filamentous morphology. Whole-genome expression analyses of cells exposed to 2.5% salt stress revealed that the expression of these cells overlapped with the expression of cells exposed to 5% salt stress, suggesting that the corresponding genes were involved in a general salt stress response. Upregulation of osmoprotectant, Na(+)/H(+), and di- and tripeptide transporters and activation of an oxidative stress response were noticeable aspects of the general salt stress transcriptome response. Activation of this response may confer cross-protection against other stresses, and indeed, increased resistance to heat and hydrogen peroxide could be demonstrated after preexposure to salt. A temporal shift between the transcriptome response and several phenotypic responses of severely salt-stressed cells was observed. After resumption of growth, these cells showed cellular filamentation, reduced chemotaxis, increased catalase activity, and optimal oxidative stress resistance, which corresponded to the transcriptome response displayed in the initial lag period. The linkage of transcriptomes and phenotypic characteristics can contribute to a better understanding of cellular stress adaptation strategies and possible cross-protection mechanisms.

Ribosomes from spores of Bacillus cereus T.

Data from analytical ultracentrifuge studies suggest that polymers of ribosomes exist in dormant spores of Bacillus cereus T.

Phenotypic heterogeneity within microbial populations at the single-cell level investigated by confocal Raman microspectroscopy.

Single cells in genetically homogeneous microbial cultures exhibit marked phenotypic heterogeneity that is considered to bolster the fitness of the whole population. Heterogeneity on the single-cell level is typically masked in conventional studies of microbial populations, which rely on data averaged across thousands or millions of cells in a sample. Here we introduce confocal Raman microspectroscopy as a method for investigating and illustrating the spatial heterogeneity of microbial cell populations. By the use of three different test organisms as model systems, we show pronounced cellular heterogeneity even in colonies cultivated under laboratory conditions.

Effect of UVA irradiance on photocatalytic and UVA inactivation of Bacillus cereus spores.

The current study is the first to delineate the contribution of photocatalysis to inactivation of Bacillus cereus endospores on dry surfaces over a broad range (0-153W/m(2)) of UVA irradiance. Inactivation of spores at low UVA irradiance (30W/m(2)) was primarily due to photocatalysis, whereas at higher UVA irradiance inactivation was primarily due to UV alone. A linear relationship between UVA irradiance and the rate of spore inactivation was observed in the absence of photocatalyst. The rate of photocatalytic inactivation was non-linear with respect to UVA irradiance, exhibiting a maximum at 30W/m(2).

Species association increases biofilm resistance to chemical and mechanical treatments.

The study of biofilm ecology and interactions might help to improve our understanding of their resistance mechanisms to control strategies. Concerns that the diversity of the biofilm communities can affect disinfection efficacy have led us to examine the effect of two antimicrobial agents on two important spoilage bacteria. Studies were conducted on single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofilms were formed on a stainless steel rotating device, in a bioreactor, at a constant Reynolds number of agitation (Re(A)). Biofilm phenotypic characterization showed significant differences, mainly in the metabolic activity and both extracellular proteins and polysaccharides content. Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde (GLUT) solutions in conjunction with increasing Re(A) were used to treat biofilms in order to assess their ability to kill and remove biofilms. B. cereus and P. fluorescens biofilms were stratified in a layered structure with each layer having differential tolerance to chemical and mechanical stresses. Dual species biofilms and P. fluorescens single biofilms had both the highest resistance to removal when pre-treated with CTAB and GLUT, respectively. B. cereus biofilms were the most affected by hydrodynamic disturbance and the most susceptible to antimicrobials. Dual biofilms were more resistant to antimicrobials than each single species biofilm, with a significant proportion of the population remaining in a viable state after exposure to CTAB or GLUT. Moreover, the species association increased the proportion of viable cells of both bacteria, comparatively to the single species scenarios, enhancing each other's survival to antimicrobials and the biofilm shear stress stability.

Structural Basis for Cell-Wall Recognition by Bacteriophage PBC5 Endolysin.

Phage endolysins are hydrolytic enzymes that cleave the bacterial cell wall during the lytic cycle. We isolated the bacteriophage PBC5 against Bacillus cereus, a major foodborne pathogen, and describe the molecular interaction between endolysin LysPBC5 and the host peptidoglycan structure. LysPBC5 has an N-terminal glycoside hydrolase 25 domain, and a C-terminal cell-wall binding domain (CBD) that is critical for specific cell-wall recognition and lysis. The crystal and solution structures of CBDs reveal tandem SH3b domains that are tightly engaged with each other. The CBD binds to the peptidoglycan in a bidentate manner via distal ß sheet motifs with pseudo 2-fold symmetry, which can explain its high affinity and host specificity. The CBD primarily interacts with the glycan strand of the peptidoglycan layer instead of the peptide crosslink, implicating the tertiary structure of peptidoglycan as the recognition motif of endolysins.

Antagonism between Bacillus cereus and Pseudomonas fluorescens in planktonic systems and in biofilms.

In the environment, many microorganisms coexist in communities competing for resources, and they are often associated as biofilms. The investigation of bacterial ecology and interactions may help to improve understanding of the ability of biofilms to persist. In this study, the behaviour of Bacillus cereus and Pseudomonas fluorescens in the planktonic and sessile states was compared. Planktonic tests were performed with single and dual species cultures in growth medium with and without supplemental FeCl3. B. cereus and P. fluorescens single cultures had equivalent growth behaviours. Also, when in co-culture under Fe-supplemented conditions, the bacteria coexisted and showed similar growth profiles. Under Fe limitation, 8 h after co-culture and over time, the number of viable B. cereus cells decreased compared with P. fluorescens. Spores were detected during the course of the experiment, but were not correlated with the decrease in the number of viable cells. This growth inhibitory effect was correlated with the release of metabolite molecules by P. fluorescens through Fe-dependent mechanisms. Biofilm studies were carried out with single and dual species using a continuous flow bioreactor rotating system with stainless steel (SS) substrata. Steady-state biofilms were exposed to a series of increasing shear stress forces. Analysis of the removal of dual species biofilms revealed that the outer layer was colonised mainly by B. cereus. This bacterium was able to grow in the outermost layers of the biofilm due to the inhibitory effect of P. fluorescens being decreased by the exposure of the cells to fresh culture medium. B. cereus also constituted the surface primary coloniser due to its favourable adhesion to SS. P. fluorescens was the main coloniser of the middle layers of the biofilm. Single and dual species biofilm removal data also revealed that B. cereus biofilms had the highest physical stability, followed by P. fluorescens biofilms. This study highlights the inadequacy of planktonic systems to mimic the behaviour of bacteria in biofilms and shows how the culturing system affects the action of antagonist metabolite molecules because dilution and consequent loss of activity occurred in continuously operating systems. Furthermore, the data demonstrate the biocontrol potential of P. fluorescens on the planktonic growth of B. cereus and the ability of the two species to coexist in a stratified biofilm structure.