Emetic Strains Research Articles

Role of water activity on sporulation traits and resistance to 915 MHz microwave in the emetic type of Bacillus cereus on rice

The objective of this study is to investigate the influence of water activity on the sporulation of emetic strains of Bacillus cereus and the subsequent susceptibility of sporulated B. cereus to 915 MHz microwave treatment. Water activity levels were manipulated in the sporulation medium by adjusting glycerol concentrations to 0 %, 3 %, 7.5 %, and 10 %, resulting in corresponding water activities of 0.996, 0.981, 0.971, and 0.960, respectively and sporulated at 30℃. These changes in water activity were intended to simulate the variations in water activity that can occur during the cultivation and processing of rice, where B. cereus is commonly found. Sporulation rates increased with higher water activity, achieving over 90 % of total cells after 3, 5, 5, and 6 days of incubation at water activities of 0.996, 0.981, 0.971, and 0.960, respectively. Resistance to microwave treatment increased with higher water activity levels during sporulation. Microwave treatment for 5 min yielded a reduction of 3.03 log CFU/g at a water activity of 0.960 and 1.98 log CFU/g at 0.996 during sporulation. Lower water activity led to higher % dipicolinic acid (DPA) release during microwave treatment, as measured using a spectrometer, indicating greater membrane damage. To investigate the morphology of spores, transmission electron microscopy was used. Spores produced at lower water activity levels were observed to be enveloped by the mother cell, whereas those sporulated at higher water activity levels were distinct from the mother cell. Additionally, spores sporulated at higher water activity levels exhibited higher wet density in Percoll gradient. The levels of germination were also compared after sporulation under different water activity medium using L-alanine. The spores evolved under low water activity exhibited higher germination level compared to those from high water activity conditions. These findings suggested that water activity conditions during sporulation induce variations in morphology, density, germination level, and resistance to 915 MHz microwave. Our experimental results indicated that, even with the same sporulation percentage, there can be differences in the maturation stage, indicating that the level of 915 MHz microwave treatment should be adjusted according to the sporulation conditions of B. cereus.

The megaplasmid pCER270 of Bacillus cereus emetic strain affects the timing of the sporulation process, spore resistance properties, and germination.

The acquisition of new mobile genetic elements, such as plasmids, is essential for the pathogenesis and adaptation of bacteria belonging to the Bacillus cereus group. This can confer new phenotypic traits and beneficial functions that enable bacteria to adapt to changing environments and colonize new ecological niches. Emetic B. cereus strains cause food poisoning linked to the production of cereulide, the emetic toxin whose synthesis is due to the presence of plasmid pCER270. In the environment, cereulide provides a competitive advantage in producing bacteria against various competitors or predators. This study demonstrates that pCER270 also regulates the sporulation process, resulting in spores with improved heat resistance and germination capacity. The transfer of plasmid pCER270 among different strains of the B. cereus group may enhance their adaptation to new environments. This raises the question of the emergence of new pathogenic strains, which could pose a serious threat to human health.

A poisonous cocktail: interplay of cereulide toxin and its structural isomers in emetic Bacillus cereus.

Food intoxications evoked by emetic Bacillus cereus strains constitute a serious threat to public health, leading to emesis and severe organ failure. The emetic peptide toxin cereulide, assembled by the non-ribosomal peptide synthetase CesNRPS, cannot be eradicated from contaminated food by usual hygienic measures due to its molecular size and structural stability. Next to cereulide, diverse chemical variants have been described recently that are produced concurrently with cereulide by CesNRPS. However, the contribution of these isocereulides to the actual toxicity of emetic B. cereus, which produces a cocktail of these toxins in a certain ratio, is still elusive. Since cereulide isoforms have already been detected in food remnants from foodborne outbreaks, we aimed to gain insights into the composition of isocereulides and their impact on the overall toxicity of emetic B. cereus. The amounts and ratios of cereulide and isocereulides were determined in B. cereus grown under standard laboratory conditions and in a contaminated sample of fried rice balls responsible for one of the most severe food outbreaks caused by emetic B. cereus in recent years. The ratios of variants were determined as robust, produced either under laboratory or natural, food-poisoning conditions. Examination of their actual toxicity in human epithelial HEp2-cells revealed that isocereulides A-N, although accounting for only 10% of the total cereulide toxins, were responsible for about 40% of the total cytotoxicity. An this despite the fact that some of the isocereulides were less cytotoxic than cereulide when tested individually for cytotoxicity. To estimate the additive, synergistic or antagonistic effects of the single variants, each cereulide variant was mixed with cereulide in a 1:9 and 1:1 binary blend, respectively, and tested on human cells. The results showed additive and synergistic impacts of single variants, highlighting the importance of including not only cereulide but also the isocereulides in routine food and clinical diagnostics to achieve a realistic toxicity evaluation of emetic B. cereus in contaminated food as well as in patient samples linked to foodborne outbreaks. Since the individual isoforms confer different cell toxicity both alone and in association with cereulide, further investigations are needed to fully understand their cocktail effect.

Effect of Temperature, pH, and aw on Cereulide Synthesis and Regulator Genes Transcription with Respect to Bacillus cereus Growth and Cereulide Production.

Bacillus cereus is a food-borne pathogen that can produce cereulide in the growth period, which causes food poisoning symptoms. Due to its resistance to heat, extreme pH, and proteolytic enzymes, cereulide poses a serious threat to food safety. Temperature, pH, and aw can influence cereulide production, but there is still a lack of research with multi-environmental impacts. In this study, the effects of temperature (15~45 °C), pH (5~8), and aw (0.945~0.996) on the emetic reference strain B. cereus F4810/72 growth, cereulide production, relevant ces genes (cesA, cesB, cesP), and transcription regulators genes (codY and abrB) expression at transcription level were studied. B. cereus survived for 4~53 h or grew to 6.85~8.15 log10 CFU/mL in environmental combinations. Cereulide accumulation was higher in mid-temperature, acidic, or high aw environments. Increased temperature resulted in a lower cereulide concentration at pH 8 or aw of 0.970. The lowest cereulide concentration was found at pH 6.5 with an increased aw from 0.970 to 0.996. Water activity had a strong effect on transcriptional regulator genes as well as the cesB gene, and temperature was the main effect factor of cesP gene expression. Moreover, environmental factors also impact cereulide synthesis at transcriptional levels thereby altering the cereulide concentrations. The interaction of environmental factors may result in the survival of B. cereus without growth for a period. Gene expression is affected by environmental factors, and temperature and pH may be the main factors influencing the correlation between B. cereus growth and cereulide formation. This study contributed to an initial understanding of the intrinsic link between the impact of environmental factors and cereulide formation and provided valuable information for clarifying the mechanism of cereulide synthesis in combined environmental conditions.

The potential of lactose to inhibit cereulide biosynthesis of emetic Bacillus cereus in milk

This study aims to investigate the potential role of lactose on cereulide biosynthesis by emetic Bacillus cereus in dairy matrices. The cereulide yields in whole milk and lactose-free milk were investigated using the emetic reference strain F4810/72. To eliminate the influence of complex food substrates, the LB medium model was further used to characterize the effect of lactose on cereulide produced by F4810/72 and five other emetic B. cereus strains. Results showed that the lactose-free milk displayed a 13-fold higher amount of cereulide than whole milk, but the cereulide level could be reduced by 91 % when the lactose content was restored. The significant inhibition of lactose on cereulide yields of all tested B. cereus strains was observed in LB medium, showing a dose-dependent manner with inhibition rates ranging of 89–98 %. The growth curves and lactose utilization patterns of all strains demonstrated that B. cereus cannot utilize lactose as a carbon source and lactose might act as a signal molecule to regulate cereulide production. Moreover, lactose strongly repressed the expression of cereulide synthetase genes (ces), possibly by inhibiting the key regulator Spo0A at the transcriptional level. Our findings highlight the potential of lactose as an effective strategy to control cereulide production in food.

S-layer is a key element in metabolic response and entry into the stationary phase in Bacillus cereus AH187

Bacillus cereus is a food-borne Gram-positive pathogen. The emetic reference strain B. cereus AH187 is surrounded by a proteinaceous surface layer (S-layer) that contributes to its physico-chemical surface properties, and promotes its adhesion in response to starvation conditions. The S-layer produced by B. cereus AH187 is composed of two proteins, SL2 and EA1, which are incorporated at different growth stages. Here, we showed that deletion of the genes encoding SL2 and EA1 produced viable cells, but decreased the glucose uptake rate at the start of growth, and induced extensive reorganization of the cellular and exoproteomes upon entry into the stationary phase. As a consequence, stationary cells were less resistant to abiotic stress. Taken together, our data indicate that the S-layer is crucial but comes at a metabolic cost that modulates the stationary phase response. SignificanceThe emetic strains of Bacillus cereus are known to cause severe food poisoning, making it crucial to understand the factors contributing to their selective enrichment in foods. Most emetic strains are surrounded by a crystalline S-layer, which is a costly protein structure to produce. In this study, we used high-throughput proteomics to investigate how S-layer synthesis affects the allocation of cellular resources in the emetic B. cereus strain AH187. Our results demonstrate that the synthesis of the S-layer plays a crucial role in the pathogen's ability to thrive under stationary growth phase conditions by modulating the stress response, thereby promoting its lifestyle as an emetic pathogen. We conclude that the synthesis of the S-layer is a critical adaptation for emetic B. cereus to successfully colonize specific niches.

Two novel antimicrobial peptides against vegetative cells, spores and biofilm of Bacillus cereus

Bacillus cereus is a ubiquitous bacterium that causes food-associated gastrointestinal diseases. The ability to form spores and biofilm makes this bacterium highly resistant to antimicrobial agents and food preservatives, posing persistent risks to consumers and the food industry. The excessive use of antibiotics and chemical preservatives has led to the emergence of resistant B. cereus strains, impacting the safety of food. Therefore, there is demand for new antimicrobial agents to combat B. cereus. The use of antimicrobial peptides (AMPs) for food preservation has attracted attention because they can preserve food quality with low possibility of bacteria acquiring resistance. In this research, a series of peptide derivatives based on the previously reported peptide D51 (sequence FLFRVASKVFPALIGKFKKK) was designed and synthesized. Two of the peptides, D51-P11G and D51-P11K, exhibited satisfactory antimicrobial (MICs: 8 μg/mL) activities toward B. cereus, including diarrheal and emetic reference strains. Peptides D51-P11G and D51-P11K showed good physiological stability, high safety and a low tendency to induce bacterial resistance. Meanwhile, at 16 μg/mL, D51-P11G and D51-P11K could inhibit spore germination and biofilm formation. Studies of the antibacterial mechanism suggested that D51-P11G and D51-P11K exert their antibacterial activities mainly via a membrane-lytic mechanism, but they also bound to genome DNA of the bacteria. Notably, D51-P11G and D51-P11K (32 μg/mL) could control the growth of B. cereus in boiled rice and minced meat, the antimicrobial activities were almost the same as or even better than nisin A (300 μg/mL). These findings suggest that D51-P11G and D51-P11K are ideal candidates for application against vegetative cells, spores, and biofilm of B. cereus.

Susceptibility of emetic and enterotoxigenic Bacillus cereus grown at high temperature to disinfectants

The prevalence of emetic strains in food products is rare; however, infection with these may be fatal to the vulnerable population. Bacterial control of the emetic Bacillus cereus strains is still unclear. This study aimed to assess the influence of high temperature on the disinfection of emetic and enterotoxigenic B. cereus. Emetic B. cereus strains survived up to 50 °C; the lag time and maximum growth rate were higher at 42 °C than those at 30 °C. Compared to enterotoxigenic B. cereus, all emetic food strains showed higher minimum inhibitory concentrations and minimum bactericidal concentrations for sodium hypochlorite and citric acid. The disinfectant susceptibility of the emetic B. cereus OS-05 strain incubated at a higher temperature did not increase and was maintained at the highest MBC value. In all emetic B. cereus strains, enterotoxin gene expression was upregulated at 42 °C and 45 °C. Increased ces gene expression was also found in emetic B. cereus strains GP-15 and OS-05, with upregulation of 128- and 820-fold at 42 °C. Thus, emetic B. cereus grown at high temperatures may resist common disinfectants of the food industry. The findings may help control B. cereus in food or the food processing industry.

Dynamic Profile of S-Layer Proteins Controls Surface Properties of Emetic Bacillus cereus AH187 Strain

Many prokaryotes are covered by a two-dimensional array of proteinaceous subunits. This surface layers (S-layer) is incompletely characterized for many microorganisms. Here, we studied Bacillus cereus AH187. A genome analysis identified two genes encoding the S-layer proteins SL2 and EA1, which we experimentally confirmed to encode the two protein components of the S-layer covering the surface of B. cereus. Shotgun proteomics analysis indicated that SL2 is the major component of the B. cereus S-layer at the beginning of exponential growth, whereas EA1 becomes more abundant than SL2 during later stages of stationary growth. Microscopy analysis revealed the spatial organization of SL2 and EA1 at the surface of B. cereus to depend on their temporal-dynamics during growth. Our results also show that a mutant strain lacking functional SL2 and EA1 proteins has distinct surface properties compared to its parental strain, in terms of stiffness and hydrophilicity during the stationary growth phase. Surface properties, self-aggregation capacity, and bacterial adhesion were observed to correlate. We conclude that the dynamics of SL2 and EA1 expression is a key determinant of the surface properties of B. cereus AH187, and that the S-layer could contribute to B. cereus survival in starvation conditions.

A Flagella Hook Coding Gene flgE Positively Affects Biofilm Formation and Cereulide Production in Emetic Bacillus cereus.

Bacillus cereus, an important foodborne pathogen, poses a risk to food safety and quality. Robust biofilm formation ability is one of the key properties that is responsible for the food contamination and food poisoning caused by B. cereus, especially the emetic strains. To investigate the mechanism of biofilm formation in emetic B. cereus strains, we screened for the mutants that fail to form biofilms by using random mutagenesis toward B. cereus 892-1, an emetic strain with strong biofilm formation ability. When knocking out flgE, a flagellar hook encoding gene, the mutant showed disappearance of flagellar structure and swimming ability. Further analysis revealed that both pellicle and ring presented defects in the null mutant compared with the wild-type and complementary strains. Compared with the flagellar paralytic strains Δ motA and Δ motB, the inhibition of biofilm formation by Δ flgE is not only caused by the inhibition of motility. Interestingly, Δ flgE also decreased the synthesis of cereulide. To our knowledge, this is the first report showing that a flagellar component can both affect the biofilm formation and cereulide production in emetic B. cereus, which can be used as the target to control the biohazard of emetic B. cereus.

Predicting B. cereus growth and cereulide production in dairy mix

This study aims to quantify growth and cereulide production by Bacillus cereus and their potential correlation in an intermediate dairy wet-mix. Systematic experiments were carried out using the emetic reference strain F4810/72 in the suboptimal range of temperature of 12 °C to 20 °C. Growth and cereulide kinetic parameters were estimated and the three parameters (i) time to first cereulide quantification (tcer), (ii) maximum specific growth rates (μmax) and (iii) cereulide production rates (k) were modelled as a function of temperature. As temperature increased, growth lag time and tcer were shorter while microbial increase and cereulide production happened earlier, and at higher rates. Maximum concentration of cells and maximum cereulide concentration proved to be temperature-independent, reaching the average values of 7.9 ± 0.3 log10(CFU/mL) and 2.6 ± 0.2 log10(ng.g−1) respectively. Moreover, the time to reach the widely used threshold of 5 log10CFU/mL (t5log) was tested against tcer, and this suggested that this threshold can be used with increased confidence at lower temperatures to assure toxin is not quantified in this matrix. The average tcer were equal to 314 h, 118 h, 73 h and 45 h for 12 °C, 15 °C, 18 °C and 20 °C respectively. A validation study was performed using independent data sets obtained with the same strain in other dairy matrices. The microbial growth models presented good predictive power even when extrapolated beyond the temperature range of construction. Nevertheless, the models proposed for prediction of toxin production over time presented limitations, especially for food matrices that deviate significantly from the original matrix for which the model was developed, making cereulide predictions less accurate. Our findings suggest that similar modelling approaches can be used to predict growth, time to first cereulide quantification as well as cereulide formation over time for a specific matrix, but that matrix-extrapolations are more suitable for growth than for cereulide.

Phenotypic homogeneity of emetic Bacillus cereus isolates in China.

Emetic Bacillus cereus strains produce a potent cereulide cytotoxin, which can cause acute and fatal cases of food poisoning. We isolated 18 emetic B. cereus strains from a food poisoning event, and from clinical and non-random food surveillance in China and phenotypic characteristics of haemolysis, starch hydrolysis, salicin fermentation, gelatin liquefaction, cytotoxicity, and susceptibility to antibiotics were assessed. All isolates were positive for haemolysis and gelatin liquefaction, and negative for starch hydrolysis and salicin fermentation. Their haemolytic potentials were intermediate to Bacillus anthracis and B. cereus ATCC 14579 (a non-emetic strain). All isolates were cytotoxic to CHO, Hep-2, and Vero cells, and were sensitive to ampicillin. The homogeneous phenotypes of emetic isolates from China are similar to the corresponding traits of European and Japanese isolates that have been characterized, suggesting highly similar phenotypes of emetic B. cereus worldwide.

Prevalence, virulence genes, and genetic diversity of Bacillus cereus isolated from convenience food.

In times when there is a growing interest in ready-to-eat food (RTEF), the presence of potentially pathogenic bacteria, including the toxigenic psychrotolerant bacilli from the B. cereus group, on this type of carrier may pose a real threat to the health of consumers. A significant part of RTEF is represented by vegetable products and food products made with them. The increased production of convenience foods has resulted in their international turnover growing. When coupled with a rising percentage of persons from risk groups (YOPI), including the elderly or immunocompromised, this may mean increased health risks posed by the so-called "novel pathogens", like the toxigenic psychrotolerant B. cereus sensu lato. Food samples were analyzed for the presence and count of putative B. cereus according to the Polish Standard PN-EN ISO 7932:2005. All genetic analyses were conducted using a qualitative real-time PCR. The presence of B. cereus sensu lato was confirmed in 130 out of the 192 samples of convenience foods, at contamination levels ranging from 1.65 to 3.32 log CFU/g. Among the strains confirmed to belong to the B. cereus group, 23 were identified as emetic B. cereus. The analysis of each strain's ability to grow at temp. 4-10°C demonstrated that 4.9% and 12.7% of the isolates were able to grow at 4°C and 6°C, respectively. In turn, 15.2% were able to grow at 8°C, and 36.3% at 10°C. None of the psychrotrophic strains possessed genes typical of B. weihenstephanensis. The group of psychrotrophic B. cereus included potentially toxigenic strains being carriers of genes that determine the synthesis of the following toxins: NHE, HBL, CytK, and cereulide. Some of them were potent enough to produce more than one toxin. The analyses conducted in this study demonstrate that the psychrotolerant strains of B. cereus (including the toxigenic ones) are frequent microbiological contaminants of RTEF products offered in retail. The presence of emetic strains from the B. cereus group, which are able to grow in a wide range of temperatures and produce enterotoxins and enzymes with the characteristics of toxins, in ready-to-eat foods may pose a real threat to consumer health.

Modeling Bacillus cereus Growth and Cereulide Formation in Cereal-, Dairy-, Meat-, Vegetable-Based Food and Culture Medium.

This study describes the simultaneous Bacillus cereus growth and cereulide formation, in culture medium and cereal-, dairy-, meat-, and vegetable-based food matrices. First, bacterial growth experiments were carried out under a wide range of temperatures (from 9 to 45°C), using the emetic reference strain F4810/72, in the above-mentioned matrices. Then, the generated data were put in a modeling framework where the response variable was a vector of two components: the concentration of B. cereus and that of its toxin, cereulide. Both were considered time-, temperature- and matrix-dependent. The modeling was carried out in a series of steps: the parameters fitted in one step became the response variable of the following step. Using the square root link function, the maximum specific growth rate of the organism and the time to the appearance of quantifiable cereulide were modeled against temperature by cardinal parameters models (CPM), for each matrix. Finally, a validation study was carried out on an independent data set obtained in the same matrices and using various Bacillus cereus strains. Results showed that both growth and toxin-formation depended on the food matrix and on the environment but not in the same way. Thus, the matrix (culture medium), where the highest growth rate of B. cereus was observed, was not the medium where the shortest time to quantifiable cereulide occurred. While the cereal-based matrix generated the smallest growth rates (0.41-times smaller than culture medium did), quantifiable cereulide appeared in it at earlier times compared to the other tested matrices. In fact, three groups of matrices could be distinguished based on their ability to support cereulide formation (1) the cereal-based matrix (highest), (2) the culture medium and the dairy-based matrix (intermediate), and (3) the meat- and vegetable-based matrices (lowest). This ranking between the matrices is quite different from that based on their suitability to the growth of the organism. Our models can be used in HACCP studies, to improve shelf-life predictions and, generally, microbiological food safety assessments of products for which B. cereus is the main concern.

Evaluation of MALDI-ToF Mass Spectrometry for Rapid Detection of Cereulide From Bacillus cereus Cultures.

Bacillus cereus plays an often unrecognized role in food borne diseases. Food poisoning caused by this pathogen is manifested by either diarrhea or emesis. Due to the relatively high prevalence of emetic toxin cereulide associated food poisoning, methods for simple and reliable detection of cereulide producing strains are of utmost importance. Recently, two different studies reported on the application of MALDI-ToF MS for either the differentiation of emetic and non-emetic strains of B. cereus or for direct detection of cereulide from bacterial colony smears. However, for implementation of cereulide detection using MALDI-ToF MS in routine microbiological diagnostics additional investigations on the sensitivity and specificity as well as on the fitting into common workflows for bacterial identification are needed. These aspects prompted us to investigate open issues and to test sample preparation methods, commonly used for microbial identification for their suitability to detect the emetic toxin from bacteria. Based on our experimental findings we propose a workflow that allows identification of B. cereus and sensitive detection of cereulide in parallel, using linear-mode MALDI-ToF MS equipment. The protocol was validated in a blinded study and is based on the well-established ethanol/formic acid extraction method. Cereulide is detected in the ethanol wash solution of samples identified as B. cereus as peaks at m/z 1175 and 1191. Peak position difference of 16 Th (Thomson) indicates detection of the sodium and potassium adducts of cereulide. This sample treatment offers possibilities for further characterization by more sophisticated LC-MS-based methods. In summary, the ease of use and the achieved level of analytical sensitivity as well as the wide-spread availability of MALDI-ToF MS equipment in clinical microbiological laboratories provides a promising tool to improve and to facilitate routine diagnostics of B. cereus associated food intoxications.

Groundwater promotes emergence of asporogenic mutants of emetic Bacillus cereus.

Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments.

Goat Colostrum—Source of Toxigenic Bacillus Cereus

Abstract The aim of this study was to evaluate the toxigenic potential of Bacillus cereus strains isolated from frozen goat colostrum. Of the 50 phenotypically suspected B. cereus isolates, 39 (78.0 %) were confirmed as B. cereus by the polymerase chain reaction (PCR) method based on the gyrB gene detection. In these isolates, genes encoding the production of haemolysin BL (Hbl), a complex of non-haemolytic enterotoxins (Nhe) and emetic toxin were detected by the PCR method. In 36 (92.3 %) confirmed B. cereus isolates, genes encoding at least one type of toxins of interest were detected. In all toxigenic isolates, we found the presence of genes for Nhe production, and in 16 (41.0 %) of the isolates, genes encoding both Nhe and haemolysin BL were shown. Eight (20.5 %) of the emetic strains of B. cereus were identified. The emetic toxin production gene was always detected simultaneously with genes encoding non-haemolytic enterotoxin production. The ability to produce BL haemolysin and non-haemolytic enterotoxins were confirmed by the immunochromatographic method. In summary, goat colostrum can be a significant source of toxigenic strains of B. cereus.

Complete genome sequence of bacteriophage Deep-Purple, a novel member of the family Siphoviridae infecting Bacillus cereus.

Bacteriophage Deep-Purple, isolated from an agricultural soil in Belgium, lyses the emetic Bacillus weihenstephanensis strain LH002 and exhibits a lytic activity against 55% of emetic Bacillus cereus and B. weihenstephanensis strains. Deep-Purple is able to complete its lytic cycle within 45 min and is stable to a large range of pHs and temperatures below 60°C. It possesses an icosahedral head of about 63 nm in diameter and a non-contractile tail of approximately 165 nm in length. The genome of this newly classifiable Siphoviridae family member is 36,278 bp long, with a G+C content of 38.36% and 40 putative CDSs. Most CDSs do not display similarity with other B. cereus group phages supporting the idea that Deep-Purple belongs to a new and currently uncharacterised Siphoviridae subfamily.

Detection of toxin genes and randomly amplified polymorphic DNA typing of Bacillus cereus isolates from infant milk formulas

AbstractBacillus cereus, a ubiquitous human foodborne pathogen, can persist in different environment, including dry conditions. In this study, we explored the genetic diversity of B. cereus isolates from infant milk formulas. Out of 83 samples, B. cereus was confirmed in 26 (31.3%). Isolates were resistant to penicillin G and ampicillin (100%), trimethoprim‐sulfamethoxazole (84.6%), and erythromycin (3.8%). Ent, hbl, and cer genes were detected in 21 (80.8%), 2 (7.7%), and 11 (42.3%) isolates, respectively. Randomly amplified polymorphic DNA (RAPD) analysis, using set of three primers, provided an overall genetic distance of 95% between B. cereus isolates. A group of seven emetic strains and another two enterotoxic strains were indistinguishable by RAPD, revealing their high clonality. In conclusion, we detected a high proportion of pathogenic B. cereus isolates in infant milk formulas. The majority of isolates harboring both cer and ent genes showed high clonality, pointing food processing environment as probable contamination source.Practical applicationsDetection of Bacillus cereus in powdered infant milk formulas, the prevalence of emetic and diarrhegenic genes, and especially the simultaneous presence of both types of genes in the same isolate, confirm the importance of B. cereus as foodborne pathogen. The consumption of contaminated formula may cause serious health problems in babies, which emphasizes the need to improve B. cereus monitoring and characterization. The clonal relationship between strains, revealed by RAPD analysis, may point to the source of infection and the corresponding prevention measures to improve food safety. Moreover, the identical RAPD profile of cer positive strains obtained with AG15 primer enables the application of this analysis in screening for emetic B. cereus isolates.

Surveillance of Bacillus cereus Isolates in Korea from 2012 to 2014.

ObjectivesTo investigate the prevalence and toxin production characteristics of non-emetic and emetic Bacillus cereus strains isolated via the laboratory surveillance system in Korea.MethodsA total of 667 B. cereus strains were collected by the Korea National Research Institute of Health laboratory surveillance system from 2012 to 2014. The collected strains were analyzed by geographical region, season, patient age, and patient sex. Additionally, the prevalence rates of enterotoxin and emetic toxin genes were evaluated.ResultsThe isolation rate of B. cereus strains increased during the summer, but the isolation rate was evenly distributed among patient age groups. Emetic toxin was produced by 20.2% of the isolated strains. The prevalence rates of five enterotoxin genes (entFM, nheA, cytK2, hblC, and bceT) were 85.0, 78.6, 44.5, 36.6, and 29.7%, respectively, among non-emetic strains and 77.8, 59.3, 17.8, 11.9 and 12.6%, respectively, among emetic strains. Thus, the prevalence rates of all five enterotoxin genes were lower in emetic B. cereus.ConclusionThe prevalence of enterotoxin genes differed between non-emetic and emetic B. cereus strains. Among emetic B. cereus strains, the prevalence rates of two enterotoxin genes (cytK2 and hblC) were lower than those among the non-emetic strains. In both the emetic and non-emetic strains isolated in Korea, nheA and entFM were the most prevalent enterotoxin genes.