Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by ...the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-γ-D-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.
B. cereus are of particular interest in food safety and public health because of their capacity to cause food spoilage and disease through the production of various toxins. The aim of this study was ...to determine the prevalence, virulence factor genes and antibiotic resistance profile of B. cereus sensu lato isolated from cattle grazing soils and dairy products in Ghana. A total of 114 samples made up of 25 soil collected from cattle grazing farm land, 30 raw milk, 28 nunu (yoghurt-like product) and 31 woagashie (West African soft cheese). Ninety-six B. cereus sensu lato isolates from 54 positive samples were screened by PCR for the presence of 8 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK and entFM), and one emetic gene (ces). Phenotypic resistance to 15 antibiotics were also determined for 96 B. cereus sensu lato isolates.
About 72% (18 of 25 soil), 47% (14 of 30 raw milk), 35% (10 of 28 nunu) and 39% (12 of 31 woagashi) were positive for B. cereus sensu lato with mean counts (log
cfu/g) of 4.2 ± 1.8, 3.3 ± 2.0, 1.8 ± 1.4 and 2.6 ± 1.8 respectively. The distribution of enterotoxigenic genes revealed that 13% (12/96 isolates) harboured all three gene encoding for haemolytic enterotoxin HBL complex genes (hblA, hblC and hblD), 25% (24/96 isolates) possessed no HBL gene, whereas 63% (60/96 isolates) possessed at least one of the three HBL genes. All three genes encoding for non-haemolytic enterotoxin (nheA, nheB and nheC) were detected in 60% (57/96) isolates, 14% (13/96) harboured only one gene, 19% (18/96) whereas 8% possessed none of the NHE genes. The detection rates of cytk, entFM, and ces genes were 75, 67 and 9% respectively. Bacillus cereus s. l. isolates were generally resistant to β-lactam antibiotics such as ampicillin (98%), oxacillin (92%), penicillin (100%), amoxicillin (100%), and cefepime (100%) but susceptible to other antibiotics tested.
Bacillus cereus s. l. is prevalent in soil, raw milk and dairy products in Ghana. However, loads are at levels considered to be safe for consumption. Various enterotoxin genes associated with virulence of B. cereus are widespread among the isolates.
The inactivation of bacterial spores generally proceeds faster and at lower temperatures when heat treatments are conducted under high pressure, and high pressure high temperature (HPHT) processing ...is, therefore, receiving an increased interest from food processors. However, the mechanisms of spore inactivation by HPHT treatment are poorly understood, particularly at moderately elevated temperature. In the current work, we studied inactivation of the spores of Bacillus cereus F4430/73 by HPHT treatment for 5min at 600MPa in the temperature range of 50–100°C, using temperature increments of 5°C. Additionally, we investigated the effect of the natural antimicrobial carvacrol on spore germination and inactivation under these conditions. Spore inactivation by HPHT was less than about 1logunit at 50 to 70°C, but gradually increased at higher temperatures up to about 5logunits at 100°C. DPA release and loss of spore refractility in the spore population were higher at moderate (≤65°C) than at high (≥70°C) treatment temperatures, and we propose that moderate conditions induced the normal physiological pathway of spore germination resulting in fully hydrated spores, while at higher temperatures this pathway was suppressed and replaced by another mechanism of pressure-induced dipicolinic acid (DPA) release that results only in partial spore rehydration, probably because spore cortex hydrolysis is inhibited. Carvacrol strongly suppressed DPA release and spore rehydration during HPHT treatment at ≤65°C and also partly inhibited DPA release at ≥65°C. Concomitantly, HPHT spore inactivation was reduced by carvacrol at 65–90°C but unaffected at 95–100°C.
•HPHT at 50 to 100°C inactivated B. cereus spores by 1 to 5logunits.•DPA release and loss of spore refractility were higher at ≤65°C than at ≥70°C.•Carvacrol strongly suppresses DPA release and loss of spore refractility at ≤65°C.•Carvacrol partly suppresses DPA release and loss of spore refractility at ≥65°C.•Carvacrol suppresses spore inactivation by HPHT treatment at 65–90°C.
The effect of Plant Growth Promoting Rhizobacteria (Bacillus sp.) and silver nanoparticles on Zea mays was evaluated. The silver nanoparticles were synthesized from Tagetes erecta (Marigold) leaf and ...flower extracts, whereas PGPR isolated from spinach rhizosphere. The silver nanoparticles (AgNPs) were purified using ultra centrifugation and were characterized using UV-Vis spectroscopy at gradient wavelength and also by High Resolution Transmission Electron microscopy (HRTEM). The average particles size of AgNPs was recorded approximately 60 nm. Almost all potential isolates were able to produce Indole Acetic Acid (IAA), ammonia and Hydrogen cyanide (HCN), solubilized tricalcium phosphate and inhibited the growth of Macrophomina phaseolina in vitro but the isolate LPR2 was found the best among all. On the basis of 16S rRNA gene sequence, the isolate LPR2 was characterized as Bacillus cereus LPR2. The maize seeds bacterized with LPR2 and AgNPs individually showed a significant increase in germination (87.5%) followed by LPR2 + AgNPs (75%). But the maximum growth of root and shoot of maize plant was observed in seeds coated with LPR2 followed by AgNPs and a combination of both. Bacillus cereus LPR2 and silver nanoparticles enhanced the plant growth and LPR2 strongly inhibited the growth of deleterious fungal pathogen. Therefore, LPR2 and AgNPs could be utilized as bioinoculant and growth stimulator, respectively for maize.
Summary
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.
Bacillus cereus can exist as vegetative cells, spores, and biofilms in food-processing environment, posing a big challenge for the food industry. The objective of this study was to examine the ...inactivation efficacy of slightly acidic electrolyzed water (SAEW) in combination with benzalkonium chloride (BAC) and mild heat treatment (50 and 60 °C) on B. cereus strains (ATCC 10987 and ATCC 14579). The inactivation efficacy of SAEW was found to be largely dependent on available chlorine concentration (ACC) level and exposure time as well as B. cereus strains and growth conditions. SAEW with ACC of 40 ppm reduced ATCC 10987 and ATCC 14579 vegetative cells to the non-detection limit within 30 s. and 1 min, respectively. Combination treatment with SAEW+60 °C for 10 min resulted in reductions of ATCC 10987 spores, ATCC 14579 spores, and ATCC 10987 biofilms at 0.76 logCFU/ml, 0.59 logCFU/ml, and 1.28 logCFU/cm2, respectively. While, treatment with SAEW+BAC + 60 °C for 10 min resulted in reductions of ATCC 10987 spores, ATCC 14579 spores, and ATCC 10987 biofilms at 1.91 logCFU/ml, 1.98 logCFU/ml, and 2.62 logCFU/cm2, respectively. The inactivation kinetics under different ACC of SAEW and in combination with BAC and mild treatment were determined by Weibull model. The calculated adjusted correlation coefficients (R2adj) and root mean sum of squared error (RMSE) values for all curves were found to be ranges from 0.95–0.99 and 0.04–0.23, respectively, indicating that the Weibull model precisely predicted the inactivation kinetics of B. cereus during SAEW in combination with BAC and mild heat treatments. These results suggest that SAEW in combination with BAC and mild heat may be used as an effective cleaning strategy against B. cereus in the food contact surfaces.
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•Disinfection efficacy of SAEW largely depends on growth conditions of B. cereus.•Additional heat treatment is effective on B. cereus biofilms.•SAEW combined with BAC has synergistic effect on B. cereus spores and biofilms.•Combined treatment of SAEW+BAC + 60 °C showed higher effect on biofilms than spores.
Bacillus cereus has long been recognized as an important pathogen in foodborne poisoning worldwide. Fresh vegetables are often contaminated with enterotoxigenic B. cereus and have been implicated as ...a vehicle for the transmission of this bacterium. This study reports on the occurrence, virulence gene profile, and antibiotic resistance of B. cereus in fresh vegetables. Of 102 examined samples, 48 (47%) of the samples were contaminated with B. cereus (>1 log CFU/g) and 7 (6.8%) of the samples showed more than 3 log CFU/g. In total, 118 B. cereus isolates were examined for the virulence genes nheA, nheB, nheC, hblA, hblC, hblD, cytK, and entFM and for resistance to antibiotics. Of these B. cereus isolates, 70% harbored nheA, nheB, nheC, and cytK. Eighteen (80%) of 21 isolates from bell peppers possessed eight enterotoxin genes. B. cereus isolates were susceptible to imipenem, vancomycin, gentamicin, erythromycin, ciprofloxacin, and chloramphenicol, whereas 22.4% of isolates from garlic chives, 48.7% from perilla leaf, and 40.5% from romaine lettuce showed antibiotic resistance to rifampin and 6% of isolates from garlic chives exhibited resistance to tetracycline. Three isolates from garlic chives were resistant to both tetracycline and rifampin. Raw vegetables were revealed to be major sources of B. cereus containing multiple toxin genes and exhibiting antibiotic resistance. Therefore, the potential health risks of consuming these vegetables raw or undercooked should not be underestimated. This study provides basic information for monitoring the antibiotic resistance and toxigenicity of B. cereus in the food chain during vegetable distribution and for developing food safety management to reduce the contamination with and transmission of B. cereus.
Sigma 54 is a transcriptional regulator predicted to play a role in physical interaction of bacteria with their environment, including virulence and biofilm formation. In order to study the role of ...Sigma 54 in Bacillus cereus, a comparative transcriptome and phenotypic study was performed using B. cereus ATCC 14579 WT, a markerless rpoN deletion mutant, and its complemented strain. The mutant was impaired in many different cellular functions including low temperature and anaerobic growth, carbohydrate metabolism, sporulation and toxin production. Additionally, the mutant showed lack of motility and biofilm formation at air-liquid interphase, and this correlated with absence of flagella, as flagella staining showed only WT and complemented strain to be highly flagellated. Comparative transcriptome analysis of cells harvested at selected time points during growth in aerated and static conditions in BHI revealed large differences in gene expression associated with loss of phenotypes, including significant down regulation of genes in the mutant encoding enzymes involved in degradation of branched chain amino acids, carbohydrate transport and metabolism, flagella synthesis and virulence factors. Our study provides evidence for a pleiotropic role of Sigma 54 in B. cereus supporting its adaptive response and survival in a range of conditions and environments.
In this study, a new vancomycin (Van)-modified poly-l-lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a ...multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d-alanyl-d-alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus, the entFM, cesB, cer, and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 103 cfu/mL, with a dynamic range of detection at 103 to 107 cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 101 cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria.
The goal of this study was to identify Cd-resistant bacterial strains with endurance capacity and to evaluate their ability to remove cadmium ions from cadmium-polluted water. The Bacillus cereusS5 ...strain identified in this study had the closest genetic relationship with B. cereus sp. Cp1 and performed well in the removal of Cd2+ions from solution. The results showed that both the live and dead biomasses of the Cd2+-tolerant B. cereus S5 strain could absorb Cd2+ ions in solution but that the live biomass of the B. cereus S5 strain outperformed the dead biomass at lower Cd2+concentrations. An analysis of the cadmium tolerance genes of B. cereus S5 identified ATPase genes that were associated with cadmium tolerance and involved in the ATP pumping mechanism. The FTIR spectra revealed the presence of amino, carboxyl and hydroxyl groups on the pristine biomass and indicated that the cadmium ion removal ability was related to the structure of the strain. The maximum absorption capacity of the B. cereus S5 strain in viable spore biomass was 70.16 mg/g (dry weight) based on a pseudo-second-order kinetic model fit to the experimental data. The Langmuir and Langmuir-Freundlich isotherm adsorption models fit the cadmium ion adsorption data well, and the kinetic curves indicated that the adsorption rate was second-order. For Cd2+ concentrations (mg/L) of 1-109 mg/L, good removal efficiency (>80%) was achieved using approximately 3.48-10.3 g/L of active spore biomass of the B. cereus S5 strain. A cadmium-tolerant bacteria-activated carbon-immobilized column could be used for a longer duration and exhibited greater treatment efficacy than the control column in the treatment of cadmium-polluted water. In addition, a toxicity assessment using mice demonstrated that the biomass of the B. cereus S5 strain and its fermentation products were non-toxic. Thus, the isolated B. cereus S5 strain can be considered an alternative biological adsorbent for use in emergency responses to severe cadmium pollution and in the routine treatment of trace cadmium pollution.
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