Rapidly fatal infection with Bacillus cereus/thuringiensis: genome assembly of the responsible pathogen and consideration of possibly contributing toxins.

Bloodstream infection with Bacillus cereus/thuringiensis can be life threatening, particularly in patients who are severely immunocompromised. In this report we describe a case that progressed from asymptomatic to fatal over approximately 5 hours despite extensive resuscitation efforts. We identify the pathogen and assemble its genome, in which we find genes for toxins that may have contributed to the precipitous demise. In the context of this and other cases we discuss the possible indication for rapid appropriate antibiotic administration and potentially antitoxin treatment or toxin removal in fulminant illness in immunocompromised patients.

The cytotoxic potential of Bacillus cereus strains of various origins.

B. cereus is a human pathogen associated with food poisoning leading to gastrointestinal disorders, as well as local and severe systemic infections. The pathogenic spectrum of B. cereus ranges from strains used as probiotics in humans to lethal highly toxic strains. In this study, we gathered a collection of 100 strains representative of the pathological diversity of B. cereus in humans, and characterized these strains for their cytotoxic potential towards human cells. We analyzed the correlation between cytotoxicity to epithelial and macrophage cells and the combination of 10 genes suspected to play a role during B. cereus virulence. We highlight genetic differences among isolates and studied correlations between genetic signature, cytotoxicity and strain pathological status. We hope that our findings will improve our understanding of the pathogenicity of B. cereus, thereby making it possible to improve both clinical diagnosis and food safety.

Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors.

Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.

Bacillus cereus group: genetic aspects related to food safety and dairy processing / Grupo do Bacillus cereus: aspectos genéticos relacionados à segurança alimentar e ao processamento de derivados lácteos

Bacillus cereus group includes not pathogenic and high pathogenic species. They are considered as a risk to public health due to foodborne diseases and as an important cause of economic losses to industries due to production of spoilage enzymes. Some researches have been performed in order to assess the possible factors that contribute to put public health into risk because of consumption of food contaminated with viable cells or toxins which have complex mechanisms of production. The control of these bacteria in food is difficult because they are resistant to several processes used in industries. Thus, in this way, this review focused on highlighting the risk due to toxins production by bacteria from B. cereus group in food and the consequences for food safety and dairy industries.(AU)

Diversas espécies fazem parte do grupo de Bacillus cereus, desde algumas apatogênicas até outras com alta patogenicidade. Consistem em risco à saúde pública decorrentes de toxinfecções alimentares, além de causarem importantes perdas econômicas para as indústrias em virtude da produção de enzimas deteriorantes. O controle da contaminação em alimentos por esses micro-organismos é difícil, visto que são resistentes a vários tratamentos utilizados pelas indústrias. Assim, diante do exposto, esta revisão objetivou fornecer informações em relação aos aspectos genéticos desse grupo de bactérias e seus mecanismos de produção de toxinas, além de ressaltar a importância e as novas estratégias de controle para as companhias alimentícias e de laticínios.(AU)

Caracterização da intoxicação alimentar causada pelo Bacillus cereus: uma revisão / Characterization of food poisoning caused by Bacillus cereus: a review

Neste trabalho foi realizada uma revisão com o objetivo de demonstrar a importância do Bacillus cereus, o perfil toxigênico desse grupo de bactérias formadoras de esporos, que tem sido coligada a surtos de intoxicação alimentar, as metodologias utilizadas para sua determinação, identificar os fatores de virulência das toxinas emética e diarréica, bem como formas para evitar a intoxicação e os tratamentos para prevenção. A presença de B. cereus tem sido detectada em equipamentos e utensílios, mostrando que são fontes potenciais de transmissão do micro-organismo para os alimentos e também a importância da higienização correta dos equipamentos.

Lactobacillus delbrueckii subsp lactis CIDCA 133 modulates response of human epithelial and dendritic cells infected with Bacillus cereus.

It is known that probiotic microorganisms are able to modulate pathogen virulence. This ability is strain dependent and involves multiple interactions between microorganisms and relevant host's cell populations. In the present work we focus on the effect of a potentially probiotic lactobacillus strain (Lactobacillus delbrueckii subsp. lactis CIDCA 133) in an in vitro model of Bacillus cereus infection. Our results showed that infection of intestinal epithelial HT-29 cells by B. cereus induces nuclear factor kappa B (NF-κB) pathway. Noteworthy, the presence of strain L. delbrueckii subsp.lactis CIDCA 133 increases stimulation. However, B. cereus-induced interleukin (IL)-8 production by epithelial cells is partially abrogated by L. delbrueckii subsp. lactis CIDCA 133. These findings suggest that signalling pathways other than that of NF-κB are involved. In a co-culture system (HT-29 and monocyte-derived dendritic cells), B. cereus was able to translocate from the epithelial (upper) to the dendritic cell compartment (lower). This translocation was partially abrogated by the presence of lactobacilli in the upper compartment. In addition, infection of epithelial cells in the co-culture model, led to an increase in the expression of CD86 by dendritic cells. This effect could not be modified in the presence of lactobacilli. Interestingly, infection of enterocytes with B. cereus triggers production of proinflammatory cytokines by dendritic cells (IL-8, IL-6 and tumour necrosis factor alpha (TNF-α)). The production of TNF-α (a protective cytokine in B. cereus infections) by dendritic cells was increased in the presence of lactobacilli. The present work demonstrates for the first time the effect of L. delbrueckii subsp. lactis CIDCA 133, a potentially probiotic strain, in an in vitro model of B. cereus infection. The presence of the probiotic strain modulates cell response both in infected epithelial and dendritic cells thus suggesting a possible beneficial effect of selected lactobacilli strains on the course of B. cereus infection.

Antibacterial Activity and Action Mechanism of the Essential Oil from Enteromorpha linza L. against Foodborne Pathogenic Bacteria.

Foodborne illness and disease caused by foodborne pathogenic bacteria is continuing to increase day by day and it has become an important topic of concern among various food industries. Many types of synthetic antibacterial agents have been used in food processing and food preservation; however, they are not safe and have resulted in various health-related issues. Therefore, in the present study, essential oil from an edible seaweed, Enteromorpha linza (AEO), was evaluated for its antibacterial activity against foodborne pathogens, along with the mechanism of its antibacterial action. AEO at 25 mg/disc was highly active against Bacillus cereus (12.3-12.7 mm inhibition zone) and Staphylococcus aureus (12.7-13.3 mm inhibition zone). The minimum inhibitory concentration and minimum bactericidal concentration values of AEO ranged from 12.5-25 mg/mL. Further investigation of the mechanism of action of AEO revealed its strong impairing effect on the viability of bacterial cells and membrane permeability, as indicated by a significant increase in leakage of 260 nm absorbing materials and K⁺ ions from the cell membrane and loss of high salt tolerance. Taken together, these data suggest that AEO has the potential for use as an effective antibacterial agent that functions by impairing cell membrane permeability via morphological alternations, resulting in cellular lysis and cell death.

Secreted Compounds of the Probiotic Bacillus clausii Strain O/C Inhibit the Cytotoxic Effects Induced by Clostridium difficile and Bacillus cereus Toxins.

Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus, by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB, encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea.

Bactericidal Mechanism of Bio-oil Obtained from Fast Pyrolysis of Pinus densiflora Against Two Foodborne Pathogens, Bacillus cereus and Listeria monocytogenes.

Foodborne bacteria are the leading cause of food spoilage and other related diseases. In the present study, the antibacterial activity of bio-oil (BO) manufactured by fast pyrolysis of pinewood sawdust (Pinus densiflora Siebold and Zucc.) against two disease-causing foodborne pathogens (Bacillus cereus and Listeria monocytogenes) was evaluated. BO at a concentration of 1000 µg/disc was highly active against both B. cereus (10.0-10.6 mm-inhibition zone) and L. monocytogenes (10.6-12.0-mm inhibition zone). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration values of BO were 500 and 1000 µg/mL, respectively, for both pathogens. At the MIC concentration, BO exhibited an inhibitory effect on the viability of the bacterial pathogens. The mechanism of action of BO revealed its strong impairing effect on the membrane integrity of bacterial cells, which was confirmed by a marked release of 260-nm absorbing material, leakage of electrolytes and K(+) ions, and reduced capacity for osmoregulation under high salt concentration. Scanning electron microscopy clearly showed morphological alteration of the cell membrane due to the effect of BO. Overall, the results of this study suggest that BO exerts effective antibacterial potential against foodborne pathogens and can therefore potentially be used in food processing and preservation.

Iron acquisition in Bacillus cereus: the roles of IlsA and bacillibactin in exogenous ferritin iron mobilization.

In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.

Bacillus cereus bacteremia and multiple brain abscesses during acute lymphoblastic leukemia induction therapy.

Bacillus cereus can cause serious infections in immunosuppressed patients. This population may be susceptible to B. cereus pneumonia, bacteremia, cellulitis, and rarely cerebral abscess. Here we report an 8-year-old boy undergoing induction therapy for acute lymphoblastic leukemia who developed multifocal B. cereus cerebral abscesses, highlighting the propensity for B. cereus to develop cerebral abscesses. A review of the literature over the past 25 years identified another 11 cases (3 children and 8 adults) of B. cereus cerebral abscess in patients undergoing cancer therapy. B. cereus cerebral abscesses were associated with a high mortality rate (42%) and significant morbidity. Notably, B. cereus bacteremia with concomitant cerebral abscess was associated with induction chemotherapy for acute leukemia in both children and adults (10 of 12 case reports). Our case report and review of the literature highlights the propensity for B. cereus to develop cerebral abscess(es). Therefore, early consideration for neuroimaging should be given for any neutropenic cancer patient identified with B. cereus bacteremia, in particular those with acute leukemia during induction therapy.

Bacillus cereus immune escape: a journey within macrophages.

During bacterial infection, professional phagocytes are attracted to the site of infection, where they constitute a first line of host cell defense. Their function is to engulf and destroy the pathogens. Thus, bacteria must withstand the bactericidal activity of professional phagocytes, including macrophages to counteract the host immune system. Bacillus cereus infections are characterized by bacteremia despite the accumulation of inflammatory cells at the site of infection. This implies that the bacteria have developed means of resisting the host immune system. Bacillus cereus spores survive, germinate, and multiply in contact with macrophages, eventually producing toxins that kill these cells. However, the exact mechanism by which B. cereus evades immune attack remains unclear. This review addresses the interaction between B. cereus and macrophages, highlighting, in particular, the ways in which the bacteria escape the microbicidal activities of professional phagocytes.

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

Certhrax toxin, an anthrax-related ADP-ribosyltransferase from Bacillus cereus.

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD(+) with high affinity (K(D) = 52.3 ± 12.2 µM). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K(D) = 1.7 ± 0.2 µM, K(i) = 1.8 ± 0.4 µM) and PJ34 (K(D) = 5.8 ± 2.6 µM, K(i) = 9.6 ± 0.3 µM)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD(+) binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.

Bacillus cereus y su papel en las intoxicaciones alimentarias / Bacillus cereus and food poisoning

Introducción: Bacillus cereus es una bacteria Gram positiva, habitante frecuente de un amplio número de ambientes. Esta bacteria es la responsable del síndrome emético y del diarréico, y además se ha identificado vinculada a otras enfermedades como endocarditis, endoftalmitis, por lo que se le reconoce su carácter de patógeno oportunista. Objetivo: revisar información sobre las toxinas producidas por B cereus y cuáles y cómo están relacionadas con las intoxicaciones alimentarias, así como abordar otros factores responsables de la virulencia de esta bacteria. Fuente de datos: para recopilar la información necesaria se utilizó el acceso a Internet mediante los buscadores Google y Scirus. Los artículos que se examinaron incluyeron revisiones del tema, artículos de investigación y capítulos de libros extraídos como acceso libre o solicitados a los autores. Síntesis de los datos: se profundizó en el conocimiento sobre la toxina emética y la diarreica, la sintomatología clínica, el tiempo de aparición, así como los alimentos involucrados en cada síndrome, lo cual sirve de ayuda al clínico para determinar las causas de la toxoinfección. Se hace un bosquejo general de la microbiología de esta bacteria y otros factores que explican su virulencia y otras implicaciones en la salud humana. Conclusiones: El conocimiento obtenido permite reconocer a B. cereus como una bacteria con importancia clínica, lo que evita considerarla solo como un contaminante ambiental

Introduction: Gram positive bacterium Bacillus cereus is widely distributed in the environment. This bacterium is responsible for two food poisoning syndromes (emetic and diarrheal) and also it has been related to other diseases such as endocarditis and endophthalmitis; therefore it is recognized as opportunist pathogen. Objective: to review the information about Bacillus cereus toxins and its connection with food poisoning, as well as to address others factors influencing this bacterium's virulence. Data sources: for collecting this information, search engines such as Google and Scirus. Reviewed articles included research articles, systematic reviews on this topic and free accessed book chapters, or requested from the authors. Data synthesis: the review delved into Knowledge on diarrheal and emetic toxins, clinical symptomatology and time of occurrence, as soon as foodstuffs involved in each syndrome. This helps the clinician to look for the causes of toxin infections. A general overview of Bacillus cereus microbiology and others factors that explain its virulence and other implications in the human health was given. Conclusions: the acquired knowledge shows that Bacillus cereus is one bacterium with clinical importance rather than a mere environmental pollutant

Critical illness polyneuropathy and myopathy caused by Bacillus cereus sepsis in acute lymphoblastic leukemia.

We report a pediatric case of critical illness polyneuropathy and myopathy caused by Bacillus cereus sepsis during acute lymphoblastic leukemia therapy. A 15-year-old boy developed B. cereus sepsis and multiple organ failure on the 19th day after initiation of chemotherapy, and multidisciplinary treatment was started. Treatment was effective and septic shock with multiple organ failure remitted. He was weaned from a respirator on day 23 after the onset of sepsis, but complete flaccid paralysis of the 4 extremities occurred. His compound muscle action potential and F-wave occurrence were reduced on a nerve conduction test. The number of motor units was markedly decreased, and the amplitude and duration of individual motor units were low and short, respectively, on electromyography. Cerebrospinal fluid was normal. On the basis of these findings, he was diagnosed with critical illness polyneuropathy/myopathy. He underwent intensive rehabilitation and recovered the ability to walk 3 months after onset. He was discharged 1 year after the initiation of chemotherapy, and remission has been maintained without inconvenience to daily living activities for 3 years since disease onset.

Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages.

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.

Ability of Bacillus cereus group strains to cause food poisoning varies according to phylogenetic affiliation (groups I to VII) rather than species affiliation.

Cytotoxic activity levels of culture filtrates and toxin distributions varied according to the phylogenetic group (I to VII) within the Bacillus cereus group, suggesting that these groups are of different clinical significance and are more suitable than species affiliations for determining food poisoning risk. A first-line, simple online tool (https://www.tools.symprevius.org/Bcereus/english.php) to assign strains to the different phylogenetic groups is presented.

Cytotoxicity of the Bacillus cereus Nhe enterotoxin requires specific binding order of its three exoprotein components.

This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted beta-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic beta-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.