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.

Implication of a Key Region of Six Bacillus cereus Genes Involved in Siroheme Synthesis, Nitrite Reductase Production and Iron Cluster Repair in the Bacterial Response to Nitric Oxide Stress.

Bacterial response to nitric oxide (NO) is of major importance for bacterial survival. NO stress is a main actor of the eukaryotic immune response and several pathogenic bacteria have developed means for detoxification and repair of the damages caused by NO. However, bacterial mechanisms of NO resistance by Gram-positive bacteria are poorly described. In the opportunistic foodborne pathogen Bacillus cereus, genome sequence analyses did not identify homologs to known NO reductases and transcriptional regulators, such as NsrR, which orchestrate the response to NO of other pathogenic or non-pathogenic bacteria. Using a transcriptomic approach, we investigated the adaptation of B. cereus to NO stress. A cluster of 6 genes was identified to be strongly up-regulated in the early phase of the response. This cluster contains an iron-sulfur cluster repair enzyme, a nitrite reductase and three enzymes involved in siroheme biosynthesis. The expression pattern and close genetic localization suggest a functional link between these genes, which may play a pivotal role in the resistance of B. cereus to NO stress during infection.

Dr. NO and Mr. Toxic - the versatile role of nitric oxide.

Nitric oxide (NO) is present in various organisms from humans, to plants, fungus and bacteria. NO is a fundamental signaling molecule implicated in major cellular functions. The role of NO ranges from an essential molecule to a potent mediator of cellular damages. The ability of NO to react with a broad range of biomolecules allows on one hand its regulation and a gradient concentration and on the other hand to exert physiological as well as pathological functions. In humans, NO is implicated in cardiovascular homeostasis, neurotransmission and immunity. However, NO can also contribute to cardiovascular diseases (CVDs) or septic shock. For certain denitrifying bacteria, NO is part of their metabolism as a required intermediate of the nitrogen cycle. However, for other bacteria, NO is toxic and harmful. To survive, those bacteria have developed processes to resist this toxic effect and persist inside their host. NO also contributes to maintain the host/microbiota homeostasis. But little is known about the impact of NO produced during prolonged inflammation on microbiota integrity, and some pathogenic bacteria take advantage of the NO response to colonize the gut over the microbiota. Taken together, depending on the environmental context (prolonged production, gradient concentration, presence of partners for interaction, presence of oxygen, etc.), NO will exert its beneficial or detrimental function. In this review, we highlight the dual role of NO for humans, pathogenic bacteria and microbiota, and the mechanisms used by each organism to produce, use or resist NO.

Advanced Methods for Detection of Bacillus cereus and Its Pathogenic Factors.

Bacillus cereus is an opportunistic foodborne pathogen causing food intoxication and infectious diseases. Different toxins and pathogenic factors are responsible for diarrheal syndrome, like nonhemolytic enterotoxin Nhe, hemolytic enterotoxin Hbl, enterotoxin FM and cytotoxin K, while emetic syndrome is caused by the depsipeptide cereulide toxin. The traditional method of B. cereus detection is based on the bacterial culturing onto selective agars and cells enumeration. In addition, molecular and chemical methods are proposed for toxin gene profiling, toxin quantification and strain screening for defined virulence factors. Finally, some advanced biosensors such as phage-based, cell-based, immunosensors and DNA biosensors have been elaborated to enable affordable, sensitive, user-friendly and rapid detection of specific B. cereus strains. This review intends to both illustrate the state of the B. cereus diagnostic field and to highlight additional research that is still at the development level.

Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria.

Foodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification.

Correction: Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis.

[This corrects the article DOI: 10.1371/journal.ppat.1002629.].

Virulence Analysis of Bacillus cereus Isolated after Death of Preterm Neonates, Nice, France, 2013.

After the deaths of 2 preterm neonates with Bacillus cereus systemic infection in the same intensive care unit, we investigated the pathogenic potential of this bacterium. Genetic and virulence analysis indicated the neonates were infected with 2 different strains with a virulence potential similar to environmental strains, indicating likely patient immune response failure.

Bacillus cereus-induced food-borne outbreaks in France, 2007 to 2014: epidemiology and genetic characterisation.

The aim of this study was to identify and characterise Bacillus cereus from a unique national collection of 564 strains associated with 140 strong-evidence food-borne outbreaks (FBOs) occurring in France during 2007 to 2014. Starchy food and vegetables were the most frequent food vehicles identified; 747 of 911 human cases occurred in institutional catering contexts. Incubation period was significantly shorter for emetic strains compared with diarrhoeal strains A sub-panel of 149 strains strictly associated to 74 FBOs and selected on Coliphage M13-PCR pattern, was studied for detection of the genes encoding cereulide, diarrhoeic toxins (Nhe, Hbl, CytK1 and CytK2) and haemolysin (HlyII), as well as panC phylogenetic classification. This clustered the strains into 12 genetic signatures (GSs) highlighting the virulence potential of each strain. GS1 (nhe genes only) and GS2 (nhe, hbl and cytK2), were the most prevalent GS and may have a large impact on human health as they were present in 28% and 31% of FBOs, respectively. Our study provides a convenient molecular scheme for characterisation of B. cereus strains responsible for FBOs in order to improve the monitoring and investigation of B. cereus-induced FBOs, assess emerging clusters and diversity of strains.

Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.

Necrotrophism is a quorum-sensing-regulated lifestyle in Bacillus thuringiensis.

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading.

Pathogenic potential of Bacillus cereus strains as revealed by phenotypic analysis.

The Bacillus cereus pathogenic spectrum ranges from strains used as probiotics to human-lethal strains. However, prediction of the pathogenic potential of a strain remains difficult. Here, we show that food poisoning and clinical strains can be differentiated from harmless strains on the basis of host colonization phenotypes.

Bacillus anthracis protease InhA regulates BslA-mediated adhesion in human endothelial cells.

To achieve widespread dissemination in the host, Bacillus anthracis cells regulate their attachment to host endothelium during infection. Previous studies identified BslA (Bacillus anthracis S-layer Protein A), a virulence factor of B. anthracis, as necessary and sufficient for adhesion of vegetative cells to human endothelial cells. While some factors have been identified, bacteria-specific contributions to BslA mediated adhesion remain unclear. Using the attenuated vaccine Sterne 7702 strain of B. anthracis, we tested the hypothesis that InhA (immune inhibitor A), a B. anthracis protease, regulates BslA levels affecting the bacteria's ability to bind to endothelium. To test this, a combination of inhA mutant and complementation analysis in adhesion and invasion assays, Western blot and InhA inhibitor assays were employed. Results show InhA downregulates BslA activity reducing B. anthracis adhesion and invasion in human brain endothelial cells. BslA protein levels in ΔinhA bacteria were significantly higher than wild-type and complemented strains showing InhA levels and BslA expression are inversely related. BslA was sensitive to purified InhA degradation in a concentration- and time-dependent manner. Taken together these data support the role of InhA regulation of BslA-mediated vegetative cell adhesion and invasion.

Iron regulates Bacillus thuringiensis haemolysin hlyII gene expression during insect infection.

Bacillus thuringiensis (Bt) is a spore-forming entomopathogen broadly used in agriculture crop. The haemolysin HlyII is an important Bt virulence factor responsible for insect death. In this work, we focused on the regulation of the hlyII gene throughout the bacterial growth in vitro and in vivo during insect infection. We show that hlyII regulation depends on the global regulator Fur. This regulation occurs independently of HlyIIR, the other known regulator of hlyII gene expression. Moreover, we show that hlyII is highly expressed when iron is depleted in vivo. As HlyII induces haemocyte and macrophage death, which are involved in the sequestration of iron upon infection, HlyII may induce host cell death to allow bacteria to gain access to iron.

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.

The pathogenic biomarker alcohol dehydrogenase protein is involved in Bacillus cereus virulence and survival against host innate defence.

Bacillus cereus is a spore forming bacteria recognized among the leading agents responsible for foodborne outbreaks in Europe. B. cereus is also gaining notoriety as an opportunistic human pathogen inducing local and systemic infections. The real incidence of such infection is likely underestimated and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We have recently analyzed a large strain collection of varying pathogenic potential. Screening for biomarkers to differentiate among clinical and non-clinical strains, a gene encoding an alcohol dehydrogenase-like protein was identified among the leading candidates. This family of proteins has been demonstrated to be involved in the virulence of several bacterial species. The relevant gene was knocked out to elucidate its function with regards to resistance to host innate immune response, both in vitro and in vivo. Our results demonstrate that the adhB gene plays a significant role in resistance to nitric oxide and oxidative stress in vitro, as well as its pathogenic ability with regards to in vivo toxicity. These properties may explain the pathogenic potential of strains carrying this newly identified virulence factor.

New genetic biomarkers to differentiate non-pathogenic from clinically relevant Bacillus cereus strains.

OBJECTIVES: Bacillus cereus is responsible for food poisoning and rare but severe clinical infections. The pathogenicity of strains varies from harmless to lethal strains. However, there are currently no markers, either alone or in combination, to differentiate pathogenic from non-pathogenic strains. The objective of the study was to identify new genetic biomarkers to differentiate non-pathogenic from clinically relevant B. cereus strains. METHODS: A first set of 15 B. cereus strains were compared by RNAseq. A logistic regression model with lasso penalty was applied to define combination of genes whose expression was associated with strain pathogenicity. The identified markers were checked for their presence/absence in a collection of 95 B. cereus strains with varying pathogenic potential (food-borne outbreaks, clinical and non-pathogenic). Receiver operating characteristic area under the curve (AUC) analysis was used to determine the combination of biomarkers, which best differentiate between the "disease" versus "non-disease" groups. RESULTS: Seven genes were identified during the RNAseq analysis with a prediction to differentiate between pathogenic and non-pathogenic strains. The validation of the presence/absence of these genes in a larger collection of strains coupled with AUC prediction showed that a combination of four biomarkers was sufficient to accurately discern clinical strains from harmless strains, with an AUC of 0.955, sensitivity of 0.9 and specificity of 0.86. CONCLUSIONS: These new findings help in the understanding of B. cereus pathogenic potential and complexity and may provide tools for a better assessment of the risks associated with B. cereus contamination to improve patient health and food safety.

Identification of a New Pathogenicity Island Within the Large pAH187_270 Plasmid Involved in Bacillus cereus Virulence.

OBJECTIVES: Bacillus cereus is responsible for food poisoning and rare but severe clinical infections. The pathogenicity of B. cereus strains varies from harmless to lethal strains. The objective of this study was to characterize three B. cereus isolates isolated from the same patient and identify their virulence potentials. METHODS: Three isolates of B. cereus were isolated from various blood samples from a patient who developed sepsis following a central venous catheter infection. The three isolates were compared by WGS, genotyping and SNP analysis. Furthermore, the isolates were compared by phenotypical analysis including bacterial growth, morphology, germination efficacy, toxin production, antibiotic susceptibility and virulence in an insect model of infection. RESULTS: According to WGS and genotyping, the 3 isolates were shown to be identical strains. However, the last recovered strain had lost the mega pAH187_270 plasmid. This last strain showed different phenotypes compared to the first isolated strain, such as germination delay, different antibiotic susceptibility and a decreased virulence capacity towards insects. A 50- kbp region of pAH187_270 plasmid was involved in the virulence potential and could thus be defined as a new pathogenicity island of B. cereus. CONCLUSIONS: These new findings help in the understanding of B. cereus pathogenic potential and complexity and provide further hints into the role of large plasmids in the virulence of B. cereus strains. This may provide tools for a better assessment of the risks associated with B. cereus hospital contamination to improve hygiene procedure and patient health.

Highly sensitive detection of Campylobacter spp. In chicken meat using a silica nanoparticle enhanced dot blot DNA biosensor.

Paper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/µL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.

Bacillus cereus Induces Severe Infections in Preterm Neonates: Implication at the Hospital and Human Milk Bank Level.

Human breast milk (HBM) is a source of essential nutrients for infants and is particularly recommended for preterm neonates when their own mother's milk is not available. It provides protection against infections and decreases necrotizing enterocolitis and cardiovascular diseases. Nevertheless, HBM spoilage can occur due to contamination by pathogens, and the risk of a shortage of HBM is very often present. B. cereus is the most frequent ubiquitous bacteria responsible for HBM being discarded. It can contaminate HBM at all stages, from its collect point to the storage and delivery. B. cereus can induce severe infection in newborns with very low birth weight, with sometimes fatal outcomes. Although the source of contamination is rarely identified, in some cases, HBM was suspected as a potential source. Even if the risk is low, as infection due to B. cereus in preterm infants should not be overlooked, human milk banks follow strict procedures to avoid contamination, to accurately identify remaining bacteria following pasteurization and to discard non-compliant milk samples. In this review, we present a literature overview of B. cereus infections reported in neonates and the suspected sources of contamination. We highlight the procedures followed by the human milk banks from the collection of the milk to its microbiological characterization in Europe. We also present improved detection and decontamination methods that might help to decrease the risk and to preserve the public's confidence in this vital biological product for infants whose mothers cannot breastfeed.

Nitric Oxide Impacts Human Gut Microbiota Diversity and Functionalities.

The disruption of gut microbiota homeostasis has been associated with numerous diseases and with a disproportionate inflammatory response, including overproduction of nitric oxide (NO) in the intestinal lumen. However, the influence of NO on the human gut microbiota has not been well characterized yet. We used in vitro fermentation systems inoculated with human fecal samples to monitor the effect of repetitive NO pulses on the gut microbiota. NO exposure increased the redox potential and modified the fermentation profile and gas production. The overall metabolome was modified, reflecting less strict anaerobic conditions and shifts in amino acid and nitrogen metabolism. NO exposure led to a microbial shift in diversity with a decrease in Clostridium leptum group and Faecalibacterium prausnitzii biomass and an increased abundance of the Dialister genus. Escherichia coli, Enterococcus faecalis, and Proteus mirabilis operational taxonomic unit abundance increased, and strains from those species isolated after NO stress showed resistance to high NO concentrations. As a whole, NO quickly changed microbial fermentations, functions, and composition in a pulse- and dose-dependent manner. NO could shift, over time, the trophic chain to conditions that are unfavorable for strict anaerobic microbial processes, implying that a prolonged or uncontrolled inflammation has detrimental and irreversible consequences on the human microbiome. IMPORTANCE Gut microbiota dysbiosis has been associated with inflammatory diseases. The human inflammatory response leads to an overproduction of nitric oxide (NO) in the gut. However, so far, the influence of NO on the human gut microbiota has not been characterized. In this study, we used in vitro fermentation systems with human fecal samples to understand the effect of NO on the microbiota: NO modified the microbial composition and its functionality. High NO concentration depleted the microbiota of beneficial butyrate-producing species and favored potentially deleterious species (E. coli, E. faecalis, and P. mirabilis), which we showed can sustain high NO concentrations. Our work shows that NO may participate in the vicious circle of inflammation, leading to detrimental and irreversible consequences on human health.