Alien species invasion of deep-sea bacteria into mouse gut microbiota.

INTRODUCTION: Deep sea has numerous bacteria which dominate in the biomass of deep-sea sediments. Some deep-sea bacteria may possess the capacity to destroy mammal health by the alteration of gut microbiota, acting as potential pathogens. OBJECTIVES: Pathogenic bacteria are great threats to human health. However, the ultimate origin of pathogenic bacteria has not been intensively explored. In this study, therefore, the influence of deep-sea bacteria on the gut microbiota was evaluated on a global scale. METHODS: The bacteria isolated from each of 106 deep-sea sediment samples were transplanted into mice in our study to assess the infectiousness of deep-sea bacteria. RESULTS: The results showed that some bacteria from deep sea, an area that has existed since the earth was formed, could proliferate in mouse gut. Based on the infectious evaluation of the bacteria from each of 106 deep-sea sediments, the bacteria isolated from 13 sediments invaded the gut bacterial communities of mice, leading to the significant alteration of mouse gut microbiota. Among the 13 deep-sea sediments, the bacteria isolated from 9 sediments could destroy mouse health by inducing glucose metabolism deterioration, liver damage and inflammatory symptom. As an example, a bacterium was isolated from deep-sea sediment DP040, which was identified to be Bacillus cereus (termed as Bacillus cereus DP040). Bacillus cereus DP040 could invade the gut microbiota of mice to change the gut microbial structure, leading to inflammatory symptom of mice. The deep-sea sediments containing the bacteria destroying the health of mice were distributed in hydrothermal vent, mid-ocean ridge and hadal trench of the Indian Ocean, the Atlantic Ocean and the Pacific Ocean. CONCLUSION: Our findings demonstrate that deep sea is an important origin of potential pathogenic bacteria and provide the first biosecurity insight into the alien species invasion of deep-sea bacteria into mammal gut microbiota.

Characterization of a Bacillus cereus strain associated with a large feed-related outbreak of severe infection in pigs.

AIMS: Bacillus cereus is often responsible for foodborne diseases and both local and systemic infections in humans. Cases of infection in other mammals are rather rare. In this study, we report a B. cereus feed-related outbreak that caused the death of 6234 pigs in Italy. METHODS AND RESULTS: Massive doses of a Gram-positive, spore-forming bacterium were recovered from the animal feed, faeces of survived pigs and intestinal content of dead ones. The B. cereus MM1 strain was identified by MALDI-TOF MS and typified by RAPD-PCR. The isolate was tested for the production of PC-PLC, proteases, hemolysins and biofilm, for motility, as well as for the presence of genes encoding tissue-degrading enzymes and toxins. Antimicrobial resistance and pathogenicity in Galleria mellonella larvae were also investigated. Our results show that the isolated B. cereus strain is swimming-proficient, produces PC-PLC, proteases, hemolysins, biofilm and carries many virulence genes. The strain shows high pathogenicity in G. mellonella larvae. CONCLUSIONS: The isolated B. cereus strain demonstrates an aggressive profile of pathogenicity and virulence, being able to produce a wide range of determinants potentially hazardous to pigs' health. SIGNIFICANCE AND IMPACT OF STUDY: This study highlights the proficiency of B. cereus to behave as a devastating pathogen in swine if ingested at high doses and underlines that more stringent quality controls are needed for livestock feeds and supplements.

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.

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems.

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin's mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl's ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus.

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.

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.

Genotype and virulence gene analyses of Bacillus cereus group clinical isolates from the Chinese softshell turtle (Pelodiscus sinensis) in Taiwan.

Chinese softshell turtles (Pelodiscus sinensis) (CST) are susceptible to infections by bacteria belonging to the Bacillus cereus group (Bcg). Bcg includes several closely related species, two of which, B. cereus and B. thuringiensis, are pathogens of aquatic animals or insects. In the present study, we collected 57 Bcg isolates obtained from diseased CST from 2016 to 2019 in Kaohsiung and Pingtung, the areas with the most CST farms in Taiwan. All isolates were divided into four genotypes with two restriction enzymes, SmaI and NotI, by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Representative isolates from each genotype were subjected to phylogenetic tree analysis using 16S rDNA and pyruvate carboxylase genes as phylogenetic markers, and these CST isolates appeared in different clades. PCR was performed targeting six selected virulence genes, four of which were detected in CST isolates, including cytotoxin K (1/57), hblC of the haemolysin BL complex (46/57), nheA of the non-haemolytic enterotoxin complex (52/57) and enterotoxin FM (57/57), whereas cereulide synthetase and cereulide peptide synthase-like genes were not detected in any isolates.

The Bacillus virulome in endophthalmitis.

Bacillus cereus is recognized as a causative agent of gastrointestinal syndromes, but can also cause a devastating form of intraocular infection known as endophthalmitis. We have previously reported that the PlcR/PapR master virulence factor regulator system regulates intraocular virulence, and that the S-layer protein (SlpA) contributes to the severity of B. cereus endophthalmitis. To better understand the role of other B. cereus virulence genes in endophthalmitis, expression of a subset of factors was measured at the midpoint of disease progression in a murine model of endophthalmitis by RNA-Seq. Several cytolytic toxins were expressed at significantly higher levels in vivo than in BHI. The virulence regulators codY, gntR, and nprR were also expressed in vivo. However, at this timepoint, plcR/papR was not detectable, although we previously reported that a B. cereus mutant deficient in PlcR was attenuated in the eye. The motility-related genes fla, fliF, and motB, and the chemotaxis-related gene cheA were detected during infection. We have shown previously that motility and chemotaxis phenotypes are important in B. cereus endophthalmitis. The sodA2 variant of manganese superoxide dismutase was the most highly expressed gene in vivo. Expression of the surface layer protein gene, slpA, an activator of Toll-like receptors (TLR)-2 and -4, was also detected during infection, albeit at low levels. Genes expressed in a mouse model of Bacillus endophthalmitis might play crucial roles in the unique virulence of B. cereus endophthalmitis, and serve as candidates for novel therapies designed to attenuate the severity of this often blinding infection.

A virulent Bacillus cereus strain from deep-sea cold seep induces pyroptosis in a manner that involves NLRP3 inflammasome, JNK pathway, and lysosomal rupture.

Recent studies indicate that the Bacillus species is distributed in deep-sea environments. However, no specific studies on deep-sea Bacillus cereus have been documented. In the present work, we isolated a B. cereus strain, H2, from the deep-sea cold seep in South China Sea. We characterized the pathogenic potential of H2 and investigated H2-induced death of different types of cells. We found that H2 was capable of tissue dissemination and causing acute mortality in mice and fish following intraperitoneal/intramuscular injection. In vitro studies revealed that H2 infection of macrophages induced pyroptosis and activation of the NLRP3 inflammasome pathway that contributed partly to cell death. H2 infection activated p38, JNK, and ERK, but only JNK proved to participate in H2-triggered cell death. Reactive oxygen species (ROS) and intracellular Ca2+ were essential to H2-induced activation of JNK and NLRP3 inflammasome. In contrast, lysosomal rupture and cathepsins were required for H2-induced NLRP3 inflammasome activation but not for JNK activation. This study revealed for the first time the virulence characteristics of deep-sea B. cereus and provided new insights into the mechanism of B. cereus infection.

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.

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.

The Food Poisoning Toxins of Bacillus cereus.

Bacillus cereus is a ubiquitous soil bacterium responsible for two types of food-associated gastrointestinal diseases. While the emetic type, a food intoxication, manifests in nausea and vomiting, food infections with enteropathogenic strains cause diarrhea and abdominal pain. Causative toxins are the cyclic dodecadepsipeptide cereulide, and the proteinaceous enterotoxins hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe) and cytotoxin K (CytK), respectively. This review covers the current knowledge on distribution and genetic organization of the toxin genes, as well as mechanisms of enterotoxin gene regulation and toxin secretion. In this context, the exceptionally high variability of toxin production between single strains is highlighted. In addition, the mode of action of the pore-forming enterotoxins and their effect on target cells is described in detail. The main focus of this review are the two tripartite enterotoxin complexes Hbl and Nhe, but the latest findings on cereulide and CytK are also presented, as well as methods for toxin detection, and the contribution of further putative virulence factors to the diarrheal disease.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Bacillus cereus: Epidemiology, Virulence Factors, and Host-Pathogen Interactions.

The toxin-producing bacterium Bacillus cereus is an important and neglected human pathogen and a common cause of food poisoning. Several toxins have been implicated in disease, including the pore-forming toxins hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE). Recent work revealed that HBL binds to the mammalian surface receptors LITAF and CDIP1 and that both HBL and NHE induce potassium efflux and activate the NLRP3 inflammasome, leading to pyroptosis. These mammalian receptors, in part, contribute to inflammation and pathology. Other putative virulence factors of B. cereus include cytotoxin K, cereulide, metalloproteases, sphingomyelinase, and phospholipases. In this review, we highlight the latest progress in our understanding of B. cereus biology, epidemiology, and pathogenesis, and discuss potential new directions for research in this field.

Gastric Ulceration and Immune Suppression in Weaned Piglets Associated with Feed-Borne Bacillus cereus and Aspergillus fumigatus.

As a multifactorial cause, gastric ulceration-mediated diarrhea is widely prevalent in the weaned piglets, impairing pig health and economic benefits. With full implementation of antibiotic stewardship programs in China, Bacillus cereus (B. cereus) and Aspergillus fumigatus (A. fumigatus) were identified frequently in porcine feedstuffs and feeds of the animal industry. Association between feed-borne B. cereus and frequent diarrhea remains unclear. In the present study, we conducted a survey of B. cereus and A. fumigatus from feeds and feedstuffs in pig farms during hot season. Interestingly, B. cereus, B. subtilis, B. licheniformis and B. thuringinesis were isolated and identified from piglets' starter meals to sow feeds, accounting for 56.1%, 23.7%, 13.7% and 6.5%, respectively. Obviously, both B. cereus and B. subtili were dominant contaminants in the survey. In an in vitro study, Deoxynivalenol (DON) contents were determined in a dose-dependent manner post fermentation with B. cereus (405 and DawuC). Subsequently, 36 weaned piglets were randomly assigned to four groups and the piglets simultaneously received the combination of virulent B. cereus (Dawu C) and A. fumigatus while animals were inoculated with B. cereus (Dawu C), A. fumigatus or PBS as the control group. Clinically, piglets developed yellow diarrhea on day 5 and significant reductions of relative body weight were observed in the B. cereus group, and co-infection group. More importantly, IgG titers against Classical swine fever virus (CSFV) and Porcine epidemic diarrhea (PED) were reduced dramatically during 14-day observation in co-infection group, the B. cereus (Dawu C) group or the A. fumigatus group. However, lower Foot and mouth disease (FMD) -specific antibodies were reduced on day 7 compared to those of the control group. Additionally, lower lymphocyte proliferations were found in the B. cereus group and the co-infection group compared to the control group. Postmortem, higher lesions of gastric ulceration were observed in the B. cereus group and the co-infection group from day 7 to day 14 compared with those of the A. fumigatus group and the control group. Compared to the A. fumigatus group, higher DON contents were detected in the stomach inoculated with B. cereus and the co-infection with A. fumigatus. In conclusion, our data support the hypothesis that B. cereus might be associated with severe diarrhea by inducing gastric ulcerations and A. fumigatus might aggravate immune suppression, threating a sustainable swine industry. It is urgently needed to control feed-borne B. cereus contamination.

The Bacillus cereus Food Infection as Multifactorial Process.

The ubiquitous soil bacterium Bacillus cereus presents major challenges to food safety. It is responsible for two types of food poisoning, the emetic form due to food intoxication and the diarrheal form emerging from food infections with enteropathogenic strains, also known as toxico-infections, which are the subject of this review. The diarrheal type of food poisoning emerges after production of enterotoxins by viable bacteria in the human intestine. Basically, the manifestation of the disease is, however, the result of a multifactorial process, including B. cereus prevalence and survival in different foods, survival of the stomach passage, spore germination, motility, adhesion, and finally enterotoxin production in the intestine. Moreover, all of these processes are influenced by the consumed foodstuffs as well as the intestinal microbiota which have, therefore, to be considered for a reliable prediction of the hazardous potential of contaminated foods. Current knowledge regarding these single aspects is summarized in this review aiming for risk-oriented diagnostics for enteropathogenic B. cereus.

Bacillus cereus Decreases NHE and CLO Exotoxin Synthesis to Maintain Appropriate Proteome Dynamics During Growth at Low Temperature.

Cellular proteomes and exoproteomes are dynamic, allowing pathogens to respond to environmental conditions to sustain growth and virulence. Bacillus cereus is an important food-borne pathogen causing intoxication via emetic toxin and/or multiple protein exotoxins. Here, we compared the dynamics of the cellular proteome and exoproteome of emetic B. cereus cells grown at low (16 °C) and high (30 °C) temperature. Tandem mass spectrometry (MS/MS)-based shotgun proteomics analysis identified 2063 cellular proteins and 900 extracellular proteins. Hierarchical clustering following principal component analysis indicated that in B. cereus the abundance of a subset of these proteins-including cold-stress responders, and exotoxins non-hemolytic enterotoxin (NHE) and hemolysin I (cereolysin O (CLO))-decreased at low temperature, and that this subset governs the dynamics of the cellular proteome. NHE, and to a lesser extent CLO, also contributed significantly to exoproteome dynamics; with decreased abundances in the low-temperature exoproteome, especially in late growth stages. Our data therefore indicate that B. cereus may reduce its production of secreted protein toxins to maintain appropriate proteome dynamics, perhaps using catabolite repression to conserve energy for growth in cold-stress conditions, at the expense of virulence.

Structural Modeling of Cell Wall Peptidase CwpFM (EntFM) Reveals Distinct Intrinsically Disordered Extensions Specific to Pathogenic Bacillus cereus Strains.

The emergence of B. cereus as an opportunistic food-borne pathogen has intensified the need to distinguish strains of public health concern. The heterogeneity of the diseases associated with B. cereus infections emphasizes the versatility of these bacteria strains to colonize their host. Nevertheless, the molecular basis of these differences remains unclear. Several toxins are involved in virulence, particularly in gastrointestinal disorders, but there are currently no biological markers able to differentiate pathogenic from harmless strains. We have previously shown that CwpFM is a cell wall peptidase involved in B. cereus virulence. Here, we report a sequence/structure/function characterization of 39 CwpFM sequences, chosen from a collection of B. cereus with diverse virulence phenotypes, from harmless to highly pathogenic strains. CwpFM is homology-modeled in silico as an exported papain-like endopeptidase, with an N-terminal end composed of three successive bacterial Src Homology 3 domains (SH3b1-3) likely to control protein-protein interactions in signaling pathways, and a C-terminal end that contains a catalytic NLPC_P60 domain primed to form a competent active site. We confirmed in vitro that CwpFM is an endopeptidase with a moderate peptidoglycan hydrolase activity. Remarkably, CwpFMs from pathogenic strains harbor a specific stretch of twenty residues intrinsically disordered, inserted between the SH3b3 and the catalytic NLPC_P60 domain. This strongly suggests this linker as a marker of differentiation between B. cereus strains. We believe that our findings improve our understanding of the pathogenicity of B. cereus while advancing both clinical diagnosis and food safety.

Cereulide Synthetase Acquisition and Loss Events within the Evolutionary History of Group III Bacillus cereus Sensu Lato Facilitate the Transition between Emetic and Diarrheal Foodborne Pathogens.

Cereulide-producing members of Bacillus cereussensu lato group III (also known as emetic B. cereus) possess cereulide synthetase, a plasmid-encoded, nonribosomal peptide synthetase encoded by the ces gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by emetic B. cereus has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characterize group III B. cereussensu lato genomes which possess ces (ces positive) alongside their closely related, ces-negative counterparts (i) to assess the genomic diversity encompassed by emetic B. cereus and (ii) to identify potential ces loss and/or gain events within the evolutionary history of the high-risk and medically relevant sequence type (ST) 26 lineage often associated with emetic foodborne illness. Using all publicly available ces-positive group III B. cereussensu lato genomes and the ces-negative genomes interspersed among them (n = 159), we show that emetic B. cereus is not clonal; rather, multiple lineages within group III harbor cereulide-producing strains, all of which share an ancestor incapable of producing cereulide (posterior probability = 0.86 to 0.89). Members of ST 26 share an ancestor that existed circa 1748 (95% highest posterior density [HPD] interval = 1246.89 to 1915.64) and first acquired the ability to produce cereulide before 1876 (95% HPD = 1641.43 to 1946.70). Within ST 26 alone, two subsequent ces gain events were observed, as well as three ces loss events, including among isolates responsible for B. cereussensu lato toxicoinfection (i.e., "diarrheal" illness).IMPORTANCEB. cereus is responsible for thousands of cases of foodborne disease each year worldwide, causing two distinct forms of illness: (i) intoxication via cereulide (i.e., emetic syndrome) or (ii) toxicoinfection via multiple enterotoxins (i.e., diarrheal syndrome). Here, we show that emetic B. cereus is not a clonal, homogenous unit that resulted from a single cereulide synthetase gain event followed by subsequent proliferation; rather, cereulide synthetase acquisition and loss is a dynamic, ongoing process that occurs across lineages, allowing some group III B. cereussensu lato populations to oscillate between diarrheal and emetic foodborne pathogens over the course of their evolutionary histories. We also highlight the care that must be taken when selecting a reference genome for whole-genome sequencing-based investigation of emetic B. cereussensu lato outbreaks, since some reference genome selections can lead to a confounding loss of resolution and potentially hinder epidemiological investigations.

Traceability of potential enterotoxigenic Bacillus cereus in bee-pollen samples from Argentina throughout the production process.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.