Bacillus cereus is a ubiquitous soil bacterium responsible for two types of food-associated gastrointestinal diseases. While the emetic syndrome is caused by the cyclic depsipeptide cereulide, proteinaceous enterotoxins provoke the diarrheal disease. Here, an overview on the distribution of the main toxin genes/operons ces (encoding cereulide), hbl (encoding the tripartite hemolysin BL), nhe (encoding the tripartite non-hemolytic enterotoxin), and cytK (encoding the single protein cytotoxin K) within the B. cereus group is given.
Bacillus cereus is estimated to be responsible for 1.4%–12% of all food poisoning outbreaks worldwide. In the European Union, bacterial toxins (Clostridium, Staphylococcus and B. cereus) accounted for 17.7% (2016) and 15.9% (2017) of all registered food- and water-borne outbreaks, which ranked them second behind Salmonella. With 98 registered outbreaks in the EU in 2018, B. cereus toxins ranked in fifth place behind Salmonella, Campylobacter, the norovirus and Staphylococcus toxins. Among these was also one large food poisoning outbreak with more than 100 affected persons. Furthermore, six fatal cases were attributed to bacterial toxins (Clostridium botulinum, Clostridium perfringens and B. cereus) .
Basically, B. cereus is responsible for two types of gastrointestinal diseases. The emetic kind of illness is mainly characterized by nausea and emesis, which appear as soon as half an hour after consumption of the contaminated food and are clinically indistinguishable from intoxications with Staphylococcus aureus enterotoxins. In this classical food intoxication, the emetic toxin cereulide is pre-formed during vegetative growth of B. cereus in foodstuffs and the consumption of the bacteria is not necessary . Indeed, there are several reports of outbreaks where only the cereulide toxin was detected in the food, but no bacteria could be isolated. Nevertheless, it is generally thought that at least 103–105 B. cereus per g food are needed to produce cereulide in disease-provoking concentrations . Cereulide is a cyclic dodecadepsipeptide with a molecular weight of 1.2 kDa. The basic repeated amino acid sequence [D-O-Leu D-Ala L-O-Val D-Val]3 is extremely stable towards heat, acid or digestive enzymes and, thus, the toxin can hardly be removed or inactivated. Usually, the emetic form of disease is self-limiting and symptoms disappear after 6–24 h. Nevertheless, some severe and fatal outbreaks mostly related to liver failure are reported . Due to the ubiquitous nature of the pathogen and its production of highly resistant spores, B. cereus is frequently found in various kinds of food. Historically, starchy foodstuffs such as rice or pasta are connected to food intoxications with emetic B. cereus, but more recently evidence is growing that emetic B. cereus are much more volatile than once thought. The comprehensive analysis of a total of 3654 food samples obtained from suspected food-borne illnesses with a preliminary report of vomiting, collected over a period of seven years, revealed that emetic B. cereus strains were detected in a broad diversity of foods, including vegetables, fruit products, sauces, soups, and salads as well as milk and meat products.
The second, diarrheal form of food poisoning is also associated with a variety of different foodstuffs. This form of disease manifests mainly in diarrhea and abdominal cramps, similar to food poisoning by Clostridium perfringens type A. Symptoms occur after approximately 8–16 h. This incubation time is typical for toxico-infections, in which the toxins are produced by viable bacteria inside the human intestine. Unlike cereulide, enterotoxins pre-formed in foods most likely do not contribute to the disease, as they are considered sensitive towards heat, acids or proteases. Thus, vegetative B. cereus and, especially, spores must be consumed. The infective dose is estimated between 105–108 cfu/g or 104–109 cfu/g vegetative cells or spores. The course of disease is mainly mild and—after approximately 12–24 h—self-limiting. Fatal outbreaks are only very rarely reported. A food infection with enteropathogenic B. cereus can be seen as a multifactorial process, as a number of individual steps have to be considered before the onset of the disease, including prevalence and survival of B. cereus in different foodstuffs, survival of the stomach passage, germination of spores, active movement towards and adhesion to the intestinal epithelium, enterotoxin production under intestinal conditions, as well as the influence of consumed foods and the intestinal microbiota on these processes.
B. cereus is a ubiquitous soil bacterium and can thus be found worldwide in the ground, in dust, or on different foods. Early studies pointed to an occurrence of diarrheal or emetic outbreaks according to country-specific dietary habits, with the emetic form manifesting in Great Britain or Japan, and the diarrheal form rather in Northern Europe or the USA. Lately, both syndromes have been reported from all over the world. Basically, emetic strains are found less frequently in foods as well as in the environment than enteropathogenic strains. In a multitude of studies, new isolates were screened for the presence of the toxin genes nhe (ABC), hbl (CDAB), cytK (1,2), entFM, and ces. In some studies, the presence of bceT (enterotoxin T) was also assessed; however, its enterotoxic capacity is disproven. Virulence/enterotoxin gene patterns are compiled for B. cereus which has been mainly isolated from foods, but also from clinical, soil and environmental samples worldwide. Generally, those patterns are highly diverse.
Common distribution of the toxin genes is approximately 85%–100% nhe (ABC), approximately 40%–70% hbl (CDA), approximately 40%–70% cytK-2, very few ces+, typically no cytK-1+, and—if tested—approximately 60%–100% entFM, which has been detected in studies from Europe ], South America, North America, Asiaand Africa. Nevertheless, in some studies, a connection was established between toxin gene patterns and geographical location of the isolates. Drewnowska et al. found that strains possessing nheA, hblA and cytK-2 were predominant in regions with arid hot climate, and were comparably rare in continental cold climates. This is supported by other studies suggesting that geographic origin might have an impact on the conservation of hblA among B. cereus populations. Zhang et al. also claim a “regional feature for toxin gene distribution”.
Besides geographical location, toxin gene patterns seem to be also influenced by the kind of foodstuffs analyzed. For instance, Berthold-Pluta et al. found higher prevalence of nhe+ and hbl+, but lower prevalence of ces+ strains in food products of animal than of plant origin. Rossi et al. showed that strains from dairy products had significantly lower cytK-2 and hblCDA prevalence than strains from equipment or raw milk, and Hwang and Park found hbl in >95% of tested ready-to-eat (RTE) foods, but only in 30% of infant formulas. Furthermore, the prevalence of cytK-2 was comparably low in the latter food.
Studies were also conducted comparing food related and food poisoning related strains. Santos et al. showed that food poisoning strains had a higher occurrence and higher genetic diversity of plcR-papR, nheA, cytK-2, plcA, and gyrB genes than strains isolated from soil or foods. CytK and the combination hbl-nhe-cytK were more often found among food poisoning related than among food related strains
Generally, all B. cereus isolates can be categorized into seven different toxin profiles: A (nhe+, hbl+, cytK+), B (nhe+, cytK+, ces+), C (nhe+, hbl+), D (nhe+, cytK+), E (nhe+, ces+), F (nhe+), and G (cytK+). In fact, the hbl genes alone or a combination of ces and hbl have only been reported for the very few emetic Bacillus weihenstephanensis isolates described so far. There are further studies showing “unusual” results, particularly low or no prevalence of nhe or extraordinarily high prevalence of hbl or ces, which must be interpreted cautiously, especially as nhe is well known for its molecular heterogeneity. Thus, the choice of detection methods, especially primer pairs for nhe, can have a crucial influence on the results.
However, it has to be mentioned that the presence of enterotoxin genes or a certain toxin gene profile does not necessarily allow conclusions on the toxic activity of a B. cereus isolate. In our own studies, we chose pairs of strains with an identical toxin gene profile, but one strain exhibited high and the other low toxic activity both under routine laboratory and simulated intestinal growth conditions. The reasons for this are so far not completely understood, but it is believed that highly variable and strain-specific mechanisms in toxin gene transcription, posttranscriptional and posttranslational modification and protein secretion are involved.
In many of the studies mentioned in Section 2.1, often only B. cereus sensu lato (s. l.) strains are investigated, meaning there is no differentiation between the members of the B. cereus group. In routine microbiological diagnostics, only “presumptive” B. cereus are detected on selective culture media according to international standards (ISO 7932:2005-03). The B. cereus group comprises at least eight species: B. anthracis, B. cereus sensu stricto (s. s.), B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, B. cytotoxicus and B. toyonensis. Additionally, more and more species such as B. wiedmannii, B. bingmayongensis, B. gaemokensis, B. manliponensis, and others are described. Generally, they exhibit high genetic similarities and, thus, it has been suggested that they be considered as one species or to completely change the taxonomic nomenclature of the B. cereus group. Species definition is historically based on phenotypes or clinical and economical relevance. While the unique characteristics of B. anthracis, emetic B. cereus and B. thuringiensis are located on plasmids, the enterotoxins are chromosome-coded and can thus be present throughout the B. cereus group. This is particularly problematic for the assessment of B. thuringiensis, which is frequently used as biopesticide worldwide. B. thuringiensis has been isolated from a variety of foodstuffs and the presence of the enterotoxin genes nhe, hbl and cytK-2 has been shown, with similar percentages as for B. cereus, while ces genes have not been found. Enterotoxin production and cytotoxic activity have also been shown , and B. thuringiensis could therefore be involved in food poisoning outbreaks. Consequently, it was debated whether the B. thuringiensis-associated biopesticides represent a risk for public health. To clarify this question, there is a demand for simple methods enabling a clear discrimination between B. cereus and B. thuringiensis in routine food and clinical diagnostics as well as for unequivocal identification of the strains used as biopesticides.
Next to B. cereus and B. thuringiensis, further species of the B. cereus group were isolated from foods and the presence of enterotoxin genes was proven, such as B. anthracis, B. mycoides, B. pseudomycoides, B. toyonensis , and B. weihenstephanensis . It has also been shown that Bacillus spp. outside the B. cereus group can harbor one or more enterotoxin genes . For instance, Mäntynen and Lindström found hblA+ B. pasteurii DSM 33, B. smithii DSM 459, and Bacillus sp. DSM 466 . Nhe and/or hbl genes were also detected in B. amyloliquefaciens, B. circulans, B. lentimorbis, and B. pasteurii . On the other hand, From et al. found no enterotoxin genes outside the B. cereus group in the strains analyzed .
According to MLST (multi-locus sequence typing), AFLP (amplified fragment length polymorphism) and whole genome sequencing, the B. cereus group was first assigned to three phylogenetic groups (clades), then seven (panC types), and later nine, which do not correlate with species definition. Prevalence of enterotoxin genes and their profiles were also compared to phylogenetic groups. B. cereus isolates from dairy products in Brazil with approximately 50% cytK-2 and hbl, and approximately 85% nhe were mostly assigned to phylogenetic group III. Group IV and V showed significantly higher prevalence of hblCDA and group IV showed additionally higher prevalence of cytK-2 . In another study on dairy isolates, strains of clade IIIc had no hblCDA operon, while strains of clade IV carried it and produced the Hbl toxin, whereas strains of clade VI carried the gene but did not produce the toxin . Furthermore, a broad distribution of enterotoxin genes among seven phylogenetic clades, in which dairy-associated isolates were divided, was shown . Okutani et al. investigated the genomes of 44 B. cereus group isolates from soil, animal and food poisoning cases in Japan. Strains were assigned to four different panC types and five different clades. The nhe operon was found in all strains tested, while ces was detected only in the food poisoning strains. When the presence or absence of virulence-associated genes was statistically analyzed, the majority of soil and animal isolates was part of overlapping clusters, while three of the four food poisoning isolates formed a distinct cluster . Furthermore, the hbl and the ces genes were significantly correlated with the phylogenetic group. Several further studies suggested that the toxic potential of B. cereus s. l. strains depends rather on the phylogenetic group than on the species.