Bacillus cereus – Culture; Identification of Bacillus cereus; Molecular diagnosis of genes codifying for toxins (PCR and sequencing)
Bacillus cereus is a ubiquitous bacterium of the family Bacillaceae that includes gram-positive rod shape bacteria that are large and sporulated, with a central or subterminal ellipsoidal spore. This bacterium is frequently found in soil, dust, vegetables, cereals, flours, spices, medicinal plants, natural waters, and in some raw or processed foods, such as rice-based dishes, such as fried rice, based on pasta, based on meat products such as chicken or dairy products, among others.
This species is currently considered a group (Bacillus cereus group) in which six species are included: B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides and B. weihenstephanensis. Some of these species are mobile by peritrichous flagella, but others are non-mobile, and even in the species considered mobile there are non-mobile variants, so this characteristic cannot condition their identification. They are facultative aerobic-anaerobic bacteria, gram-positive although in aged cultures, the Gram stain can be variable, as occurs with B. cereus. From the metabolic point of view it has catalase, reduces nitrates to nitrites, gives the Voges Proskauer reaction, ferments glucose, sucrose, salicin and glycerol, does not ferment mannitol, nor arabinose and produces lecithinase. The characteristics of lecithinase production and the absence of mannitol fermentation are used for the design of some differential selective media.
B. cereus can produce enterotoxins. Food poisoning by this bacterium can occur when the food is prepared and is stored without adequate refrigeration for several hours before consumption or reaches a concentration of > 105 to 108 CFU/g or mL. Foods implicated in outbreaks include cooked meats and vegetables, boiled or fried rice, vanilla sauces, custards, soups, and raw vegetable sprouts. The consumption of food contaminated with Bacillus cereus has been implicated in two types of manifestations: 1) first and best known, the diarrheal syndrome, characterized by abdominal pain and non-bloody watery diarrhea, after an incubation period of 8 to 16 hours after the ingestion of a contaminated food, with a duration of the manifestations of 12 to 24 hours; 2) second, the emetic syndrome, characterized by an acute manifestation with nausea and vomiting, which appears after 1 to 5 hours of consumption of the contaminated food, and the general absence of diarrhea. Both syndromes are due to the production of exotoxins by B. cereus and in general they are mild processes, although cases have been described that have required hospitalization, and some cases have even been described with death.
The diarrheal syndrome is caused by three thermolabile enterotoxins, so this syndrome requires the intake of food contaminated with spores, which once in the small intestine will germinate and release the enterotoxins during their growth. The enterotoxins described in Bacillus cereus are: two protein complexes of hemolysin BL (HBL) and non-hemolytic enterotoxin (NHE: Non-hemolytic enterotoxin); Cytotoxin K (CytK); and FM enterotoxin (EntFM).
Some of these enterotoxins have been described in other Bacillus species (Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus mojavensis and Bacillus fusiformis), so it should be taken into account in the absence of Bacillus cereus, and the presence of some other species.
The emetic syndrome is caused by a thermostable toxin, called cereulide, which corresponds to a non-ribosomal cyclic peptide (DO-Leu-D-Ala-LO-Val-L-Val)3, encoded by the ces gene of the (Non-ribosomal peptide synthetase gene). This toxin is ingested preformed into food, and is toxic for the mitochondria by acting as an ionophore (captured and transporter) for potassium ions.
For Bacillus cereus to cause these syndromes, it is required that:
- the food is highly contaminated (concentrations of 106 to 108 CFU/g or mL).
- the food has been subjected to insufficient heat treatment to destroy its spores.
In order to detect their presence and carry out their counting, culture media containing inhibitors should be used to prevent the development of other microorganisms present in the sample and evidencing a differential characteristic such as lecithinase and the absence of fermentation of mannitol. Examples of these culture media are the Mossel´s MYP agar (Mannitol-Egg Yolk-Polymixin Agar; Agar MYPA), that has been considered the standard medium, although with little scarce selectivity, so it does not totally inhibit the accompanying flora and can be masked the development of B. cereus. To be able to detect it requires at least the presence of 10 CFU/g or per mL of B. cereus. Other authors recommend the Bacara medium, which is a selective and differential chromogenic medium that facilitates the growth and identification of B. cereus, inhibiting the growth of the accompanying flora. The colonies appear pink-orange surrounded by a zone of precipitation by the lecithinase activity on the lecithin contained in the culture medium. In order to identify the species Bacillus cereus requires a differential diagnosis using several metabolic tests that exclude the other species of the Bacillus cereus group: B. cereus, B. thuringiensis, B. anthracis, B. mycoides, and B. weihenstephanensis.
The diarrheal syndrome is mediated by several exotoxins, encoded by different genes. To detect the production of exotoxins, cell cultures were used that are exposed to the supernatants of Bacillus cereus cultures. However, genomic amplification (PCR) methods have now been developed to detect the genes that produce these toxins once a Bacillus cereus isolate has been obtained in culture. Commercial tests (kits) have also been developed to detect toxins, but all have the problem of being based on an antigen-antibody reaction using antibodies that react with protein toxin. Since there are several different toxins, the existing kits do not detect all of them, so their sensitivity is not complete.
Cell methods: To detect toxins through the damaging effect they cause in cell cultures in vitro, the following have been used: McCoy cell culture, CHO cell cultures (Chinesse Hamster Ovary), or Hep-2 cell cultures. A cytopathic effect on cell proliferation or vacuolization occurs in cell cultures.
Immunological methods: commercial tests (kits) to detect the enterotoxin of Bacillus cereus do not have an acceptance because they do not identify the toxin detected. The Oxoid kit (Bacillus cereus Enterotoxin-Reverse Passive Latex Agglutination-RPLA) detects the L2 component of the hemolysin BL, and the Tecra kit (Bacillus diarrhoeal Enterotroxin Visual Immunoassay) apparently detects two non-toxic proteins.
Molecular methods: each of the enterotoxins would be constituted by several subunits that in turn are encoded by different genes. Therefore, in order to carry out a complete study, each of the genes that can be made by the different components must be detected, since strains have been found in which all the genes of each of the toxins are not detected. These genes are:
- HBL complex (hemolytic enterotoxin). This toxin is a three-component hemolysin (binding protein B-binding-encoded by hblB gene, and lytic components L1 and L2 encoded by hblC and hblD genes, respectively):
- Gen hblB
- Gen hblC
- Gen hblD
- NHE complex (non-haemolytic enterotoxin), with three components, none of them hemolytic:
- Gen nheA
- Gen nheB
- Gen nheC
- Cytotoxin K (cytK)
- FM Enterotoxin (EntFM)
- Enterotoxin T
- Gen bceT
- Citotoxina K (cytK)
- Enterotoxina FM (EntFM)
- Enterotoxina T
- Gen bceT
Tests carried out in IVAMI:
- Qualitative culture in liquid culture media and subsequent sowing in plates of differential selective culture medium.
- Quantitative culture in differential selective culture medium.
- Identification of the species Bacillus cereus by sequencing the 16S rRNA gene.
- Detection of genes coding for the toxins involved in emetic syndrome and diarrheal syndrome.
Emetic syndrome toxin:
- Cereulide (genes of the non-ribosomal peptide synthetase)
Toxins of diarrheal syndrome:
- Enterotoxin HBL (hemolytic enterotoxin)
- Gen hblC to detect component L1.
- Enterotoxin NHE (non-hemolytic enterotoxin)
- nheB gene to detect component B.
- Cytotoxin K (cytK)
- FM Enterotoxin (EntFM)
- Enterotoxin T
- Gen bceT.
- Any type of food suspected of causing intoxication, both fresh (without cooking) and cooked.
Conservation and shipment of the sample:
- Refrigerated (preferred) for less than 2 days.
- Frozen: more than 2 days.
- Cultivation for isolation: 48 to 72 hours.
- PCR for molecular targets: 48 hours.
Cost of the test:
- Cultivation for isolation and microscopic observation with a presumptive result of the presence of Bacillus cereus: Consult to firstname.lastname@example.org.
- Quantitative culture for Bacillus cereus in elective and differential environments: Consult to email@example.com.
- Cultivation, microscopic observation and identification by sequencing of the 16S RNA gene: Consult to firstname.lastname@example.org.
- Culture for isolation, microscopic observation and detection of genes coding for toxins (each gene): Consult to email@example.com.