Bacillus subtilis (Bacillus subtilis species group): qualitative and quantitative culture, identification and detection of toxins  

Bacillus subtilis is considered a group of very similar bacteria (Bacillus subtilis Group), which would include the following species: Bacillus subtilis sensu stricto, B. amyloliquefaciens, B. licheniformis, B. atrophaeus, B. vallismortis, B. pumilus, B. donorensis, B. mojavensis, B. axarquiensis, B. malacitensis, tequilensis B. and B. venezuelensis). This group of Gram - positive bacteria, aerobic and spore forming, is very ubicuitario (water, soil in its upper layers, aquatic sediments, air, plant residues, digestive tract of animals and humans, ...). In people have come to be in concentrations up to ^{April 10} spores / gram, which seems too abundant to proceed ingestion and rather indicate that he would be to multiply in the gut. The species can be difficult to differentiate because there are very few phenotypic or biochemical characteristics that distinguish them . Moreover most of these species have high homology in the 16S rRNA gene, sometimes over 99%, so the sequencing of this gene does not allow to differentiate. Only methods allow DNA-DNA hybridization find homologies lower than 70%, by differences in other genomic regions, which has led some authors have recommended phylogenetic analysis of multiple genetic loci (multilocus) to differentiate. Bacillus subtilis species the sensu stricto has 72% homology with Bacillus cereus. One of the differences of these various species Bacillus cereus is their inability to grow below 10 ° C, while Bacillus cereus if you can do.

Being very ubicuitarias bacteria, and found in the upper soil layers and in the digestive tract of animals, is relatively easy to contaminate fresh food, particularly those that can make contact with the ground, and the vegetable. They can also contaminate dairy products, meat, baby food, rice dishes, spices and cereals). Its spores can survive cooking, procedures pasteurizing milk or fruit juices, and preparing homemade meals, so the spores can germinate later and multiply vegetatively when environmental conditions are conducive him.

Generally they considered nonpathogenic species, and have been used as probiotics in healthy individuals. However, they have described isolated or grouped in outbreaks with digestive symptoms, such as gastroenteritis, due to the consumption of foods that have proliferated cases, and attributed to the ingestion of a preformed toxin in food (emetic syndrome), or the generation toxin to digestive level (diarrheic syndrome). However, information on food poisoning species Bacillus subtilis group are rare and has only been involved in occasional outbreaks. Some of these species have been found in infections of immunocompromised patients.

Regarding food, food poisoning outbreaks attributable to this group of species have also been very casual. It described the production of an extracellular toxin, subtilisin (Bacillus subtilis) of low toxigenicity and only related to the development of allergic reactions in individuals who work with industrial crops of this species. Also described the production of a thermostable protein toxin, the amilosina, (Bacillus amyloliquefaciens), it would be forming ion channels in cell membranes. Other toxins would be a thermostable lipopeptides as lichepysina A (Bacillus licheniformis) described in a fatal case; the pumilacidina (Bacillus pumilus) in a severe case of food poisoning; and surfactin B (Bacillus subtilis and Bacillus mojavensis)

Symptoms of food poisoning Bacillus subtilis are very similar to those caused by poisoning Bacillus cereus, which causes a diarrheal and emetic box, the first of which related to the number of ingested bacteria, and the second related to the amount of emetic toxin ingested. The onset of symptoms could be as early as 10 minutes of ingestion and may last up to 2 days. Its relation to the production of boxes of food poisoning occur when a concentration of 10 ⁶ CFU / gram of food is reached (for other species of Bacillus, such as Bacillus cereus would suffice to reach the number of 10 ⁵ cells / gram).  

Diagnostic methods

Diagnosing cases of food intoxicacón is based on qualitative detection, and better yet, quantitative, their presence by methods enrichment culture (qualitative culture), and counting (quantitative culture), identification and detection its toxigenic capacity.

Methods of qualitative and quantitative crop: These methods aim to demonstrate its presence and its concentration, as when food poisoning has implications in concentration must be less than 10 ⁶ CFU / mL or per gram.  

Detection of toxigenic capacity: various types of methods can be used to detect the production of toxins by this group of bacteria.

• Methods Cell culture: Cell culture Hep-2 cells and CHO (Chinese Hamster Ovary) cells, have been used to highlight the presence of these toxins in culture filtrates of these bacteria, by detecting the effect produced in cells in culture by adding culture filtrates of this bacterium. The effect is determined by reducing metabolic activity in cell cultures exposed to the filtrates containing the toxin.

• Immunological reactions based methods: These methods marketed (kits), using specific antibodies against enterotoxigenic some of the components that can produce these bacteria. As methods for detecting one or other of the enterotoxigenic components may fail when the isolated strain produces a different enterotoxin detected by the kit used. There is scientific publications show that only 36% of cases of positive cases by cytotoxicity assays, which are immunological assays (Beatie SH, Williams AG. Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. With an improved citotoxicity Lett Appl Microbiol 1999 assay, 28.. 221-225).

• Molecular Methods: these methods detect any of the genes encoding enterotoxins: HBL enterotoxin hemolytic (HBLA genes, hblC, hblD); T enterotoxin (BceT gene), or other. Enterotoxins produced by Bacillus species corresponds to one of the following types:

• • HBL complex (hemolytic enterotoxin). This toxin is a hemolysin three components (B -binding- binding protein encoded by gene HBLA and lytic components L1 and L2 gene - encoded hblC and hblD, respectively):
    • • Gen HBLA
      • Gen hblB
      • Gen hblC
      • Gern hblD
  • NHE complex (non - hemolytic enterotoxin) with three components, none of them Hemolytic:
    • • Gen nheA
      • Gen nheB
      • Gen nhec
  • Cytotoxin K (CYTK)
  • Enterotoxin FM (EntFM)
  • enterotoxin T
    • • Gen bceT  

Tests in IVAMI:

• Qualitative culture media and cultivation liquid plating medium selective culture differential
• Quantitative cultures with differential selective culture
• Identification of the group of species Bacillus subtilis by sequencing 16S rRNA gene.
• Detection of genes encoding toxins involved in the emetic syndrome and diarrhea syndrome, by PCR amplification of the corresponding genes:
  • Cereulide (ces gene ribosomal peptide synthetase-no)
  • HBL enterotoxin (hemolytic enterotoxin)
    • HblC to detect L1 gene component.
    • NHE enterotoxin (non-hemolytic enterotoxin)
      • NheB gene to detect the component B.
  • Cytotoxin K (CYTK)
  • Enterotoxin FM (EntFM)
  • enterotoxin T
    • • Gen bceT.
  • Detection of toxin production by exposure of cultured Hep-2 cells or CHO cells, culture filtrates, with metiltetrazolio reduction assay (MTT).  

Recommended sample:

• Any suspicion of poisoning caused both fresh (uncooked) and cooked food.  

Preservation and shipment of sample:

• Refrigerated (preferred) for less than 2 days.
• Frozen: over 2 days.  

Delivery term:

• Culture for isolation: 48 to 72 hours.
• Molecular identification sequencing: 48 hours
• Detection of toxin producing genes by PCR: 48 hours.
• Detection of toxin production by exposure to cell culture filtrates cultvos: 5 days.  

Cost of testing:

• Consult ivami@ivami.com