Food Testing- Bacillus species. Dr Roy Betts Head of Microbiology Campden BRI, Chipping Campden. UK

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1 Food Testing- Bacillus species. Dr Roy Betts Head of Microbiology Campden BRI, Chipping Campden. UK

2 Who are Campden BRI? Independent Food Research Organisation Membership based with over 2400 members International Client Base- 75 countries 350 staff 3500m 2 laboratories, 3000m 2 processing areas Microbiology 45 Staff ISO 9001 certified, ISO for many tests

3 Our Locations Hungary Chipping Campden Nutfield

4 Bacillus -----Why Test foods? Bacillus spp. Spore formers- can survive cooking Some are food spoilage organisms Some are human pathogens

5 Food Spoilage E.g. Rope in bread- B.subtilis Spoilage of heat processed foods Spore survival & growth e.g. Geobacillus stearothermophilus

6 Food Pathogens B.cereus Emetic toxin (cereulide)- forms in food, heat resistant, vomiting Diarrhoeal-forms during vegetative growth in the gut. Other Bacillus spp. Public Health England 2009 note: subtilis, licheniformis, pumilis, amyloliquifaciens as an issue (despite EFSA 2013 Qualified Presumption of Safety) Some UK companies are looking for total Bacillus numbers not B.cereus due to this.

7 Bacillus cereus Group B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis, B. toyonensis, and B. anthracis Difficulty to differentiate using current standard test methods B.anthracis: non-haemolytic and non-motile Very generally in foods, presumptive B.cereus: >10 5 seen as an health issue < 10 3 seen as acceptable

8 Reactions of Various Bacillus spp. Species Lecithinase Motility Penicillin susceptibility Crystal formation Bacillus anthracis -/w - S - Bacillus cereus + + R - Bacillus megaterium - + R - Bacillus mycoides + - R - Bacillus thuringiensis + + R + Bacillus circulans - + R - Bacillus coagulans - + R - Bacillus licheniformis - + R - Bacillus pumilus - + R - Bacillus subtilis - + R - Bacillus sphaericus - + R -

9 B.cereus group differentiation US FDA Feature B. cereus B. thuringiensis B. mycoides B. weihenstephab. anthracis nensis Gram reaction + (a) Catalase Motility +/ (b) +/ (c) + Reduction of nitrate Tyrosine + + +/ + (d) decomposed Lysozymeresistant Egg yolk reaction Anaerobic utilization of glucose VP reaction Acid produced from mannitol Hemolysis (Sheep RBC) (d) Known pathogenicity e /characteristic produces enterotoxins endotoxin crystals pathogenic to insects rhizoidal growth growth at 6 C; no growth at 43 C pathogenic to animals and humans

10 EN/ISO Horizontal method for the enumeration of presumptive Bacillus cereus Colony count technique at 30 C US FDA BAM test is the basically the same Note: Presumptive test the confirmatory stage does not enable the distinction of B. cereus from other closely related but less commonly encountered Bacillus species, such as B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides. Test Result MYP agar (9.4.1) Formation of pink colonies surrounded by precipitate - Haemolysis- sheep blood agar Result: Count of presumptive B.cereus

11 Previous Versions of ISO amended1997 Count of B.cereus Same agar- same presumptive count Then confirmation by mannitol/egg yolk/polymyxin (MYP) agar medium, glucose fermentation, Voges-Proskauer reaction and nitrate reduction Result given as a count of B.cereus.

12 B.cereus on MYP Agar

13 B.cereus under the microscope

14 B.thuringiensis

15 A revised ISO 7932? ISO/TC34/SC9 WG20 Considering use of the parasporal crystal Differentiation of cereus and thuringiensis

16 MALDI ToF MS Identification Bruker Maldi biotyper *Bacillus anthracis, cereus, mycoides, pseudomycoides, thuringiensis and weihenstephanensis are closely related and members of the Bacillus cereus group. Discrimination between these species is difficult at this level of investigation and caution should be used in the assignment of a sample to a single species.

17 Future Differentiation of Strains Previously Phenotypic/ecological methods Bacillus cereus sensu lato B. cereus B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis Molecular Methods Species boundaries difficult to define 16S rdna sequencing MLST PFGE (can separate anthracis from cereus/thuringiensis)

18 DNA Sequencing Solutions DNA sequencing could offer an ultimate way of identifying isolates It can identify strain to strain variations. Many ways to do it An Example:

19 B.cereus emetic strain 75% B.cereus group sequence mapped against B.cereus

20 B.cereus diarrhoeal strain 42% B.cereus sequence mapped against B.cereus. 9% mapped against B.thuringiensis.

21 B.thuringiensis- isolated as presumptive B.cereus from a vegetable crop 75% B.cereus group sequence mapped against B.thuringiensis 1% mapped against B.cereus

22 Final Thoughts B.cereus group is difficult to differentiate using current standard methods (B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis, B. toyonensis, and B. anthracis) The ISO method is for presumptive B.cereus, it can only identify anthracis out of the other species. In the future 7932 may have a method for presence of crystal included. DNA based methods could provide an answer Issue is getting good reference strains in a curated database, against which to compare Developed methods must be robustly validated (e.g. ISO based approaches) Sequencing offers a potential way of splitting the B.cereus group Depends on a good database Could identify exact strains