Food Testing- Bacillus species.

Dr Roy Betts

Head of Microbiology

Campden BRI, Chipping Campden. UK

Who are Campden BRI?• Independent Food Research Organisation

• Membership based with over 2400 members

• International Client Base- 75 countries

• 350 staff

• 3500m2 laboratories, 3000m2 processing areas

• Microbiology 45 Staff

• ISO 9001 certified, ISO 17025 for many tests

Chipping Campden

Nutfield

Hungary

Our Locations

Bacillus -----Why Test foods?

• Bacillus spp.

– Spore formers- can survive cooking

– Some are food spoilage organisms

– Some are human pathogens

Food Spoilage

• E.g. Rope in bread- B.subtilis

• Spoilage of heat processed foods

– Spore survival & growth e.g. Geobacillus

stearothermophilus

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.

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:

– >105 seen as an health issue

– < 103 seen as acceptable

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 -

B.cereus group differentiation US FDAFeature B. cereus B. thuringiensis B. mycoides B. weihenstepha

nensis

B. anthracis

Gram reaction +(a) + + + +

Catalase + + + + +

Motility +/−(b) +/− −(c) + −

Reduction of

nitrate

+ + + + +

Tyrosine

decomposed

+ + +/− + −(d)

Lysozyme-

resistant

+ + + + +

Egg yolk

reaction

+ + + + +

Anaerobic

utilization of

glucose

+ + + + +

VP reaction + + + + +

Acid produced

from mannitol

− − − − −

Hemolysis

(Sheep RBC)

+ + + + −(d)

Known

pathogenicitye

/characteristic

produces

enterotoxins

endotoxin

crystals

pathogenic to

insects

rhizoidal growth growth at 6°C;

no growth at

43°C

pathogenic to

animals and

humans

EN/ISO 7932-2004• 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

Previous Versions of ISO 7932-1993 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.

B.cereus on MYP Agar

B.cereus under the microscope

B.thuringiensis

A revised ISO 7932?

• ISO/TC34/SC9 WG20

• Considering use of the parasporal crystal

• Differentiation of cereus and thuringiensis

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.

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)

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:

B.cereus emetic strain75% B.cereus group

sequence mapped

against B.cereus

B.cereus diarrhoeal strain42% B.cereus sequence

mapped against B.cereus.

9% mapped against

B.thuringiensis.

B.thuringiensis- isolated as presumptive

B.cereus from a vegetable crop75% B.cereus group

sequence mapped

against

B.thuringiensis

1% mapped against

B.cereus

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 7932-2004 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 16140

based approaches)

• Sequencing offers a potential way of splitting the B.cereus group

– Depends on a good database

– Could identify exact strains