Numerous types of foods have been associated with food poisoning and
a lot of cases have been linked to heat treated foods. Bacillus cereus group is involved
in many outbreaks due to the consumption of cooked food. B. cereus is present in
most raw foods and spores have been found also in packaging materials representing
a contamination source for treated foods. Storage temperature is a critical point for
processed foods in relation with spore germination. While vegetative cells die
during heat treatment, spores can survive and germinate under not limiting conditions.
B. cereus food poisoning is principally associated with temperature abuse during
the storage of cooked foods. B. cereus may cause illness through the production
of a high variety of toxins and enzymes, including a necrotizing enterotoxin,
an emetic toxin, phospholipases, proteases and haemolysin inducing also nongastro-
intestinal (e.g. systemic or pulmonary) infections. Enterotoxins could also
be produced from B. circulans, B. lentus and B. mycoides, B. thuringiensis and B.
anthracis closely related to B. cereus. Many cases are confused with those caused
by other pathogens. An accurate evaluation of potentially contaminated foods lead
to the utilization of fast and sensitive methods in food monitoring to satisfy safety
requirements. Classical methods which use enrichment methods due to the usual
small number of B. cereus present in food, are time consuming. Moreover, recent
data showed proteases and chitinases contribute to virulence leading to the need
for suitable means of differentiating members of the B. cereus group during the
monitoring of potentially contaminated foods. Some authors experienced difficulties
distinguishing strains considered to be members of the “B. cereus group” such as
B. mycoides, B. thuringiensis and B. anthracis either using classical or molecular
methods. Identification by classical microbiological methods fails due to horizontal
gene transfer from B. cereus group members. Characters responsible for some
phenotypic characteristics can be lost from one species and can be acquired from another one causing confusion in taxonomy based upon phenotypic characters
evaluation. Different molecular approaches were able to differentiate B. anthracis
from B. cereus but failed to differentiate. B. cereus from B. thuringiensis. Even with
recent improvements in the molecular methods used for microbial phylogeny, B.
cereus, B. mycoides and B. thuringiensis were referred to as B. cereus when 16S
rRNA sequences were used for the differentiation. Genetic similarity between B.
cereus, B. thuringiensis and B. anthracis has been investigated by means of DNADNA
reassociation, multilocus enzyme electrophoresis (MEE) which compare
the allozyme patterns of 10–20 genes, Pulsed Field Gel Electrophoresis (PFGE),
Randomly Amplified Polymorphic DNA (RAPD)-PCR, repetitive extragenic
palindromic (rep)-PCR, Real Time PCR, Microarrays. Genotypic approaches
tend to be less dependent on bacterial growth variables, less time consuming and
useful for determining phylogenetic relationships between microbial isolates and
for assigning strains in specific groups. Molecular methods using PCR are useful
in specific detection of B. cereus from plate isolates and for PCR application on
microbial DNA extracted from food. Species identification can be achieved by
specific couple of primers annealing gyrB gene sequence. To decrease the detection
limit of B. cereus in food the type of treatment applied before DNA extraction
is fundamental, as it allows the increase of the amplification reaction efficiency.
Detection limit for artificially contaminated foods was 50 cells g−1 for boiled rice,
5 × 102 cells g−1 for minced meat, 30 cells g−1 for salad, 20 cells mL−1 for pasteurized
milk and <10 cells mL−1 for concentrated coffee. RE technique applied
onto amplification products allowed differentiation between B. cereus and B.
thuringiensis for the 81 sample tested using Hinf I endonuclease. RAPD-PCR and
rep-PCR used for microbial typing gave different results. Cluster analyes for rep-
PCR was more effective in discriminating between B. cereus and B. thuringiensis
strains than RAPD-PCR. Strains were grouped in 14 clusters, from A to P using
70% similarity by RAPD-PCR, and in ten clusters from A to L by rep-PCR using
80% similarity in fingerprinting analysis.
PCR-TTGE and PCR-DGGE are methods used for single point mutations detection
among DNA sequences. Amplicons obtained for B. cereus and B. thuringiensis
strains were analyzed by this molecular technique to differentiate strains
among the two species identified previously by the PCR-RE method. B. cereus
strains were divided in seven levels whereas B. thuringiensis strains in two levels.
The distribution of PCR products in each gel indicate high variability either within
B. thuringiensis or B. cereus. No clear differentiation was obtained by TTGE
between the two species considered. Horizontal gene transfer between strains of
these different “species” makes it impossible to delineate discrete levels. A desirable
goal is to differentiate the organisms detected in food products and patients
to understand the real contribution of B. cereus and B. thuringiensis to human
infections, either due to food consumption or to hospitalization, using fast and
sensitive protocols.
