Environmental Microbiology (2008) 10(4), 851–865
Ecological diversification in the Bacillus cereus Group
Marie-Hélène Guinebretière,1*
Fabiano L. Thompson,2,3 Alexei Sorokin,4
Philippe Normand,5 Peter Dawyndt,2,6
Monika Ehling-Schulz,7 Birgitta Svensson,8
Vincent Sanchis,9 Christophe Nguyen-The,1
Marc Heyndrickx10 and Paul De Vos2
1
Institut National de la Recherche Agronomique,
UMR A408 Sécurité et Qualité des Produits d’Origine
Végétale, INRA, Domaine Saint-Paul, Site Agroparc,
F-84914 Avignon Cedex 9, France.
2
Laboratory for Microbiology, and BCCM/LMG Bacteria
Collection, Ghent University, Ghent 9000, Belgium.
3
Federal University of Rio de Janeiro, Institute of
Biology, Department of Genetics, Ilha do fundão, Caixa
postal 68011, CEP 21944-970, Rio de Janeiro, Brazil.
4
Institut National de la Recherche Agronomique,
Laboratoire de Génétique Microbienne, Centre de
Recherche de Jouy-en-Josas, 78352 Jouy-en-Josas
cedex, France.
5
Université Lyon 1, CNRS, UMR 5557, Ecologie
Microbienne, Lyon, F-69003 Villeurbanne, France.
6
Department of Applied Mathematics and Computer
Science, Ghent University, Krijgslaan 281, S9 B-9000
Ghent, Belgium.
7
Lehrstuhl für Mikrobielle Ökologie, Department für
Grundlagen der Biowissenschaften, Technische
Universität München, Weihenstephaner Berg 3,
D-85354 Freising, Germany.
8
Swedish Dairy Association, Scheelevaegen 18, 223 63
Lund, Sweden.
9
Institut National de la Recherche Agronomique, Unité
Génétique Microbienne et Environnement, INRA, La
Minière, 78285 Guyancourt, Cedex, France.
10
Ministry of the Flemish Community, Center for
Agricultural Research, Department of Animal Product
Quality, Brusselsesteenweg 370, B-9090 Melle,
Belgium.
Summary
The Bacillus cereus Group comprises organisms that
are widely distributed in the environment and are of
health and economic interest. We demonstrate an
‘ecotypic’ structure of populations in the B. cereus
Received 24 June, 2007; accepted 15 October, 2007. *For
correspondence. E-mail guinebre@avignon.inra.fr; Tel. (+33) 432 72
25 24; Fax (+33) 432 72 24 92.
© 2007 The Authors
Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd
doi:10.1111/j.1462-2920.2007.01495.
Group using (i) molecular data from Fluorescent
Amplified Fragment Length Polymorphism patterns,
ribosomal gene sequences, partial panC gene
sequences, ‘psychrotolerant’ DNA sequence signa-
tures and (ii) phenotypic and descriptive data from
range of growth temperature, psychrotolerance and
thermal niches. Seven major phylogenetic groups
(I to VII) were thus identified, with ecological differ-
ences that provide evidence for a multiemergence of
psychrotolerance in the B. cereus Group. A moderate
thermotolerant group (VII) was basal to the meso-
philic group I, from which in turn distinct thermal
lineages have emerged, comprising two mesophilic
groups (III, IV), an intermediate group (V) and two
psychrotolerant groups (VI, II). This stepwise evolu-
tionary transition toward psychrotolerance was par-
ticularly well illustrated by the relative abundance
of the ‘psychrotolerant’ rrs signature (as defined by
Pruss et al.) copies accumulated in strains that varied
according to the phylogenetic group. The ‘psychro-
tolerant’ cspA signature (as defined by Francis et al.)
was specific to group VI and provided a useful way to
differentiate it from the psychrotolerant group II. This
study illustrates how adaptation to novel environ-
ments by the modification of temperature tolerance
limits has shaped historical patterns of global eco-
logical diversification in the B. cereus Group. The
implications for the taxonomy of this Group and for
the human health risk are discussed.
Introduction
Organisms of the Bacillus genus are ubiquitous bacteria
found both above and below the Earth’s surface. The
Bacillus cereus Group strains display a wide diversity. Six
closely related species have so far been described in this
taxonomic Group: Bacillus anthracis, Bacillus thuringien-
sis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus
weihenstephanensis and B. cereus (sensu stricto).
Some literature data indicate that these species are not
strictly based on genomic divergence (Guinebretiere and
Sanchis, 2003; Helgason et al., 2004; Hill et al., 2004;
Priest et al., 2004), but rather on subjective consideration
of practical usefulness such as virulence (B. anthracis,
B. thuringiensis), physiology (B. weihenstephanensis),
morphology (B. mycoides and B. pseudomycoides) and
ill-defined features (B. cereus sensu stricto) (Pirttijarvi
et al., 2000; Guinebretiere and Sanchis, 2003). Theses
852 M.-H. Guinebretière et al.
species range in practical importance. Bacillus anthracis
is known to be the causal agent of anthrax. Bacillus
cereus (sensu stricto) includes pathogenic strains that
may cause food poisoning, eye infections and periodontal
disease in humans (Drobniewski, 1993; Helgason et al.,
2000). By contrast, certain B. cereus strains are used as
probiotics. Spore and crystal toxin preparations from
B. thuringiensis are used as commercial biopesticides
(Schnepf et al., 1998). Bacillus weihenstephanensis,
B. mycoides and B. pseudomycoides have not been
described as food poisoning agents, but their toxigenic
potential remains uncertain (Pruss et al., 1999a; Stenfors
et al., 2002).
Strains of the B. cereus Group sensu lato seem to adapt
to very different habitats, from cold to hot thermal environ-
ments, spanning alpine to temperate soils (von Stetten
et al., 1999), and refrigerated foods to dehydrated foods
(Pirttijarvi et al., 1998; Guinebretiere and Nguyen-The,
2003). As a consequence, a very broad thermal range for
growth temperature in the B. cereus Group strains has
been recorded in the literature, ranging from 4°C to 50°C
(Anderson, 1997). Although the range of growth tempera-
ture varies between strains, it has not yet been associated
with particular species or groups, except for the psychro-
tolerant species B. weihenstephanensis whose growth
range has been first defined from below 7°C up to 43°C
(Lechner et al., 1998) and later revised from 7°C up to 38°C
(von Stetten et al., 1999). Adaptation to this very broad
range of growth temperatures in the B. cereus Group has
important implications for the Food Industry. Psychrotoler-
ant strains are a recurrent problem in perishable foods
during refrigerated storage and distribution, particularly in
dairy products (Larsen and Jorgensen, 1997; Te Giffel
et al., 1997) and cooked chilled foods (Carlin et al., 2000;
Guinebretiere et al., 2001), where they may induce spoil-
age or raise safety concerns. Mesophilic B. cereus strains
can persist on hot dairy equipment such as pasteuriser
(Svensson et al., 2000). They can also survive the drastic
processing of dehydrated foods and subsequently con-
taminate diverse foodstuffs via dehydrated ingredients
(Becker et al., 1994; Guinebretiere and Nguyen-The,
2003). Numerous cases of food poisoning have been
attributed to cooked foods or infant foods containing boiled
or reconstituted dehydrated ingredients, e.g. spaghetti,
rice, noodles, macaroni, milk powder, infant formula and
dehydrated potato purées (Holmes et al., 1981; Kramer
and Gilbert, 1989; Shinagawa, 1990).
Temperature is one of the most important environmen-
tal factors to which microorganisms have to respond. The
existence of diverse growth temperature ranges in the
B. cereus Group and psychrotolerant strains that cannot
be identified as B. weihenstephanensis (Stenfors and
Granum 2001), suggests there may be different ecologi-
cal populations associated with this character, not directly
Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
related to the species currently defined in the B. cereus
Group. One hypothesis is that in the B. cereus Group,
distinct genetic groups can possess their own growth
range specificities. Addressing this hypothesis might give
microbiologists a better understanding of extant B. cereus
Group diversity and its special ability to adapt to widely
diverse habitats. Thus, the objectives of the present work
were (i) to establish the existence of thermal groups
based on a genotypic structure and (ii) to position psy-
chrotrophs, food pathogens and the current phenotypic
species in this ‘ecotypic structure’.
For this purpose, we undertook an extensive character-
ization of 425 independent strains selected to cover the
global phenotypic and genotypic diversity of the entire
B. cereus Group, including all six currently defined species
and, in particular B. weihenstephanensis, B. mycoides
and B. pseudomycoides, of which little is known. MLST is
generally an excellent tool for genetic population studies,
but to characterize 425 strains and avoid biases due to
potentially inadequate MLST schemes, we chose instead
to associate a technique based on genome-wide blind
markers, the Fluorescent Amplified Fragment Length Poly-
morphism (fAFLP), and the phylogeny of ribosomal genes
and of an appropriate housekeeping gene, the panC gene.
Fluorescent Amplified Fragment Length Polymorphism is a
reliable tool that has successfully been adapted to the
study of strains in the B. cereus Group (Keim et al., 1997;
Ticknor et al., 2001; Hill et al., 2004). Derived from a pre-
vious study (Candelon et al., 2004), the panC gene
sequence analysis showed high congruence with fAFLP
results in preliminary tests. While ribosomal sequence data
were useful for distinguishing moderately divergent
populations of B. cereus Group into separate sequence
clusters, panC gene provided a better opportunity for dis-
tinguishing very closely related ecological populations.
Using this global approach, we reconstituted the global
genotypic and phylogenetic structure of the whole
B. cereus Group, without a priori and biases, and assessed
the robustness of this model using different clustering
methods and bootstrap analyses. Finally, the resulting
phylogenetic groups were characterized for their growth
temperature limits and for the presence of known ‘psychro-
tolerant signatures’ (Francis et al., 1998; Pruss et al.,
1999b) to define their thermal growth properties.
Results
Reconstructing the phylogenetic structure of the
B. cereus Group strains
Fluorescent Amplified Fragment Length Polymorphism
banding patterns were found to consist of a mean of
68 ⫾ 6 fragments in the size range of 50–536 bp. Com-
parison and classification of data using the Dice similarity
© 2007 The Authors
 B. cereus ecological diversification 853
c
% strains with : Total No. of strains received as : No. of strains with No. of food
A B Genetic Genetic rrs cspA Range of
No. of Bm Bm Rhizoidal Growth poisoning
50 60 70 80 90 100
subgroup group a b growth T°C Bc Bt Ba Bw Bp d
sign sign strains (p) (m) colonies at 7°C strains
BC10 19 0 0 0 0 0 17 2 19 0 0
I 14 0 > 10-43 <
BC13 9 0 0 0 0 0 5 4 9 0 0

BC6 II 88 0 > 7-40 < 33 26 7 0 0 0 0 0 0 25 7

BC12 15 9 6 0 0 0 0 0 0 0 2

BC05 34 33 1 0 0 0 0 0 0 0 18
III 37 0 > 15-45 <
BC09 19 18 1 0 0 0 0 0 0 0 4

BC08 28 10 5 12 0 0 1 0 0 0 5

BC04 0 36 13 23 0 0 0 0 0 0 0 8

BC03 IV 59 0 > 10-45 < 36 26 9 0 0 1 0 0 0 0 11

BC07 0 29 8 21 0 0 0 0 0 0 0 3

BC11 V 94 0 > 8-40 < 17 6 11 0 0 0 0 0 0 1 2

BC01 100 104 9 12 0 22 61 0 0 57 101 0

VI 100 > 5-37 <

BC02 100 39 8 0 0 11 17 3 0 20 35 0

BC14 VII 0 0 > 20-50 < 2 2 0 0 0 0 0 0 0 0 1

Fig. 1. Genetic diversity of the B. cereus group.

A. Simplified dendrogram showing the genotypic relationships between bacterial strains of the B. cereus group, based on fAFLP data analysis.
The percentage similarities (represented by the horizontal scale) were calculated using the Dice similarity coefficient and clustering by UPGMA
(Sneath and Sokal, 1973). The resulting fAFLP groups (DU groups) formed at 60% similarity level are represented in grey. Numbers on
branches are bootstrap values. The genetic subgroups (BC01 to BC14) are schematically represented in black on the dendrogram and are
issue from a non-hierarchical classification using the Bin Class software. BpT, B. pseudomycoides DSM 12442T = lMG 18993T; Ba, B. anthracis
CEB 94–0040; BcT, B. cereus ATCC 14579T = lMG 6923T; BtT, B. thuringiensis CIP 53.137T = lMG 7138T; BwT, B. weihenstephanensis WSBC
10204T = lMG 18989T; BmT, B. mycoides CIP 103472T = lMG 7128T.
B. Distribution and characteristics of strains in each genetic group. a‘psychrotolerant’ rrs sequence signature; b‘psychrotolerant’ cspA sequence
signature; cBc, B. cereus; Bt, B. thuringiensis; Ba, B. anthracis; Bw, B. weihenstephanensis; Bm, B. mycoides; Bp, B. pseudomycoides (p):
psychrotolerant (m): mesophilic; daccording to the rapid screening test.

coefficient and the Unweighted Pair Group Method of
Arithmetic Averages (UPGMA) clustering method gener-
ated 12 robust groups at a 60% similarity level (DU groups
represented in grey in Fig. 1) among the 425 strains of the
B. cereus Group (Table 2, Fig. 1). This cut-off level (60%)
represented the maximum level for which all resulting
fAFLP groups with no exception were highly robust,
showing bootstrap values above 80% (Fig. 1). BinClass
classification performed on the same data set generated
14 groups (BC groups, shown in black in Fig. 1). Despite
the major conceptual difference between the two
clustering approaches (one hierarchical and one non-
hierarchical method), the obtained results are highly
congruent (Table 1). New relationships among strains
within each DU group were disclosed by the BinClass
software, delimiting robust subgroups within each DU
group (Table 1). Finally, taking into account the two clas-
sification methods, we established a genetic structure of
the B. cereus Group, which was composed of seven
major genetic groups (I to VII), themselves subdivided into
14 genetic subgroups (Table 1, Fig. 1). The good overall
correspondence between the two classification methods
demonstrates the high robustness of the seven major
genetic groups and subgroups. In what follows, this geno-
typic structure represented by seven major genetic
groups will be used as reference.

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Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
 854 M.-H. Guinebretière et al.

The genetic group I is a well-separated and robust

Major genetic
entity (Fig. 1) validated by high bootstrap values (above

groupsb
95%), along with the small genetic group VII (bootstrap
values 100%) and a third complex composed of the five

VII
IV

VI
III

V
II
I
remaining genetic groups II, III, IV, V and VI (bootstrap
Total of
strains

26
values 100%). In this complex, the relationship between
2
31
3
2
94
101
17
139
5
3
2
425
the five genetic groups and their subgroups remained
unresolved. panC phylogeny (Fig. 2) strongly correlates
BC14

201.3
565.7
with the major genetic groups defined by AFLP analysis,
1d

2
7
4
confirming the robustness (bootstrap values > 90%) of the

106.4
327.5
five major genetic groups (II, III, IV, V and VI) and high-
BC13

9
lighting relatedness between subgroups inside each
genetic group. It also confirmed that the genetic group II
BC10

132.7
429.8
17
2

19
was phylogenetically close to the genetic group III, and
the genetic group IV to the genetic group V. The phyloge-
BC06

206.3
604.2 netic relationships between the five genetic groups (II, III,
31
2

33

IV, V and VI) were not completely resolved, notably the

position of the phylogenetic group VI in this set.
BC09

177.6
522.0
2
17

19

The position of group VI in the topology shown by the

fAFLP dendrogram (Fig. 1) showed it clearly separated
BC12

184.2
541.0

from groups II, III, IV, V. Furthermore, the group VII

15

15

seemed to be basal to all groups. To elucidate this, a

phylogeny of the 16S (rrs) gene and Intergenic tran-
BC08

152.3
470.0
28

28

scribed spacer situated between the rrs and the 23S (rrl)
genes (ITS1) (Normand et al., 1996) was constructed
BC05

157.1
485.5

(Fig. 3), based on sequence analysis from one to two

34

34

representative strains of each group (the strains closest

BC07

184.5
559.3

to the HMO of BC classes). A strain (AF2), not included

29

29

in the AFLP study but presenting similar genetic traits

as strains of group VII (Fagerlund et al., 2007), was
Table 1 Congruence of the BinClass classification and Dice-UPGMA clustering methods.

BC04

166.8
523.4
36

36

included in this study to check its phylogenetic related-

Groups obtained from the clustering method Dice-UPGMA at 60% similarity level.

ness with group VII strains. The resulting phylogenic

BC03

179.9
523.6

topology of the different groups posits group VII as basal

36

36

to the others, supported by strong bootstrap results. In

Major genetic groups issue from the combination of the two methods.

total coherence with fAFLP topology, the next to emerge

149.6
466.5
BC11

17

17

is group I (supported by a low bootstrap value of 51%)

and then, the group VI (supported also by a low boot-
BC02

188.6
561.3

strap value of 43%). The remaining groups (II, III, IV and

35
1
3

39

V) are difficult to position relative to one another

because of the insufficient resolution of the marker
BC01

160.4
505.1
100
4

104

Groups obtained from BinClass Classification.

chosen. The topologies yielded by the more resolutive

panC and fAFLP analyses are slightly different, thus we
Bina class

conclude that theses topologies are more representative

than the conserved rrs gene for the closely related
Strains excluded from the study.

groups II, III, IV and V.

Only three strains have been clustered to group VII
Average Shannon code length

among over 400 strains, themselves selected among

thousand of strains around the world. This attests of the
scarceness of group VII strains. Their scarceness can be
Average distortion

due to (i) a declining population or to (ii) an uncommon

Total of strains
AFLP groupc

specific niche that allows isolated multiplication of strains.

For the following experiments, we thus considered these
DU01
DU02
DU03
DU04
DU05
DU06
DU07
DU08
DU09
DU10

DU12
DU11

three independent strains as representative of group VII in

b.

d.

spite of this low number.

a.

c.

© 2007 The Authors

Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
 B. cereus ecological diversification 855
a
Table 2. Range of growth temperature recorded for each genetic group.

% strains with positive growth at:

Phylogenetic
group 4°C 5°C 7°C 8°C 10°C 15°C 20°C 35°C 37°C 40°C 43°C 45°C 50°C 55°C

VII 0 0 0 0 0 0 100 100 100 100 100 100 100 0

III 0 0 0 0 0 100 100 100 100 100 100 85 0 0
IV 0 0 0 0 100 100 100 100 100 100 83 58 0 0
I 0 0 0 0 75 100 100 100 100 100 25 0 0 0
V 0 0 0 14 100 100 100 100 100 100 0 0 0 0
II 0 0 73 87 100 100 100 100 100 100 0 0 0 0
VI 0 40 100 100 100 100 100 100 86 0 0 0 0 0

a. Strains affiliated to groups II to VII were significantly (P < 0.05) associated to a specific range of growth (R): group VII to R01 (20–50°C), group
III to R02 (15–45°C) and R03 (15–43°C), group IV to R05 (10–45°C) and R06 (10–43°C), group V to R07 (10–40°C), group II to R08 (8–40°C)
and R09 (7–40°C), and group VI to R10 (7–37°C), R11 (5–37°C) and R12 (5–35°C). Strains of group I exhibited more variable R, globally not
significantly different from those of groups IV or from those of group V, ranging from R04 (15- 40°C) to R07.

Ranges of growth temperatures
In our high throughput screening, psychrotrophs (strains
able to grow at temperatures ⱕ 7°C) were restrictively
recovered in two phylogenetic groups out of seven. In
group VI, 100% of strains were able to grow at 7°C (Fig. 1)
and 40% at 5°C, whereas in genetic group II 76% of
strains were able to grow at 7°C and 0% at 5°C. Interest-
ingly, these psychrotrophs belong to two distant phyloge-
netic groups (II and VI).
In our second experiment, strains affiliated to groups II
to VII differed significantly in growth range characteristics
(P < 0.05, see Table 2), whereas strains of group I had
similar growth range characteristics (R04 to R07) as those
from groups IV (R05, R06) or from group V (R07). The
genetic group VII had the particularity of being composed
of strains able to grow between 20°C to 50°C and should
thus be considered as a moderate thermotolerant group.
In all cases, while the breadth of temperature range did
not significantly change, we observed that more psychro-
tolerant groups have lost the ability to grow at higher
temperatures. Enhanced psychrotolerance may have
come at the cost of increased thermal specialization for
some phylogenetic groups such as the psychrotolerant
groups VI and II.
Thermal groups and habitats
Habitats represent different ‘thermal niches’ depending on
the thermal conditions they offer to bacteria (Table 3).
Strains of groups VI, II, V (growth profiles R07 to R12, see
Table 2) were significantly (P < 0.05) distributed in thermal
niches 1 (temperatures with high fluctuations frequently
covering low temperatures (ⱕ 8°C), whereas strains of
groups III and VII (growth profiles R01 to R03) were
significantly (P < 0.05) associated with thermal niches 3
(warm temperatures: mammals, dehydrated/starched
foods). Strains of groups IV (growth profiles R05 to R06)
seemed to form intermediate broad population signifi-
cantly (P < 0.05) distributed in thermal niches 2 (inter-
mediate temperatures) and thermal niches 3. Thus, all
phylogenetic populations are able to colonize foods, but
they are preferentially distributed according to the fit
between their thermal properties and the thermal condi-
tions to which food or their transformation is associated.
This selective association probably explains the fact that,
in a previous work, the B. cereus population of the warm
parts of the cheese dairy process was found to be differ-
ent from that of cold part of the process (Pirttijarvi et al.,
1998). Similar results were also recorded for B. cereus
genotypes on a purée processing line (Guinebretiere and
Nguyen-The, 2003).
In the different thermal niches and particularly in
thermal niches 1 and 2 (soils, rivers, plants, insects, . . .),
the moderate thermotolerant strains of group VII probably
are in competition with the more adapted strains of groups
I to VI, which could explain their scarceness. Their pres-
ence in dehydrated foods may be explained by the selec-
tive effect of high heat treatments during processing and
by the absence of competitive bacterial growth in the
finished product due to low aw.
Distribution of thermal cspA and rrs genotypes
The psychrotolerant cspA genotype as defined by Francis
and colleagues (1998) was found only in the genetic
group VI with 100% cspA signature-positive strains
(Fig. 1). Thus, the ‘psychrotolerant’ cspA signature must
be considered a specific characteristic of genetic group
VI. The three strains of the minor fAFLP DU11 group (see
Table 1) that were found close to group VI possessed the
mesophilic cspA genotype.
The psychrotolerant rrs signature defined by Pruss and
colleagues (1999b) was detected in all groups, except
group VII. We observed a differential distribution accord-
ing to the genetic group (Fig. 4B). The psychrotolerant
groups II and VI and the intermediate group V contained

© 2007 The Authors

Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
856 M.-H. Guinebretière et al.

© 2007 The Authors

Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
 B. cereus ecological diversification 857
Fig. 2. Phylogenetic relationships between bacterial strains of the B. cereus group, based on panC sequences analysis. Strains selected
for this analysis were representative of the fAFLP genetic groups II, III, IV, V and VI. The branching pattern was generated by
Neighbour-Joining method (Saitou and Nei, 1987). Numbers on branches are bootstrap values above 50%. Bar, 0.01 nucleotide substitution
per site. (1) B. anthracis CEB 94–0040; (2) B. weihenstephanensis WSBC 10204T = lMG 18989T; (3) B. mycoides CIP 103472T = lMG 7128T;
(4) B. thuringiensis CIP 53.137T = lMG 7138T; (5) B. cereus ATCC 14579T = lMG 6923T. The corresponding genetic groups and their subgroups
to which the strains belong are mentioned on branches and on the right side of the figure respectively.

a high number of strains carrying this rrs signature in
abundance (subtypes P, P/m, P/M, Fig. 4B). In contrast,
groups III and I contained a low number of strains with this
signature and in low amount (p/M). Group IV contained a
low part of its strains (18%) with a significant amount of rrs
signature (subtype P/M), in coherence with the fact that
this population is distributed among diverse thermal
niches (2, 3 and in a low proportion 1).
Pruss and colleagues (1999b) hypothesized that this
adaptive mutation allows two different states for the rrs
RNA molecule of the B. cereus group and that this may
lead to two different structure of the 30S subunit, one of
these structures allowing translation initiation at low
temperature, and the other one allowing it at higher
temperature. They stipulate that the ability of a strain to
grow at low or high temperature may be influenced by the
ratio of these two structures. This is in coherence with our
results and this may explain why extension of the lower
thermal limit in the B. cereus group was accompanied by
a trade-off with performance at higher thermal limits.
Position of the current phenotypic species
All the mesophilic rhizoidal colony-forming strains
(received as B. mycoides or B. pseudomycoides) were
clustered in phylogenetic group I, whereas all the psy-
chrotolerant rhizoidal colony-forming strains (received
with an identification as B. mycoides) were found in

Fig. 3. Phylogeny of the B. cereus Group based on rrs and ITS1 genes sequence analysis, and species of the B. cereus group associated to
each group. The Phylogenetic tree was generated by the Neighbour-Joining method (Saitou and Nei, 1987). Numbers on branch nodes are
bootstrap values above 50%. The bar indicates 0.005 nucleotide substitution per site. Highly similar topologies were obtained using the
Maximum-Parsimony (Kluge and Farris, 1969) method and Maximum-Likelihood method (Felsenstein, 1981). (P) indicates the Psychrotolerant
group; (I) the Intermediate group with moderate psychrololerance characteristics; (M) the Mesophilic group and (MT) the moderate
thermotolerant group. Species associated to each genetic group are mentioned on the right hand of the figure. For group VI, B. cereus VI
strains were undistinguishable from B. weihenstephanensis strains and thus were considered as B. weihenstephanensis.

© 2007 The Authors

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858 M.-H. Guinebretière et al.
Table 3. Distribution of strains according to the origin and according to the genetic group (in percentage of the total number of strains in each
group).a

Thermal Group I Group II Group III Group IV Group V Group VI Group VII
Origin nicheb (28)c (33) (96) (101) (17) (143) (3)

% from natural environment (soil, water, air, plants) 1, 2 40 40 12 29 41 35

% from Animals:
Mammals, feces 3 9 23 2
Insects 2 1 16 1
% from the Dairy Industry:
Dairy environment 1, 2 28 3 1 22
Raw milk 1, 2 14 7 1 8
% from foods:
Chilled pasteurized milk 1 7 3 4 6 21
Chilled pasteurized vegetables 1 1 2 5
Cooked foods with vegetables as major ingredient 3 6 2 4 6
Cooked foods with meat as major ingredient 3 6 8 7 6
Milk products (cheese, cream, ice cream . . .) 2, 1 3 3 2 1
Diverse cooked foods 2, 3 3 1 1
Raw vegetables 1, 2 18 1 2 6 2
Dehydrated foods (starch or proteins) 3 11 8 70
Herbes and spices 3 3 30
Starched foods (rice, pasta . . .) 3 3 11 10 6
Unknown source 11 9 7 16 29 4
Food poisoning foods 0 21 33 22 12 0 50

a. Absence of numbers indicates: Nb. = 0.

b. 1: highly fluctuating temperatures frequently encompassing low temperatures; 2: intermediate temperatures; 3: warm temperatures
(see Experimental procedures)
c. Parenthetical numbers indicate the total number of strains in each group.

genetic group VI (Fig. 1). This topology can be explained
by the fact that most strains received as B. mycoides
(including reference strains) were identified before the
description of the B. pseudomycoides species. The mor-
phological similarities observed between the two species
and difficulties in differentiating the two species with
simple methods (only some levels in 12:0 iso and 13:0
anteiso fatty acids have been proposed for differentiating
between the two species (Nakamura, 1998) may have
favoured the non-revision of strain identity. In our study,
we clearly show that the B. pseudomycoides species cor-
responds to phylogenetic group I (Fig. 1), a separate phy-
logenetic group composed of mesophilic rhizoidal colony-
forming strains that can be easily distinguished from
the psychrotolerant rhizoidal colony-forming strains
(B. mycoides ‘sensu stricto’, group VI) by the absence of
‘psychrotolerant’ cspA signature.
Bacillus cereus and B. thuringiensis species were
highly polyphyletic, being components of the phylogenetic
groups II, III, IV, V and VI (Fig. 1, Table S1). These
species thus cover the entire spectrum of growth tem-
peratures observed in the B. cereus Group. When they
belonged to the same genetic group, they differed by the
presence of the ‘cry-carrying’ plasmid.
By contrast, B. mycoides ‘sensu stricto’ (psychrotolerant
rhizoidal colony-forming strains) and B. weihenstephanen-
sis were clustered together in the same phylogenetic group
(VI) (Fig. 1). In phylogenetic group VI these two species did
not form separate entities and had similar psychrotoler-
ance properties. Group VI also contained 20% of strains
received as B. cereus or B. thuringiensis (Fig. 1). It
therefore appears that psychrotolerant organisms of the
B. cereus Group, despite their restrictive phylogenetic
position in groups II and VI, may be B. weihenstephanen-
sis, B. mycoides strains as well as B. cereus and B. thur-
ingiensis strains. Bacillus cereus strains clustered into
group VI are not distinguishable from B. weihenstephan-
ensis strains by any known characters and should be
considered as B. weihenstephanensis based on the
current identification keys.
The 12 strains of B. anthracis were recovered in
genetic group III, and belonged to a well-delimited and
homogenous phylogenetic subgroup BC08. Interestingly,
in contrast to the monomorphic character usually attrib-
uted to B. anthracis species, the phylogenetic subgroup
BC08 also contained 11 strains received as B. cereus
strains and five strains received as B. thuringiensis
strains.
Position of the food pathogens
Because of their ubiquitous nature, B. cereus Group
strains may be readily recovered in foods. In this study,
the food poisoning strains (49 strains connected to

© 2007 The Authors

Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
 B. cereus ecological diversification 859

A Fig. 4. Presence of the ‘psychrotolerant’ rrs

sequence signature in strains and distribution
according to the genetic group.
A. Illustration of the five patterns (P, P/m,
P/M, p/M, M) obtained for strains during the
experiment and of the three resulting
genotypes (P, psychrotrophic; I, intermediate;
M, mesophilic). P: two bands (351 bp, 122 bp)
resulting from the SspI digestion of amplicons
473bp containing 100% rrs copies with the sequence
351bp signature; P/m: three bands (473 bp, 351 bp,
122 bp) resulting from the SspI digestion of
amplicons containing a majority of rrs copies
122bp with the sequence signature; P/M: three
bands (473 bp, 351 bp, 122 bp) resulting from
the SspI digestion of amplicons containing
around 50% of rrs copies with the sequence
P P/m P/M p/M M signature; p/M: three bands (473 bp, 351 bp,
122 bp) resulting from the SspI digestion of
amplicons containing a minority of rrs copies
with the sequence signature; M: one band
(473 bp) resulting from amplicons that does
P I M not contain rrs copies with the sequence
signature.
B B. Percentage of strains with ‘psychrotolerant’
rrs sequence signature according to the
100% genetic group (I to VII). Number of tested
strains: 22 for group I, 32 for group II, 27 for
90% group III, 22 for group IV, 17 for group V, 143
80% for group VI and 2 for group VII. Results for
the subtypes P, P/m, P/M, p/M, M are detailed
70% in the table in abscissa of the figure.
60%
% 50% P
40% I
30% M
20%
10%
0%
VII I III IV V II VI Group
P 0 0 0 0 0 10 87
P
P/m 0 0 0 0 12 16 10

I P/M 0 0 0 18 47 50 3

p/M 0 14 37 41 35 12 0
M
M 100 86 63 41 6 12 0

diarrhoeal syndrome and 17 strains connected to emetic
syndrome) were positioned across genetic groups II, III,
IV, V and VII (Fig. 1). The diarrhoeal strains were
present in all these five groups, whereas the emetic
strains were only distributed in the mesophilic group III
inside subgroup BC05. It is noteworthy that among the
143 strains that were clustered in the psychrotolerant
group VI, none have been connected with food poison-
ings. Stenfors and colleagues (2002) showed that most
B. weihenstephanensis strains are not cytotoxic on vero
cells and grow only with difficulty at 40°C (a temperature
close to 37°C). In the conditions that were used here,
the strains of the phylogenetic group VI, including
B. weihenstephanensis strains and psychrotolerant rhiz-
oidal strains (B. mycoides ‘sensu stricto’ ), did not grow
at 37°C or grew slowly compared with the strains of
other phylogenetic groups. This temperature constraint
and other stresses, including that implied by the intesti-
nal bacteria, greatly decrease the risk of intoxication by
such strains. This also may explain why food poisoning
strains are statistically not associated to this group. By
contrast, the psychrotolerant strains of genetic group II
can be pathogenic, such as the strains NVH 0861/00,
RIVM BC 120, RIVM BC 126.

© 2007 The Authors

Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
860 M.-H. Guinebretière et al.
Discussion
Psychrotolerance and evolution of the B. cereus group
The phylogenetic branching order of the different thermal
lineages provides information on the direction of pheno-
typic change during cladogenesis. The most thermotoler-
ant lineage is basal (groups VII), while strains with lower
thermotolerance or greater psychrotolerance branch later
along the phylogeny (Figs 1 and 3). This indicates a tran-
sition from a moderately thermotolerant status (group VII)
to a mesophilic status (group I). This was followed by a
shift in growth ranges for the most recently appeared
groups (II, III, IV, V and VI), resulting in specialization for
groups II and VI (psychrotrophic groups). This strongly
supports the hypothesis of a multiemergence of psychro-
tolerance in the B. cereus Group. The differential pres-
ence of the rrs signature in all groups shows that all
groups (I to VI) appear to be genetically concerned by this
evolution but the relative quantity of this signature accu-
mulated in strains indicates that, in this respect, the
groups do not evolve at the same rate.
We may speculate that the emergence of more cold-
adapted populations corresponds to the colonization of
new or different environments for which B. cereus organ-
isms had to adapt. According to the index reference of
Ochman and Wilson (1987), group VII should have
diverged 125 millions years ago. One hypothesis is that
this group was initially associated to a rare or restricted/
isolated thermal niche that supported moderately high
temperatures (up to 50°C) such as hot springs, volcanic
warm soil, etc. . . . The transition to a mesophilic status
may correspond to the colonization of lower thermal
niches such as diverse soils, rivers and animals (insects,
mammals). An argument for this is the major genome
expansions observed for the sequenced strains of groups
III, IV and VI (Lapidus et al., 2007), similar to the signifi-
cant modification of genome size that had previously been
associated to a modification of habitat for Frankia sp.
strains (Normand et al., 2007). Furthermore, the associa-
tion between the thermal niches and the genetic groups,
strongly correlates the stepwise pattern of diversification
observed for the clade made of groups II to VI with the
appearance of diverse biological niches and thus with
different ‘thermal’ niche histories for these genetic groups.
The thermal niches associated to the mesophilic and
psychrotolerant groups may act as microgeographical
separations, preventing competition between each type of
population, so that they are able to independently evolve
at different rates for psychrotolerance. According to the
rrs-ITS1 sequence phylogeny, the psychrotolerant group
VI that appeared nearly at the same time as the clade
made up of groups II to V, seems to be characterized by
a higher mutation rate. Although the mutation rate data
was recorded on a limited number of strains, this seems
Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
well correlated with the higher evolutionary status of
group VI for the psychrotolerant signature and with its
higher level of adaptation to low temperatures. A similar
correlation between the mutation rate and the level of
adaptation has been previously shown for the pathogenic
Escherichia coli (Matic et al., 1997).
In conclusion, the global evolution of the B. cereus
Group seems to be strongly determined by ecological
adaptations; the extension of ecological tolerance limits,
in particular temperature tolerance, may have been an
important mechanism by which strains of the different
groups (I to VII) adapted to novel environments and his-
torically by which these groups have diverged. This rich
adaptive faculty guarantees development of new evolu-
tionary lines and long-term maintenance of the B. cereus
Group sensu lato.
Practical applications for detecting B. cereus Group
strains in foods
The present work provides an important complement to
the existing phylogenetic topologies (Helgason et al.,
2004; Hill et al., 2004; Priest et al., 2004; Sorokin et al.,
2006) through additional data concerning the B. wei-
henstephanensis, B. mycoides and B. pseudomycoides
species, and through the discovery of a global ecotypic
structure. These results have important practical conse-
quences. A particularly original result is the existence
of two very different groups of psychrotrophs (groups II
and VI). Food manufacturers need fast and effective
methods of detection for psychrotrophs to predict the
shelf-life of perishable products more accurately and to
reduce the incidence of food poisoning by strains of the
B. cereus Group. Detection of psychrotolerant signatures
on the rrs gene (Pruss et al., 1999b) and cspA gene
(Francis et al., 1998) have been previously proposed.
Our study shows that the ‘psychrotolerant’ rrs signature
can be regarded as a general indication of a strain’s
adaptive potential, but it is not sufficient to accurately
identify group II or VI. Furthermore, the ‘psychrotolerant’
cspA signature cannot be defined as a marker for all
psychrotrophs but just for group VI strains. Finally, the
health risk is not the same for the psychrotolerant strains
depending on their genetic affiliation to group II or group
VI. The problem therefore arises of how to detect all
psychrotrophs and how to discriminate the two types of
psychrotrophs. Because the group II strains are more
prone to induce food poisoning, a method complemen-
tary to that proposed by Francis and colleagues (1998)
is needed to allow the detection of all psychrotrophs in
food.
The highly polyphyletic character of B. cereus sensu
stricto and B. thuringiensis will result in highly different
characters for strains inside each species. We thus
© 2007 The Authors

propose to add the phylogenetic number (I to VII) to the
species name when identifying strains: B. cereus II, III, IV,
V and VII and B. thuringiensis II, III, IV, V and VI. Tmin and
Tmax, as well as rhizoidal morphology of colony and cspA
signature provide a first intention approach for differenti-
ating strains of the different phylogenetic groups I to VII.
The origin of food ingredients and the temperature
applied during processing of foods and storage will have
a differential selective effect on the bacterial strains of the
B. cereus Group, tightly related to their specific thermal
characteristics. Then for consumers, the health hazards
do not concern all ingested strains with the same degree
of risk. Strains of the different phylogenetic groups will not
have the same potential to develop under intestinal con-
ditions, the strains of phylogenetic groups VII, III and IV
probably being the best performers.
Currently, the public health authorities do a clear dis-
tinction between B. cereus sensu stricto (recognized as
a potential food poisoning agent of risk group 2) and
the species B. thuringiensis, B. weihenstephanensis,
B. mycoides and B. pseudomycoides (not recognized as
potential pathogenic agents). As suggested above, the
risk seems to be rather associated to the phylogenetic
group. For example a B. cereus II or a B. thuringiensis
VI will not have the same food poisoning potential as,
respectively, a B. cereus VI or a B. thuringiensis III.
Therefore, we recommend that risk groups, instead of
being defined by reference to the current phenotypic
species, be defined by reference to the seven major
phylogenetic groups established here. It is certain also
that some additional studies are needed to quantitatively
characterize the pathogenic potential conferred by each
group.
Experimental procedures
Bacterial strains and their origins
We randomly selected 425 independent strains from different
laboratories and international collections in Europe and the
USA (see Table S1). These strains covered a broad spectrum
of different geographic origins, substrate origins, RAPD
profiles, serotypes and virulence characteristics. To get a
complete coverage of the B. cereus group, strains from
all six currently defined species were studied. Also, strains
representative of species (B. cereus ATCC 14579T,
B. thuringiensis CIP 53137T, B. mycoides DSM 2048T,
B. pseudomycoides DSM 12442T, B. weihenstephanensis
WSBC 10204T and B. anthracis CEB 94–0040) were
included. Given their known monomorphic characteristics, we
restricted the number of B. anthracis and emetic strains to
represent the maximum diversity observed for each entity
according to tandem repeat polymorphism (Le Fleche et al.,
2001) or RAPD pattern (Ehling-Schulz et al., 2005).
In order to determine the position of food poisoning strains,
we included 49 strains connected to diarrhoeal syndrome and
17 strains connected to emetic syndrome (see Table S1).
© 2007 The Authors
Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
B. cereus ecological diversification 861
Screening of psychrotolerant strains and rhizoidal
colony-forming strains
Psychrotolerant strains were detected by testing growth at
7°C and at 5°C. Given the high number of strains (425
strains), the experiment was conducted as a rapid screen-
ing test. The test was performed on J-agar Petri dishes
(Claus and Berkeley, 1986) in two independent experi-
ments. A colony from a fresh culture (24 h) on J-agar was
picked up and inoculated by streaking on a J-agar plate.
Growth was conducted in incubators at 30°C ⫾ 0.5 for
48 h (control of viability), at 7.1°C ⫾ 0.4 for 21 days and
at 5.1°C ⫾ 0.5 for 21 days. The type strains WSBC
10204T = LMG 18989T and ATCC 14579T = LMG 6923T were
used as positive and negative controls respectively. For
each experiment and each incubation condition, two J-agar
Petri dishes per strain were inoculated. Strains were con-
sidered as positive for growth when colonies appeared after
the above incubation times.
The appearance of rhizoidal colony-forming strains on
J-agar after a 48 h culture at 30°C was also monitored for
each strain.
Fluorescent Amplified Fragment Length
Polymorphism analysis
Total DNA was extracted for each strain as described previ-
ously (Guinebretiere and Nguyen-The, 2003). Digestion and
ligation steps were carried out according to the protocols
of Ticknor and colleagues (2001) with slight modifications.
The selective polymerase chain reaction (PCR), capillary
electrophoresis and numerical analyses were performed as
described previously (Thompson et al., 2001).
The fAFLP banding patterns were converted into vector
format. To find a near-optimal discretization method, a
number of alternative discretization algorithms were evalu-
ated in order to preserve maximal information content of the
original fingerprint patterns (Dawyndt et al., 2005). The most
conservative vector representation resulted from a discreti-
zation by the sliding window algorithm introduced by Austin
and colleagues (2004), with a position tolerance e of 0.7%
and a resolution d of 0.1%, producing a binary vector repre-
sentation of length 994.
To obtain an unbiased view of the relationships between
the fAFLP profiles, we compared the outcomes of two differ-
ent mathematical approaches for classifying the genomic
fingerprint patterns. The first classification resulted from a
hierarchical clustering, using the Dice similarity coefficient
and applying the UPGMA (Sneath and Sokal, 1973). Bootstrap
values reflecting the robustness of a classification were
obtained from 1000 replicates (Bionumerics 3.5 software).
The second classification resulted from a non-hierarchical
clustering. It optimized a given information theoretic
expression. Indeed, the binary vector representation also
made it possible to apply a minimization of stochastic com-
plexity (Gyllenberg et al., 1998) for classification of the fAFLP
fingerprint patterns. Minimization of stochastic complexity
was performed using the BinClass software package (Gyllen-
berg et al., 2001). The robustness of the classification is
reflected by a high level of overall congruence between the
two methodologies (Dawyndt et al., 2005).
862 M.-H. Guinebretière et al.
The centroid of a class is, by definition, the vector giving
the frequencies of one’s for the different attributes. Rounding
off each component of the centroid to the nearest binary
value (0 or 1) gives the hypothetical mean organism (HMO)
(Sneath and Sokal, 1973). As a measure of the heterogeneity
of a class, we chose its distortion, which is the average
number of bits by which the members of the class differ from
the HMO. An alternative quantifier for describing the hetero-
geneity of a class is the Shannon code length (Cover and
Thomas, 1991), which we also included.
panC gene sequence analysis
The panC gene was chosen from a pilot experiment on mul-
tiple locus sequencing of B. cereus strains (Candelon et al.,
2004). This gene was sequenced for 67 selected strains,
representing AFLP groups. Polymerase chain reaction ampli-
fication and sequencing procedures were carried out as pre-
viously described (Candelon et al., 2004).
The consensus sequences were compared using the
multiple alignment program CLUSTAL ¥ 1.8 (Thompson et al.,
1997). A phylogenetic tree was constructed using the
Neighbour-Joining (NJ) method (Saitou and Nei, 1987), and
NJplot software (Perriere and Gouy, 1996) was used to gen-
erate a graphic representation of the resulting tree. Bootstrap
estimates (Felsenstein, 1985) were obtained from 1000
replicates.
rrs gene and Intergenic transcribed spacer rrs – rrl
genes (ITS1) sequence analysis
The rrs and ITS1 DNA genes of 12 representative strains,
corresponding to the strains nearest to the HMO of each BC
class, were amplified by PCR, using the primers described by
Normand and colleagues (1996). The PCR reactions and
amplifications were performed in a final volume of 90 ml,
following the conditions described previously (Guinebretiere
et al., 2001).
The amplification products were purified with a Spin PCR
purification kit (ROCHE diagnostic, Mannheim, Germany)
and were sequenced by MWG (Martinsried, Germany). Five
primers were used in the sequencing reaction to obtain com-
plete rrs gene and ITS1 sequence (about 1800 bp): S6, S10,
S15, S17 and FGPL 132 (Normand et al., 1996; Guinebre-
tiere et al., 2001).
To determine the phylogeny of the representative strains
for which the entire rrs-ITS1 was sequenced, phylogenetic
trees were constructed by (i) the NJ method (Saitou and Nei,
1987) using the two-parameter substitution rate method of
Kimura (1980) (ii) the Maximum-Parsimony method (MP)
(Kluge and Farris, 1969) and (iii) the Maximum-Likelihood
method (ML) (Felsenstein, 1981). Bootstrap estimates
(Felsenstein, 1985) were obtained from 1000 replicates for
both the NJ and MP methods. The different analyses were
implemented using the Phylo-win software (Perriere and
Gouy, 1996). Graphic representation of the resulting trees
was obtained using the NJPlot software (Perriere and Gouy,
1996). A test of comparison of mutation rates was conducted
using the RRT procedure (Robinson-Rechavi and Huchon,
2000).
Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
Targeting of thermal DNA signatures for the
discrimination of psychrotolerant genotypes
A total of 252 strains were tested for the cspA signature
sequence defined by Francis and colleagues (1998), includ-
ing all psychrotolerant strains (by reference to the screening
test) as well as a subset of mesophilic representatives of
each mesophilic fAFLP groups (20% minimum). For each of
these bacterial strains, the cspA signature was detected by a
triplex PCR assay as previously described (Francis et al.,
1998), with minor modifications (use of Goldstar DNA poly-
merase (Eurogentec), 50 ng of DNA per reaction and a 15 ml
final volume).
A total of 246 strains, including all the psychrotolerant
strains (by reference to the screening test) and mesophilic
representatives of each mesophilic fAFLP group strains (20%
minimum), were tested for the rrs ribosomal DNA signature
defined by Pruss and colleagues (1999b). The rrs ribosomal
DNA signature was characterized for each strain using the
PCR-RFLP protocol described by Pruss and colleagues
(1999b), with minor modifications (use of Goldstar DNA poly-
merase (Eurogentec) and a 15 ml final volume). The resulting
PCR product representing a partial DNA fragment of rrs gene
(473 bp) was submitted to digestion by SspI restriction
enzyme. A 10 ml aliquot of the digestion mixture (containing
5 U SspI, 2 mg BSA and SspI buffer) was added directly to the
15 ml of PCR product and incubated at 37°C for 4 h. The
resulting DNA fragments were separated on 2% agarose gel.
The ‘psychrotolerant’ rrs signature was detected by the pres-
ence of two DNA fragments (351 bp and 122 bp) after SspI
digestion (Pruss et al., 1999b), whereas in the absence of
the targeted restriction site, only one band of 473 bp was
present. As the relative number of rrs copies with this signa-
ture correlates with the relative intensity of the characteristic
351 bp band as compared with the 473 bp band (Pruss et al.,
1999b), we established three main thermal genotypes
(M = mesophilic genotype, I = intermediate genotype,
P = psychrotolerant genotype) and five thermal subtypes
(P, P/m, P/M, p/M, M) (see Fig. 4A).
Growth temperature ranges
A subset of 75 strains representative of each fAFLP group
and thermal genotype (strains that have similar psychrotoler-
ant signatures and growth properties) was tested for growth
ability at extreme temperature. Growth was determined using
nutrient agar and J-agar slants, as described in the Bergey’s
Manual of Systematic Bacteriology (Claus and Berkeley,
1986). Systematic growth tests at 7°C, 8°C, 37°C and 43°C
were included, in addition to the conventional tests at 5°C
intervals described in the Bergey’s Manual, by reference to
the upper limit (7°C) for defining a psychrotolerant strain
(Francis et al., 1998), the standard temperature used by
several Food Safety Agencies in European countries (8°C),
the temperature of human body (37°C), and a taxonomic
criterion (43°C) used for B. cereus Group (Lechner et al.,
1998). For the tests at low temperatures, bath temperatures
were stabilized at 7, 8, 10, 15 or 20°C in an RTE-111 Re-
frigerated Bath/Circulator (Neslab Instruments, Newington,
USA), while for the higher temperature tests, bath tempera-
tures were stabilized at 35, 37, 40, 43, 45, 50 or 55°C in a
© 2007 The Authors

PolyStat 55 heating circulating bath (Bioblock Scientific).
Temperature fluctuation was not greater than ⫾0.2°C for all
tested temperatures. Tubes were immersed in the water
baths at the appropriate temperatures until equilibrated, and
then inoculated. Cultures growths were examined after 3 and
5 days for the high temperature tests and every week up to
21 days for the low temperature tests.
The type strains of B. weihenstephanensis (WSBC
10204T), B. licheniformis (CIP 52.71T) and B. circulans
(LMG 6926T) were used as negative or positive controls for
the extreme conditions of growth (7°C, 8°C, 50°C, 55°C).
Statistics
For comparison of the eco-physiological characteristics of
strains, we established different key variables. Growth range
(R) of each strain was defined by qualitative data correspond-
ing to the association of its minimal growth temperature (Tmin)
and maximum growth temperature (Tmax). Twelve different
values in total were recorded for R among our strains sample:
R01 (20–50°C), R02 (15–45°C), R03 (15–43°C), R04
(15–40°C), R05 (10–45°C), R06 (10–43°C), R07 (10–40°C),
R08 (8–40°C), R09 (7–40°C), R10 (7–37°C), R11 (5–37°C),
R12 (5–35°C). Concerning habitats of strains, we categorized
the origin of strains according to the thermal niche to which
they were associated: The thermal niche #1 corresponded to
highly fluctuating temperatures frequently covering low tem-
peratures (ⱕ 8°C); it included soils/air/rivers/plant surface of
Northern Europe, cold surfaces of dairy equipment, pasteur-
ized and refrigerated foods (milk, vegetables . . .), raw veg-
etables conserved at low temperature. The thermal niche #2
corresponded to intermediate temperatures with low fluctua-
tions; it included insects, Asian or pacific soils, indoor air,
transformed or raw foods not refrigerated and not heat-
treated (milk products such as cheese, vegetables), surfaces
of equipment not associated with refrigeration, cakes. The
thermal niche #3 corresponded to warm temperatures,
including mammals, faeces, dehydrated foods (starch, pro-
teins, herbs and spices), cooked foods (warm meals, rice,
pasta . . .).
Tests of independence and particularly the significance per
case (Fisher’s exact test) were carried out using the software
package XLSTAT 2007.7 to determine to what extent the
different categories of the above variables were significantly
associated to one or the other of the different genetic groups.
Nucleotide sequence accession numbers
Nucleotide sequence data have been submitted to the EMBL/
GenBank databases under Accession Nos AM747044 to
AM747097, DQ301053, DQ301054, DQ301062, DQ301066,
DQ301068, DQ301078, DQ301089, DQ301090, DQ301137,
DQ301426, DQ301433, AY224725 for the partial panC gene
sequences and AM747220 to AM747234 for the rrs-ITS1
sequences.
Acknowledgements
This work was supported by the European Commission
(QLK1-CT-2001–00854) and by specific funds from the Uni-
© 2007 The Authors
Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 851–865
B. cereus ecological diversification 863
versity of Ghent, the Mission des Relations Internationales of
INRA (Paris, France) and the CEPIA and MICA Departments
of INRA. We are grateful to the Bijzonder Onderzoeksfonds
(BOF) of Ghent, the BCCMTM/LMG Bacteria Collection
(Ghent) and the FAPESP (Brazil) for their contribution.
Strains of interest were kindly provided by the USDA
(USA), the DSM (Germany) and the ‘Centre du Bouchet’
(France).
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Supplementary material
The following supplementary material is available for this
article online:
Table S1. Strains from this study and the genetic groups to
which they belong.
This material is available as part of the online article from
http://www.blackwell-synergy.com