The hidden lifestyles of Bacillus cereus and relatives

Environmental Microbiology (2003)

5

(8) 631ndash640

copy 2003 Society for Applied Microbiology and Blackwell Publishing Ltd

Blackwell Science LtdOxford UKEMIEnvironmental Microbiology1462-2920Blackwell Publishing Ltd 20035

8631640

Review Article

The hidden lifestyles of B cereus and relativesG B Jensen B M Hansen J Eilenberg and J Mahillon

Received 29 November 2002 accepted 17 March 2003 Forcorrespondence E-mail gbjamidk Tel (

+

45) 3916 5244 Fax (

+

45)3916 5201

Minireview

The hidden lifestyles of

Bacillus cereus

and relatives

G B Jensen

1

B M Hansen

2

J Eilenberg

3

and J Mahillon

4

1

National Institute of Occupational Health Lersoslash Parkalle 105 2100 Copenhagen Denmark

2

National Environmental Research Institute Frederiksborgvej 399 4000 Roskilde Denmark

3

The Royal Veterinary and Agricultural University Thorvaldsensvej 40 1871 Frederiksberg C Denmark

4

Laboratory of Food and Environmental Microbiology Universiteacute Catholique de Louvain Place Croix du Sud 212 B-1348 Louvain-la-Neuve Belgium

Summary

Bacillus cereus sensu lato

the species group com-prising

Bacillus anthracis

Bacillus thuringiensis

and

B cereus

(

sensu stricto

) has previously been scruti-nized regarding interspecies genetic correlation andpathogenic characteristics So far little attention hasbeen paid to analysing the biological and ecologicalproperties of the three species in their natural envi-ronments In this review we describe the

B cereussensu lato

living in a world on its own all

B cereussensu lato

can grow saprophytically under nutrient-rich conditions which are only occasionally found inthe environment except where nutrients are activelycollected As such members of the

B cereus

grouphave recently been discovered as common inhabit-ants of the invertebrate gut We speculate that allmembers disclose symbiotic relationships withappropriate invertebrate hosts and only occasionallyenter a pathogenic life cycle in which the individualspecies infects suitable hosts and multiplies almostunrestrained

Introduction

The

Bacillus cereus

group a very homogeneous clusterwithin the

Bacillus

genus comprises six recognized spe-cies

B cereus

B thuringiensis

B anthracis

B mycoides

B pseudomycoides

and

B weihenstephanensis

These

species are closely related but their precise phylogeneticand taxonomic relationships are still debated Recent databased on multilocus enzyme electrophoresis (MEE)(Helgason

et al

2000) and DNA sequence variations ofthe 16S

-

23S internal transcribed spacers (Daffonchio

et al

2000) suggested that

B anthracis

B thuringiensis

and

B cereus sensu stricto

are members of a singlespecies

B cereus sensu lato

Whereas intensive workhas been performed to decipher their genetic relationship(Harrell

et al

1995 Helgason

et al

2000 Hansen

et al

2001 Chen and Tsen 2002) less attention has been paidto comparing the biological and ecological properties ofthe three species in their natural environments The mainpurpose of this review is to elucidate the ecological andbiological properties of

B cereus

ie the three species

Banthracis

B thuringiensis

and

B cereus

with specialfocus on interactions with other organisms Furthermoreto the extent of the limited information available thespecies

B mycoides

B pseudomycoides

and

B weihen-stephanensis

are also included in this analysis

Properties of

Bacillus anthracis

Bacillus anthracis

is the causative agent of anthrax whichis primarily a disease in mammals including man (forrecent reviews see Mock and Fouet 2001) Apart frombeing one of the oldest known diseases described as oneof the Egyptian plagues in the time of Moses many of theecological and epidemiological questions about anthraxare still unanswered Anthrax has been linked withendemic soil environments long before

B anthracis

wasidentified as the causative agent (Rayer 1850 Davaine1863)

The virulence of

B anthracis

is based on the presenceof two virulence plasmids pXO1 (1817 kbp) and pXO2(948 kbp) The plasmid pXO1 encodes three toxic factorsthe protective antigen (PA) the lethal factor (LF) and theoedema factor (EF) (Bhatnagar and Batra 2001) Thesecomponents associate into two bipartite exotoxins PA-LFand PA-EF The plasmid pXO2 encodes a poly D glutamicacid capsule enabling the bacterium to withstand phago-cytosis The loss of pXO2 renders the cells incapable ofestablishing an infection ie the bacterium becomesattenuated a trait that is the basis of the Sterne vaccine

632

G B Jensen B M Hansen J Eilenberg and J Mahillon


Environmental Microbiology

5

631ndash640

strain Both plasmids have been sequenced recently(Okinaka

et al

1999ab)Current models of

B anthracis

ecology rely on its patho-genicity ie the spores are ingested by herbivores theanimals becomes infected and the bacteria proliferate inthe lymphoid glands concomitantly expressing the exotox-ins which ultimately leads to the death of the animal (seeFig 1) Once the animal is dead the vegetative cells of

B anthracis

having reached a serum concentration of

gt

10

7

cells ml

-

1

will be outcompeted by anaerobic bacteriafrom the gastrointestinal tract through antagonistic inter-actions (Dragon and Rennie 1995) The environmentalfate of the spore is not known in detail The spores willsurvive lsquoindefinitelyrsquo in dry and protected environmentsHowever exposure to sunlight for 4 h has a significantnegative effect on the survival of the spores (Lindequeand Turnbull 1994) Furthermore photoinduced repair ofUV damage is absent in

B anthracis

spores of the Sternevaccine strain (Knudson 1986) A few reports stated thatspores could actually germinate in nature when conditionsare favourable Van Ness (1971) reported that soil pHabove 60 and temperatures above 155

infin

C favour out-breaks of anthrax The term lsquoincubator areasrsquo has beenintroduced to describe puddles in which decaying grassand other organic matter constitute the nutrients neces-sary for the germination of

B anthracis

spores Howeverthe study by Van Ness (1971) did not show actual growthof

B anthracis

in these incubator areas and other studiesindicated that

B anthracis

has very specific growthrequirements making it very unlikely for the spores togerminate outside a host (Minett and Dhanda 1941) It isalso noteworthy that growth of

B anthracis

outside a hostoften leads to loss of virulence caused by loss of theplasmid carrying the capsule gene (pXO2) ndash an argument

that further reduces the incubator area theory As a con-sequence Dragon and Rennie (1995) renamed the areaslsquostorage areasrsquo

Tabaniid flies (horse and deer flies from for instancethe genera

Tabanus

and

Chrysops

) have been reportedto disseminate anthrax and to excrete

B anthracis

in theirfaeces up to 13 days (the average lifetime of adult tabaniidflies) after initial feeding on animals infected with anthrax(Khrisna Rao and Mohiyudeen 1958) These flies havealso shown ability to transmit anthrax even after subse-quent feeding on uninfected hosts (Krinsky 1976) In anexperiment with radioactive-labelled blood from an impalacarcass Braack and De Vos (1990) were able to showthat the faeces of carrion-feeding blowflies (

Diptera

Fam-ily Calliphoridae) were deposited in the vicinity of thecarcass on leaves and twigs The kudu antelopes in SouthAfrica normally eat leaves and twigs and could thereforebe more at risk of acquiring anthrax disseminated thisway Moreover these browsers are normally severelyaffected in anthrax epizootic episodes In laboratoryexperiments stable flies and mosquitoes have beenshown to transmit

B anthracis

after feeding on infectedanimals (Turell and Knudson 1987) Also faeces samplescollected from scavengers in the Etosha National Park inNamibia revealed

B anthracis

spores in more than halfthe samples (Lindeque and Turnbull 1994) indicating apossible route of dissemination Furthermore the samestudy showed a rapid decline in shed vegetative bacilliand failed to demonstrate multiplication of

B anthracis

inthe environment

Properties of



is generally regarded as an insect

Fig 1

An illustration of the known pathogenic life cycles of

B anthracis

and

B thuringiensis

Although a human pathogen

B cereus

has not been shown to enter a pathogenic life cycle similar to those of

B anthracis

and

B thuringiensis


B cereus

and relatives

633



5

631ndash640

pathogen because of its ability to produce large crystalprotein inclusions (

d

-endotoxins) during sporulation theonly feature that can distinguish

B thuringiensis

from

Bcereus

(Baumann

et al

1984) These inclusions whichconstitute up to 25 of the dry weight of the sporulatedcells (Agaisse and Lereclus 1995) are responsible for thebiopesticide activity of the bacterium and its target spec-ificity (van Rie

et al

1990) (see Fig 1) The genes encod-ing the insecticidal proteins are generally located on largetransferable plasmids (Kronstad

et al

1983 Gonzaacutelezand Carlton 1984) The

B thuringiensis

denominationactually comprises a considerable number of isolates cov-ering a broad range of toxins active against larvae fromdifferent insect orders especially

Lepidoptera

Diptera

and

Coleoptera

At present more than 235 delta-endot-oxin gene sequences have been described (Crickmore

et al

2002) and 82 different serotypes have beenreported (Lecadet

et al

1999) The numbers of delta-endotoxin genes are thought to grow steadily as most

Bthuringiensis

strains carry more than one delta-endotoxingene Furthermore several

B thuringiensis

strains areknown to produce vegetative insecticidal proteins (VIPs)Unlike the

d

-endotoxins the expression of which isrestricted to sporulation VIPs are expressed in the vege-tative stage of growth starting at mid-log phase as well asduring sporulation

Although

B thuringiensis

is an insect pathogen theecology of the bacteria is still somewhat of an enigmaAccording to Martin and Travers (1989)

B thuringiensis

is a ubiquitous soil microorganism but it is also found inenvironmental niches including phylloplane and insectsDescriptions of natural epizootic episodes are very rarebut were reported in the first observation of

B thuringiensis

by Ishiwata (Milner 1994) in water mills (Vankova andPurrini 1979) in a corn crop (Porcar and Caballero 2000)and in mosquito breeding habitats (Damgaard 2000) Inaddition to being organized into a structured parasporalcrystal the

d

-endotoxins can also be embedded in thespore wall Du and Nickerson (1996) found that germina-tion of spores of

B thuringiensis

ssp

kurstaki

HD-73 withCry1Ac embedded in the spore coat could be activatedby alkaline conditions whereas selected Cry-negative

Bthuringiensis

ssp

kurstaki

HD-73 could not Furthermorecry

+

spores could bind to toxin receptors in brush bordermembrane preparations a binding that also stimulatedspore germination (Du and Nickerson 1996) This phe-nomenon may in part explain the evolutionary advantageof possessing

d

-endotoxins namely the ability for

B thu-ringiensis

to germinate faster than

B cereus

and thus havea greater chance to proliferate and dominate in an insectgut even in the absence of the crystalline

d

-endotoxinsIt is important to note however that

d

-endotoxins have

per se

no apparent antimicrobial effect for enhancingcolonization efficacy (Koskella and Stotzky 2002)

Several facts andor premises on the ecological nicheoccupied by

B thuringiensis

have been reported (i)

Bthuringiensis

does not grow in soil but is deposited thereby insects (Glare and OrsquoCallaghan 2000) (ii)

B thuring-iensis

may grow in soil when nutrient conditions arefavourable (Saleh et al 1970) and (iii) it occupies thesame niche as B cereus (iv) vegetative B thuringiensisproliferates in the gut of earthworms leather jacket larvaeand in plant rhizospheres (Hendriksen and Hansen2002) (v) multiplication of B thuringiensis occurs ininsects weakened by the presence of other pathogens(Eilenberg et al 2000) and (vi) germinating B thuring-iensis ssp israelensis were found in excreted food vacu-oles of protozoa (Manasherob et al 1998)

These different possibilities are not mutually exclusiveIt is conceivable that B thuringiensis is a natural inhabit-ant of the intestinal systems of certain insects with orwithout provoking disease and eventually death Thus thebacterium is able to be released in soil and can subse-quently proliferate when conditions are propitious Hansenand Salamitou (2000) hypothesized that B thuringiensisis a natural inhabitant of the digestion system of manyinvertebrates As such if the animal is diseased the Bthuringiensis present in the digestion system can start togrow in the dyingdead carcass As nutrients become lim-ited sporulation occurs along with the production of d-endotoxins These spores and toxins can then contributeto a local epizootic in dense populations of target organ-isms The presence of B thuringiensis in the intestine ofmammals is transient indicating that the food of theseanimals has varying contents of B thuringiensis(Swiecicka et al 2002) Along the same lines long-termsheep feeding with B thuringiensis-based biopesticidepreparations (ordf 1012 spores daily for 5 months) did notharm the animals (Hadley et al 1987) Furthermorerecent studies of faecal samples from greenhouse work-ers did not show adverse effects after exposure to Bthuringiensis (Jensen et al 2002)

Rhizoid-growing and psychrotolerant bacteria

Rhizoid growth is characterized by the production of col-onies with filaments or root-like structures that may extendseveral centimetres from the site of inoculation Relativelyfew data are available on the rhizoid-growing bacteria Bmycoides and B pseudomycoides and more specificallyon their ecology As for the other members of the B cereusgroup they have been isolated from various environmentalniches including manured soils (Klimanek and Greilich1976) activated sludge arthropod guts (C Vannieuwen-burgh and J Mahillon unpublished results) or plant rhizo-sphere where they are thought to have antagonisticactivity against fungal species (Pandey et al 2001) Sim-ilarly inhibition of the pathogen Listeria monocytogenes

634 G B Jensen B M Hansen J Eilenberg and J Mahillon

copy 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Environmental Microbiology 5 631ndash640

by putative B mycoides has also been reported in silage(Irvin 1969) Although the rhizoid growth is characteristicof B mycoides non-rhizoid variants have been describedin a study of environmental isolates of B mycoides by vonWintzingerode et al (1997) it was found that fatty acidanalysis identified the majority of the isolates as Bmycoides even though they lacked the characteristic rhiz-oid growth Even less information has been gathered onB weihenstephanensis which regroups part of the psy-chrotolerant B cereus isolates (Lechner et al 1998 Sten-fors and Granum 2001) except for their wide distributionin natural habitats (von Stetten et al 1999)

The hidden life cycle of B cereus

One major consequence of the lack of knowledge on theecology of B cereus is that pleomorphism of B cereushas not been given much attention This could result partlyfrom the general notion of modern microbiologists thatbacteria only occasionally show slight morphological vari-ation In the very early days of microbiology the study ofmicroorganisms was almost exclusively restricted tomicroscopical observations and hence surprisinglydetailed observations were made then

Bacillus cereus is a well-known food poisoning bacte-rium B cereus causes two distinct types of food poison-ing characterized either by diarrhoea and abdominal pain(diarrhoeal syndrome) or by nausea and vomiting (emeticsyndrome) The latter has often been associated with friedrice Apart from food poisoning cases there are only afew reports on intestinal carriage of B cereus Turnbulland Kramer (1985) reported seasonal changes in theisolation of B cereus ranging from 243 in the winter to43 in the summer from faecal samples from 120 schoolchildren Ghosh (1978) reported the presence of Bcereus in 100 samples from 711 adults (14) Bothpapers stated that because of the omnipresence of Bcereus in many food products the bacteria are inevitablyingested in small numbers and thus contribute to thetransitory intestinal flora

A place to look for this bacterium in its natural niche isthe gut microflora of invertebrates In certain arthropodsthe lsquointestinal stagersquo of B cereus has been shown to befilamentous the so-called Arthromitus stage In fact thisfilamentous stage of the bacterium was discovered indifferent soil-dwelling arthropods as early as 1849 (Leidy1849) The filamentous forms of B cereus have beenstudied in continuous cultures (Wahren et al 1967) andhave lately been proposed as the normal intestinal stageof B cereus sensu lato in soil-dwelling insects (Marguliset al 1998) Furthermore colonization of mosquito larvaeand various soil-dwelling pests by B cereus has beenobserved (Feinberg et al 1999 Luxananil et al 2001Wenzel et al 2002) (see Fig 1)

Other circumstantial evidence supports the data on Bcereus colonization of insect gut systems In aphidsDasch et al (1984) reported that the introduction of pen-icillin had little effect on growth and as evident fromTable 1 the majority of the members of the B cereusgroup are known to produce b-lactamases In one casethe symbiont of Cletus signatus (a hemipteran insect) isidentified as B cereus var signatus (Singh 1974) A highfrequency of vegetative B cereus and B mycoides hasbeen found in the gut of the earthworm Lumbricus terres-tris (B M Hansen and N B Hendriksen unpublishedresults)

Gene transfer in the environment

Interestingly earthworms are known to contribute to genetransfer activity with gut passage being a prerequisite forDNA transfer (Daane et al 1996 Thimm et al 2001)Other insects have been shown to promote gene transferand transfer of B thuringiensis plasmids has been observedin lepidopteran larvae (Jarrett and Stephenson 1990 Tho-mas et al 2000 2001) It is therefore tempting to envisagethe continuous exchange of B thuringiensis plasmids asthese phenotypevirulence plasmids are easily transferableby transduction mobilization or conjugation (Reddy et al1987 Green et al 1989 Stepanov et al 1989 Jensenet al 1996) The actual exchange of DNA is further cor-roborated by the data presented in Table 1 eg the repliconof the virulence plasmid pXO2 is almost identical to thereplicon found on the conjugative plasmid pAW63 from Bthuringiensis ssp kurstaki HD-73 (Wilcks et al 1999)Other genotypical features such as the presence of geneticmarkers for phospholipase C and non-haemolytic entero-toxin genes characteristic of B cereus further substantiatethe close relationship among these species Furthermoreserotyping of B thuringiensis has revealed that the numberof cross-reacting H-antigens among B cereus strains isincreasing (Lecadet et al 1999)

However the case of B anthracis has its own particu-larities It now seems likely that B anthracis does notsimply stem from the superposition of the virulence genesborne by the pXO1 and pXO2 plasmids on the chromo-somic background of an opportunistic bacteria B cereusRecent studies have indeed indicated that the lsquoemer-gencersquo of B anthracis as a specialized animal and humanpathogen has most probably proceeded through a step-wise reciprocal adaptation between its chromosomal andextrachromosomal genomes (Mignot 2002) This hasresulted in a finely tuned gene regulation of different oper-ons and regulons such as those involved in sporulationgermination haemolytic activity capsule formation or exo-toxin expression These complex genomic cross-talks arethought to be mediated by an arsenal of gene regulatorsamong which are the plasmid-encoded PagR and AtxA

The hidden lifestyles of B cereus and relatives 635


Table 1 Selected phenotypic genotypic and ecological features of B anthracis B thuringiensis and B cereus

B anthracis B thuringiensis B cereus

PhenotypePenicillin resistant(b-lactamase production)

11 of tested B anthracisstrains showed resistance topenicillin G (Cavallo et al 2002)

Yes 1 of tested strains showed no resistance to ampicillin(Rusul and Yaacob 1995)

Haemolytic activitya

(on sheep erythrocytes)Weak haemolysis by somestrains of B anthracis(Drobniewski 1993Guttmann and Ellar 2000)

Yes Few haemolysis-negative mutants have been isolated

Motility Isolated monoflagellar B anthracis have beendescribed (Liang and Yu 1999)Occasional motile strains (Brownand Cherry 1955)

Spontaneous flagella-minusmutants of B thuringiensiscan be readily isolated

4 of tested strains showed no motility (Logan and Berkeley 1984) Occasional isolation of non-motile variants (Brown and Cherry 1955)

Crystalline parasporalinclusions

No 6 of tested strains showed noinclusions (Logan and Berkeley 1984)

No

Mucoid colony(capsule synthesis)

Yes No No

Gamma phage sensitivity Several rare B anthracis are refractory (Abshire et al 2001)

No B cereus ATCC4342 susceptible(Abshire et al 2001)

Chitinase activity Activity was not found in B anthracis Guttmann and Ellar 2000)

Yes Yes

GenotypepXO1 Yes Sequence homology to pBtoxis

of Bt ssp israelensis (Berry et al 2002)

B cereus (ATCC 43881) shows high homology to an unknown ORF of pXO1 (co-ordinates 121815ndash122327) (Okinaka et al 1999b Pannucci et al 2002)

IS231 from Bt ssp finitimus found in pXO1(Okinaka et al 1999b)B thuringiensis ssp kurstaki(ATCC 33679) shows high homology to an unknownORF in pXO1 (base numbers 121815ndash122327) (Okinaka et al 1999b Pannucci et al 2002)

pXO2 Growth of B anthracis outside a host often leads to loss of pXO2

The replicons of pAW63 fromBt ssp kurstaki are almostidentical to that of pXO2 (Wilcks et al 1999)

Phospholipase C +(Mignot et al 2001 G B Jensen unpublished results)

+ +

nheA gene (accessionno Y19005)

+ (Mignot et al 2001 G B Jensen unpublished results)

+ +

EcologyHost range(toxin specific)

Vertebrates InvertebratesSpecific toxins are only activeagainst a limited number of related invertebrate hosts

Not known

Distribution Worldwide but many areas not yet studied

Worldwide but lack of successin isolating in Antarctica (Wasano et al 1999)

Worldwide

Prevalence in hosts Endemic in AfricaAsia Generally low natural levels ofinfection occasional epidemicsamong mosquitoes and insectsin stored product environment(Milner 1994)

Present in invertebrates (gut system) but not regarded as a disease of invertebrates

a Note that at least four distinct haemolytic protein complexes can participate in the haemolytic activity



(Guignot et al 1997 Mignot et al 2001 Mignot 2002)For instance the pleiotrophic regulator PlcR that regulatesseveral virulence functions in B cereus (Gohar et al2002) is inactive in B anthracis because of a nonsensemutation The introduction of a functional PlcR in Banthracis activates several B cereus-like virulence func-tions which are not normally expressed in B anthracis(Mignot et al 2001) This is in agreement with the dataof Bonventre (1965) who found that in contrast to Bcereus filtrates from liquid cultures of B anthracis werenot toxic to animal tissue culture cells

Table 1 lists the textbook characteristics of each mem-ber of the B cereus group together with exceptions foundin the literature These data are intended to display boththe close relationship among the species and subse-quently the possible pitfalls of data misinterpretationThus B anthracis seems to constitute a narrow group ofhighly similar strains which have only recently been dis-tinguished genetically (Jackson et al 1999 Ticknor et al2001) Consequently and as the most significant differ-ences are plasmid encoded it seems appropriate to(p)reserve the name B anthracis for B cereus strainspossessing the pXO1 and pXO2 plasmids Likewise

emetic B cereus strains constitute a narrow group ofbacteria most of which belong to the B cereus H-1 sero-type Furthermore strains that produce the emetic toxindo not show expression of enterotoxins and starch hydro-lytic activity (Agata et al 1996 Pirttijarvi et al 2000)

Conclusion

The presence of B anthracis in both vultures and variousbiting insects reveals multiple routes of recycling of Banthracis Whether there is de facto colonization of theintestinal systems of both the vultures and the insects orthe observations cited here resulted from transient expo-sures resulting from feeding habits is still debatable How-ever the carnivorous nature of the Tabanus larvae mayequip the adult fly with an intestinal flora comprising anymember(s) of the B cereus group and although much ofthe data on anthrax transmission by tabaniid flies is exper-imental the importance of tabaniid flies in natural out-breaks is conceivable According to previously presenteddata B cereus can enter a filamentous stage in which itcolonizes a variety of insects In this context it is sug-gested as illustrated in Fig 2 that members of the B

Fig 2 A supposed model in which the mem-bers of the B cereus group experience two life cycles one type in which the bacteria live in a symbiotic relation with their invertebrate host(s) and another more infrequent life cycle in which the bacteria can multiply rapidly in another infected insect host or a mammal



cereus group experience two types of life cycles one inwhich the bacteria live in a symbiotic relation with theirinvertebrate host(s) and another more infrequent lifecycle in which the bacteria can multiply rapidly in anotherand infected host (invertebrate or vertebrate) The rela-tionship between the two types of life cycle has not yetbeen documented experimentally but some indicationsexist In the case of a pathogenic relationship the inver-tebrate host from the symbiotic relationship becomes thevector of the disease

For example a recent study showed that female mos-quitoes are attracted to culture filtrates of B thuringiensisfor ovipositioning (Poonam et al 2002) It is possible thatthese and other insects could have a preference for ovi-positioning in areas where B thuringiensis is frequentlylocated ie soil (Martin and Travers 1989) activatedsludge (Mizuki et al 2001) water (Ichimatsu et al 2000Maeda et al 2000) and the lsquostorage areasrsquo mentionedearlier subsequently giving the larvae a possibility ofbeing fitted with an intestinal flora consisting of membersof the B cereus group These bacteria can then providetheir host with enhanced capabilities for instance degrad-ing cellulose (Wenzel et al 2002)

Further studies on the ecology of B anthracis Bcereus and B thuringiensis will hopefully not only shedlight on the working models proposed here They will alsoenable us to set up better controlling programmes thatcould cope with different objectives One objective is toavoid B anthracis outbreaks especially in risk areasOther objectives are to improve the biotechnological useof B thuringiensis and consequently obtain better controlof insect pests

Although experimental evidence is still missing it islikely that the rhizoid-growing bacteria share part of thehorizontal gene pool of the B cereus senso lato groupusing plasmid conjugation phage transduction or DNAtransformation Consequently it remains to be seenwhether and how these still cryptic bacteria participatedirectly or indirectly in the various life cycles of the othermembers of the B cereus group

Acknowledgements

We are indebted to Tacircm Mignot for his inspiring PhD thesisWe are thankful to Lars Andrup for fruitful discussions andcritical reading of the manuscript JM is a research associ-ate at the National Fund for Scientific Research (FNRSBelgium)

References

Abshire TG Brown JE Teska JD Allan CM RedusSL and Ezzell JW (2001) Validation of the use ofgamma phage for identifying Bacillus anthracis In Pro-

ceedings of the Fourth International Conference onAnthrax 10ndash13 June St Johnrsquos College Annapolis MD

Agaisse H and Lereclus D (1995) How does Bacillus thu-ringiensis produce so much insecticidal crystal protein JBacteriol 177 6027ndash6032

Agata N Ohta M and Mori M (1996) Production of anemetic toxin cereulide is associated with a specific classof Bacillus cereus Curr Microbiol 33 67ndash69

Baumann L Okamoto K Unterman BM Lynch MJand Baumann P (1984) Phenotypic characterization ofBacillus thuringiensis and Bacillus cereus J InvertebrPathol 44 329ndash341

Berry C OrsquoNeil S Ben Dov E Jones AF Murphy LQuail MA et al (2002) Complete sequence and organi-zation of pBtoxis the toxin-coding plasmid of Bacillus thu-ringiensis subsp israelensis Appl Environ Microbiol 685082ndash5095

Bhatnagar R and Batra S (2001) Anthrax toxin Crit RevMicrobiol 27 167ndash200

Bonventre PF (1965) Differential cytotoxicity of Bacillusanthracis and Bacillus cereus culture filtrates J Bacteriol90 284ndash285

Braack LE and De Vos V (1990) Feeding habits and flightrange of blow-flies (Chrysomyia spp) in relation to anthraxtransmission in the Kruger National Park South AfricaOnderstepoort J Vet Res 57 141ndash142

Brown ER and Cherry WB (1955) Specific identificationof Bacillus anthracis by means of a variant bacteriophageJ Infect Dis 96 34ndash39

Cavallo JD Ramisse F Girardet M Vaissaire J MockM and Hernandez E (2002) Antibiotic susceptibilities of96 isolates of Bacillus anthracis isolated in France between1994 and 2000 Antimicrob Agents Chemother 46 2307ndash2309

Chen ML and Tsen HY (2002) Discrimination of Bacilluscereus and Bacillus thuringiensis with 16S rRNA and gyrBgene based PCR primers and sequencing of their anneal-ing sites J Appl Microbiol 92 912ndash919

Crickmore N Zeigler DR Schnepf E Van Rie JLereclus D Baum J et al (2002) Bacillus thuringiensistoxin nomenclature [wwwdocument] URL httpwwwbiolssusxacukHomeNeil_CrickmoreBtIndexhtml

Daane LL Molina JAE Berry EC and Sadowsky MJ(1996) Influence of earthworm activity on gene transferfrom Pseudomonas fluorescens to indigenous soil bacte-ria Appl Environ Microbiol 62 515ndash521

Daffonchio D Cherif A and Borin S (2000) Homoduplexand heteroduplex polymorphisms of the amplified riboso-mal 16S-23S internal transcribed spacers describegenetic relationships in the lsquoBacillus cereus grouprsquo ApplEnviron Microbiol 66 5460ndash5468

Damgaard PH (2000) Natural occurrence and dispersal ofBacillus thuringiensis in the environment In Ento-mopathogenic Bacteria from Laboratory to Field Applica-tion Charles J-F Delecluse A and Nielsen-LeRouxC (eds) Dordrecht Kluwer Academic Publishers pp 23ndash40

Dasch GA Weiss E and Chang K (1984) Endosymbiot-ics of insects In Bergeyrsquos Manual of Systematic Bacteriol-ogy Krieg NR (ed) Baltimore Williams amp Wilkins pp811ndash833



Davaine C (1863) Recherches sur les infuoires du sangdans la maladie de la pustule maligne C R Acad Sci 601296ndash1299

Dragon DC and Rennie RP (1995) The ecology ofanthrax spores tough but not invincible Can Vet J 36295ndash301

Drobniewski FA (1993) Bacillus cereus and related spe-cies Clin Microbiol Rev 6 324ndash338

Du C and Nickerson KW (1996) Bacillus thuringiensisHD-73 spores have surface-localized cry1ac toxin physio-logical and pathogenic consequences Appl Environ Micro-biol 62 3722ndash3726

Eilenberg J Damgaard PH Hansen BM PedersenJC Bresciani J and Larsson R (2000) Natural coprev-alence of Strongwellsea castrans Cystosporogenes deli-aradicae and Bacillus thuringiensis in the host Deliaradicum J Invertebr Pathol 75 69ndash75

Feinberg L Jorgensen J Haselton A Pitt A Rudner Rand Margulis L (1999) Arthromitus (Bacillus cereus) sym-bionts in the cockroach Blaberus giganteus dietary influ-ences on bacterial development and population densitySymbiosis 27 104ndash123

Ghosh AC (1978) Prevalence of Bacillus cereus in thefaeces of healthy adults J Hyg 80 233ndash236

Glare TR and OrsquoCallaghan M (2000) Bacillus Thuringien-sis Biology Ecology and Safety Chichester UK JohnWiley amp Sons

Gohar M Oslashkstad OA Gilois N Sanchis V Kolstoslash ABand Lereclus D (2002) Two-dimensional electrophoresisanalysis of the extracellular proteome of Bacillus cereusreveals the importance of the PlcR regulon Proteomics 2784ndash791

Gonzaacutelez JM Jr and Carlton BC (1984) A large trans-missible plasmid is required for crystal toxin production inBacillus thuringiensis variety israelensis Plasmid 11 28ndash38

Green BD Battisti L and Thorne CB (1989) Involvementof Tn4430 transfer of Bacillus anthracis plasmids mediatedby Bacillus thuringiensis plasmid pXO12 J Bacteriol 171104ndash113

Guignot J Mock M and Fouet A (1997) AtxA activatesthe transcription of genes harbored by both Bacillus anthra-cis virulence plasmids FEMS Microbiol Lett 147 203ndash207

Guttmann DM and Ellar DJ (2000) Phenotypic andgenotypic comparisons of 23 strains from the Bacilluscereus complex for a selection of known and putative Bthuringiensis virulence factors FEMS Microbiol Lett 1887ndash13

Hadley WM Burchiel SW McDowell TD Thilsted JPHibbs CM Whorton JA et al (1987) Five-month oral(diet) toxicityinfectivity study of Bacillus thuringiensisinsecticides in sheep Fundam Appl Toxicol 8 236ndash242

Hansen BM and Salamitou S (2000) Virulence of Bacillusthuringiensis In Entomopathogenic Bacteria from Labora-tory to Field Application Charles J-F Delecluse A andNielsen-LeRoux C (eds) Dordrecht Kluwer AcademicPublishers pp 41ndash64

Hansen BM Leser TD and Hendriksen NB (2001)Polymerase chain reaction assay for the detection of Bacil-lus cereus group cells FEMS Microbiol Lett 202 209ndash213

Harrell LJ Andersen GL and Wilson KH (1995)

Genetic variability of Bacillus anthracis and related spe-cies J Clin Microbiol 33 1847ndash1850

Helgason E Okstad OA Caugant DA Johansen HAFouet A Mock M et al (2000) Bacillus anthracis Bacil-lus cereus and Bacillus thuringiensis ndash one species on thebasis of genetic evidence Appl Environ Microbiol 662627ndash2630

Hendriksen NB and Hansen BM (2002) Long-term sur-vival and germination of Bacillus thuringiensis var kurstakia field trial Can J Microbiol 48 256ndash261

Ichimatsu T Mizuki E Nishimura K Akao T Saitoh HHiguchi K and Ohba M (2000) Occurrence of Bacillusthuringiensis in fresh waters of Japan Curr Microbiol 40217ndash220

Irvin AD (1969) The inhibition of Listeria monocytogenesby an organism resembling Bacillus mycoides present innormal silage Res Vet Sci 10 106ndash108

Jackson PJ Hill KK Laker MT Ticknor LO and KeimP (1999) Genetic comparison of Bacillus anthracis and itsclose relatives using amplified fragment length polymor-phism and polymerase chain reaction analysis J ApplMicrobiol 87 263ndash269

Jarrett P and Stephenson M (1990) Plasmid transferbetween strains of Bacillus thuringiensis infecting Galleriamellonella and Spodoptera littoralis Appl Environ Microbiol56 1608ndash1614

Jensen GB Andrup L Wilcks A Smidt L and PoulsenOM (1996) The aggregation-mediated conjugation sys-tem of Bacillus thuringiensis subsp israelensis host rangeand kinetics Curr Microbiol 33 228ndash236

Jensen GB Larsen P Jacobsen BL Madsen B SmidtL and Andrup L (2002) Bacillus thuringiensis in fecalsamples from greenhouse workers after exposure to Bthuringiensis-based pesticides Appl Environ Microbiol 684900ndash4905

Khrisna Rao NS and Mohiyudeen S (1958) Tabanus fliesas transmitters of anthrax a field experience Ind Vet J 35248ndash253

Klimanek E and Greilich J (1976) [To the ecology of Bacil-lus cereus var mycoides (Flugge) in loess black earth inrelation to fertilization] Zentralbl Bakteriol ParasitenkdInfektionskr Hyg 131 66ndash71

Knudson GB (1986) Photoreactivation of ultraviolet-irradi-ated plasmid-bearing and plasmid-free strains of Bacillusanthracis Appl Environ Microbiol 52 444ndash449

Koskella J and Stotzky G (2002) Larvicidal toxins fromBacillus thuringiensis subspp kurstaki morrisoni (straintenebrionis) and israelensis have no microbicidal or micro-biostatic activity against selected bacteria fungi and algaein vitro Can J Microbiol 48 262ndash267

Krinsky WL (1976) Animal disease agents transmitted byhorse flies and deer flies (Diptera Tabanidae) J Med Ento-mol 13 225ndash275

Kronstad JW Schnepf HE and Whiteley HR (1983)Diversity of location for Bacillus thuringiensis crystal pro-tein genes J Bacteriol 154 419ndash428

Lecadet MM Frachon E Dumanoir VC Ripouteau HHamon S Laurent P and Thiery I (1999) Updating theH-antigen classification of Bacillus thuringiensis J ApplMicrobiol 86 660ndash672

Lechner S Mayr R Francis KP Pruss BM Kaplan T



Wiessner-Gunkel E et al (1998) Bacillus weihenstephan-ensis sp nov is a new psychrotolerant species of theBacillus cereus group Int J Syst Bacteriol 48 1373ndash1382

Leidy J (1849) Enterobrus a new genus of ConfervaceaeProc Acad Nat Sci Philadelphia 4 225ndash233

Liang X and Yu D (1999) Identification of Bacillus anthra-cis strains in China J Appl Microbiol 87 200ndash203

Lindeque PM and Turnbull PC (1994) Ecology and epi-demiology of anthrax in the Etosha National Park NamibiaOnderstepoort J Vet Res 61 71ndash83

Logan NA and Berkeley RCW (1984) Identification ofBacillus strains using the API system J Gen Microbiol 1301871ndash1882

Luxananil P Atomi H Panyim S and Imanaka T (2001)Isolation of bacterial strains colonizable in mosquito larvalguts as novel host cells for mosquito control J BiosciBioeng 92 342ndash345

Maeda M Mizuki E Nakamura Y Hatano T and OhbaM (2000) Recovery of Bacillus thuringiensis from marinesediments of Japan Curr Microbiol 40 418ndash422

Manasherob R Bendov E Zaritsky A and Barak Z(1998) Germination growth and sporulation of Bacillusthuringiensis subsp israelensis in excreted food vacuolesof the protozoan Tetrahymena pyriformis Appl EnvironMicrobiol 64 1750ndash1758

Margulis L Jorgensen JZ Dolan S Kolchinsky RRainey FA and Lo SC (1998) The Arthromitus stageof Bacillus cereus intestinal symbionts of animals ProcNatl Acad Sci USA 95 1236ndash1241

Martin PAW and Travers RS (1989) Worldwide abun-dance and distribution of Bacillus thuringiensis isolatesAppl Environ Microbiol 55 2437ndash2442

Mignot T (2002) Les proteacuteines de couches S et le reacutegulon-PlcR de Bacillus anthracis implications physiologiques eteacutevolutives ThesisDissertation Pasteur Institute ParisFrance

Mignot T Mock M Robichon D Landier A Lereclus Dand Fouet A (2001) The incompatibility between the PlcR-and AtxA-controlled regulons may have selected a non-sense mutation in Bacillus anthracis Mol Microbiol 421189ndash1198

Milner RJ (1994) History of Bacillus thuringiensis AgricEcosyst Environ 49 9ndash13

Minett FC and Dhanda MR (1941) Multiplication of Banthracis and Cl chauvaei in soil and in water Ind J VetSci Anim Husb 11 308ndash328

Mizuki E Maeda M Tanaka R Lee DW Hara MAkao T et al (2001) Bacillus thuringiensis a commonmember of microflora in activated sludges of a sewagetreatment plant Curr Microbiol 42 422ndash425

Mock M and Fouet A (2001) Anthrax Annu Rev Microbiol55 647ndash671 647ndash671

Okinaka R Cloud K Hampton O Hoffmaster A Hill KKeim P et al (1999a) Sequence assembly and analysisof pX01 and pX02 J Appl Microbiol 87 261ndash262

Okinaka RT Cloud K Hampton O Hoffmaster AR HillKK Keim P et al (1999b) Sequence and organizationof pXO1 the large Bacillus anthracis plasmid harboring theanthrax toxin genes J Bacteriol 181 6509ndash6515

Pandey A Palni LM and Bisht D (2001) Dominant fungiin the rhizosphere of established tea bushes and their

interaction with the dominant bacteria under in situ condi-tions Microbiol Res 156 377ndash382

Pannucci J Okinaka RT Sabin R and Kuske CR(2002) Bacillus anthracis pXO1 plasmid sequence conser-vation among closely related bacterial species J Bacteriol184 134ndash141

Pirttijarvi TS Andersson MA and Salkinoja-SalonenMS (2000) Properties of Bacillus cereus and other bacillicontaminating biomaterial-based industrial processes IntJ Food Microbiol 60 231ndash239

Poonam S Paily KP and Balaraman K (2002) Oviposi-tion attractancy of bacterial culture filtrates response ofCulex quinquefasciatus Mem Inst Oswaldo Cruz 97 359ndash362

Porcar M and Caballero P (2000) Molecular and insecti-cidal characterization of a Bacillus thuringiensis strain iso-lated during a natural epizootic J Appl Microbiol 89 309ndash316

Rayer P (1850) Inoculation du sang de rate C R Soc BiolParis 2 141ndash144

Reddy A Battisti L and Thorne CB (1987) Identificationof self-transmissible plasmids in four Bacillus thuringiensissubspecies J Bacteriol 169 5263ndash5270

van Rie J McGaughey WH Johnson DE Barnett BDand van Mellaert H (1990) Mechanism of insect resis-tance to the microbial insecticide Bacillus thuringiensisScience 247 72ndash74

Rusul G and Yaacob NH (1995) Prevalence of Bacilluscereus selected foods and detection of enterotoxin usingTECRA-VIA and BCET-RPLA Int J Food Microbiol 25131ndash139

Saleh SM Harris RF and Allen ON (1970) Fate ofBacillus thuringiensis soil effect of soil pH and organicamendment Can J Microbiol 16 677ndash680

Singh G (1974) Endosymbiotic microorganisms in Cletussignatus Walker (Coreidae Heteroptera) Experientia 301406ndash1407

Stenfors LP and Granum PE (2001) Psychrotolerantspecies from the Bacillus cereus group are not necessarilyBacillus weihenstephanensis FEMS Microbiol Lett 197223ndash228

Stepanov AS Puzanova OB Gavrilov SV Brandzi-shevskii I Bragin IV and Anisimov PI (1989) [Trans-duction and conjugation transfer of the pXO2 plasmid inBacillus anthracis] Mol Gen Mikrobiol Virusol Dec 39ndash43

von Stetten F Mayr R and Scherer S (1999) Climaticinfluence on mesophilic Bacillus cereus and psychrotoler-ant Bacillus weihenstephanensis populations in tropicaltemperate and alpine soil Environ Microbiol 1 503ndash515

Swiecicka I Fiedoruk K and Bednarz G (2002) Theoccurrence and properties of Bacillus thuringiensis isolatedfrom free-living animals Lett Appl Microbiol 34 194ndash198

Thimm T Hoffmann A Fritz I and Tebbe CC (2001)Contribution of the earthworm Lumbricus rubellus (Annel-ida Oligochaeta) to the establishment of plasmids in soilbacterial communities Microb Ecol 41 341ndash351

Thomas DJI Morgan JAW Whipps JM and Saun-ders JR (2000) Plasmid transfer between the Bacillusthuringiensis subspecies kurstaki and tenebrionis labora-tory culture and soil and in lepidopteran and coleopteranlarvae Appl Environ Microbiol 66 118ndash124



Thomas DJ Morgan JA Whipps JM and SaundersJR (2001) Plasmid transfer between Bacillus thuringiensissubsp israelensis strains in laboratory culture river waterand dipteran larvae Appl Environ Microbiol 67 330ndash338

Ticknor LO Kolsto AB Hill KK Keim P Laker MTTonks M and Jackson PJ (2001) Fluorescent amplifiedfragment length polymorphism analysis of NorwegianBacillus cereus and Bacillus thuringiensis soil isolatesAppl Environ Microbiol 67 4863ndash4873

Turell MJ and Knudson GB (1987) Mechanical transmis-sion of Bacillus anthracis by stable flies (Stomoxys calci-trans) and mosquitoes (Aedes aegypti and Aedestaeniorhynchus) Infect Immun 55 1859ndash1861

Turnbull PC and Kramer JM (1985) Intestinal carriage ofBacillus cereus faecal isolation studies in three populationgroups J Hyg (London) 95 629ndash638

Van Ness GB (1971) Ecology of anthrax Science 1721303ndash1307

Vankova J and Purrini K (1979) Natural epizooties causedby bacilli of the species Bacillus thuringiensis and Bacilluscereus Z Angew Entomol 88 216ndash221

Wahren A Holme T Haggmark A and Lundquist PG(1967) Studies on filamentous forms of Bacillus cereusstrain T J Gen Microbiol 49 59ndash65

Wasano N Imura S and Ohba M (1999) Failure torecover Bacillus thuringiensis from the Luumltzow-HolmBay region of Antarctica Lett Appl Microbiol 28 49ndash51

Wenzel M Schonig I Berchtold M Kampfer P andKonig H (2002) Aerobic and facultatively anaerobic cellu-lolytic bacteria from the gut of the termite Zootermopsisangusticollis J Appl Microbiol 92 32ndash40

Wilcks A Smidt L Oslashkstad OA Kolstoslash A-B MahillonJ and Andrup L (1999) Replication mechanism andsequence analysis of the replicon of pAW63 a conjugativeplasmid from Bacillus thuringiensis J Bacteriol 181 3193ndash3200

von Wintzingerode F Rainey FA Kroppenstedt RM andStackebrandt E (1997) Identification of environmentalstrains of bacillus mycoides by fatty acid analysis and spe-cies-specific 16s rDNA oligonucleotide probe FEMSMicrobiol Ecol 24 201ndash209

632

G B Jensen B M Hansen J Eilenberg and J Mahillon



5

631ndash640

strain Both plasmids have been sequenced recently(Okinaka

et al

1999ab)Current models of

B anthracis

ecology rely on its patho-genicity ie the spores are ingested by herbivores theanimals becomes infected and the bacteria proliferate inthe lymphoid glands concomitantly expressing the exotox-ins which ultimately leads to the death of the animal (seeFig 1) Once the animal is dead the vegetative cells of

B anthracis

having reached a serum concentration of

gt

10

7

cells ml

-

1

will be outcompeted by anaerobic bacteriafrom the gastrointestinal tract through antagonistic inter-actions (Dragon and Rennie 1995) The environmentalfate of the spore is not known in detail The spores willsurvive lsquoindefinitelyrsquo in dry and protected environmentsHowever exposure to sunlight for 4 h has a significantnegative effect on the survival of the spores (Lindequeand Turnbull 1994) Furthermore photoinduced repair ofUV damage is absent in

B anthracis

spores of the Sternevaccine strain (Knudson 1986) A few reports stated thatspores could actually germinate in nature when conditionsare favourable Van Ness (1971) reported that soil pHabove 60 and temperatures above 155

infin

C favour out-breaks of anthrax The term lsquoincubator areasrsquo has beenintroduced to describe puddles in which decaying grassand other organic matter constitute the nutrients neces-sary for the germination of

B anthracis

spores Howeverthe study by Van Ness (1971) did not show actual growthof

B anthracis

in these incubator areas and other studiesindicated that

B anthracis

has very specific growthrequirements making it very unlikely for the spores togerminate outside a host (Minett and Dhanda 1941) It isalso noteworthy that growth of

B anthracis

outside a hostoften leads to loss of virulence caused by loss of theplasmid carrying the capsule gene (pXO2) ndash an argument

that further reduces the incubator area theory As a con-sequence Dragon and Rennie (1995) renamed the areaslsquostorage areasrsquo

Tabaniid flies (horse and deer flies from for instancethe genera

Tabanus

and

Chrysops

) have been reportedto disseminate anthrax and to excrete

B anthracis

in theirfaeces up to 13 days (the average lifetime of adult tabaniidflies) after initial feeding on animals infected with anthrax(Khrisna Rao and Mohiyudeen 1958) These flies havealso shown ability to transmit anthrax even after subse-quent feeding on uninfected hosts (Krinsky 1976) In anexperiment with radioactive-labelled blood from an impalacarcass Braack and De Vos (1990) were able to showthat the faeces of carrion-feeding blowflies (

Diptera

Fam-ily Calliphoridae) were deposited in the vicinity of thecarcass on leaves and twigs The kudu antelopes in SouthAfrica normally eat leaves and twigs and could thereforebe more at risk of acquiring anthrax disseminated thisway Moreover these browsers are normally severelyaffected in anthrax epizootic episodes In laboratoryexperiments stable flies and mosquitoes have beenshown to transmit

B anthracis

after feeding on infectedanimals (Turell and Knudson 1987) Also faeces samplescollected from scavengers in the Etosha National Park inNamibia revealed

B anthracis

spores in more than halfthe samples (Lindeque and Turnbull 1994) indicating apossible route of dissemination Furthermore the samestudy showed a rapid decline in shed vegetative bacilliand failed to demonstrate multiplication of

B anthracis

inthe environment

Properties of



is generally regarded as an insect

Fig 1

An illustration of the known pathogenic life cycles of

B anthracis

and

B thuringiensis

Although a human pathogen

B cereus

has not been shown to enter a pathogenic life cycle similar to those of

B anthracis

and

B thuringiensis


B cereus

and relatives

633



5

631ndash640


d


B thuringiensis

from

Bcereus

(Baumann

et al


et al


et al


B thuringiensis


Lepidoptera

Diptera

and

Coleoptera


et al


et al


Bthuringiensis


B thuringiensis


d


Although

B thuringiensis


B thuringiensis


B thuringiensis


d


B thuringiensis

ssp

kurstaki


Bthuringiensis

ssp

kurstaki


+


d


B thu-ringiensis


B cereus


d


d

-endotoxins have

per se



B thuringiensis


Bthuringiensis


B thuring-iensis































No


Yes No No




Yes Yes








+ +



+ +



Not known



Worldwide









Conclusion









Acknowledgements


References










































































































B cereus

and relatives

633



5

631ndash640


d


B thuringiensis

from

Bcereus

(Baumann

et al


et al


et al


B thuringiensis


Lepidoptera

Diptera

and

Coleoptera


et al


et al


Bthuringiensis


B thuringiensis


d


Although

B thuringiensis


B thuringiensis


B thuringiensis


d


B thuringiensis

ssp

kurstaki


Bthuringiensis

ssp

kurstaki


+


d


B thu-ringiensis


B cereus


d


d

-endotoxins have

per se



B thuringiensis


Bthuringiensis


B thuring-iensis































No


Yes No No




Yes Yes








+ +



+ +



Not known



Worldwide









Conclusion









Acknowledgements


References



































































































































No


Yes No No




Yes Yes








+ +



+ +



Not known



Worldwide









Conclusion









Acknowledgements


References
























































































































No


Yes No No




Yes Yes








+ +



+ +



Not known



Worldwide









Conclusion









Acknowledgements


References














































































































Conclusion









Acknowledgements


References















































































































Acknowledgements


References
































































































































































































































































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The hidden lifestyles of Bacillus cereus and relatives

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