Bacillus anthracis: Molecular taxonomy, population ... - CyberLeninka

Infection, Genetics and Evolution 11 (2011) 1218–1224

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Review

Bacillus anthracis: Molecular taxonomy, population genetics, phylogeny and patho-evolution Paola Pilo, Joachim Frey * Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, La¨nggassstrasse 122, Postfach, CH-3001 Bern, Switzerland

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 February 2011 Received in revised form 18 May 2011 Accepted 18 May 2011 Available online 26 May 2011

Bacillus anthracis, the etiological agent of anthrax, manifests a particular bimodal lifestyle. This bacterial species alternates between short replication phases of 20–40 generations that strictly require infection of the host, normally causing death, interrupted by relatively long, mostly dormant phases as spores in the environment. Hence, the B. anthracis genome is highly homogeneous. This feature and the fact that strains from nearly all parts of the world have been analysed for canonical single nucleotide polymorphisms (canSNPs) and variable number tandem repeats (VNTRs) has allowed the development of molecular epidemiological and molecular clock models to estimate the age of major diversifications in the evolution of B. anthracis and to trace the global spread of this pathogen, which was mostly promoted by movement of domestic cattle with settlers and by international trade of contaminated animal products. From a taxonomic and phylogenetic point of view, B. anthracis is a member of the Bacillus cereus group. The differentiation of B. anthracis from B. cereus sensu stricto, solely based on chromosomal markers, is difficult. However, differences in pathogenicity clearly differentiate B. anthracis from B. cereus and are marked by the strict presence of virulence genes located on the two virulence plasmids pXO1 and pXO2, which both are required by the bacterium to cause anthrax. Conversely, anthrax-like symptoms can also be caused by organisms with chromosomal features that are more closely related to B. cereus, but which carry these virulence genes on two plasmids that largely resemble the B. anthracis virulence plasmids. ß 2011 Elsevier B.V. All rights reserved.

Keywords: Bacillus anthracis Identification Subtyping Phylogeny Patho-evolution

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Taxonomy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular mechanisms of pathogenicity and host–pathogen interactions . B. anthracis strain typing and molecular epidemiology . . . . . . . . . . . . . . . . Global genetic population structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Geographical spread of B. anthracis clusters and sublineages . . . . . . . . . . . Pathogenic evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Bacillus anthracis, the causative agent of anthrax, is a sporulating Gram-positive bacterium. Anthrax primarily affects ruminants, although other mammals may also succumb to the disease but less

* Corresponding author. Tel.: +41 31 631 2430; fax: +41 31 631 2634. E-mail address: [email protected] (J. Frey). 1567-1348/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2011.05.013

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frequently. Humans generally acquire anthrax from infected animals or as the result of occupational or nutritional exposure to contaminated animal products. The disease is endemic worldwide but occurs only sporadically in countries where drastic eradication measures have been established and implemented (WHO, 1998). The potential use of B. anthracis spores as a bioterrorism and warfare agent has strongly promoted research on the molecular mechanisms of virulence, forensic methods to trace the origin of specific strains and phylogenetic lineages of B.

P. Pilo, J. Frey / Infection, Genetics and Evolution 11 (2011) 1218–1224

anthracis (Keim et al., 2004, 2009; Moayeri and Leppla, 2004; Van Ert et al., 2007a; Koehler, 2009; van der Goot and Young, 2009). An important biological feature of B. anthracis is the alternation between the vegetative phase and the spore phase. This bacterium is principally found in the environment as a spore, where it is metabolically dormant and does not replicate. However, it has been observed that B. anthracis can grow vegetatively in autoclaved, rich soil after being cured of one of the virulence plasmids pXO2. This indicates that certain virulence attributes not only are advantageous for B. anthracis to infect and multiply in the host, but also confer host restriction (Saile and Koehler, 2006). Generally, B. anthracis solely replicates during short periods of infection, which are terminated by host death or by clearance of the bacteria by the immune system or therapeutic agents. Genetic evolution of B. anthracis is therefore limited to the short vegetative periods from infection to host death, which is estimated to cover 20–40 generations (Keim et al., 2004). Due to the long dormant spore periods, this pathogen evolves very slowly, in contrast to most other bacteria with similar generation times. For this reason, B. anthracis is genetically and phenotypically extremely homogeneous. Conventional methods such as serology, biochemical tests and phage typing are not appropriate to differentiate among strains (WHO, 1998). The advent of rapid sequencing methods has resulted in the availability of several complete B. anthracis genome sequences (Pearson et al., 2004). These data allowed the identification, by genome comparison, of several genetic markers for discrimination of strains and the study of population genetics of this bacterium over long time periods (Keim et al., 2009). This review will give a summary of more recent knowledge acquired via these new molecular methods into the molecular taxonomy, population genetics, phylogeny and patho-evolution of B. anthracis.

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clonal and are restricted to clade 1 along with the highly virulent emetic toxin-producing B. cereus (Didelot et al., 2009). B. anthracis can be identified using a battery of specific bacteriological tests that examine colony morphology, capsule staining, lack of hemolysis, sensitivity to g-phage, sensitivity to penicillin and motility (WHO, 1998). Nevertheless, some strains may show variability in terms of phenotype, complicating the exact identification and differentiation between B. anthracis and other types of B. cereus, although these two species show entirely different pathological manifestations (Klee et al., 2006; Ross et al., 2009; Beesley et al., 2010). B. anthracis, a severe zoonotic pathogen of ruminants, causes life threatening anthrax. Nonetheless, strains with characteristics of B. cereus have been isolated from animals with clinical anthrax (P. Pilo, unpublished; Klee et al., 2006). Hence, several established genetic markers for B. anthracis, such as the chromosomal locus Ba813 (Patra et al., 1996) or the gene sap encoding the S-layer protein (Ryu et al., 2003), can result in ambiguous diagnostic identification of B. anthracis (Ramisse et al., 1999). The key virulence determinants that differentiate pathogenic species of the B. cereus group in their pathogenicity and host specificity represent relatively small portions of their genomes. These genome segments are harboured by mobile elements and were likely acquired by gene transfer from other species rather than evolved by adaptation in their respective hosts. In B. anthracis, virulence is determined by two plasmids named pXO1 (toxin plasmid) and pXO2 (capsule plasmid) (Fig. 1), which under certain circumstances are capable of being transferred between related species, particularly among B. cereus clade 1 (Ruhfel et al., 1984; Pannucci et al., 2002a; Hoffmaster et al., 2004; Klee et al., 2006; Didelot et al., 2009; Hu et al., 2009).

2. Taxonomy

3. Molecular mechanisms of pathogenicity and host–pathogen interactions

Bacterial taxonomy includes both phenotypic and genotypic characteristics and leads to the biological binomial nomenclature based on the genus (generic name) followed by the species (specific name) (Woese, 1994; Prescott et al., 1999; Moore et al., 2010). B. anthracis belongs to the phylum Firmicutes, the family Bacillaceae, the genus Bacillus and the Bacillus cereus group. The latter consists of six members: B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B. weihenstephanensis. These species are closely related and have been traditionally separated into different species based on phenotypic characteristics, pathogenicity, clinical symptoms, host preference and ecological niche (Rasko et al., 2005). Most of these bacteria are of economic, environmental, medical and biodefence importance. While the main pathogenic characteristics of this group of bacteria can be attributed to toxins that are generally encoded on plasmids, the chromosomes of the different members of the B. cereus group show a high level of synteny and protein similarity with little difference in gene content (Ko et al., 2004; Rasko et al., 2005). The identification of individual species of the B. cereus group is therefore complex, and some isolates display unusual biochemical and/or physiological properties that complicate their accurate distinction (Klee et al., 2006; Ross et al., 2009; Beesley et al., 2010). Phylogenetically, the B. cereus group of bacteria is divided into 3 distinct clades (Priest et al., 2004; Ko et al., 2004; Helgason et al., 2004; Didelot et al., 2009) that show partial barriers to interclade gene flow (Didelot et al., 2009). Most pathogenic B. cereus strains are evenly distributed among clades 1 and 2. The insect pathogen B. thuringiensis is primarily found in clade 2. Clade 3 contains B. cereus, B. mycoides and B. weihenstephanensis (Helgason et al., 2000, 2004; Priest et al., 2004; Sorokin et al., 2006; Didelot et al., 2009). Strains of B. anthracis, in contrast to the other species, are strictly

The key virulence factors in B. anthracis are encoded on the two virulence plasmids pXO1 (182 kb) and pXO2 (96 kb) (Mikesell et al., 1983; Uchida et al., 1985). Plasmid pXO1 encodes two binary toxins: lethal factor (LF), a zinc metalloprotease, and oedema factor (EF), a calmodulin-dependent adenylate-cyclase. These toxins share the same adhesion subunit, known as protective antigen (PA), that binds to the cellular receptors tumour endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2) (Bradley et al., 2001). After binding, PA is cleaved by furin and oligomerizes and binds LF or EF for transport into cells via membrane lipid rafts (Abrami et al., 2003) (Fig. 1). Once inside the cell, LF exerts its toxic activity by cleavage of the N-terminal portion of mitogen-activated protein kinase kinase (MAPKK) causing cell lysis or induction of pro-inflammatory cytokines leading to vascular collapse, shock and death (Moayeri et al., 2003) (Fig. 1). In the host cell, EF catalyses the conversion of ATP to cAMP, deregulating a broad spectrum of cytokine responses in the host (Tang and Guo, 2009) (Fig. 1). During early infection and germination, when doses are sub-lethal, LT and EF seem to suppress the cellular immune and cytokine responses allowing bacterial outgrowth and dispersion throughout the host body (Moayeri and Leppla, 2004). The plasmid pXO2 encodes the enzymes that synthesize the poly-D-glutamic acid capsule. This virulence factor is necessary for survival in macrophages, which is a crucial step in the course of disease. The poly-D-glutamic acid capsule and the two anthrax toxins are the key phenotypes distinguishing B. anthracis from closely related species. The presence of the two plasmids pXO1 and pXO2 carrying the capsular biosynthesis genes capBCADE is currently the crucial diagnostic evidence to confirm infection with B. anthracis (WHO, 1998). The loss of one or both of these

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Fig. 1. Overview of the molecular mechanisms of virulence of Bacillus anthracis. B. anthracis (upper left) synthesizes the 4 major virulence factors: the lethal toxin zinc metalloprotease (LF), the oedema factor calmodulin-dependent adenylate cyclase (EF) and the protective antigen (PA), which is the adhesive subunit of the two toxins, all of which are encoded on plasmid pXO1, and the poly-D-glutamic acid polymer capsule (CAP) that allows survival of the bacterium in macrophages and is encoded on plasmid pXO2. PA binds to the receptors on the host cell, TEM8 and CMG2 (see text), and is subsequently cleaved by furin, allowing multimerization and binding of LF or EF. The PA:EF and PA:LF complexes are then internalized by endocytosis. In the acid environment of the endosomes, the PA:EF and PA:LF complexes are dissolved and exert their toxic activities by inhibiting oxidative burst through cAMP or release of pro-inflammatory cytokines and subsequent shock by proteolytic cleavage of MAPKK. Variable number tandem repeats (VNTRs) spread throughout the whole genome are used as molecular epidemiological markers. Abbreviations: CAP, capsule; PA, protective antigen; EF, oedema factor; LF, lethal factor; VNTR, variable number tandem repeat; MAPKK, mitogen-activated protein kinase kinase; CaM, calmodulin; cAMP, cyclic adenosine 30 ,50 mono phosphate.

plasmids attenuates the virulence of B. anthracis or leads to a lack of virulence (Mock and Fouet, 2001). While pXO1 and pXO2 are generally found in strains with a characteristic B. anthracis chromosomal background, researchers have recently isolated B. anthracis-like strains in West Africa (Ivory Coast and Cameroon) from anthrax-like cases in great apes (Klee et al., 2006). The sequence of the complete genome of one of these strains from the Ivory Coast showed chromosomal properties of B. cereus but the presence of the two virulence plasmids specific to B. anthracis (Klee et al., 2010). The authors of this report suggested naming these strains B. cereus variety anthracis. This type of strain seems to be broadly spread in this geographic area, revealing the complexity of B. anthracis taxonomy (Pilo, unpublished observations). 4. B. anthracis strain typing and molecular epidemiology Because of the characteristic slow evolutionary change of B. anthracis, largely determined by an alternating life cycle containing short replication phases and long sporulation periods, common methods for strain sub-typing such as serotyping, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) have insufficient discriminatory power to resolve

strains of various origins (Harrell et al., 1995; Keim et al., 1997; Helgason et al., 2004; Kim et al., 2005). Variable number tandem repeats (VNTR) (van Belkum et al., 1998) have been found in these species in open reading frames of genes with unknown functions and in intergenic areas on the chromosome as well as on both plasmids pXO1 and pXO2. VNTRs showed a mutation rate of approximately 105–104, resulting in sufficient copy number variation to produce length polymorphisms with satisfactory discriminatory capacity to type individual B. anthracis strains by multiple locus VNTR analysis (MLVA) (Keim et al., 2000). Using eight VNTRs (MLVA8), it was possible to classify 426 strains from diverse geographical locations into 89 MLVA genotypes that grouped into two clades (A and B) and 8 clusters (Keim et al., 2000). This VNTR typing scheme has been widely used to examine the diversity of B. anthracis strains from national strain collections and has allowed examination of strains from France (Fouet et al., 2002), Italy (Fasanella et al., 2005), Poland (Gierczynski et al., 2004), South Africa (Smith et al., 2000), Korea (Ryu et al., 2005), Western and Central Africa (Maho et al., 2006; Pilo, unpublished data), Switzerland (Pilo et al., 2008), China (Simonson et al., 2009), Bulgaria (Antwerpen et al., 2011) and Kazakhstan (Aikembayev et al., 2010). Subsequent extension of the MLVA analysis to 15 different marker loci (MLVA15) (Van Ert et al., 2007a) and to 25


different marker loci (MLVA25) (Lista et al., 2006) resulted in particularly strong resolution of the more recent population level structure of B. anthracis species. These studies suggested a relatively recent worldwide diffusion of strains belonging to clade A (Van Ert et al., 2007a), with the exception of subclade Ab which, from the current information, appears to contain only strains from Chad (Maho et al., 2006) and from other Western and Central African countries (P. Pilo, unpublished data; Lista et al., 2006). All currently known strains of subclade Ab lack anthrose, a specific sugar of B. anthracis spores (Tamborrini et al., 2011). It has to be noted that clade ‘‘E’’, as designated by Lista et al. (2006), is identical to subclade Ab (Maho et al., 2006) (Fig. 2). The particularly wide spread of clade A, with the exception of the subclade Ab, should be considered not only with regard to bacterial aspects such as genetic fitness, virulence and host adaptation capacity but also from the point of view of global trade and movement. Hence, it has been shown that strains belonging to subcluster A4 that are spread globally can be correlated, in many cases, with human anthrax due to contaminated raw cashmere wool that was processed in wool factories in many parts of the world (Pilo et al., 2008; Wattiau et al., 2008). This explains the high occurrence and wide distribution, particularly in Europe, of subcluster A4 and specifically A.Br008/009 as noted by Van Ert et al. (2007a) (Fig. 2). Therefore, it is important for epidemiological studies to distinguish between strains isolated from bovines and strains isolated from humans. Humans may become infected by animal products originating from sites that are often several thousands of miles away, such as wool imported for processing or drum skins (Pilo et al., 2008; Wattiau et al., 2008; Anaraki et al., 2008).

clades

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5. Global genetic population structure While MLVA has ideal resolving power for most epidemiological studies, single nucleotide polymorphism (SNP) analysis shows significantly less discrimination ability because of the slower evolution of these markers. However, this method defines major clonal lineages at the level of population structure, particularly for slow-evolving organisms such as B. anthracis (Keim et al., 2004; Van Ert et al., 2007b). Whole genome sequencing data from five strains of B. anthracis revealed approximately 3500 SNPs, of which about 900 could be mapped in 27 different strains and were shown to be highly robust for the design of a phylogenetic model for B. anthracis (Pearson et al., 2004). Out of these SNPs, a few canonical single polymorphisms (canSNPs) were chosen to evaluate key phylogenetic junctions to fully identify the entire genome (Van Ert et al., 2007a). The 12 slowly evolving canSNPs have an estimated mutation rate of about 1010. This analysis defined the three previously recognised major genetic lineages (clades A, B and C) and further described 12 distinct sublineages (Van Ert et al., 2007a) (Fig. 2). After coupling the MLVA15 (approximate mutation rate of VNTRs 105) to the canSNP data set in a hierarchical manner a progressive hierarchical resolving assay (PHRANA) was developed. This assay allowed analysis of a collection of 1033 B. anthracis strains from all over the world and resulted in 12 distinguished sublineages (clusters) resolved into 221 MLVA15 genotypes. Most of these genotypes belong to clade A (Van Ert et al., 2007a) (Fig. 2). Van Ert et al. developed an attractive mathematical model that allows the estimation of the divergence of the major branches of evolution of B. anthracis. This model is based on the mutation rate

SNP lineages

MLVA clusters Aβ

Aβ

WNA; A.Br.

A1 TEA; A.Br.008/009

Aα

3‘000-6‘000 ybp

A3

9‘000-17‘000 ybp

B C

13‘00026‘000 ybp

A-radiation

A2

A

A.Br.001/002 A.Br.Ames A.Br.003/004

A4

A.Br.Vollum

B1

B.Br.001/002 (Kruger B)

B2

B.Br.CNEVA

C

C.Br.A1055

Fig. 2. Schematic tree summarizing the hierarchical analysis of B. anthracis populations (adapted from the references cited in this review). MLVA clusters A, B and C are shown with their estimated historical split. Major subclusters (Aa1, Aa2, Aa3, Aa4, Ab, B1, B2 and C) are represented by triangles. The SNP lineages and sublineages are shown separately. The A radiation is indicated by a dashed rectangle.

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per site per generation (5.2 1010) as calculated from synonymous SNPs from the completely sequenced B. anthracis genomes (Vogler et al., 2002). This is based on an ungulate transmission model with an estimated infection/death/year rate of 1 or 0.5. This model suggests that clade C separates approximately 13,000– 26,000 years from the lineage containing clades A and B. In addition, clade A separated from clade B about 9000–17,000 years ago (Van Ert et al., 2007a) (Fig. 2). Furthermore, the authors of this study estimated the radiation of clade A into a large number of clusters to have occurred in the mid-Holocene, about 3000–6000 years ago (Van Ert et al., 2007a), probably due to domestication of small and large ruminants and human migration events. It should be noted that the estimated infection/death/year rate has since been reduced to 0.28 generations per year. This reduction was based on an overview of anthrax outbreaks in cattle in Northern Canada that identified 13 outbreaks in 46 years (1962–1991) (Dragon and Elkin, 2001), which would indicate that the radiation of clade A occurred about 10,000 years ago (Kenefic et al., 2009). 6. Geographical spread of B. anthracis clusters and sublineages Analysis of the dispersal of B. anthracis around the world must take into account human and animal products, such as goat and sheep hair for wool production, human and animal movements and transport of primary hosts carrying the pathogen. However, many samples stored in regional, national and international strain collections rarely contain detailed data on the epidemiological background of the infection or environmental information regarding the isolation of the strains. B. anthracis represents a model organism to track ancient epidemiological events due its strictly clonal reproductive mode and the low frequency of polymorphisms in its evolutionary molecular markers. Using a large genetic data set from an extensive strain collection and focusing on strains from wild and domestic animals, Keim’s group was able to demonstrate that the most drastic clonal expansion of B. anthracis was the spread of clade A that was tracked back to the Eurasian area where the lineage ‘Western North America’ (WNA) split from the ‘Trans-Eurasian’ lineage (TEA, A.Br.008/009), represented in cluster A1 (Fig. 2), and was introduced to North America in the pre-Columbian era (Kenefic et al., 2009). The evolutionary period during which the WNA lineage divided from the TEA ancestor was estimated by SNP analysis to be the late Pleistocene epoch (Kenefic et al., 2009). Strains from the WNA lineage were predominantly isolated between the near Arctic circle in Canada and the US-Mexican border from animal anthrax cases and could be shown by SNP analysis to have evolved into six sublineages during the epidemic spread of anthrax from the extreme north to the south of the North American sub-continent (Kenefic et al., 2009). This study represents one of the most extensive bacterial phylogeographical surveys encompassing this long time period. In contrast to this study of ‘‘natural’’ spread of B. anthracis, previous analyses indicated a very scattered spread influenced by rapid transport of B. anthracis, for example by contaminated animal products such as hair, over long distances (Van Ert et al., 2007a). Hence, it has been shown that the broad worldwide spread of cluster A4 (A.Br.Vollum) is likely due to the intensive processing of cashmere wool in many European countries and on other continents during early industrialisation. Strains of cluster A4 can indeed be traced back to cases of human anthrax in wool factory workers, indicating that the origin of this lineage may be the northern part of the Indian sub-continent (Pilo et al., 2008; Wattiau et al., 2008). Similar parameters likely also contributed to the worldwide spread of cluster A3 (A.Br.Ames, A.Br.003/004) (Simonson et al., 2009), while B.Br.CNEVA is thought to represent a newer European lineage of cattle-infecting strains (Fouet et al., 2002; Van Ert et al., 2007a; Pilo et al., 2008).

7. Pathogenic evolution The pathogenic evolution of B. anthracis should be examined in comparison to the B. cereus group of bacteria, which comprises mostly soil-dwelling saprophytes and also certain animal and human pathogens causing food poisoning, systemic infections and anthrax. The two virulence plasmids pXO1 and pXO2 (Okinaka et al., 1999) play a central role in the virulence of B. anthracis. Strains that have been cured of one plasmid are non-virulent (in case of pXO1) or highly attenuated (in case of pXO2) and can serve as live vaccines like the strain ‘Sterne’ (Sterne, 1988). Hence, the presence of pXO1 and pXO2 is key for the determination of clinically relevant strains of B. anthracis (WHO, 1998). It is hypothesised that B. anthracis derived from a B. cereus common ancestor by acquiring plasmids and various chromosomal mutations (Slamti et al., 2004; Okinaka et al., 2006; Ross et al., 2009). As replication of B. anthracis is mainly restricted to the infectious cycles within the host, survival of this bacterial species selects highly for the presence of both plasmids, which are consequently an intrinsic part of this species and must have contributed to the formation of this species. It is important to note that the two highly pathogenic members of the B. cereus group, B. anthracis and emetic B. cereus, both belong to the B. cereus clade 1 (Didelot et al., 2009; Hu et al., 2009). This clonal relationship may be due to the capacity of this cluster to support pXO1-like repX replicons that carry, in the case of B. anthracis, the genes specifying lethal toxin and oedema factor, and in case of emetic B. cereus, the emetic toxin cereulide synthetase genes, while pXO2-like plasmids can be transferred between the three clades of the B. cereus group (Hu et al., 2009). Therefore, pXO1 and pXO2 do not seem to be limited to B. anthracis. Plasmids similar to pXO1 and pXO2 have been found in related Bacillus species (Pannucci et al., 2002a,b; Hoffmaster et al., 2004; Hu et al., 2009). Anthrax-like cases in primates caused by an agent with phenotypic and chromosomal characteristics of B. cereus but containing plasmids pXO1 and pXO2 are of particular interest and have recently been analysed in detail (Klee et al., 2006, 2010). Although such strains are rarely encountered, our recent unpublished results reveal that they can also be isolated from bovines that have succumbed to anthrax in Western Africa. At a chromosomal level, species belonging to the B. cereus group are closely related (Ivanova et al., 2003). Only relatively small differences in the chromosome account for the distinctive phenotypic differences between B. cereus and B. anthracis, for example variations in the gamR gene encoding the g-phage receptor in B. anthracis, variations in the regulator plcR gene, or diversity in the anti-sigma factor responsible for the expression of the beta-lactamase genes bla1 and bla2 in B. cereus and the repression of bla1 and bla2 in B. anthracis rendering it penicillinsusceptible (Agaisse et al., 1999; Chen et al., 2003; Slamti et al., 2004; Davison et al., 2005; Gohar et al., 2008; Ross et al., 2009). This indicates that the identity of the different species at the chromosomal level is mainly due to differential gene expression or a small set of genes rather than to vast rearrangements in the genome (Read et al., 2003; Ivanova et al., 2003). 8. Conclusions Because of its life cycle that has a very low multiplication rate of 0.28–1 generation per year, B. anthracis is a highly homogeneous species and can be considered to be relatively monomorphic. This characteristic makes B. anthracis an ideal organism to study vertical evolution over extended periods of time. The use of highly stable phylogenetic markers, SNPs, has proven to be useful in identifying extended branches and key phylogenetic positions in long-term evolutionary studies, while more variable VNTR loci allow detailed subtyping of the major clades into clusters and


subclusters. This detailed subtyping allowed the discrimination of more recent events in the evolution of B. anthracis and is useful for molecular epidemiological studies. Higher resolution analysis can be performed using single nucleotide repeats (SNRs) markers that have a mutation rate in the range of 104–103 for specific outbreaks or forensic investigations (Keim et al., 2004). Virulence is an intrinsic characteristic of B. anthracis and is vital for its survival. Therefore, it is hypothesised that the acquisition of the two virulence plasmids occurred at the genesis of this species and that these plasmids have been retained until the present time. These plasmids can also be found, sometimes with modification, in related Bacillus species, resulting in highly virulent anthraxcausing strains. From a phylogenetic and taxonomic point of view, it is debatable whether such strains belong to the B. anthracis or B. cereus species; however, it seems from a medical perspective coherent to designate closely related agents that cause symptoms of anthrax with the same species name.

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