Genomic analysis of three African strains of Bacillus anthracisdemonstrates that they are part of the clonal expansion of an exclusively pathogenic bacterium
Identifieur interne : 000568 ( Pmc/Corpus ); précédent : 000567; suivant : 000569Genomic analysis of three African strains of Bacillus anthracisdemonstrates that they are part of the clonal expansion of an exclusively pathogenic bacterium
Auteurs : L. Rouli ; M. Mbengue ; C. Robert ; M. Ndiaye ; B. La Scola ; D. RaoultSource :
- New Microbes and New Infections [ 2052-2975 ] ; 2014.
Abstract
Url:
DOI: 10.1002/nmi2.62
PubMed: 25566394
PubMed Central: 4265047
Links to Exploration step
PMC:4265047Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Genomic analysis of three African strains of <italic>Bacillus anthracis</italic>demonstrates that they are part of the clonal expansion of an exclusively pathogenic
bacterium</title><author><name sortKey="Rouli, L" sort="Rouli, L" uniqKey="Rouli L" first="L" last="Rouli">L. Rouli</name><affiliation><nlm:aff id="au1"><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mbengue, M" sort="Mbengue, M" uniqKey="Mbengue M" first="M" last="Mbengue">M. Mbengue</name><affiliation><nlm:aff id="au2"><institution>Laboratoire National d'Elevage et des Recherches Vétérinaires (LNERV), Institut Sénégalais de Recherches Agricoles (ISRA)</institution><addr-line>Hann, Dakar, Senegal</addr-line></nlm:aff></affiliation></author><author><name sortKey="Robert, C" sort="Robert, C" uniqKey="Robert C" first="C" last="Robert">C. Robert</name><affiliation><nlm:aff id="au1"><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ndiaye, M" sort="Ndiaye, M" uniqKey="Ndiaye M" first="M" last="Ndiaye">M. Ndiaye</name><affiliation><nlm:aff id="au3"><institution>Laboratoire de Biologie Cellulaire, Faculté des Sciences et Techniques de l'Université Cheikh Anta DIOP de Dakar (UCAD)</institution><addr-line>Dakar, Senegal</addr-line></nlm:aff></affiliation></author><author><name sortKey="La Scola, B" sort="La Scola, B" uniqKey="La Scola B" first="B" last="La Scola">B. La Scola</name><affiliation><nlm:aff id="au1"><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Raoult, D" sort="Raoult, D" uniqKey="Raoult D" first="D" last="Raoult">D. Raoult</name><affiliation><nlm:aff id="au1"><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25566394</idno><idno type="pmc">4265047</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265047</idno><idno type="RBID">PMC:4265047</idno><idno type="doi">10.1002/nmi2.62</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000568</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Genomic analysis of three African strains of <italic>Bacillus anthracis</italic>demonstrates that they are part of the clonal expansion of an exclusively pathogenic
bacterium</title><author><name sortKey="Rouli, L" sort="Rouli, L" uniqKey="Rouli L" first="L" last="Rouli">L. Rouli</name><affiliation><nlm:aff id="au1"><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mbengue, M" sort="Mbengue, M" uniqKey="Mbengue M" first="M" last="Mbengue">M. Mbengue</name><affiliation><nlm:aff id="au2"><institution>Laboratoire National d'Elevage et des Recherches Vétérinaires (LNERV), Institut Sénégalais de Recherches Agricoles (ISRA)</institution><addr-line>Hann, Dakar, Senegal</addr-line></nlm:aff></affiliation></author><author><name sortKey="Robert, C" sort="Robert, C" uniqKey="Robert C" first="C" last="Robert">C. Robert</name><affiliation><nlm:aff id="au1"><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ndiaye, M" sort="Ndiaye, M" uniqKey="Ndiaye M" first="M" last="Ndiaye">M. Ndiaye</name><affiliation><nlm:aff id="au3"><institution>Laboratoire de Biologie Cellulaire, Faculté des Sciences et Techniques de l'Université Cheikh Anta DIOP de Dakar (UCAD)</institution><addr-line>Dakar, Senegal</addr-line></nlm:aff></affiliation></author><author><name sortKey="La Scola, B" sort="La Scola, B" uniqKey="La Scola B" first="B" last="La Scola">B. La Scola</name><affiliation><nlm:aff id="au1"><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Raoult, D" sort="Raoult, D" uniqKey="Raoult D" first="D" last="Raoult">D. Raoult</name><affiliation><nlm:aff id="au1"><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">New Microbes and New Infections</title><idno type="ISSN">2052-2975</idno><idno type="eISSN">2052-2975</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Bacillus anthracis</italic> is the causative agent of anthrax and is classified as a
‘Category A’ biological weapon. Six complete genomes of
<italic>B. anthracis</italic> (A0248, Ames, Ames Ancestor, CDC684, H0491, and Sterne) are
currently available. In this report, we add three African strain genomes: Sen2Col2, Sen3 and Gmb1.
To study the pan-genome of <italic>B. anthracis</italic>, we used bioinformatics tools, such
as Cluster of Orthologous Groups, and performed phylogenetic analysis. We found that the three
African strains contained the pX01 and pX02 plasmids, the nonsense mutation in the
<italic>plcR</italic> gene and the four known prophages. These strains are most similar to the
CDC684 strain and belong to the A cluster. We estimated that the
<italic>B. anthracis</italic> pan-genome has 2893 core genes (99% of the genome size)
and 85 accessory genes. We validated the hypothesis that <italic>B. anthracis</italic> has a
closed pan-genome and found that the three African strains carry the two plasmids associated with
bacterial virulence. The pan-genome nature of <italic>B. anthracis</italic> confirms its lack
of exchange (similar to <italic>Clostridium tetani</italic>) and supports its exclusively pathogenic
role, despite its survival in the environment. Moreover, thanks to the study of the core content
single nucleotide polymorphisms, we can see that our three African strains diverged very recently
from the other <italic>B. anthracis</italic> strains.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Scarlata, F" uniqKey="Scarlata F">F Scarlata</name></author><author><name sortKey="Colletti, P" uniqKey="Colletti P">P Colletti</name></author><author><name sortKey="Bonura, S" uniqKey="Bonura S">S Bonura</name></author><author><name sortKey="Trizzino, M" uniqKey="Trizzino M">M Trizzino</name></author><author><name sortKey="Giordano, S" uniqKey="Giordano S">S Giordano</name></author><author><name sortKey="Titone, L" uniqKey="Titone L">L Titone</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, Db" uniqKey="Wang D">DB Wang</name></author><author><name sortKey="Tian, B" uniqKey="Tian B">B Tian</name></author><author><name sortKey="Zhang, Zp" uniqKey="Zhang Z">ZP Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Koehler, Tm" uniqKey="Koehler T">TM 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<front><journal-meta><journal-id journal-id-type="nlm-ta">New Microbes New Infect</journal-id><journal-id journal-id-type="iso-abbrev">New Microbes New Infect</journal-id><journal-id journal-id-type="publisher-id">nmi2</journal-id><journal-title-group><journal-title>New Microbes and New Infections</journal-title></journal-title-group><issn pub-type="ppub">2052-2975</issn><issn pub-type="epub">2052-2975</issn><publisher><publisher-name>BlackWell Publishing Ltd</publisher-name><publisher-loc>Oxford, UK</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25566394</article-id><article-id pub-id-type="pmc">4265047</article-id><article-id pub-id-type="doi">10.1002/nmi2.62</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Genomic analysis of three African strains of <italic>Bacillus anthracis</italic>demonstrates that they are part of the clonal expansion of an exclusively pathogenic
bacterium</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Rouli</surname><given-names>L</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>MBengue</surname><given-names>M</given-names></name><xref ref-type="aff" rid="au2">2</xref></contrib><contrib contrib-type="author"><name><surname>Robert</surname><given-names>C</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ndiaye</surname><given-names>M</given-names></name><xref ref-type="aff" rid="au3">3</xref></contrib><contrib contrib-type="author"><name><surname>La Scola</surname><given-names>B</given-names></name><xref ref-type="aff" rid="au1">1</xref></contrib><contrib contrib-type="author"><name><surname>Raoult</surname><given-names>D</given-names></name><xref ref-type="aff" rid="au1">1</xref><xref ref-type="corresp" rid="cor1"></xref></contrib><aff id="au1"><label>1</label><institution>Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095</institution><addr-line>Marseille, France</addr-line></aff><aff id="au2"><label>2</label><institution>Laboratoire National d'Elevage et des Recherches Vétérinaires (LNERV), Institut Sénégalais de Recherches Agricoles (ISRA)</institution><addr-line>Hann, Dakar, Senegal</addr-line></aff><aff id="au3"><label>3</label><institution>Laboratoire de Biologie Cellulaire, Faculté des Sciences et Techniques de l'Université Cheikh Anta DIOP de Dakar (UCAD)</institution><addr-line>Dakar, Senegal</addr-line></aff></contrib-group><author-notes><corresp id="cor1"><bold>Corresponding author:</bold> D. Raoult, URMITE, UMR CNRS 7278, IRD 198, INSERM U1095,
Faculté de Médecine, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France,
<bold>E-mail:</bold><email>didier.raoult@gmail.com</email></corresp><fn><p>Funding Information: This work was supported by the Mediterranean Infection Foundation.</p></fn></author-notes><pub-date pub-type="ppub"><month>11</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>28</day><month>9</month><year>2014</year></pub-date><volume>2</volume><issue>6</issue><fpage>161</fpage><lpage>169</lpage><history><date date-type="received"><day>24</day><month>6</month><year>2014</year></date><date date-type="rev-recd"><day>04</day><month>7</month><year>2014</year></date><date date-type="accepted"><day>08</day><month>8</month><year>2014</year></date></history><permissions><copyright-statement>© 2014 The Authors. <italic>New Microbes and New Infections</italic>published by John Wiley & Sons Ltd on behalf of the European Society of Clinical Microbiology
and Infectious Disease.</copyright-statement><copyright-year>2014</copyright-year><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This is an open access article under the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided the original work is properly
cited.</license-p></license></permissions><abstract><p><italic>Bacillus anthracis</italic> is the causative agent of anthrax and is classified as a
‘Category A’ biological weapon. Six complete genomes of
<italic>B. anthracis</italic> (A0248, Ames, Ames Ancestor, CDC684, H0491, and Sterne) are
currently available. In this report, we add three African strain genomes: Sen2Col2, Sen3 and Gmb1.
To study the pan-genome of <italic>B. anthracis</italic>, we used bioinformatics tools, such
as Cluster of Orthologous Groups, and performed phylogenetic analysis. We found that the three
African strains contained the pX01 and pX02 plasmids, the nonsense mutation in the
<italic>plcR</italic> gene and the four known prophages. These strains are most similar to the
CDC684 strain and belong to the A cluster. We estimated that the
<italic>B. anthracis</italic> pan-genome has 2893 core genes (99% of the genome size)
and 85 accessory genes. We validated the hypothesis that <italic>B. anthracis</italic> has a
closed pan-genome and found that the three African strains carry the two plasmids associated with
bacterial virulence. The pan-genome nature of <italic>B. anthracis</italic> confirms its lack
of exchange (similar to <italic>Clostridium tetani</italic>) and supports its exclusively pathogenic
role, despite its survival in the environment. Moreover, thanks to the study of the core content
single nucleotide polymorphisms, we can see that our three African strains diverged very recently
from the other <italic>B. anthracis</italic> strains.</p></abstract><kwd-group><kwd>African strains</kwd><kwd><italic>Bacillus anthracis</italic></kwd><kwd>pan-genome</kwd><kwd>pathogen</kwd><kwd>specialization</kwd></kwd-group></article-meta></front><body><sec><title>Introduction</title><p>Blackleg, or anthrax, was the first disease to be linked to a specific microbe by Davaine in 1863
and the first animal infection for which a vaccine was proposed (by Pasteur, 1881) <xref rid="b1" ref-type="bibr">1</xref>. In 1876, Koch discovered the first bacterium, <italic>Bacillus
anthracis</italic>, which had the capacity to transform into spores <xref rid="b1" ref-type="bibr">1</xref><italic>. Bacillus anthracis</italic> belongs to the phylum Firmicutes and is part of the
<italic>Bacillus cereus</italic> group <xref rid="b2" ref-type="bibr">2</xref>. It is a
Gram-positive, spore-forming soil bacterium <xref rid="b2" ref-type="bibr">2</xref> that is a
facultative anaerobe <xref rid="b3" ref-type="bibr">3</xref>, able to survive in extreme and
unfriendly environmental conditions, such as high levels of radiation or extreme temperature <xref rid="b2" ref-type="bibr">2</xref>, and remain viable in the soil over a long period of time <xref rid="b4" ref-type="bibr">4</xref>. <italic>Bacillus anthracis</italic> is the causative agent of the
zoonotic disease anthrax <xref rid="b2" ref-type="bibr">2</xref>, to which cattle and horses are
particularly sensitive <xref rid="b1" ref-type="bibr">1</xref>. Human infections can occur through
the ingestion or inhalation of spores or through contact with the skin <xref rid="b2" ref-type="bibr">2</xref>. Four clinical syndromes for anthrax disease exist <xref rid="b4" ref-type="bibr">4</xref>: cutaneous anthrax (95% of reported cases), gastrointestinal anthrax due to
contaminated food, inhalational anthrax and injectional anthrax. <italic>Bacillus anthracis</italic>is classified as a ‘Category A’ biological weapon <xref rid="b2" ref-type="bibr">2</xref>. Due to the stability of its spores, its high pathogenicity and lethality, and its
capacity to be inhaled <xref rid="b5" ref-type="bibr">5</xref>, the weaponization of
<italic>B. anthracis</italic> by US <xref rid="b1" ref-type="bibr">1</xref> and Soviet armed
forces has led to two series of cases, as reported by a US Army investigator and identified through
genomic analysis <xref rid="b6" ref-type="bibr">6</xref>. Additionally, an anthrax epidemic caused
by atmospheric contamination from a military laboratory occurred in 2001 <xref rid="b1" ref-type="bibr">1</xref>,<xref rid="b7" ref-type="bibr">7</xref>, leading to an increase in research about
<italic>B. anthracis</italic> and anthrax <xref rid="b5" ref-type="bibr">5</xref> and
prompting the emergence of a new detection system <xref rid="b2" ref-type="bibr">2</xref>.</p><p>Compared with <italic>B. cereus</italic>, <italic>B. anthracis</italic> has a
nonsense mutation in the <italic>plcR</italic> gene at position 640 <xref rid="b7" ref-type="bibr">7</xref> and four prophage regions. <italic>Bacillus anthracis</italic> strains can be classified
into three different lineages (A, B or C) <xref rid="b8" ref-type="bibr">8</xref> based on
multilocus variable number tandem repeat (VNTR) analysis (MLVA) <xref rid="b9" ref-type="bibr">9</xref>–<xref rid="b11" ref-type="bibr">11</xref>. Lineage A, which is found worldwide, is
divided into four sub-lineages: A1, A2, A3 and A4. Lineage B is divided into two sub-lineages, B1
and B2, with B1 found in southern Africa and B2 in southern and Eastern Europe. Lineage C is
rare.</p><p>The first pan-genomic study was published in 2005 on <italic>Streptococcus agalactiae</italic><xref rid="b9" ref-type="bibr">9</xref>, and the number of such studies has increased rapidly since
then. A pan-genome is defined as the pool of all genes present in all the studied genomes of a
species, which allows for a comparison between different species or strains. The pan-genome can be
divided into different parts: the core genome (genes present in all the genomes), accessory genes
(genes present in several genomes but not all) and unique genes (genes present in only one of the
studied isolates). Additionally, the pan-genome can be closed or open, depending on the capacity of
the species to acquire new genes <xref rid="b9" ref-type="bibr">9</xref> and the age of the initial
clone. The capacity of the species to gain genes can be evaluated by studying its mobilome. Its age
can be determined using the single nucleotide polymorphism (SNP) observation process in the core
genome <xref rid="b10" ref-type="bibr">10</xref>–<xref rid="b12" ref-type="bibr">12</xref>.</p><p>African strains of <italic>B. anthracis</italic> from Gambia and Senegal have not yet been
studied <xref rid="b13" ref-type="bibr">13</xref>. In this study, we compiled the <italic>B.
anthracis</italic> pan-genome based on three African strains (two from Senegal and one from Gambia)
and six reference genomes (Ames <xref rid="b14" ref-type="bibr">14</xref>, Ames Ancestor <xref rid="b12" ref-type="bibr">12</xref>, A0248, CDC684 <xref rid="b15" ref-type="bibr">15</xref>, H9401
<xref rid="b13" ref-type="bibr">13</xref> and Sterne). The Ames strain is the non-virulent version
of the Ames Ancestor strain and was the strain found in letters in the USA in 2001. Virulence is
linked to the presence of two plasmids, pX01 and pX02, which carry genes encoding for toxins, as
well as the capsule <xref rid="b4" ref-type="bibr">4</xref>. Our work demonstrates that the three
African strains are very closely related to the other strains, especially CDC684, contain the pX01
and pX02 plasmids, belong to the worldwide lineage A and have a closed pan-genome.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title>Bacteriological studies</title><p>Organ harvesting was conducted at different sites according to biosecurity procedures. Cultures
were grown in a liquid medium consisting of a nutrient broth (Liofilchem s.r.l., #610037, Roseto
degli Abruzzi, Italy). The bacteriological study procedure included the following steps. After
seeding on trypticase soy agar (Bio-Rad, #64946, Marnes-la-Coquette, France), the bacterial mixture
was incubated at 37°C for 24 h. The bacteria were then streaked on Columbia sheep
blood agar (blood culture) and incubated as previously described. Isolates were assessed for Gram
positivity using Gram staining and were seeded for biochemical characteristic studies.</p><p>Eight isolates suspected as possibly being <italic>B. anthracis</italic> were sent to the URMITE
laboratory. Identification was performed using a system based on the molecular detection of the
<italic>pag</italic> gene under previously described conditions <xref rid="b14" ref-type="bibr">14</xref>. The production of acetyl methyl carbinol, as well as the fermentation of certain sugars,
was assessed on isolates identified as <italic>B. anthracis,</italic> using previously reported
methods <xref rid="b15" ref-type="bibr">15</xref>. Cultural, morphological and biochemical
characteristics were studied in detail using conventional methods. The experimental pathogenicity of
the <italic>B. anthracis</italic> strains was assessed using strain BALB/c white mice (age
6 months). These mice were offered by the Pasteur Laboratory institute of Dakar.
Intraperitoneal inoculations of 500 μL of the isolated
<italic>B. anthracis</italic> cultures was administered (culture at 104 CFU/mL), and
the animals were monitored for 24 h. Autopsies were performed post-mortem, and Giemsa-stained
smears of the spleen were observed using a microscope at 100 × magnification after
immersion.</p><p>In the pathogenicity experiments that were performed on white mice, two experimental mice
received 500 μL of the bacterial culture intraperitoneally and two control mice
received only 500 μL of physiological buffer. These mice were monitored in the
laboratory.</p><p>When the mice died, an autopsy was performed and <italic>B. anthracis</italic> was
identified by examining Giemsa-stained smears of the spleen <xref rid="b15" ref-type="bibr">15</xref>.</p></sec><sec><title>Sequencing</title><p>Sequencing of the Sen2col2, Sen3 and Gmb1 strains of <italic>B. anthracis</italic> were performed
using the SOLiD 4_Life Technology's New Generation Sequencing technology. The paired end
library was constructed from 1 μg of purified genomic DNA from each strain. The
sequencing was carried out to 50 × 35 base pairs (bp) using
SOLiD™ V4 chemistry on one full slide associated with 96 other projects on an Applied
Biosystems SOLiD 4 machine (Applied Biosystems, Foster City, CA, USA). All 96 genomic DNA samples
were barcoded with the module 1–96 barcodes provided by Life Technologies (Paisley, UK). The
libraries were pooled in equimolar ratios, and emPCR (PCR by emulsion) was performed according to
the manufacturer's specifications, using templated bead preparation kits on the EZ bead
automated Emulsifier, Amplifier and Enricher E80 system for full-scale coverage. A total of 708
million P2-positive beads were loaded onto the flow cell for the run, and the output read length was
85 bp, as expected (50 × 35 bp). The three
<italic>B. anthracis</italic> genomes (Sen2col, Sen3 and Gmb1) were sequenced through
3.2<sup>E</sup> + 6, 3.1<sup>E</sup> + 6 and
3.9<sup>E</sup> + 6 barcode reads, which led to 273, 262 and 382 Mb of
data, respectively. The global sequencing of these three genomes resulted in 917 Mb of
data.</p><p>For each of the three strains, we performed a mapping against the Ames reference strain though
CLC workbench software. For mapping we used relatively stringent parameters (length fraction of 0.7
and similarity fraction of 0.8).</p></sec><sec><title>Basic genomic data</title><p>The complete genomic sequences of the six references strains are available on NCBI: Ames <xref rid="b16" ref-type="bibr">16</xref> (NC_003997.3), Ames Ancestor <xref rid="b12" ref-type="bibr">12</xref> (NC_007530.2), A0248 (NC_012659.1), CDC684 <xref rid="b17" ref-type="bibr">17</xref>(NC_012581.1), H9401 <xref rid="b13" ref-type="bibr">13</xref> (NC_017729.1) and Sterne
(NC_005945.1). The sequences of the plasmids pX01 and pX02 from A0248, Ames Ancestor, CDC684 and
H9401 are also available on NCBI: NC_012656.1, NC_012655.1, NC_007322.2, NC_007323.3, NC_012579.1,
NC_012577.1, NC_017726.1, NC_017727.1, respectively. H9401 came from Korea, Sterne came from the UK,
and the four other strains came from the USA. Our strains of interest came from Senegal (Sen2Col2
[PRJEB1516] and Sen3 [PRJEB1517]) and Gambia (Gmb1
[PRJEB1518]) and were isolated in 2010. Sen2Col2 was isolated from the lungs of a
6-year-old ostrich. Sen3 was isolated from the lungs, liver, spleen and blood of a Touabire sheep.
Finally, Gmb1 was isolated from the blood of a trypanotolerant zebu cow. The sequences of these
three African strains and their plasmids were obtained from the SOLiD data.</p></sec><sec><title>Genomic analysis</title><sec><title>Determination of gene functions and the B. anthracis mobilome</title><p>To study genomic content and perform functional analysis, we first used CAMERA <xref rid="b18" ref-type="bibr">18</xref>, a bioinformatics portal that can perform several types of
analysis. In this portal, we performed cluster of orthologous groups (COG) analysis <xref rid="b19" ref-type="bibr">19</xref> to assign functional annotations to proteins, which were
classified into categories (the list is available at <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/COG/old/palox.cgi?fun=all">http://www.ncbi.nlm.nih.gov/COG/old/palox.cgi?fun=all</ext-link>). Next, we investigated metabolic
pathways using the Kyoto Encyclopedia of Genes and Genomes <xref rid="b20" ref-type="bibr">20</xref>data and the data generated KEGG by the KAAS <xref rid="b21" ref-type="bibr">21</xref> (KEGG
Automatic Annotation Server) online tool. In KEGG, the proteins were classified into classes and
subclasses. We also used RAST <xref rid="b22" ref-type="bibr">22</xref> to annotate the new strains
and their plasmids and to find the mobilome. Regarding the mobilome, we also used CRISPRfinder <xref rid="b23" ref-type="bibr">23</xref> and PHAST <xref rid="b24" ref-type="bibr">24</xref>. We used MeV
<xref rid="b25" ref-type="bibr">25</xref>,<xref rid="b26" ref-type="bibr">26</xref> (Multi
Experiment Viewer) to best visualize the distribution of the accessory genes and to perform
hierarchical clustering on the COG and KEGG data.</p></sec><sec><title>Phylogeny, MLVA and orthology</title><p>We performed a global genome alignment using MAUVE <xref rid="b27" ref-type="bibr">27</xref> and
a multiple alignment with MEGA5 <xref rid="b28" ref-type="bibr">28</xref> (using the ClustalW
algorithm), followed by a tree reconstruction (distance or neighbour joining method). Using MAUVE
and its backbone output file, we calculated the ratio of the size of the core genome to that of the
pan-genome to evaluate the closed or open nature of the pan-genome.</p><p>We were also interested in determining the lineages of the three African strains. For lineage
analysis, we used the MLVA8 system <xref rid="b10" ref-type="bibr">10</xref>, which is composed of
eight VNTR loci: <italic>vrrA</italic>, <italic>vrrB1</italic>, <italic>vrrB2</italic>,
<italic>vrrC1</italic>, <italic>vrrC2, CG3, pXO1-AAT</italic> and <italic>pXO2-AT</italic><xref rid="b29" ref-type="bibr">29</xref>. The NCBI and the MLVA databases were also helpful for
lineage analysis.</p><p>Next, we used OrthoMCL <xref rid="b30" ref-type="bibr">30</xref> with default parameters to
obtain a list of orthologs and determine the pan-genome composition (core, accessory and unique
genes) of <italic>B. anthracis</italic>. We also investigated the SNPs present in the core genome by
phylogeny, performing a maximum likelihood tree (phyml) with 100 bootstrap iterations. For the core
genome tree and the transition/transversion bias, we used MEGA5. Finally, based on the core SNPs
tree, we created a time tree based on the Reltime method, developed by Tamura
<italic>et al</italic>. in 2012 <xref rid="b31" ref-type="bibr">31</xref>. This tree allowed
the estimation of the divergence time in millions years and was built using the MEGA 6 software.</p></sec></sec></sec><sec sec-type="results"><title>Results</title><sec><title>General features and phylogeny</title><p>For each of the three strains, we obtained more than 6 million reads from the SOLiD sequencing.
Average coverage was approximately 30, and maximum coverage could reach 3000. The genomes of the
three African <italic>B. anthracis</italic> strains, Sen2Col2, Sen3 and Gmb1, were the same size as
the other known strains (5.23 Mb), and they had the same average number of proteins
(approximately 5300) and the same G+C% value. The three genomes were deposited at EMBL
with the accession numbers CAVC010000000, CAVD010000000 and CAVE010000000, respectively. The MAUVE
alignment revealed no rearrangements and a high conservation among all genomes. Examination of the
<italic>plcR</italic> gene revealed a nonsense mutation typical to <italic>B. anthracis</italic><xref rid="b7" ref-type="bibr">7</xref> at position 640 and the four known prophage regions in the
three new strains. The low values of the branches on the phylogenetic tree (not shown) indicated
high similarities between the strains. We also examined the <italic>rpoB</italic> gene but could not
perform a phylogenetic analysis because the sequences of all nine strains were identical. Regarding
the plasmids, we found that pX01 and pX02 in the three Africans strains were the same size
(0.18 Mb and 0.094 Mb, respectively) and contained the same average number of genes
(195 and 102, respectively). There were no gaps in the new plasmid sequences. The MAUVE alignments
(Fig.<xref ref-type="fig" rid="fig01">1</xref>) illustrate the similarities between the plasmids.
The pX01 plasmids from CDC684 and the three African strains exhibit an inversion, and the pX02
plasmids of the three African strains are identical to that of CDC684. Additionally, based on the
analysis of the phylogenetic trees (Fig.<xref ref-type="fig" rid="fig02">2</xref>a,b), the two
plasmids of the African strains are closely related to those from strain CDC684.</p><fig id="fig01" position="float"><label>Figure 1</label><caption><p>G192: Sen2Col2, G193: Sen3 and G194: Gmb1. MAUVE global alignment on pX02 plasmids.</p></caption><graphic xlink:href="nmi20002-0161-f1"></graphic></fig><fig id="fig02" position="float"><label>Figure 2</label><caption><p>Numbers on the branches correspond to branch length. (a) Tree based on the pX01 plasmid. (b) Tree
based on the pX02 plasmid.</p></caption><graphic xlink:href="nmi20002-0161-f2"></graphic></fig><p>We defined the profile of the eight VNTR loci for the three new strains, and the tree shows that
these African strains again clustered with CDC684 (Fig.<xref ref-type="fig" rid="fig03">3</xref>).
Using the values of the eight loci and comparing them to the work of Keim et al. <xref rid="b10" ref-type="bibr">10</xref>, we found that the three African strains belong to lineage A.
More specifically, we found that these strains belong to lineage A4, the same lineage as CDC684 and
one other African strain <xref rid="b10" ref-type="bibr">10</xref>.</p><fig id="fig03" position="float"><label>Figure 3</label><caption><p>Multi locus VNTR analysis tree. Colours correspond to lineages: light blue, A1a; dark blue, A1b;
red, A2; light green, A3a; dark green, A3b; blue green, other A3; pink, A4.</p></caption><graphic xlink:href="nmi20002-0161-f3"></graphic></fig></sec><sec><title>Genomic comparison</title><sec><title>KEGG and COG</title><p>The three African strains are identical to each other and to CDC684 at a functional level.
Fig.<xref ref-type="fig" rid="fig04">4</xref> represents the clustering of the strains based only on
the difference in the KEGG distribution. Divergences are as follows: one difference with Sterne
(ko02020), two with H9401 (ko03010 and ko00300), and multiple differences with Ames and Ames
Ancestor (ko00260, ko00300, ko00440, ko02010, ko02020, ko02040), and A0248 (ko00562, ko01040,
ko00300, ko00340, ko00440, ko02010, ko02020, ko02040, ko05100). When we examined these details, we
noticed two groups: one group contains A0248, Ames and Ames Ancestor, and the other group contains
the other strains. There were no differences between the pX01 and pX02 plasmids of the African
strains and those of the other strains (data not shown). We looked in detail at categories related
to the genetic systems for DNA surveillance because they might be a part of the slow evolution of
these bacteria. DNA replication and nucleotide excision repair were complete and active. With regard
to homologous recombination, the ruvC gene is absent in all strains. For mismatch repair, mutH, the
unique enzyme for the incision step, is absent. Hence, this pathway is incomplete.</p><fig id="fig04" position="float"><label>Figure 4</label><caption><p>Analysis of Kyoto Encyclopaedia of Genes and Genomes (KEGG) results. Hierarchical clustering of
the strains based only on the difference in the KEGG distribution. Colours depend on the number of
proteins implied in each category for each strain. The scale is presented in the figure.</p></caption><graphic xlink:href="nmi20002-0161-f4"></graphic></fig><p>The COG data showed similar results. First, as shown in Fig.<xref ref-type="fig" rid="fig05">5</xref>, a cluster with Ames, Ames Ancestor and A0248 (group 1) and another cluster with the other
strains (group 2) was found. Moreover, we can see uniformity between the strains inside each of the
COG categories, except in the E (amino acid transport and metabolism), G (carbohydrate transport and
metabolism), P (inorganic ion transport and metabolism) and M (cell wall/membrane/envelope
biogenesis) categories, where there is, for group 1, approximately 5% less protein function
as group 2. These small differences can be observed as slight modifications of the colours in
Fig.<xref ref-type="fig" rid="fig05">5</xref>.</p><fig id="fig05" position="float"><label>Figure 5</label><caption><p>Clustering of the strains based on the distribution of all the Cluster of Orthologous Groups
categories. J, translation, ribosomal structure and biogenesis; K, transcription; L, replication,
recombination and repair; B, chromatin structure and dynamics; D, cell cycle control, cell division,
chromosome partitioning; O, post-translational modification, protein turnover, chaperones; M, cell
wall/membrane/envelope biogenesis; N, cell motility and secretion; P, inorganic ion transport and
metabolism; T, signal transduction mechanisms; U, intracellular trafficking, secretion and vesicular
transport; V, defence mechanisms; W, extracellular structures; Z, cytoskeleton; C, energy production
and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F,
nucleotide transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and
metabolism; Q, secondary metabolite biosynthesis, transport and catabolism; R, general function
prediction only; S, function unknown. Colours depend on the number of proteins implied in each
category for each strain. The scale is presented in the figure.</p></caption><graphic xlink:href="nmi20002-0161-f5"></graphic></fig></sec><sec><title>Pan-genome, SNPs and mobilome</title><p>The mobilome was examined, and the same trend was found in all of the strains: two transposases,
one or two confirmed CRISPRs and numerous proteins/associated proteins from phages or prophages. We
also examined the mobilome in the plasmids. All of the pX01 plasmids contained five transposases,
one phage and no CRISPRs. All of the pX02 plasmids contained two transposases and no CRISPRs.</p><p>The pan-genome is composed of 2893 core genes, seven unique genes and 85 accessory genes. First,
we considered the unique genes. We found five in Sterne (two not found on NCBI, one conserved
hypothetical protein, an EmrB/QacA family drug resistance transporter and a zinc-binding
dehydrogenase), one in CDC684 (not found on NCBI) and one in H9401 (a yfeT DNA-binding
transcriptional regulator), but all were false positives, as they were eventually identified in
other <italic>B. anthracis</italic> strains available on the NCBI database. Next, we carefully
examined the 85 accessory genes. The three African strains and CDC684 contained almost all of the
accessory genes. Half of the accessory genes were annotated as hypothetical proteins. The
hierarchical clustering again resulted in the same two groups found using COG and KEGG (one with
Ames, Ames Ancestor and A0248 and the second with the other strains). Moreover, we calculated the
core/pan-genome ratio and found that the core genome represented 99% of the pan-genome
(Table<xref ref-type="table" rid="tbl1">1</xref>), again indicating the high rate of conservation
among the nine strains. Finally, we studied the SNPs at the core genomic level. We found
approximately 3800 SNPs between the nine strains, with 2911 in the core. Among the three African
strains, there are 1500 SNPs in the core and 1120 among the six reference strains. We can see that
the three African strains cluster together. Moreover, we have a transition/transversion bias of 0.32
between the nine strains. The very small rate of SNPs, low transition/transversion bias and very
high ratio of the core genome function to the pan-genome indicates that
<italic>B. anthracis</italic> evolves very slowly. To go further, we generated a
maximum-likelihood tree based on SNPs of the core of the nine <italic>B. anthracis</italic>strains, together with eight <italic>B. cereus</italic>, three <italic>Bacillus
thuringiensis</italic> and, as an outgroup, one <italic>Bacillus cytotoxicus</italic> (Fig.<xref ref-type="fig" rid="fig06">6</xref>a) strain. This tree clearly showed that our three African
strains evolved differently from the other six strains of <italic>B. anthracis</italic> and
that is an ancestral difference. Moreover, we obtained good bootstraps, so we could be confident
about this cluster. Finally, we generated a time tree (Fig.<xref ref-type="fig" rid="fig06">6</xref>b) to estimate the divergence time between each cluster. On Fig.<xref ref-type="fig" rid="fig06">6</xref>b, the number on branches are indicated in millions of years. For instance,
between the African cluster and <italic>B. anthracis</italic> H9401, the estimated divergence
time is approximately 360 years. Moreover, the estimated divergence time between the
<italic>B. anthracis</italic> cluster and <italic>B. cereus</italic> AH820 (n15 on
Fig.<xref ref-type="fig" rid="fig06">6</xref>b) is approximately 12 000 years. For
comparison, in a work in 1999, Achtman <italic>et al</italic>. first estimated the divergence
time between <italic>Yersinia pestis</italic> and <italic>Yersinia pseudotuberculosis</italic> to be
between 1500 and 20 000 years. Then, in 2004 <xref rid="b32" ref-type="bibr">32</xref>, they looked at different pathovars of <italic>Y. pestis</italic>. They found the
estimated divergence time between an Orientalis strain (CO92) and a Medievalis strain (KIM) to be
approximately 6500 years.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><p>Pan-genomes of human clonal pathogens</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" valign="bottom" rowspan="1" colspan="1">Species</th><th align="left" valign="bottom" rowspan="1" colspan="1">Genome<break></break>used</th><th align="left" valign="bottom" rowspan="1" colspan="1">Lifestyle</th><th align="left" valign="bottom" rowspan="1" colspan="1">Intracellular</th><th align="left" valign="bottom" rowspan="1" colspan="1">Niche</th><th align="left" valign="bottom" rowspan="1" colspan="1">%</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>Bacillus anthracis</italic></td><td align="left" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1">Allopatric</td><td align="left" rowspan="1" colspan="1">No</td><td align="left" rowspan="1" colspan="1">Soil</td><td align="left" rowspan="1" colspan="1">99</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Rickettsia rickettsii</italic></td><td align="left" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">Allopatric</td><td align="left" rowspan="1" colspan="1">Yes</td><td align="left" rowspan="1" colspan="1">Ticks</td><td align="left" rowspan="1" colspan="1">99</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Chlamydia trachomatis</italic></td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">Allopatric</td><td align="left" rowspan="1" colspan="1">Yes</td><td align="left" rowspan="1" colspan="1">Human</td><td align="left" rowspan="1" colspan="1">99</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Rickettsia prowazekii</italic></td><td align="left" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">Allopatric</td><td align="left" rowspan="1" colspan="1">Yes</td><td align="left" rowspan="1" colspan="1">Human</td><td align="left" rowspan="1" colspan="1">100</td></tr></tbody></table><table-wrap-foot><fn><p>The % column corresponds to the core/pan-genome ratio.</p></fn></table-wrap-foot></table-wrap><fig id="fig06" position="float"><label>Figure 6</label><caption><p>Single nucleotide polymorphisms of the core content based tree. The percentage on the branches
corresponds to bootstraps. This is a PhyML tree with 100 bootstrap iterations. Colours correspond to
the species. Purple is <italic>Bacillus anthracis</italic>, pink is our three African
<italic>B. anthracis</italic> strains, blue is <italic>Bacillus cereus</italic> and brown is
<italic>Bacillus thuringiensis</italic>. (a) Numbers on branches correspond to bootstrap values. (b)
Time tree based on the previous tree. Numbers on the branches are the divergence time, given in
million years.</p></caption><graphic xlink:href="nmi20002-0161-f6"></graphic></fig></sec></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>In comparing the three African strains to the other <italic>B. anthracis</italic> strains,
we noticed that all of the African strains carried the two virulence plasmids (pX01 and pX02), the
<italic>plcR</italic> nonsense mutation and four prophages. We also determined that these strains
were more closely related to CDC684 at the functional, sequence, plasmid and lineage levels. In this
work, we validate the finding that, as previously shown <xref rid="b9" ref-type="bibr">9</xref>, the
pan-genome of <italic>B. anthracis</italic> is very narrow and that
<italic>B. anthracis</italic> is clonal. To have confidence in our study, we used many
different tools to compare and validate our results. All of the tools yielded the same results: the
addition of the three African strains did not change the nature of the
<italic>B. anthracis</italic> pan-genome (2893 core genes and 85 accessory genes), which had
a core/pan-genome ratio of 99%. This core/pan-genome ratio is very close to those from the
other human clonal pathogens (Table<xref ref-type="table" rid="tbl1">1</xref>), such as
<italic>Rickettsia rickettsii</italic>. However, there is discordance between the presence of a
mobilome (some phages) and the fact that <italic>B. anthracis</italic> has a closed
pan-genome. Nevertheless, <italic>B. anthracis</italic> is derived from the
<italic>B. cereus</italic> group, a sympatric species that is not intracellular. Therefore,
<italic>B. anthracis</italic> may have become entirely allopatric. Moreover, based on the
core genome, we found very few SNPs (896), a very small transition/transversion bias (0.32) and no
CRISPRs. Hence, we can hypothesize that <italic>B. anthracis</italic> is an ancient clone
that evolved slowly. Indeed, the work of Mancini and Ippolito <xref rid="b33" ref-type="bibr">33</xref> describes the history of anthrax disease and suggested that the first case may date back
to antiquity. However, calculating the age of this type of bacterium is difficult due to its life
cycle (a short vegetative phase of 20–40 generations and a long spore phase) <xref rid="b9" ref-type="bibr">9</xref>. <italic>Bacillus anthracis</italic> does not replicate in the
spore phase, and when it is in tissues, it may replicate as a pathogen to avoid living in sympatry.
This behaviour is similar to <italic>Clostridium tetani</italic><xref rid="b34" ref-type="bibr">34</xref>, a sporulating, anaerobic bacterium that resides in the
soil and is pathogenic for humans and animals. We recently obtained a new genome of
<italic>C. tetani</italic> and found that it is also very close to the reference genome <xref rid="b35" ref-type="bibr">35</xref>. Therefore, the <italic>B. anthracis</italic> pathogen
evolves very slowly compared with other species with similar generation times <xref rid="b18" ref-type="bibr">18</xref>,<xref rid="b19" ref-type="bibr">19</xref>. We believe that the lack of gene
transfer and defence mechanisms (CRISPRs) observed in intracellular bacteria <xref rid="b36" ref-type="bibr">36</xref> suggests that <italic>B. anthracis</italic> multiplies only as a pathogen
and that its life in soil is exclusively dormant (Fig.<xref ref-type="fig" rid="fig07">7</xref>).</p><fig id="fig07" position="float"><label>Figure 7</label><caption><p>Summary of the <italic>Bacillus anthracis</italic> lifestyle.</p></caption><graphic xlink:href="nmi20002-0161-f7"></graphic></fig><p>There are different types of pathogens. Opportunist pathogens, such as <italic>Pseudomonas
aeruginosa</italic>, live in the environment but are not specialized. Real pathogens, such as
<italic>B. anthracis</italic>, <italic>Y. pestis</italic><xref rid="b37" ref-type="bibr">37</xref>, <italic>C. tetani</italic> and <italic>Tropheryma
whipplei</italic>, are specialized and have a small and closed pan-genome. Each of these pathogens
has a core/pan-genome ratio of more than 90%. Real pathogens can survive in the soil or water
but cannot multiply outside their niche. A pan-genome study, as opposed to a virulence study, can
help to determine if a species is a real, specialized pathogen.</p></sec><sec><title>Conclusion</title><p>Due to the lengthy spore phase of its life cycle, <italic>B. anthracis</italic> evolved
very slowly and has a very narrow pan-genome, despite its apparent soil ecological niche. We found
that the three African strains examined belong to lineage A (worldwide lineage), specifically
lineage A4, similar to CDC684 and another previously characterized African strain. Pan-genome
analysis allowed us to assess the lifestyle of this pathogen and confirmed its allopatric, highly
specialized lifestyle. Our studied African strains diverged very recently from the other
<italic>B. anthracis</italic> strains.</p></sec></body><back><sec><title>Conflict of Interest</title><p>None declared.</p></sec><ref-list><title>References</title><ref id="b1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Scarlata</surname><given-names>F</given-names></name><name><surname>Colletti</surname><given-names>P</given-names></name><name><surname>Bonura</surname><given-names>S</given-names></name><name><surname>Trizzino</surname><given-names>M</given-names></name><name><surname>Giordano</surname><given-names>S</given-names></name><name><surname>Titone</surname><given-names>L</given-names></name></person-group><article-title>The return of anthrax. From bioterrorism to the zoonotic cluster of Sciacca
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