Characterization of Clinically-Attenuated Burkholderia
mallei by Whole Genome Sequencing: Candidate Strain
for Exclusion from Select Agent Lists
Steven E. Schutzer1*, Linda R. K. Schlater2, Catherine M. Ronning3, David DeShazer4, Benjamin J. Luft5,
John J. Dunn6, Jacques Ravel7,8, Claire M. Fraser-Liggett7,8, William C. Nierman3,9
1Department of Medicine, University of Medicine and Dentistry - New Jersey Medical School, Newark, New Jersey, United States of America, 2United States Department
of Agriculture, Ames, Iowa, United States of America, 3J. Craig Venter Institute, Rockville, Maryland, United States of America, 4U.S. Army Medical Research Institute of
Infectious Diseases, Fort Detrick, Maryland, United States of America, 5Department of Medicine, State University of New York, Stony Brook, New York, United States of
America, 6Biology Department, Brookhaven National Laboratory, Upton, New York, United States of America, 7Institute for Genome Sciences, Department of Medicine,
University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 8Institute for Genome Sciences, Department of Microbiology and Immunology,
University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 9Department of Biochemistry and Molecular Biology, The George Washington
University School of Medicine, Washington, D. C., United States of America
Background: Burkholderia mallei is an understudied biothreat agent responsible for glanders which can be lethal in humans
and animals. Research with this pathogen has been hampered in part by constraints of Select Agent regulations for safety
reasons. Whole genomic sequencing (WGS) is an apt approach to characterize newly discovered or poorly understood
Methodology/Principal Findings: We performed WGS on a strain of B. mallei, SAVP1, previously pathogenic, that was
experimentally infected in 6 equids (4 ponies, 1 mule, 1 donkey), natural hosts, for purposes of producing antibodies.
Multiple high inocula were used in some cases. Unexpectedly SAVP1 appeared to be avirulent in the ponies and mule, and
attenuated in the donkey, but induced antibodies. We determined the genome sequence of SAVP1 and compared it to a
strain that was virulent in horses and a human. In comparison, this phenotypic avirulent SAVP1 strain was missing multiple
genes including all the animal type III secretory system (T3SS) complex of genes demonstrated to be essential for virulence
in mice and hamster models. The loss of these genes in the SAVP1 strain appears to be the consequence of a multiple gene
deletion across insertion sequence (IS) elements in the B. mallei genome. Therefore, the strain by itself is unlikely to revert
naturally to its virulent phenotype. There were other genes present in one strain and not the other and vice-versa.
Conclusion/Significance: The discovery that this strain of B. mallei was both avirulent in the natural host ponies, and did not
possess T3SS associated genes may be fortuitous to advance biodefense research. The deleted virulence-essential T3SS is
not likely to be re-acquired naturally. These findings may provide a basis for exclusion of SAVP1 from the Select Agent
regulation or at least discussion of what else would be required for exclusion. This exclusion could accelerate research by
investigators not possessing BSL-3 facilities and facilitate the production of reagents such as antibodies without the
restraints of Select Agent regulation.
Citation: Schutzer SE, Schlater LRK, Ronning CM, DeShazer D, Luft BJ, et al. (2008) Characterization of Clinically-Attenuated Burkholderia mallei by Whole Genome
Sequencing: Candidate Strain for Exclusion from Select Agent Lists. PLoS ONE 3(4): e2058. doi:10.1371/journal.pone.0002058
Editor: Frederick M. Ausubel, Massachusetts General Hospital, United States of America
Received February 4, 2008; Accepted March 13, 2008; Published April 30, 2008
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: Work was funded in part by several grants from the National Institutes of Health (N01-AI30071, U01 AI056480, AI063757). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
An initial approach today to a newly discovered pathogen is to
perform whole genome sequencing (WGS). This same approach is
relevant for investigations of an understudied high-consequence
pathogen such as Burkholderia mallei, the cause of glanders. This
zoonotic bacterium can kill humans and animals [1,2]. It is
classified as a Category B biothreat agent and it has been used as a
biological warfare agent [2–8]. Its ease of use against civilians and
lack of countermeasures has compelled several agencies to rank it
very high on their priority lists of biothreat agents[9,10]. Its ease of
use as a biothreat agent is illustrated by the physician who, even in
1915, grew it out of his Washington DC area home, and
distributed it for an attack against US horses destined for Europe
as critical transportation in World War I. Humans were also
subject to infection. We remain highly vulnerable to this agent
because there is no rapid diagnostic assay, no distinctive diagnostic
signs, and the incubation period is short, a few days. In addition
there is no vaccine, no infection-induced immunity, and limited
reliable in vivo data on antibiotic efficacy. Overall, our general
PLoS ONE | www.plosone.org1April 2008 | Volume 3 | Issue 4 | e2058
knowledge of this understudied pathogen and its disease is limited
[2,12–17]. Our desire to decrease our vulnerabilities to this
pathogen, as part of the national biodefense efforts, is hampered,
in part, by constraints of the Select Agent regulation and the need
for BSL-3 facilities. These are appropriately in place for safety
reasons, however, the availability of a suitable attenuated
surrogate strain would be desirable as it could accelerate B. mallei
related biodefense research in many non-BSL-3 laboratories.
B. mallei is a Gram-negative non-motile aerobic bacteria
with a genome of approximately 6 Mb organized in two circular
chromosomes. In addition to infecting humans, B. mallei can cause
acute or chronic fatal contagious zoonotic infections in its natural
equine host, such as horses, donkeys, and mules, with a very low
infectious dose. Two major potential routes of infection for a
biologic attack are aerosol and cutaneous contact. Gastrointestinal
ingestion is a common mode of natural infection in equines. The
incubation period is typically between 3–6 days but may be longer.
An example of a phenotypic highly virulent strain is B. mallei
strain ATCC 23344 (China 7). It is highly virulent in its natural
hosts, equines, in humans, and in mice and hamster mod-
els[14,19–21]. This strain of B. mallei contains an animal type III
secretion system (T3SS) gene complex which is essential for
virulence[15,22]. Genome sequencing (WGS) and analysis of this
strain identified a number of other putative virulence factors
whose function was supported by comparative genome hybridiza-
tion and expression profiling of the bacterium in hamster liver in
vivo. Numerous insertion sequence elements that have
mediated extensive deletions and rearrangements of the genome
relative to the B. pseudomallei genome were found. As part of our
interest to study mechanisms responsible for virulence we
performed WGS and comparative genomic analysis on strains of
B. mallei in which we had closely linked information from an actual
clinical infection in its natural host or a suitable animal model
rather than using an archived strain lacking this information. We
intended to compare the gene content of phenotypic virulent to
avirulent strains to identify candidate virulence genes. One
serendipitous event and an unexpected finding led to what may
be a fortuitous observation. The serendipitous event occurred
when five of six equids were infected, but not made ill, with what
was a previously pathogenic strain of B. mallei, designated as
SAVP1. At the time of inoculation this strain was still believed to
be pathogenic. A sixth equid, a donkey, developed clinical signs
only after a massive exposure. The strain appeared avirulent even
when administered in escalating doses. The other unexpected
finding came while performing comparative genomic analysis
between SAVP1 and the strain of B. mallei that was currently
behaving as virulent in humans and horses. This finding is both
informational and fortuitous because it may open the door for
SAVP1 to be classified as suitably avirulent for exclusion from
Select Agent lists thereby expanding our biodefense research
In vivo characterization of phenotypic avirulent strain of
B. mallei in natural equine hosts
Based upon our premise that relevant pathogenic differences
between strains may be revealed by WGS of a bona-fide
phenotypic avirulent strain of B. mallei, we wanted to select a
strain that was previously evaluated in the natural host. This was
accomplished by selecting a strain (SAVP1) that had previously
caused disease in a mule (in India) and surprisingly did not
produce overt disease when inoculated, first orally and subse-
quently intravenously, into a mule and 4 ponies, even at escalating
doses approaching 109colony forming units (CFU) per ml. The
prime purpose of the initial experiment, carried out by one of us
(LKS) more than twenty years ago, was to develop a diagnostic
assay for B. mallei as part of the US Department of Agricultures
mission to help prevent the disease from entering the United States
through horses. It was not meant as a controlled infectivity study,
consequently full clinical description and detailed history of the
strain are not available. Notes indicate 4 equids (4 ponies, a mule,
and a donkey) were exposed by pharyngeal spray twice to
increasing doses of B. mallei. When none of the equids became ill or
seroconverted by 30 days post exposure, they were challenged
intravenously with 5 ml of heavy suspension of organisms. The
extreme exposure produced clinical signs only in the donkey, a
species known to be more susceptible than horses and mules to B.
mallei. The donkey was euthanized for humane reasons. This
donkey received a high intravenous dose, approaching believed to
have approached 5 ml of 109cfu/ml – thus the symptoms may
have been related to endotoxin. Clinical signs in the ponies and the
mule were either minimal (fever) or nonexistent. All 6 equids
produced antibodies to B mallei as a result of the intravenous
exposure. The expected clinical signs of morbid infection were not
present in the ponies or the mule. At the time of the necropsy,
there was little evidence of systemic pathology. This included the
absence of the common sinopulmonary findings of purulent nasal
discharge, ulcerations, pleural effusion, pulmonary edema, con-
gestion, or pneumonia. The sole pulmonary involvement was
restricted to a few minimal bronchopulmonary granulomatous
lesions which did not grow B. mallei on culture. From a
microbiologic vantage it was not possible to ascertain if current
avirulence was an attenuation effect that arose from passage in the
mule before it was grown in culture and re-inoculated or if it was
an effect of the in vitro culture. What is important here is the
observation that this particular strain had avirulent/attenuated
behavior in this natural host experiment. We performed WGS on
DNA extracted from the same or near (low-passage) generation
growth of the B. mallei used in this experiment.
Detection of genes present or absent in an avirulent
strain from a natural equine host in comparison to a
known virulent strain
The SAVP1 strain was subjected to WGS and the resulting data
compared those from the virulent ATCC 23344 strain which
was know to have caused recent near-death in a human and five
horses that had to be euthanized[19,21]. A one way analysis
interrogated what genes were present in ATCC 23344 but lacking
in the SAVP1 strain. This analysis took all of the ATCC 23344
coding sequences (CDS) and aligned them against the SAVP1
genomic sequence, identifying 582 ORFs that were present in
ATCC 23344 but not SAVP1, the vast majority of which exist
within a few large gene clusters (See Table 1, Supplementary
Table S1, and Figure 1). In contrast, all of the genes shared
between the two isolates are at least 99% identical over 99% or
more of their length, as determined by blastn.
The most noteworthy difference was the loss of all the animal
T3SS associated genes in an apparent IS mediated deletion
containing contiguous genes (two sets of T3SS: one consists of 23
genes from BMA_A1530-A1552 (the Bsa genes) and a second set
from BMA_A1625-A1637 consists of 13 genes, Genbank accession
numbers, ATCC 23344 small chromosome- CP000011 and
SAVP1- CP000525). These genes are located within the largest
of the ATCC 23344-specific gene clusters mentioned above. The
absence of animal type T3SS alone could explain its avirulence in
the natural host. Differences in the ATCC 23344 and SAVP1
genomes result from multiple inversions and deletions of genome
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segments at insertion sequences (IS), particularly IS407A elements
(Fig. 1 and 2). SAVP1 has lost approximately 610 kb of DNA due
to IS mediated deletions (see Supplemental Table 1 for genes not
present in SAVP1). Most of the lost genes are on chromosome 2, a
replicon that encodes more accessory functions than chromosome
1. Notable in comparing presence of genes on SAVP1 but not on
ATCC 23344 include multiple fimbriae/pili genes and several
hypothetical genes (See Figure 2). The rearrangements detailed
here demonstrate the plasticity of the B. mallei genome and suggest
that IS mediated deletions may have a profound effect on the
relative virulence of B. mallei strains.
Thus, based on our current knowledge, the strain is unlikely to
undergo a natural restoration to a virulent state by passage in an
animal host, in contrast to the situation if there were only
reparable replication errors or point mutations in a single or a few
The general public remains at risk to the pathogenic effects of B.
mallei until we can develop reliable therapeutics, vaccines or other
protective measures, and rapid diagnostics. We would have an
opportunity to develop these countermeasures more quickly if we
could identity attenuated strains of B. mallei and employ the strains
in selected experiments. Unlike anthrax, where we have effective
diagnostics, approved therapy, and vaccines, none of these
resources are available for B. mallei. This means that those most
capable of advancing research in this field are at high occupational
risk, without effective contingency measures, should an infection
be suspected or actually occur. In addition to occupational risk,
two other factors that impede research are the requirement to use
BSL-3 facilities and the Select Agent regulations[23,24]. Fortu-
nately, there are provisions within the Select Agent Program that
Table 1. Type III secretion proteins encoded by genes present in ATCC 23344 but absent in SAVP1.
Locus Annotation59 end39 end
BMA_A1520 type III secretion chaperone BicP1650045 1649590
BMA_A1532 type III secretion chaperone BicA16623751661830
BMA_A1533 type III secretion system protein BsaZ1663706 1662471
BMA_A1534 type III secretion system protein BsaY1664480 1663710
BMA_A1535 type III secretion system protein BsaX1664753 1664499
BMA_A1536type III secretion system protein BsaW 16654691664789
BMA_A1537 type III secretion system protein BsaV 16664421665459
BMA_A1540type III secretion system protein BsaS 1669435 1668125
BMA_A1541 type III secretion system protein BsaR1669839 1669432
BMA_A1542 type III secretion system protein BsaQ16719231669851
BMA_A1543type III secretion system protein BsaP16730801671959
BMA_A1544 type III secretion system protein BsaO16748971673077
BMA_A1545type III secretion system transcriptional regulator BsaN 1675726 1674968
BMA_A1547 type III secretion system protein BsaM16761201677406
BMA_A1548type III secretion system protein BsaL 16774031677672
BMA_A1550type III secretion system BasJ 16780341678984
BMA_A1551 type III secretion apparatus protein OrgA/MxiK1678981 1679568
BMA_A1552 type III secretion apparatus protein, HrpE/YscL family16795371680331
BMA_A1602 type III secretion outer membrane pore, YscC/HrcC family17409181739119
BMA_A1613 type II/III secretion system family protein17540931752303
BMA_A1625type III secretion inner membrane protein, authentic frameshift 17638341762891
BMA_A1627 type III secretion inner membrane protein SctS1765159 1764896
BMA_A1628type III secretion inner membrane protein SctR 1765850 1765200
BMA_A1629type III secretion inner membrane protein SctQ17671351765837
BMA_A1630type III secretion inner membrane protein SctV17698431767771
BMA_A1631type III secretion protein, YscU/HrpY family1770922 1769840
BMA_A1632type III secretion protein, HrpB1/HrpK family17711581771733
BMA_A1633 type III secretion protein HrpB217717471772163
BMA_A1634 type III secretion inner membrane protein SctJ, authentic frameshift17721661773012
BMA_A1635type III secretion protein HrpB417730091773686
BMA_A1636type III secretion inner membrane protein SctL17736711774387
BMA_A1637type III secretion apparatus H+-transporting two-sector ATPase1774423 1775712
All genes are located on chromosome II and appear to be present in two basically contiguous segments: BMA_A1520-BMA_A1552 and BMA_A1625-BMA_A1637. Genes
with Bsa are part of the animal pathogen-like T3SS. A complete list of the genes present in ATCC 23344 but missing from SAVP1 is given in Supplementary Table S1.
Avirulent Burkholderia mallei
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enable a particular microbial strain to removed or excluded.
Certain strains of microbes have already been excluded from the
list and not subject to the requirements of 42 CFR Part 73 and 9
CFR Part 121 if used in basic or applied research, as positive
controls, for diagnostic assay development, proficiency testing, or
for the development of vaccines and therapeutics. Examples now
excluded are certain strains of Yersinia pestis, Bacillus anthracis strains
devoid of both plasmids pX01 and pX02, Bacillus anthracis strains
devoid of the plasmid pX02 (e.g., Bacillus anthracis Sterne,
pX01+pX02-), Brucella abortus Strain 19 and strain RB51 (vaccine
strains), Coxiella burnetii, Francisella tularensis subspecies novicida, and
Francisella tularensis subspecies holartica LVS (live vaccine strain).
However, the regulations go back into effect if there is any
reintroduction of factor(s) associated with virulence or other
manipulations that restore the virulence or diminish the
attenuation. Therefore, as might be done with excluded strains
above, we believe it is prudent to monitor for any possible
reversions with the SAVP1 during in vivo experiments. The same
caveat applies to restoration experimentation, if it could be done,
to provide more definitive evidence of that absence of T3SS as
necessary and sufficient for attentuation.
We believe it is preferable to pair the strain with clinical cases in
a natural-host infection whether that occurs naturally or by
experiment. The closer the strain isolate is to the case the better,
Figure 1. Comparison of the small chromosomes of B. mallei ATCC 23344 (top) and B. mallei SAVP1 (bottom) using ACT and the
whole genome alignment MUMmer. Regions of similarity, rearrangements, and deletions are readily apparent between these two strains. The
red and blue bands represent the forward and reverse matches, respectively. The orange horizontal bars represent the regions of ATCC 23344 that
are missing in SAVP1. The locations of the animal pathogen-like T3SS (T3SS ap) and the plant pathogen-like (T3SS pp) gene clusters that are present
in ATCC 23344, but absent in SAVP1, are indicated by arrows. The ATCC 23344 small chromosome (CP000011) is 2.32 Mb and the SAVP1 small
chromosome (CP000525) is 1.73 Mb.
Figure 2. Comparison of the large chromosomes of B. mallei ATCC 23344 (top) and B. mallei SAVP1 (bottom) using ACT and the
whole genome alignment MUMmer. Regions of similarity, rearrangements, and deletions are readily apparent between these two strains. The
red and blue bands represent the forward and reverse matches, respectively. The ATCC 23344 large chromosome (CP000010) is 3.51 Mb and the
SAVP1 large chromosome (CP000526) is 3.49 Mb.
Avirulent Burkholderia mallei
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rather than an isolate from multiple in vitro passages in cultures.
Misleading data might result by randomly selecting an isolate from
a strain collection without the associated clinical information. In
the current example we happened to identify the loss of animal
T3SS genes in a strain associated with avirulent phenotypic
behavior in its natural host. Because the T3SS is so essential to
export of virulent factors, and this was unexpectedly found by
WGS of SAVP1, there is increased confidence that experiments
with this isolate may be accomplished with a higher degree of
safety that with the virulent strain. Loss of some of the other 582
ORFs could be additive to this effect. Presence of other genes
which are on SAVP1 and not on ATCC 23344 could also
contribute to attenuated behavior. Though this strain would no
longer be our choice for comparative genomic studies to detect
subtle genetic differences that may account for virulence, its ability
to evoke antibody responses confers it with great potential for
other types of biodefense research as well as potential vaccines in
equids and humans. Other avirulent strains may also prove to be
candidates for Select Agent regulation exclusion[16,17].
In summary, our investigation into virulence factors using WGS
on clinically-associated strains of B. mallei led to an unexpected
finding. This finding may serve to eventually have at least one
strain, SAVP1, removed from select agent constraints. Its research
utility could be assessed. We believe there are comparable
situations with other biothreat agents. We hope that the example
of our finding with SAVP1 will engender discussion among public
health and regulatory agencies, academia, and the private sector
that will favorably impact our biodefense research efforts.
Materials and Methods
The genomes of B. mallei were sequenced and assembled by
random shotgun method as described.
Coding Sequence (CDS) Prediction and Gene
Open reading frames (ORFs) likely to encode proteins (CDSs)
were identified by using GLIMMER. Identified CDSs were
annotated by manual curation of the outputs of a variety of
similarity searches. Searches of the predicted coding regions were
performed with BLASTP, as described. The protein–protein
matches were aligned with blast_extend_repraze, a modified
Smith-Waterman algorithm that maximally extends regions of
similarity across frameshifts. Gene identification is facilitated by
searching against a database of nonredundant bacterial proteins
(nraa) developed at The Institute for Genomic Research (TIGR)
and curated from the public archives GenBank, Genpept, Protein
Information Resource, and SwissProt. Searches matching entries
in nraa have the corresponding role, gene common name, percent
identity and similarity of match, pairwise sequence alignment, and
taxonomy associated with the match assigned to the predicted
coding region and stored in the database. CDSs were also
analyzed with two sets of hidden Markov models constructed for a
number of conserved protein families from PFAM and TIGR-
FAM. Regions of the genome without CDSs and CDSs without a
database match were reevaluated by using BLASTX as the initial
search, and CDSs were extrapolated from regions of alignment.
Finally, each putatively identified gene was assigned to one of 113
ATCC 23344 - SAVP1 Comparative Analysis
All CDSs from ATCC 23344 were aligned against the whole
genome sequence of SAVP1 with the Program to Assemble
Spliced Alignments (PASA) . PASA first summons BLAT
to align the CDSs to the genome and then validates each
alignment by requiring a minimum 95% sequence identity over at
least 90% of the gene length. Alignments failing BLAT validation
are then realigned using sim4 and revalidated using the same
criteria. All ATCC 23344 CDSs that could not be aligned were
thus assumed to be absent from SAVP1. Similar analyses were
applied to reverse strain comparisons.
The experimental infection of equids was performed under the
auspices and regulations of the United States Department of
Agriculture on Plum Island.
Found at: doi:10.1371/journal.pone.0002058.s001 (0.08 MB
Table of Genes present in ATCC23344 but absent
We express our gratitude to Wolfgang F. Fricke for assistance with design
of the figures.
Conceived and designed the experiments: CF SS BL LS JD. Performed the
experiments: LS WN CR. Analyzed the data: CF SS JR DD BL LS JD
WN CR. Contributed reagents/materials/analysis tools: DD LS WN.
Wrote the paper: CF SS DD BL LS JD WN CR. Other: Figure
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