Genomic Analysis of a Mycobacterium Bovis Bacillus Calmette-Guérin Strain Isolated from an Adult Patient with Pulmonary Tuberculosis
Genomic Analysis of a Mycobacterium Bovisbacillus Calmette-Gurin Strain Isolated from an Adult Patient with Pulmonary Tuberculosis
Xuming Li 0 1
Liping Chen 0 1
Yongqiang Zhu 0 1
Xia Yu 0 1
Jun Cao 0 1
Rui Wang 0 1
Xinyan Lv 0 1
Jin He 0 1
Aizhen Guo 0 1
Hairong Huang 0 1
Huajun Zheng 0 1
Siguo Liu 0 1
0 1 Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agriculture , Harbin 150001, China , 2 Stake Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University , Wuhan, Hubei 430070, China , 3 Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai , Shanghai 201203, China , 4 National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory on Drug-resistant Tuberculosis Research, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing Chest Hospital, Capital Medical University , Beijing101149, China , 5 Laboratory of Medical Foods, Shanghai Institute of Planned Parenthood Research , 2140 Xie-Tu Road, Shanghai 200032 , China
1 Academic Editor: Anil Kumar Tyagi, University of Delhi , INDIA
For years, bacillus Calmette-Gurin (BCG) has served as the unique vaccine against tuberculosis and has generally been regarded as safe. However, a clinical strain labeled 3281 that was isolated from a TB patient was identified to be BCG. Via the combination of nextgeneration sequencing (NGS) and comparative genomic analysis, unique 3281 genetic characteristics were revealed. A region containing the dnaA and dnaN genes that is closely related to the initial chromosome replication was found to repeat three times on the BCG Pasteur-specific tandem duplication region DU1. Due to the minimum number of epitopes in BCG strains, 3281 was inferred to have a high possibility for immune evasion. Additionally, variations in the virulence genes and predictions for potential virulence factors were analyzed. Overall, we report a pathogen that has never previously been thought to be pathogenic and initial insights that are focused on the genetic characteristics of virulent BCG.
Funding: This work was supported by the Major
State Basic Research Development Program of
China (973 program, No. 2012CB518800), the
National Natural Science Foundation of China (Nos.
During the 20 years since the WHO declared that tuberculosis (TB) is a global public health
emergency, great efforts have been made to control and eradicate this diseaseworldwide.
Globally, the TB mortality rate has fallen by 45% since 1990. Although considerable progress has
been made in these years,an estimated 8.6 million individuals stilldevelop TB, and 1.3 million
31201920, 31272538), Bank of clinical resourceson
Tuberculosis (D09050704640000), and the
Transmission Mode of Tuberculosis project of the
National Key Program of Mega Infectious Diseases
Competing Interests: The authors have declared
that no competing interests exist.
die from the disease every year. As one of the three killers of humans, TB remains a
current major global health problem.Furthermore, one-third of the world population is latently
infected with Mycobacterium tuberculosis (MTB), which makes the eradication of this this
disease more difficult.
With the development of genomics and high-throughput sequencing technology, scientists
have sought to disclose the secret garden of TB via the use of genomic methods[3,4]. H37Rv
is a laboratoryvirulent MTB strain whose genome was the first to be completely sequenced,
and it has typically been used as areference strain in comparative genomic research. The sole
available TB vaccine, bacillus Calmette-Gurin (BCG), was derived from Mycobacterium bovis
(M.bovis); the virulence of this mycobacterium was attenuated in the laboratory via cultivation
on potatoglycerol medium, and this vaccine can only supply sufficient protection for children.
However, this vaccine is incapable ofproviding the same efficacy for adolescents and adults
[1,5]. Furthermore, the continual process of the subculturingof BCG in laboratories around the
world has led to the generation of daughter strains, and the protective efficacies against these
strains has been shown to varyacross laboratories and epidemiological investigations.To
define the molecular basis of the attenuation of BCGs and the variation among daughter
strains, comparative genomics research has been performed. Comparisons of BCG to M.
bovisrevealed that several genes associated with virulence were lost.Further studies identified
twotandem duplications, DU1 and DU2,which were shown to vary across all of the BCG
vaccine strains. In addition to these major mutations, it has been demonstrated that single
nucleotide polymorphisms (SNPs) might also play significant roles in the attenuation and
variation of BCGs[13,14].
In our study, a strain labeled 3281, which was derived from an adult TB patient who
reported having never been inoculated with a TB vaccine and was determined to be free of HIV
infection, was screened and identified to be BCG. Our interest was aroused by the question
how BCG turned into a pathogen despitebeing regarded as safe for years. Thepresent research
compared a virulent BCG isolate withBCG vaccines.
The strain 3281 was isolated from a 33 year old male, who lived in Hebei province, which is a
none-animal-husbandry regionlocated in northern China. The patient worked in a commercial
company which was not involved with livestock. The patient had never previously been
diagnosed with tuberculosis and there was no known tuberculosis case among his family members
or friends. The patient reported a cough and expectorate for less than 3 weeks before he
consulted a doctor. The chest X-ray and CT demonstrated sign of pneumonia. Three consecutive
sputa were all Acid-Fast Bacilli (AFB) positive while the M. bovis BCG strain was cultured from
all of the sputa. Given these reason, we suggested that the M. bovis BCG strain might be the
pathogen of this pneumonia patient.
This isolate 3281 belonged to a predominant spoligotype (SB0120) which was frequently
reported both among human bovine TB and among cattle. This spoligotype is similar to the
spoligotype of the vaccine strain BCG type, and four strains out of the 14 M. bovis strains
isolated from cattle in China during 2007 and 2008 had the same spoligotype.
MIC(minimal inhibitory concentration)testing
Mycobacterium tuberculosis susceptibility to 12 first- and second-line drugs were performed
using Trek Sensitre MYCOTB MIC plate (MYCOTB; Trek Diagnostic Systemes, Cleveland,
OH), with incubation at 37C for 30 days. The MIC was recorded as the lowest antibiotic
concentration that reduced visible growth (Table 1). The result showed that BCG 3281 showed
a higher resistance to Ethionamide (5g/ml) than BCG Pasteur (2.5g/ml), M.bovis(1.2g/ml)
and H37Rv (0.6g/ml). Meanwhile, BCG 3281 showed similar resistance to Isoniazid as M.
bovis (0.12g/ml), twice that of BCG Pasteur and H37Rv (0.06g/ml). In addition, the
resistance to Para-aminosalicylic acid, Kanamycin, Ofloxacin and Moxifloxacin of BCG 3281 was
different with BCG Pasteur, indicating that BCG 3281 was not a traditional BCG strain.
General genomic features
The size of the BCG 3281genome was 4,410,431 bp (Fig 1), and the sequencing error was less
than 1/Mb. Thus far, BCG 3281 has the largest genome size in terms of the genomes of BCG
that have been completed. The genome of 3281 is 135,909 bps larger than that of BCG Pasteur
(Table 2). A total of 4,186 CDSs were identified by glimmer-prediction and reference
genealignment. Among these CDSs, 3,079 might be COG categories with e-values 1e-5. No
credible prophage was found, despite the finding that prophage genes produced four hits in the
BCG 3281 genomevia phage-finder. Due to the polymorphic G+C-rich sequences
(PGRSs), most of which consist of enzymes involved in lipogenesis and lipolysisandthe
ProGlu(PE) motif-Pro-Pro-Glu(PPE) motifgene family, BCG 3281s GC-content was as much as
65.6%, which is similar to the GC contents of MTB and M. bovis. Forty-fivetRNA operons
were predicted by tRNAscan-SE,and one rRNAoperon was located by RNAmmer[19,20].
Genomic comparison with M.bovis and the four BCG strains revealed that the regions of
difference (RDs)that contain virulence genes that were lost in the BCGswere also absent in
3281. Compared to the other BCGs and M. bovis, 35 BCG 3281-specific single nucleotide
polymorphisms (SNPs) were identified (Fig 1), and 23of these SNPs produced nonsynonymous
variations. Additionally, nineindels (threeinsertions and sixdeletions) were found to be
exclusive to BCG 3281, and fourother deletions were shared only with M. bovis only. A total of 20
genes were affected by the 23 nonsynonymous variations (S1 Table), and 50 genes were
affected by the 13 indels (Table 3 and S2 Table).
Unique genomic features of the BCG strains
Thirteen years of laboratory cultivation have caused great differences in virulence between the
progeny and the original strainand resulted in the attenuated virulence and sufficient reserved
Fig 1. Circular representation of the M. bovis BCG 3281 chromosome. The outer black circle shows the coordinate. Moving inward, the next two circles
show forward and reverse strand CDS, respectively, with colors representing the functional classification, the next circle shows RD(red) and DU (orange),
followed by the 3281 unique SNP with nonsynonymous blue and synonymous red, then is the tRNA (blue) and rRNA (purple), final two are GC-content and
GC-skew by using a 10-kb window.
antigenicity for protection against TB. Comparative genomic analyses have revealed massive
discrepancies between BCG and M. bovis. The most significant two events were the loss of the
RD1 regions that contain a specialized secretion system that is strongly associated with
pathogenic ability[11,21]and the two tandem duplications, DU1 and DU2. DU1 is restricted to BCG
Pasteur 1173P2, and DU2 is present in four different types in different BCGs[10,22].
In the genome sequence of BCG 3281, a loss of RD1and duplications in the DU1 and DU2
regions were observed, which validates this stain as BCG. In the DU1 region, a 7 kb unit that
covered six genes and crossed the oriC was found to be repeated three times (Fig 2); this
duplication is specific to BCG 3281 and has never been reported before (Table 4 and 5). The DU1 in
BCG Pasteur is 29.7 Kb, encompassing the region from Rv3910 to Rv0013, while the DU1 in
BCG 3281 is only 7.2 Kb, including the region from Rv3921c to Rv0003. BCG 3281 has three
copies of dnaA-dnaN region with functional oriC.Protein DnaA initiates chromosome
replication when accumulated to the initiation level, and multiple copies of dnaA in BCG 3281
The GS11 is the official locus of BCG 3281 given by Genbank.
Fig 2. Scheme showing the DU1 region of BCG 3281 and BCG Pasteur 1173p2. (A). The color schemes means duplicated regions. (B). Details of genes
involved in the BCG 3281 duplicated units (using H37Rv coordinate).
might help the strain increase growth rate and activate some gene expression .Thusthe
triploidfor DNA replication elements might partly contribute to the pathogenic of BCG 3281.
The DU2 zone of BCG 3281 belongs to the DU2- type, which consists of two repeat units
(41 kb and 37.5 kb) that correspondto regions 3,567,4593,608,472 and 3,671,5363,709,097 of
M. tuberculosisH37Rv that are separate and repeattwice (Table 6).
The loss of RD1 and the two identified tandem duplications in BCG 3281 confirmed that
the strain is a BCG. This result is completely contrasted with our expectation that BCG 3281
would be an M. bovis. Furthermore, the RD17 and RDpan, which are specific to BCGsand lost
in M.bovisAF2122, were also found in BCG 3281.
To ensure the accuracy of the strain identification, a SNP-based NJ phylogenetic tree was
constructed (Fig 3). The phylogenetic position of BCG 3281was located near BCG Tokyo and
far from the clinic strains, which validated 3281 as a BCG. For years, people have acknowledged
NA means not present.
dnaA, chromosomal replication initiation protein
dnaN, DNA polymerase III subunit beta
recF, recombination protein F
oxaA, inner membrane protein translocase component YidC
rnpA, ribonuclease P protein component
rpmH, 50S ribosomal protein L34
that BCG strains are safe for vaccination and have notransmissibility. Nevertheless, thestrain
3281, which was isolated from an adult patient who had not been vaccinated with a BCG, was
identified to be a BCG. We believe that the source of pathogen in this case was from the vaccine
and had mutated to acquire the ability for horizontal transmission.
Epitopes are the parts of antigens that are recognized by T-cell receptors (TCRs) and B-cell
receptor (BCRs) and play the core role in the immune response. We believed similarities between
the epitopes of BCG 3281 and M. Bovis or MTB would exist because all of these strains are
To identify the variations in the epitopes of these strains, 2,667 epitopes complied from the
Immune Epitope Database(IEDB), including 2,055 T-cell epitopes and 612 B-cell epitopes,
were selected and renamed (S3 Table).These epitopes were subsequently positively
experimentally identified by IEDB. Four complete genome BCG vaccines (i.e., BCG Pasteur 1173P2, BCG
Tokyo 172, BCG Mexico and BCG Korea 1168P) were acquired from the National Center of
Biotechnology Information (NCBI).
Only 100% identical match results were considered as the same epitopes because recent
studies have shown that human T cell epitopes of Mycobacteriumtuberculosis are evolutionarily
hyper-conserved. For comparison, 1,600 epitopes, including 1,213 T-cell epitopes and 387
B-cell epitopes, were identified in all seven strains (BCG 3281, BCG Pasteur 1173P2, BCG
Tokyo 172, BCG Mexico, BCG Korea1168P, M.bovisAF2122 and M. tuberculosis H37Rv). In
contrast, 531 epitopes, including 404 T-cell epitopes and 127 B-cell epitopes, were absent in all
seven strains. Moreover, 329 epitopes, including 290 T-cell epitopes and 39 B-cell epitopes,
were found to be lost in only BCG 3281 and other BCGs. Additionally, 44 epitopes, including
33 T-cell epitopes and 11 B-cell epitopes, located in 22 geneswere found to be missing in only
Fig 3. Phylogenetic tree of M. tuberculosis, M. Bovis and BCGS. The tree was constructed employing Neighbor-joining method. It is based on the SNPs
within 2263 core genes of the strains.
BCG 3281. When these 22 genes were examined, frameshiftswere found to have occurred in
the coding regions of 19genes and 3 genes were lost (S4 Table).
Despite sharing majorities of both T-cell and B-cell epitopes with H37Rv and M. bovis, the
BCGs obviously possess fewer epitopes (Fig 4), whichmight result in reduced protection
againstTB. In other words, fewer epitopes indicate poorer recognition of alien invadersby the
human body. Moreover, BCG 3281 had the fewest number of epitopes among the BCGs, which
amplifies the possibility for immune escape. Wen et al. found that BCG Tokyo possess the
greatest number of both T-cell and B-cell epitopes among the BCGs and thus might be the
vaccine that confers the best immune protection. We found that 62 unique epitopes of BCG
Tokyothat are locatedin two BCG Tokyo genes,JTY1991 and JTY1996, that were also present in
M.bovis and H37Rvbut absent in other BCGs. The efficiency of BCG protection might be
improved by the transduction of two genes into other BCG vaccines. No epitopes unique to 3281
among the other BCGs were identified. In one aspect, this might hint that BCG 3281 did not
obtain exogenous genetic element through lateral gene transfer, emphasizing the possibility
that pathogenic BCG 3281 might be formed through mutation from BCG vaccine. On the
other hand, epitopes that had not been experimentally identified might existed in BCG 3281
Virulence factors in BCG 3281
Variation in known virulence factors. BecauseBCG 3281 was considered to be a
pathogenic bacterium, we expected that BCG 3281would share extensive similarities with MTB and
M. bovisand possess distinct genetic differences from other BCGs, particularly with respect to
Fig 4. Epitopes in M. tuberculosis H37Rv, M. bovis AF2122 and genome finished BCGs. Duplicate epitopes were removed and only epitopes with 100%
identical matches were considered present in the strain.
virulence genes. To detect the variationsin the virulence factors, 88 virulence genes that were
identified from the Virulence Factors Database (VFDB) were selected. Blastpresults
(Table 7) revealed that 51 virulence genes were 100% identical with M. bovis and the five CG
strains,threegenes (located at RD5) were lost in both M. bovis and all of the BCGs, and
sevengenes were M. bovis-specific; the latter genes were located at RD1 and were lost in all of the
BCGs. A copy number variation (CNV) of one gene (VFG1412) was found and was located in
the DU2 region. Additionally, a frameshiftin one virulence gene (VFG2388) was found in both
M. bovis and the BCGs. Moreover, plentiful nonsynonymousmutations were identified. To our
surprise, no virulence genes were found to be specific to BCG 3281 with respect to M. bovis and
the other BCGs. Although the differences between M. bovisand BCG 3281were enormous,
these differences were found to be common characteristics of BCGs.
Possible virulence genes. Because no large variations in confirmed virulence genes were
detected within BCG 3281, a pan-genome analysis was performed to identify possiblenew
virulence factors. Via the use of the pan-genome analysis pipeline (PGAP),orthologous clusters
within the 5 BCGs were grouped (Fig 5). The pan-genome clusters consisted of 4,282
orthologs and had a core of 3,363 orthologs.Two hundred and ninety orthologclusters
+ means 100% in identit, - stands for lost, the number shows nonsynonymous mutations number or copy number.
contained 294 CDSs that were likely to be unique to 3281 and might have conferred additional
virulence to BCG 3281.
Considering the prediction discrepancyand the restrictions of the software, we searched
these 294 CDSs within the genome and re-predicted the CDS libraries of the other fourBCGs.
Ultimately, fourCDSs were proven to be 3281-specific, and all of these CDSs were generated by
indels (Table 8).
Fig 5. The Venn diagram showing core orthologs of five genome finished BCGs. Genes overlapping at least 50% length and 50% (by PGAP) of
similarity were considered orthologs.
Although BCG, which is an attenuated derivativeof M. bovis, has served for nearly 90 years as
the sole vaccine that provides protection against tuberculosis, the clinical strain 3281 was
proven to be a BCG and was found to be morbigenous. In an effort to determine the genetic
structure of BCG 3281 and determine whether a BCG could be pathogenic, we sequenced the
complete genome of BCG 3281 and compared its entire genome to four complete BCG
The GS11 is the official locus of BCG3281 given by Genbank.
genomesand the genome of M. bovis AF2122. First, to demonstrate the accuracy of the
physiological and biochemical identification results, we examined the tandem duplicationsDU1 and
DU2, which are significant characteristics of BCGs. Simultaneously, a genetic evolution
analysis of the complete BCG genomes and the genome of M. bovis was constructed. The results of
both analyses verified that strain 3281 is a BCG.
Examinations of all of the BCG genomes, includingthose of BCG Pasteur, Tokyo, Mexico,
Korea,Frappier, Glaxo, Moreau, Phipps, Prague, Sweden, China, ATCC35733, ATCC35740
and ATCC35743,revealed that none contained the 7 kb duplication in the DU1 region.The
presence of the dnaA and dnaNgenes is strongly associated with the initiation and regulation
of chromosomal replication; thus, we inferred that BCG 3281would likely be capable of
enduring greater burdens in replication.
To determine whether any identified virulence factors were unique to 3281 relative to the
other BCGs, 88 virulence genes located at H37Rv were examined; 3281-unique indels anda
single amino acid polymorphismwere located, but 3281-unique virulence factors were not found.
We believe that these variations might influence the virulence of BCG 3281 to some extent but
not so much as to convert an attenuated vaccine into a pathogenic bacterium. To identify the
possible virulence factors, a pan-genome method was applied and four BCG 3281-unique
CDSs were identified as puativevirulence genes since no other large variations in genome
structure were found.Additionally, we detected antigen epitope variationsin BCG 3281. Compared
to the other BCGs, BCG 3281 has lost more epitopes, which might intensify this strains
potential for immune escape and increase the risk of secondary infection.Overall, this study provides
initial insight into the characteristics of a pathogenic BCGthat should have significant effects
on TB vaccine research.
Materials and Methods
The mycobacterial strain used in this study was acquired from the Beijing Bio-Bankof clinical
resourceson Tuberculosis (D09050704640000)". This strain was originally isolated from an
adult male patientwho was not infected with HIV.
Genome sequencing, assembly and annotation
Through a combination of next-generation sequencing (NGS) techniques, thegenome was
sequenced with both a 454 GS-FLX system and a Hiseq2500. The 454 data were assembled with
Newbler 2.5 withcoverage of 29.6.Using Soap 1.05, the Hiseq reads were assembled with a
108.9-fold coverage. Gap closure was performed using the PCR method with the help of
ContigScapeusing the 454 assembly results. The low value dots were verified by the Hiseq
assembly results. ORFs were predicted with Glimmer 3.0.2 and replenished by reference
SNP and Phylogenetic analyse
All SNPs were identified with Mauve 2.3.1, and they were localized to CDSsvia an in-house
Perl script.The pangenomemethod was employed for the phylogenetic analysis. A core of
2,263 geneslengths of at least 0.8 and similarities of at least 0.8 was generated. The
neighborjoining tree was generated by MEGA with a bootstrap value of 1,000.
S1 Table. Details of M. bovisBCG 3281 specific SNPs.
S2 Table. Details of indels between M. bovis and genome finished BCGs.
S3 Table. . Detailed information of epitopes used in this paper.
S4 Table. . Details of the lost epitopes by BCG 3281 alone.
This work was supported by the Major State Basic Research Development Program of China
(973 program, No. 2012CB518800), the National Natural Science Foundation of China (Nos.
31201920, 31272538), Bank of clinical resources on Tuberculosis (D09050704640000), and the
Transmission Mode of Tuberculosis project of the National Key Program of Mega Infectious
Conceived and designed the experiments: AG HH HZ SL. Performed the experiments: X. Li LC
YZ. Analyzed the data: X. Li YZ HZ. Contributed reagents/materials/analysis tools: X. Li LC
YZ XY JC RW X. Lv JH AG HH HZ SL. Wrote the paper: X. Li HZ SL.
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