Characterization of a Bacillus anthracis isolate causing a rare case of fatal anthrax in a 2-year-old boy from Hong Kong.
ABSTRACT We used multiple-locus variable-number tandem repeat analysis (MLVA) and pagA sequencing to genotype a Bacillus anthracis isolate from a fatal case of human anthrax in Hong Kong. The isolate has a unique MLVA genotype, is related to the Sterne and Ames strains, and is consistent with genotypes identified in China.
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ABSTRACT: Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (pag) was sequenced from 26 of the most diverse B. anthracis strains to identify potential variation in the toxin and to further our understanding of B. anthracis evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of pag from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the pag corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in B. anthracis has occurred with little or no horizontal transfer between the different strains.Journal of Bacteriology 05/1999; 181(8):2358-62. · 3.83 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2005, p. 1992–1994
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 4
Characterization of a Bacillus anthracis Isolate Causing a Rare Case of
Fatal Anthrax in a 2-Year-Old Boy from Hong Kong
Danny T. L. Cheung,1Kai Man Kam,1Kong Lung Hau,1Tak Kong Au,1Chung K. Marston,2
Jay E. Gee,2Tanja Popovic,2Matthew N. Van Ert,3Leo Kenefic,3Paul Keim,3,4
and Alex R. Hoffmaster2*
Department of Health, Hong Kong Special Administrative Region, Hong Kong, China1; Centers for Disease Control
and Prevention, Atlanta, Georgia2; Department of Biological Sciences, Northern Arizona University,
Flagstaff, Arizona 86011-56403; and Pathogen Genomics Division, The Translational Genomics
Research Institute (TGen), 445 N. Fifth Street, , Phoenix, Arizona 850044
Received 27 September 2004/Returned for modification 8 November 2004/Accepted 19 December 2004
We used multiple-locus variable-number tandem repeat analysis (MLVA) and pagA sequencing to genotype
a Bacillus anthracis isolate from a fatal case of human anthrax in Hong Kong. The isolate has a unique MLVA
genotype, is related to the Sterne and Ames strains, and is consistent with genotypes identified in China.
Human anthrax, which can include cutaneous, gastrointesti-
nal, and inhalation forms, is caused by Bacillus anthracis. The
type of anthrax is directly related to the route of infection.
Inhalation anthrax, which was historically associated with tex-
tile mill workers and most recently with bioterrorism, occurs
upon inhaling spores. Cutaneous anthrax is generally the result
of infection via a skin abrasion while handling contaminated
animal products, while gastrointestinal anthrax occurs upon
eating contaminated meat. Human anthrax is very rare world-
wide, and only three cases have been reported in Hong Kong
in the last 20 years.
On 27 May 2003, a 2-year-old boy, who was in apparent good
health prior to this illness, became ill with fever and anorexia
as main symptoms. His condition rapidly deteriorated 3 days
later with abdominal pain, irritability, vomiting, and shortness
of breath. He was pronounced dead shortly after admission to
a hospital. B. anthracis was isolated from both cerebrospinal
fluid and blood. Postmortem examination revealed the pres-
ence of gram-positive rods in mesenteric lymph nodes and
brain tissues. Although pathological findings suggested an oral
route of transmission, an extensive epidemiological investiga-
tion and culturing of food and environmental samples from the
victim’s home, nursery, playgrounds, restaurants, supermar-
kets, and meat suppliers that might have been linked to the
victim’s activities were all negative for B. anthracis.
We used multiple-locus variable-number tandem repeat
(VNTR) analysis (MLVA) and pagA sequencing (protective
antigen [PA] genotype) to molecularly characterize the isolate
from this case in an attempt to gain insight into its potential
origin. MLVA determines the copy number of VNTRs at eight
genetic loci (six chromosomal and one on each of the two
virulence plasmids, pXO1 and pXO2). MLVA has been used
to differentiate 426 B. anthracis isolates into 89 distinct geno-
types and has been used to study the ecology of anthrax and to
subtype isolates during the 2001 bioterrorism-associated an-
thrax outbreak in the United States (1, 2, 4). We used MLVA
to analyze eight VNTRs within the B. anthracis genome as
previously described (1, 2). The allele sizes (in nucleotides,
corrected to the sizes reported by Keim et al. (2), for the eight
loci were identified as vrrA ? 313, vrrB1? 229, vrrB2? 162,
vrrC1? 583, vrrC2? 532, CG3 ? 158, pXO1 ? 129, and pXO2
? 142 and resulted in a unique MLVA genotype within the
A3.b cluster as originally described by Keim et al. (2) (Fig. 1).
This cluster includes B. anthracis isolates from China in addi-
tion to well-known strains such as Ames and Sterne.
It should be noted that the pXO1 allele reported here is 3 bp
larger than the comparable allele from Keim et al. (132 versus
129 bp), based upon DNA sequence comparison. Since minor
“real size” fragment length discrepancies, based upon DNA
sequence, may exist in the sizes initially reported by Keim et al.
(2; P. Keim, personal communication), we confirmed the ac-
curacy of the genotype assignment by MLVA typing the Hong
Kong isolate in parallel with the B. anthracis isolates repre-
senting the 89 genotypes presented previously (2). In addition,
this is the first description of a 142-bp pXO2 allele (between
the 141-bp and 143-bp alleles) due to an insertion of an addi-
tional C in a poly(C) tract.
PA is one of the three anthrax toxin proteins and is key to
developing immunity to anthrax. Sequencing of the gene that
encodes PA (pagA) has been used to subtype 26 diverse B.
anthracis isolates into six PA genotypes and was also used
during the 2001 bioterrorism-associated anthrax outbreak (1,
3). Sequencing of the pagA gene (2,294 bp) was done as de-
scribed by Hoffmaster et al. (1) and identified this isolate to be
* Corresponding author. Mailing address: Centers for Disease Con-
trol and Prevention, Atlanta, GA 30333. Phone: (404) 639-0852. Fax:
(404) 639-3024. E-mail: firstname.lastname@example.org.
FIG. 1. MLVA-based dendrogram generated using unweighted-
pair group method arithmetic average cluster analysis of raw genotype
scores of 88 diverse B. anthracis strains (2) and the Hong Kong isolate.
Genotype numbers are listed as reported by Keim et al. (2). Note that
genotype 25 is not present since reanalysis of the isolate indicated that
it was missing the pX02 virulence plasmid. We have indicated the
locations of the Sterne strain, the Ames strain, and all Chinese strains.
Included next to the Chinese strains are the provinces where the
strains were isolated.
of PA genotype I. This PA genotype is the same as that of the
Sterne and Ames strains of B. anthracis (3).
MLVA and pagA sequencing of the isolate causing this rare
case of anthrax revealed that the isolate is closely related to
strains such as Sterne and Ames and is consistent with geno-
types previously identified in China. In fact, the isolate differs
from genotype 59, a Chinese strain isolated from soil in the
Guangxi province, by a single nucleotide addition in the pX02
locus (Fig. 2) (2). This is particularly interesting considering
the geographic proximity of these two regions, the inland wa-
terways that link them, and the extensive trade between Hong
Kong and Guangxi. While the genotype of this isolate appears
consistent with this region, the proximal source of the B. an-
thracis isolate that caused this case remains unknown. In the
future, if more extensive databases of subtyped B. anthracis
strains from this region become available, it may be possible to
determine the origin of this strain with greater confidence.
1. Hoffmaster, A. R., C. C. Fitzgerald, E. Ribot, L. W. Mayer, and T. Popovic.
2002. Molecular subtyping of B. anthracis and the 2001 bioterrorism-associ-
ated anthrax outbreak, United States. Emerg. Infect. Dis. 8:1111–1116.
2. Keim, P., L. B. Price, A. M. Klevytska, K. L. Smith, J. M. Schupp, R. Okinaka,
P. J. Jackson, and M. E. Hugh-Jones. 2000. Multiple-locus variable-number
tandem repeat analysis reveals genetic relationships within Bacillus anthracis.
J. Bacteriol. 182:2928–2936.
3. Price, L. B., M. Hugh-Jones, P. J. Jackson, and P. Keim. 1999. Genetic
diversity in the protective antigen gene of Bacillus anthracis. J. Bacteriol.
4. Smith, K. L., V. DeVos, H. Bryden, L. B. Price, M. E. Hugh-Jones, and P.
Keim. 2000. Bacillus anthracis diversity in Kruger National Park. J. Clin.
FIG. 2. Illustration of the mutational steps that separate the Hong
Kong isolate and the closely related A3b strains.
1994 NOTESJ. CLIN. MICROBIOL.