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Journal of Clinical Microbiology, June 2007, p. 1777-1782, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02488-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic Diversity in a Bacillus anthracis Historical Collection (1954 to 1988)

David Sue ,^, Chung K. Marston, Alex R. Hoffmaster, and Patricia P. Wilkins^*

Bacterial Zoonoses Branch, Division of Foodborne, Bacterial and Mycotic Diseases, National Center for Zoonotic, Vector-Borne and Enteric Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd., NE, Atlanta, Georgia 30333

Received 12 December 2006/ Returned for modification 17 January 2007/ Accepted 13 March 2007

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Bacillus anthracis, the etiologic agent of anthrax, has been widely described as a genetically monomorphic species. We used both multiple-locus variable-number tandem-repeat analysis (MLVA) and pagA gene sequencing to determine the genetic diversity of a historical collection of B. anthracis isolates collected from the 1950s to the 1980s from various geographic locations and sources. We sequenced the pagA gene of 124 diverse B. anthracis isolates and found all previously identified B. anthracis pagA types except type 4. Sixty-three of the 124 B. anthracis strains were identified as pagA type 6, while 44 were pagA type 5, 12 were pagA type 1, and individual isolates were identified for types 2 and 3, respectively. Two new pagA genotypes were discovered in three environmental isolates within the historical collection. Two isolates had the same new genotype, and an additional isolate produced a second new genotype. MLVA detected 22 previously described genotypes in the historical collection. In addition, 33 new MLVA genotypes were found. For 11 isolates, an MLVA genotype could not be assigned because one or more alleles did not amplify. While only two additional B. anthracis pagA types were identified, in two instances, the use of pagA sequencing discriminated isolates with the same MLVA genotype. MLVA revealed that 39 of the 124 isolates were previously undocumented genotypes and that 1 isolate was found to be in the C cluster when it was subtyped by MLVA.

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Bacillus anthracis is a gram-positive, spore-forming bacterium that belongs to the Bacillus cereus group and is the etiologic agent of anthrax. Previous studies have described B. anthracis strains as genetically monomorphic, as determined by restriction fragment analysis, PCR, and direct DNA sequence comparisons (7, 8, 13, 27). Andersen et al. first described that variable-number tandem-repeat (VNTR) sequences in the vrrA locus could be used to differentiate strains of B. anthracis (1). Keim et al. then reported on amplified fragment length polymorphism markers (13) and, later, on additional VNTR locus sequences that could be used to differentiate B. anthracis strains (14). In 2000, Keim et al. described a multiple-locus VNTR analysis (MLVA) method in which 426 B. anthracis isolates were classified into 89 distinct genotypes on the basis of eight characteristic VNTR regions (15). MLVA analysis has since been used in studies to describe B. anthracis strain diversity in isolates from South Africa (Kruger National Park), France, Poland, Italy, Korea, Georgia, and Chad (4-6, 19, 21, 26, 28). From these studies, 17 novel B. anthracis MLVA genotypes have been described since the publication of the original description of the use of MLVA for the subtyping of B. anthracis (15).

In 1999, Price et al. described limited nucleotide diversity within the pagA gene sequence, finding eight distinct pagA genotypes encoding only three phenotypes among 26 B. anthracis strains selected to represent the chromosomal diversity previously observed by amplified fragment length polymorphism analysis (25). pagA is a virulence gene located on the pXO1 plasmid of B. anthracis and encodes protective antigen (PA), an essential component of anthrax toxin (22, 23, 29, 31). During the 2001 anthrax event in the United States, MLVA and pagA sequencing were used to subtype isolates associated with the bioterrorism-related event. A total of 42 isolates were each identified as pagA sequence type 1 and MLVA genotype 62, which matched the sequence type and genotype of the Ames strain exactly (9). Two recent studies provide evidence that additional pagA sequence types exist. Hoffmaster et al. described a novel pagA sequence type in B. cereus strain G9241 that is similar to sequence type 5 but contains two additional point mutations that make it unique (11). In a study of environmental samples, Kuske et al. (17) identified a novel pagA gene sequence from a soil sample containing point mutations that were not described by Price et al. (25). These findings suggest that the sequence variation in pagA is not completely defined by the eight genotypes described in the study of Price et al. (25) and that additional sequence variation exists in this virulence gene. For this investigation, we sought to determine the level of genetic diversity within a historical collection of B. anthracis strains at the Centers for Disease Control and Prevention using MLVA genotyping and pagA sequencing.

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Bacterial isolates. For this investigation, a convenience sample of 124 B. anthracis isolates, a subset of strains recovered in a study by Marston et al. (20), were subtyped. The strains were collected between 1954 and 1989 from environmental samples and animal and human clinical specimens. All strains in this study were previously confirmed to be pXO1 and pXO2 positive by PCR (20). These isolates originate from 14 states in the United States, as well as Argentina, Haiti, England, Paraguay, Pakistan, and South Africa.

DNA isolation. DNA from each isolate was prepared by a heat lysis method, as described by Hoffmaster et al. (9). Briefly, each isolate was streaked onto Trypticase soy agar containing 5% sheep blood (Becton Dickinson Microbiology Systems, Cockeysville, MD) and incubated at 37°C. Following overnight growth, a single isolated colony was suspended into 0.22-µm-pore-size centrifugal filter units (Millipore, Bedford, MA) containing 200 µl of 10 mM Tris-HCl (pH 8.0). Following heat treatment at 95°C for 20 min, each suspension was centrifuged at room temperature through the filter unit at 6,000 x g for 2 min. All DNA lysates were stored at –20°C.

MLVA subtyping. MLVA typing was performed as described by Keim et al. (15). Briefly, six chromosomal loci (vrrA, vrrB1, vrrB2, vrrC1, vrrC2, and CG3) and two plasmid loci (pXO1-aat and pXO2-at) were amplified and labeled with one of three dyes. The products were separated by use of a capillary on an ABI 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA). The allele sizes were determined by using ABI Gene Mapper (version 3.7) software (Applied Biosystems). MLVA analysis of 24 reference strains from Keim et al. (15) was used to calibrate the allele "size calling" performed by the software.

pagA sequence typing. The amplification and sequencing of pagA were performed with the 124 B. anthracis isolates as described previously (9, 25). Briefly, pagA sequencing templates were amplified by using the Expand High Fidelity PCR system (Roche, Mannheim, Germany). Fifty-microliter PCR mixtures contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 4.0 mM MgCl₂, 0.4 mM each forward and reverse primer, 100 µM each deoxynucleotide, 2.0 U of Taq DNA polymerase (Roche), and 2 µl of bacterial lysate. The reaction mixtures were heated at 94°C for 5 min and then cycled 35 times at 94°C for 30 s, 51°C for 30 s, and 72°C for 1.5 min, with a final extension of 72°C for 5 min. The PCR amplicons were purified by using a QIAquick PCR purification kit (QIAGEN, Inc., Valencia, CA), and the resulting purified amplicons were used in the subsequent sequencing reactions.

Sequencing was performed on an Applied Biosystems 3130 genetic analyzer (Foster City, CA) by using the BigDye Terminator (version 3.1) cycle sequencing ready reaction mix and the manufacturer's instructions (Applied Biosystems).

Data analysis. pagA sequence data were analyzed by using the Lasergene 99 (DNASTAR, Madison, WI) software package, which includes the SeqMan and MegAlign programs, which were used for sequence alignments and comparisons, respectively. Nei's index of diversity (D_N) for the individual MLVA markers was calculated as 1 – (allele frequency)² (30). Simpson's index of diversity (D_S) was calculated for the MLVA genotyping and the pagA sequence typing methods, as described by Hunter and Gaston (12).

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MLVA genotypes. Among the 124 B. anthracis isolates included in this study, we identified 55 MLVA genotypes (Tables 1 and 2; Fig. 1). This includes 22 previously described genotypes (4, 5, 13) and 33 previously undescribed genotypes (Table 1). For 11/124 isolates, no MLVA genotype was assigned because one or more loci failed to amplify. Genotype 4 was the most frequently observed genotype (19/124 isolates) in this study. Many of the genotypes observed in this study belonged to the A.4.a cluster (genotypes 71 to 73 and 75 to 78). In addition, we identified a rare isolate belonging to the newly described C cluster (24). This is the third isolate identified as belonging to this cluster and the first reported isolate in the C cluster to contain both virulence plasmids, pXO1 and pXO2.

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TABLE 1. Bacillus anthracis MLVA genotypes found in a historical collection of isolates collected from 1954 to 1988 from various sources and geographic locations

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TABLE 2. Relative allele sizes for eight loci from the new B. anthracis MLVA genotypes described in this study

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FIG. 1. Cluster analysis by the unweighted pair group method with arithmetic means built from the eight-locus MLVA genotypes of 124 strains from this study, as well as a subset of reference genotypes from Keim et al. (15) *, new genotype.

Each of the 33 previously undescribed genotypes represents a new combination of previously described alleles and/or has a new allele size among the eight loci (Table 2). Among the 124 strains, we identified new allele sizes for the loci vrrB1 (289 alleles), vrrC1 (357, 369, and 655 alleles), vrrC2 (637 alleles), and pXO2-at (130 alleles). The average D_N for the eight individual MLVA loci was calculated by use of the equation described above (30) by using the locus sizes reported by Keim et al. (15), as well as the locus sizes from the genotypes described in this study. The D_N for the individual MLVA loci was 0.52, which is same D_N value reported by Keim et al. (15).

To evaluate the resolution power of MLVA genotyping to discriminate among the 124 B. anthracis strains included in this study, we calculated D_S and found that it was 0.959.

pagA sequence types. Among the 124 B. anthracis isolates used in our study, we found all previously identified B. anthracis pagA sequence types except types 4, 7, and 8 (Table 3). Over half (63/124) of the strains tested were pagA sequence type 6, while nearly all of the remaining strains were pagA type 5 (44/124). For strains that were collected from environmental sources (n = 44), approximately 70% (31/44) of the strains were pagA type 6. To evaluate the resolution power of pagA sequence typing to discriminate among the 124 B. anthracis strains included in this study, we calculated D_S and found that it was equal to 0.531.

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TABLE 3. Description of two new pagA sequence types from environmental isolates (n = 3)

Identification of two new pagA genotypes which lead to novel predicted phenotypes. Our investigation revealed two previously undescribed pagA sequence types of B. anthracis. pagA sequence type 10 was found in two independently collected isolates. One strain was isolated from a surface sample collected in 1956 from an unknown location, and the other isolate was from a water sample collected in South Africa in 1974.

The nucleotide substitution found in pagA sequence type 10, at position 196 from the ATG start site, is also found in pagA sequence type 9 (10). This substitution occurs in the region of the gene that encodes the PA20 domain of the protein. The missense mutation site leads to Ser66Pro.

The other new pagA genotype described in this study, pagA sequence type 11, was found in a single B. anthracis strain isolated from a surface sample collected in a tannery of unknown location in 1957. This new pagA type contains missense mutations at previously undescribed nucleotide positions. At nucleotide position 35, a cytosine-to-adenine substitution leads to Ala12Glu. Also, a deletion at nucleotide position 813 leads to the Ile271Leu substitution and a frame shift in the open reading frame of pagA. The pagA type 11 genotype is predicted to yield a truncated protein, as there is a premature stop codon at amino acid 292.

MLVA analysis revealed that the two strains with pagA type 10 have different MLVA genotypes. The strain isolated from a surface sample collected in a tannery in 1974 belongs to MLVA genotype 40. By contrast, the additional strain identified as pagA type 10, which was isolated from a surface sample in 1956, belongs to a recently described MLVA C cluster (genotype 133). The single pagA type 11 strain isolated was MLVA genotype 107.

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The bioterrorism-related anthrax outbreak of 2001 highlighted the importance of having rapid and discriminatory diagnostic tools for both the identification and the characterization of biological threat agents. B. anthracis MLVA and pagA sequence typing were reliable genotypic methods for strain characterization that greatly aided the epidemiological investigation, in which numerous isolates had to be evaluated within a short time (9). During such investigations, it is important to have not only powerful, discriminatory tools but also a context that allows the proper interpretation of the results. Our investigation sought to reveal new MLVA genotypes and pagA sequence types in a historical B. anthracis strain collection in order to build a broader understanding of strain diversity. We observed 33 previously unreported MLVA genotypes, including an isolate belonging to the recently described C cluster (24). This is the first identification of a wild-type (pXO1- and pXO2-positive) isolate belonging to this MLVA cluster. By analysis using the unweighted pair group method with arithmetic means, these new MLVA genotypes appear to be evenly distributed among the genotypes previously described by Keim et al. (15). Our observation of 33 new MLVA genotypes among 124 B. anthracis isolates in our historical collection suggests that a population of strains more diverse than was previously recognized exists. Among the new MLVA genotypes reported in this study are new combinations of the locus marker sizes, as well as previously undescribed allele sizes (for four of the eight loci). After calculation of D_N to include these new MLVA genotype locus marker sizes, we observed no change in the average D_N of 0.52 that was originally reported by Keim et al. (15).

The high D_S of MLVA genotyping (D_S = 0.96) calculated in this study reflects the discriminatory power of this subtyping tool for differentiation among B. anthracis strains. This measure indicates the probability that MLVA genotyping alone will successfully distinguish two unrelated strains within a population. Even as MLVA genotyping for B. anthracis has been developed to include 15 and even 25 loci, this study suggests that MLVA typing of 8 loci should provide adequate strain discrimination for most genotyping applications (16, 18). With over 130 different described B. anthracis MLVA types based on eight loci, the MLVA genotyping method remains a robust discriminatory tool. In some instances isolates from certain regions are difficult to discriminate by using MLVA genotyping of eight loci (18). In some cases additional loci may be needed to discriminate closely related isolates, such as additional VNTRs or single-nucleotide repeats.

A concern that has arisen as more laboratories have begun using MLVA is the standardization of allele calling so that data can be shared effectively between laboratories. Discrepancies between the relative allele fragment sizes for MLVA markers, as determined with fragment analysis software, and the actual sizes determined by direct sequencing of PCR products and complete genome sequencing have been reported (3, 18, 19). These differences may occur if different instruments, software, fluorescent dyes, size standards, or other reagents are used. This is particularly problematic when the alleles of loci containing very short repeats (i.e., 2 or 3 bp) are determined and compared (18). It is important to determine the observed fragment size for each VNTR allele for the specific MLVA protocols used in each laboratory to ensure that the data can be easily compared. This can be accomplished by sequencing the alleles or by analyzing a set of reference strains of known genotype and, thus, known allele sizes. The allele sizes given in this report are normalized to the sizes described by Keim et al. (15) and thus do not always represent the true sequenced sizes of the alleles.

Our investigation of pagA diversity also yielded new sequence types. Previously, the seven known pagA sequence types (excluding the Sverdlovsk isolates) represented only four distinct PA phenotypes (11, 25). However, our observation of pagA sequence types 10 and 11 increases the number of known PA phenotypes to six. We found that 9 (0.4%) of the total 2,294 nucleotide positions of the pagA open reading frame in the B. anthracis strains evaluated in this study contained sites with potential nucleotide changes. Previously, 7 changes at 2,294 nucleotide positions were described (25). Thus, the monomorphic nature of the B. anthracis chromosome also appears to be reflected in the pXO1 virulence gene, pagA, as well. Two of these newly described nucleotide mutations (35 and 196 nucleotides from the start) within pagA contain missense mutations that lead to amino acid changes at amino acids 12 and 66. These substitutions are located in the subterminal region (amino acid 12) and within the PA20 domain (amino acid 66). One of these mutations, found in pagA type 11, is predicted to translate into a truncated PA protein. Previously, Price et al. described six distinct sequence types from the complete pagA gene sequences from B. anthracis, leading to three phenotypes as the result of an unusual two missense to three synonymous mutations (25). Here, we report on three additional missense mutations found in pagA sequence types 10 and 11. Each strain appeared to be collected from water or surface samples from different locations, based on the limited archival data available. The two environmental B. anthracis isolates characterized as pagA type 10 represent phylogenetically distant MLVA genotypes 40 and 133, respectively. Three other strains in our historical collection were also characterized as MLVA genotype 40, but these isolates were pagA type 1 and no additional MLVA genotype 133 isolates were found in this study. It is not unusual for isolates with the same pagA sequence type to have different MLVA genotypes since VNTR regions mutate at a higher frequency than single-nucleotide polymorphisms (16). However, the two pagA type 10 isolates appear to be phylogenetically very distantly related (as they are members of cluster A and cluster C) (16). This suggests pagA homoplasy; however, it is unclear if the single T196C pagA point mutation shared by these seemingly otherwise unrelated isolates resulted from a similar environmental adaptation or by random chance.

The additional sites for nucleotide substitution described in our study for the pagA gene increases the ratio of potential missense mutations to synonymous mutations from the ratio of 3.8:5 reported previously to a ratio of 5:5 (25). Each of these new missense mutations is found near the start site or within the coding region for domain 1 of PA. The predicted amino acid changes are not within the highly antigenic region, where amino acid changes in pagA genotypes 1 to 6 have been described. It remains unclear if pagA types 10 and 11 would lead to changed pathogenicity. Further studies must be performed to gain a better understanding of how the translated PA protein is altered by these changes.

Although we observed two new pagA sequence types in this study, the usefulness of identifying the pagA type of an isolate for strain characterization has limited power. We found that the pagA sequence type data helped to differentiate isolates of the same MLVA type in only 2/124 isolates. However, pagA sequencing remains an important tool during biological threat investigations to identify potentially altered or engineered pagA genotypes. It remains unknown if alterations to pagA would lead to reduced protection against B. anthracis provided by the currently licensed and next-generation anthrax vaccines (2).

As we improve our understanding of the genetic diversity of B. anthracis, we not only elucidate the evolution of this pathogen but also improve our laboratory response capability during epidemiological investigations. Additional molecular epidemiology studies and the concomitant accumulation of more genotyping data will lead to a more rapid public health response.

 
* Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Road, NE, Mailstop D-11, Atlanta, GA 30333. Phone: (404) 639-3297. Fax: (404) 639-3172. E-mail: PWilkins{at}cdc.gov

Published ahead of print on 28 March 2007.

D.S. and C.K.M. contributed equally to this work.

Present address: Department of Microbiology and Immunology, School of Medicine, Emory University, Atlanta, GA 30322.

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Journal of Clinical Microbiology, June 2007, p. 1777-1782, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.02488-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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