Public Health Microbiology

Use of Long-Range Repetitive Element Polymorphism-PCR To Differentiate Bacillus anthracisStrains

Michael J. Brumlik, Urszula Szymajda, Dorota Zakowska, Xudong Liang, Rajendra J. Redkar, Guy Patra, Vito G. Del Vecchio
DOI: 10.1128/AEM.67.7.3021-3028.2001

ABSTRACT

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105B. anthracis strains of diverse geographical origins. AllB. anthracis strains produced fingerprints comprising seven to eight bands, referred to as “skeleton” bands, while one to three “diagnostic” bands differentiated between B. anthracisstrains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracisstrains.

Anthrax is often a fatal bacterial infection that results after Bacillus anthracis spores enter a suitable host either via inhalation or ingestion or through the contamination of a wound or abrasion (8). Since death occurs within a few days after the onset of symptoms, B. anthracis presents a very serious threat if deployed as a biological weapon (13). Naturally occurring human B. anthracis infections are rare and generally result from contact with contaminated animals or their products (5).

Due to the highly monomorphic nature of B. anthracis, differentiation of strains from diverse origins has proven to be very difficult. DNA fingerprinting of B. anthracis strains using ribotyping has shown some strain-to-strain variations, presumably due to changes in the organization and number of rrn loci (18). However, due to insufficient numbers of variant strains, ribotyping was found to have limited discriminatory potential.

Neither arbitrarily-primed PCR fingerprints (1) nor amplified fragment length polymorphisms fingerprints (15) have been useful in discriminating between B. anthracisstrains, although some genetic diversity has been observed. In this case, the diversity was due to the presence of a variable-number tandem repeat (14) sequence in the open reading frame (ORF)vrrA. Recently, seven additional loci containing these sequences were discovered in B. anthracis, leading to the identification of six genetically distinct groups of B. anthracis (16).

The presence of multiple copies of highly conserved repetitive extragenic palindromic (REP) sequences in DNA (12) has been identified in a number of microorganisms (28). The technique of REP-PCR takes advantage of the fact that multiple copies of these REP sequences may be found seemingly randomly distributed throughout the genomes of strains of a bacterial species. Thus, by employing a portion of a highly conserved REP sequence as a primer, it is possible to amplify DNA sequences that are located between closely situated pairs of correctly oriented REP elements.

REP-PCR has been successfully used to distinguish between strains ofCandida rugosa (22), Staphylococcus aureus (4), and Escherichia coli(6) in epidemiological studies, as well as between strains of the nonpathogenic bacterium Bacillus sporothermodurans(11). The LL-Rep1 primer (27), which hybridizes to a sequence found at the attP attachment site of the Lactococcus lactis temperate bacteriophage TP901-1 (2), has also been used to distinguish between L. lactis isolates, since they produce distinctive REP-PCR fingerprints (27).

In this report, we have developed a long-range (LR) REP-PCR method that can be used for the differentiation of B. anthracis strains. In 2 to 3 days from the time an isolate is grown in pure culture, classification of a particular B. anthracis strain of unknown etiological origin can be made. Moreover, the only equipment needed to accomplish LR REP-PCR is a thermocycler and an agarose gel electrophoresis apparatus.

MATERIALS AND METHODS

DNA preparation.DNA was isolated from over 105 strains ofB. anthracis (Table 1) as well as from at least 30 isolates of closely related species of Bacillus, including B. cereus, B. thuringiensis, B. mycoides, and also B. subtilis, using a modification of a method by Schraft and Griffiths (25). Bacterial cells were washed with 0.85% (wt/vol) NaCl and then suspended in 400 μl of 6.7% (wt/vol) sucrose supplemented with mutanolysin (25 U; Sigma-Aldrich, St. Louis, Mo.), lysozyme (400 μg; Sigma-Aldrich), and RNase A (100 μg; Sigma-Aldrich). Phase lock tubes (Eppendorf, Westbury, N.Y.) were used for the phenol-chloroform-isoamyl alcohol extractions of aqueous phase. The DNA concentrations were determined spectrophotometrically (24) prior to being diluted to 100 ng/μl in distilled water. Both the quality and quantity of the DNA were evaluated following electrophoresis in a 0.7% (wt/vol) GTG agarose gel (SeaKem; BioWhittaker, Rockland, Maine), containing ethidium bromide (0.5 μg/ml).

View this table:
Table 1.

B. anthracis isolates used in this study

LR REP-PCR.Each PCR mixture of 50 μl comprised 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.75 mM MgCl2, a 100 μM concentration of each deoxyribonucleotide triphosphate, a 0.75 μM concentration of either LL-Rep1 (5′-TAC AAA CAA AAC AAA AAC-3′) (27) or LL-Rep1LR (5′-GAT ATC TAA TAC AAA CAA AAC AAA AAC-3′), and 100 ng of template DNA. Both of these primers were derived from the nucleotide sequence surrounding theattP site of temperate lactococcal bacteriophage TP901-1 (2). The former primer has previously been used to distinguish isolates of L. lactis (27).

Once the samples were placed in the PE-9700 thermocycler, the template DNA was denatured at 94°C for 5 min. A hot-start was then performed by maintaining the temperature in the heating block at 80°C until 1.75 U of the Taq-Pwo enzyme mixture (Expand Long Template PCR system; Roche, Indianapolis, Ind.) had been added to each reaction tube. Each sample was then overlaid with 2 drops of UV-irradiated mineral oil (Sigma-Aldrich). The LR REP-PCR protocol employed 17 cycles consisting of denaturation at 94°C for 1 min, annealing at 54°C for 1 min (with an increase of 0.5°C/cycle), and extension at 68°C for 4 min. This was followed by 18 cycles consisting of denaturation at 94°C for 1 min, annealing at 62.5°C for 1 min, and extension at 68°C for 4 min (with an increase of 10 s/cycle). Following a final 7-min extension step at 68°C, loading buffer was added to the sample (1× loading buffer contained 0.01% [wt/vol] bromophenol blue, 8.0% [wt/vol] sucrose, 20 mM EDTA [pH 8.0], and 0.1% [wt/vol] sodium dodecyl sulfate). Ten microlitres of each sample was electrophoresed at 23 V for 16 h in a 1.0% (wt/vol) GTG agarose gel (SeaKem) containing ethidium bromide (0.5 μg/ml).

Cloning and sequencing of LR REP-PCR amplicons.Following LR REP-PCR, the amplicons were separated in a 1.0% (wt/vol) low-melting-temperature agarose gel (SeaPlaque; BioWhittaker). Diagnostic amplicons were excised from the gel and were purified using a QIAquick Gel Isolation kit (Qiagen; Valencia, Calif.). Cloning was carried out using the Original TA Cloning kit in conjunction with competent E. coli TOP10F′ cells (Invitrogen, Carlsbad, Calif.). Amplicons were then ligated into the pCR2.1 vector. Sequencing-grade DNA was prepared using a Plasmid Mini kit (Qiagen). At least two clones per amplicon were sequenced in each case. Nucleotide sequencing was performed using an ABI377, automated sequencer (Applied Biosystems, Boston, Mass.). The DNA sequences were compared using BLAST against the B. anthracis (Ames) genome (http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi ) and GenBank sequences (http://www.ncbi.nlm.nih.gov:80/BLAST/ ) for the possible identification of ORFs. Preliminary sequence data were obtained from The Institute for Genomic Research (http://www.tigr.org ).

RESULTS

Since the G+C content of L. lactis DNA, which is approximately 40%, is similar to that of DNA from Bacillussp., the LL-Rep1 primer was initially chosen to perform REP-PCR on selected B. anthracis strains. When LR REP-PCR using the shorter LL-Rep1 primer was performed, two distinct groups of B. anthracis strains were identified. Following electrophoresis, all the strains displayed relatively simple fingerprints consisting of six distinct amplicons (data not shown). However, the presence of an additional 3.3-kb amplicon in some fingerprints and not in others suggested that the categorization of B. anthracis strains was possible using this approach (data not shown).

In an attempt to increase the discriminating power of the technique, the length of the oligonucleotide primer was increased from 18 nucleotides (LL-Rep1) to 27 nucleotides (LL-Rep1LR). By using the longer primer, a more complex fingerprinting pattern was observed that could distinguish B. anthracis strains into five separate representative groups. Seven distinct amplicons were consistently observed in all 105 B. anthracis strains tested (Fig.1). The sizes of these amplicons were 4.6, 3.8, 3.4, 2.7, 2.1, 1.4, and 0.95 kb, respectively. An eighth amplicon, having a size of 0.75 kb, was also consistently present, although the intensity of the band varied between strains. These eight bands comprise the “skeleton” component of the fingerprint in everyB. anthracis strain examined. The presence or absence of one to three additional “diagnostic” amplicons, which had sizes of 3.3, 2.3, and 1.8 kb, respectively, facilitated the grouping of the 105 strains of B. anthracis into five categories (Fig. 1; Table1).

Fig. 1.
Fig. 1.

LR REP-PCR fingerprint patterns of B. anthracis strains representing five groups (A to E). The computer-generated skeleton map of the B. anthracisfingerprint (solid lines) is shown on the left and right sides of the gel image (left panel). The arrows denote the positions of the threeB. anthracis diagnostic amplicons. Amplicon sizes are shown on the left side of the figure. The positions of the diagnostic amplicons corresponding to each of the five categories are depicted in the right panel. Lane A, B. anthracis A3; lane B, B. anthracis Cepanzo; lane C, B. anthracis 128; lane D,B. anthracis 112; lane E, B. anthracis A41.

When optimizing the LR REP-PCR conditions, it became necessary to perform a hot-start reaction and also to incrementally increase the annealing temperature over the first 17 cycles from 54.0 to 62.5°C. This gradual increase in stringency eliminated any significant nonspecific amplification. Replicate LR REP-PCR experiments were performed on approximately one-third of all the DNA samples that were examined in this study (Table 1). Consistent results were obtained when the amount of template in the reaction mixture was maintained between 50 and 150 ng and freshly prepared DNA samples were used (data not shown).

In order to maximize the potential diversity of B. anthracisstrains chosen for this study, strains from North and South America, Europe, Africa, and Asia were examined (Table 1). However, this does not necessarily represent a global distribution. By plotting the number of B. anthracis strains that were placed into each category, a distribution was observed. Forty percent of the fingerprints corresponded to group A (42 of 105), 11% corresponded to group B (12 of 105), 21% corresponded to group C (22 of 105), 19% corresponded to group D (20 of 105), and 9% corresponded to group E (9 of 105).

As demonstrated in Fig. 2, greater diversity in the REP-PCR fingerprinting patterns was found among a collection of 10 different B. cereus and B. thuringiensis strains. Some of these strains had only a single band in common. The fingerprints of transitional non-B. anthracis strains which possessed the B. anthracischromosomal marker Ba813 but lacked both plasmids pXO1 and pXO2 (19) also showed little in common with B. anthracis fingerprints (Fig. 3).

Fig. 2.
Fig. 2.

LR REP-PCR fingerprint patterns of representativeBacillus species. The computer-generated skeleton map of theB. anthracis fingerprint (solid lines) is shown on the left side of the gel image. The arrows denote the positions of the threeB. anthracis diagnostic amplicons. Amplicon sizes are shown on the left side of the figure. Lane 1, B. anthracis Bc-575 (group A); lane 2, B. anthracis 7700 (group B); lane 3,B. cereus 14579T; lane 4, B. cereusF17289 ; lane 5, B. cereusF17202 ; lane 6, B. cereusF16959 ; lane 7, B. cereus 19637; lane 8,B. thuringiensis subsp. huazhongensis; lane 9,B. thuringiensis H34; lane 10, B. thuringiensis35646T; lane 11, B. thuringiensis B8; lane 12,B. thuringiensis 33689; lane 13, B. mycoidesT; lane 14, B. subtilisT.

Fig. 3.
Fig. 3.

LR REP-PCR fingerprint patterns of representativeBacillus sp. Ba813+ strains (pXO1−pXO2−). The computer-generated skeleton map of theB. anthracis fingerprint (solid lines) is shown on the left and right sides of the gel image. Solid arrows denote the presence of particular B. anthracis diagnostic amplicons in the neighboring lane, as opposed to broken arrows, which denote the absence of particular B. anthracis diagnostic amplicons in the neighboring lane. Amplicon sizes are shown on the left side of the figure. Lane 1, B. anthracis A6 (group D); lane 2,Bacillus sp. strain Ba813+ II/3, lane 3,Bacillus sp. strain Ba813+ 6684/63; lane 4,Bacillus sp. strain Ba813+ 74/8734; lane 5,Bacillus sp. strain Ba813+ PC1; lane 6,Bacillus sp. strain Ba813+ T5 97-77; lane 7,Bacillus sp. strain Ba813+ T11 97-79; lane 8,Bacillus sp. strain Ba813+ 11614-2; lane 9,Bacillus sp. strain Ba813+ PJ572; lane 10,Bacillus sp. strain Ba813+ 094; lane 11,Bacillus sp. strain Ba813+ T2 97-76; lane 12,Bacillus sp. strain Ba813+ I/2; lane 13,Bacillus sp. strain Ba813+ 9594/3; lane 14,Bacillus sp. strain Ba813+ S8553/2; lane 15,Bacillus sp. strain Ba813+ III-BL; lane 16,Bacillus sp. strain Ba813+ III; lane 17,Bacillus sp. strain 1B/A (pXO1−pXO2− Ba813−); lane 18, B. anthracis 7700 (group B).

The 1.8- and 3.3-kb diagnostic amplicons were cloned from B. anthracis strain A19 (group A) (Table 1), while the 2.3-kb diagnostic amplicon was cloned from B. anthracis strain Cepanzo (group B) (Table 1). Results of restriction mapping of each gel-isolated diagnostic amplicon and its corresponding cloned insert were identical, confirming that the correct LR REP-PCR amplicon was cloned and sequenced in each case (data not shown). Moreover, the restriction maps of diagnostic amplicons from different B. anthracis strains were identical to the ones directly excised from LR REP-PCR gels (data not shown). In order of increasing size, the three diagnostic amplicons encompassed a homologue of thelonB gene from B. subtilis (30; S. L. Wong, personal communication), a putative homologue of thetetB gene from Clostridium perfringens(26), and the acpA gene from B. anthracis (29) (Table2). This demonstrates that the five categories of B. anthracis described in this report are not simply the result of genetic polymorphism at a single genetic locus.

View this table:
Table 2.

Description of cloned diagnostic amplicons in B. anthracis LL-Rep1LR fingerprints

DISCUSSION

Pulsed-field gel electrophoresis has been unable to distinguish between any of the nonvirulent B. anthracis strains in our collection (M. Niemcewicz, G. Patra, R. J. Redkar, and V. G. DelVecchio, unpublished results). Others have reported similar results for B. anthracis strains of diverse geographical origins (9). B. anthracis is unusual in this regard since bacterial strains belonging to the same species have often been successfully distinguished by pulsed-field gel electrophoresis (17). A modified LR REP-PCR protocol, employing a mixture of robust “long-range” polymerases, was used to produce a fingerprint that was both reproducible and could discriminate betweenB. anthracis strains. This method allows the amplification of DNA between more distantly situated repetitive sequences.

Advantages of LR REP-PCR are the simplicity of the technique and the fact that only a thermocycler and electrophoresis apparatus are needed. Computer-assisted detection systems are unnecessary, although the use of pattern recognition software would be useful, especially if a database of patterns were to be established. Less than 2 to 3 days is needed to type the strains of B. anthracis. Roughly 9 h is needed to perform the DNA isolation, purification, and quantitation. The LR REP-PCR protocol requires approximately 5 h, and the fingerprint can be resolved on an agarose gel in as little time as 4 h at 72 V, although the best resolution requires electrophoresis at a much lower voltage (23 V for 16 h).

The presence of one to three variable or diagnostic amplicons within a skeleton of seven to eight amplicons facilitated the grouping ofB. anthracis strains into five categories (Fig. 1; Table 1). Based on the permutations and combinations of the three diagnostic amplicons, theoretically up to eight categories may exist. The strains examined in this study are of diverse geographical origins, having been isolated from five continents (Table 1). However, it is clear that a larger number of strains still need to be tested to find more examples of the under- represented groups (groups B and E) and of the theoretical groups for which we presently have no representatives. For example, the majority of the B. anthracis strains that were categorized in groups B and E came from Europe. On the other hand, strains from group D, the third-smallest group, have almost entirely been isolated in diverse parts of Asia, a continent that is many times the size of Europe.

The 3.3-kb amplicon encompasses the acpA gene, which encodes a transcriptional activator of capsule biosynthesis in B. anthracis and is crucial for virulence (29). During the course of our study, we found that all 17 of the B. anthracis strains lacking pXO2 (Table 1) produced LR REP-PCR fingerprints that did not possess the 3.3-kb diagnostic amplicon. This suggests that the 3.3-kb amplicon may have been amplified from the pXO2 plasmid.

The 1.8-kb amplicon contains an ORF (Table 2) that shows strong similarity to the lonB gene from B. subtilis(30; S. L. Wong, personal communication). While the exact role of the B. subtilis lonB gene remains unknown (S. L. Wong, personal communication), it resides next to several stress response genes (23, 30). Since the deduced amino acid sequences of the B. subtilis lonB gene product and theB. anthracis ORF share 77% sequence identity, the genes may perform a similar role in both species.

In contrast to the potential importance of the lonB andacpA genes in B. anthracis, we have no data that would suggest that the tetB homologue, which resides on the 2.3-kb amplicon, functions as a tetracycline resistance gene inB. anthracis or whether it is even expressed. Using a tetracycline disk sensitivity assay, we found that representativeB. anthracis strains from all five categories were extremely sensitive to tetracycline compared to representative B. cereus and B. thuringiensis strains (data not shown).

It stands to reason that the absence of any of the three diagnostic amplicons in LR REP-PCR fingerprints does not necessarily reflect the absence of the corresponding gene in a particular B. anthracis strain (Table 2). The three most likely explanations for observing B. anthracis fingerprints that lack a particular diagnostic amplicon include (i) nucleotide sequence differences at one of the LL-Rep1LR binding sites, (ii) the inversion of the orientation of an LL-Rep1LR binding site, or (iii) the insertion of DNA that abrogates amplification.

This technique clearly distinguishes B. anthracis strains from closely related species such as B. cereus and B. thuringiensis. A strong argument has been made that B. anthracis, B. cereus and B. thuringiensis should be reclassified as one species containing different plasmids (10). Perhaps the difficulty in differentiating B. anthracis strains is the result of B. anthracis'sactually being a subspecies of the much more polymorphic B. cereus lineage. The results of this study show that B. anthracis strains are more homogeneous than members of B. cereus and B. thuringiensis. The fingerprints ofB. mycoides and B. subtilis type strains showed the least similarity to those of the other species examined, which makes sense considering that these species are thought to have diverged from B. anthracis, B. cereus, and B. thuringiensis much earlier (7).

The evolutionary origin of the transitional non-B. anthracisBa813+ strains remains unclear. Did these organisms simply evolve from B. anthracis primarily as a result of losing the two virulence plasmids pXO1 and pXO2, or did they independently descend from the B. cereus lineage along with B. anthracis (16, 21)? The Ba813 chromosomal marker may be present in the transitional strains as a result of horizontal gene transfer (3), or it may be a vestige of the divergence of these transitional strains from B. anthracis. The LL-Rep1LR fingerprints suggest that these strains have a relatively close evolutionary relationship with respect to each other based on the large number of similar bands that were observed (Fig. 3) compared to the patterns obtained when B. cereus and B. thuringiensis strains were examined (Fig. 2). In fact, Fig. 3shows 15 of the most distinctive categories that were identified out of a total of 21 strains tested. However, as was found for the B. cereus and B. thuringiensis strains (Fig. 2), the LL-Rep1LR fingerprints produced by the transitional non-B. anthracis strains scarcely resembled those produced by theB. anthracis strains.

This is the first reported use of LR REP-PCR for the purpose of generating REP-PCR fingerprints. The longer LL-Rep1LR primer was well suited to distinguish between Bacillus species and strains. However, it must be emphasized that the power of the LL-Rep1LR primer resides in its ability to distinguish between at least five genetically distinct B. anthracis groups.

ACKNOWLEDGMENTS

This project was supported by research grant DABT63-99-1-002 from the Defense Advanced Research Projects Agency (DARPA).

We thank G. Bolus, M. Ferguson, and T. Horn for performing the DNA sequencing. The assistance of M. A. Wagner for critically reading the manuscript and Valerie Taylor for editing the manuscript is greatly appreciated. The sequencing of the B. anthracis genome was accomplished with support from the Office of Naval Research (ONR), the Department of Energy (DOE), and the National Institute of Allergy and Infectious Diseases (NIAID). We also thank W. Beyer, R. Bøhm, T. N. Brahmbhatt, J. Burans, A. Cataldi, J. Ezzel, Z. Liu, M. Mock, C. L. Turnbough, J. Vaissaire, and R. J. Zabransky for kindly offering Bacillus strains.

FOOTNOTES

    • Received 20 November 2000.
    • Accepted 11 April 2001.

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KEYWORDS

Bacillus anthracis
Polymerase Chain Reaction
Repetitive Sequences, Nucleic Acid

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