Abstract
A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis. A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.
Bacillus anthracis, B. cereus, and B. thuringiensis are closely related species but have different virulence characteristics (2, 5, 16). While B. anthracis is the etiological agent of anthrax and has recognized potential for use as a biological weapon, B. cereus is often associated with human food poisoning and B. thuringiensis produces insect toxins (13). Therefore, reliable methods for the discrimination of B. anthracis from B. cereus and B. thuringiensis are important for controlling disease outbreaks and for determining their sources, which could be naturally occurring or of malicious intent. However, the high degree of morphological and biochemical similarities shared by these three species poses great difficulties for differentiation. Pulsed-field gel electrophoresis (PFGE) has been adopted by the Centers for Disease Control and Prevention and all 50 state public health labs to establish PulseNet, a public health laboratory network for food-borne disease surveillance (15). Because B. anthracis and B. cereus infections may be food borne, a reliable PFGE method for distinguishing B. anthracis from its B. cereus and B. thuringiensis near neighbors would be useful for enhancing the public health response to food-borne outbreaks and to suspected bioterrorism. The objective of this study was to develop a PFGE method for reliably genotyping B. anthracis and distinguishing B. anthracis isolates from those of B. cereus and B. thuringiensis.
Table 1 lists the bacterial strains used in this study, which were obtained either from Dugway Proving Ground, Utah, or from in-house collaborators. A diverse panel of B. anthracis strains was chosen to represent different phylogenetic groups (9, 10). The B. cereus strains D17, 3A, S2-8, F1-15, and ATCC 4342 were isolated from food-borne sources. B. thuringiensis 97-27 has apparent pathogenic properties that are unusual for B. thuringiensis. The other two B. thuringiensis strains, HD 571 and Al Hakam, are nonpathogenic (12). The remaining B. cereus and B. thuringiensis isolates either were demonstrated to be closely related to B. anthracis by amplified fragment length polymorphism analysis and multiple-locus sequence typing or caused anthraxlike symptoms in animals or humans (B. cereus E33L, G924, and TX strains [6, 7, 8, 14]). Two B. atrophaeus samples were also included in our study to expand the scope of comparison.
TABLE 1.
Bacterial strains used in this study
| Strain or spore (location) | Species | Phylogenetic group |
|---|---|---|
| K7441 | B. anthracis | A1a |
| K3700 | B. anthracis | A1b |
| K6835 | B. anthracis | B2 |
| K1694 | B. anthracis | A3b |
| K5135 | B. anthracis | A2 |
| K2762 | B. anthracis | B1 |
| K4596 | B. anthracis | A4 |
| K8215 | B. anthracis | A3a |
| A2084 | B. anthracis | A3d |
| K0610 | B. anthracis | A3b |
| K4834 | B. anthracis | A3a |
| K1340 | B. anthracis | A1a |
| K2478 | B. anthracis | B2 |
| K9002 | B. anthracis | A1b |
| K0123 | B. anthracis | A3a |
| K1285.1 | B. anthracis | A3a |
| K1256 | B. anthracis | A3a |
| K2802 | B. anthracis | A3d |
| K6428 | B. anthracis | A3a |
| K7948 | B. anthracis | A1b |
| K4241 | B. anthracis | A1a |
| K2484 | B. anthracis | A3a |
| K4516 | B. anthracis | A3a |
| K7222 | B. anthracis | A4 |
| K7038 | B. anthracis | A3a |
| ATCC 4342 | B. cereus | |
| G9241 | B. cereus | |
| D17 | B. cereus | |
| 3A | B. cereus | |
| S2-8 | B. cereus | |
| F1-15 | B. cereus | |
| TX 102 | B. cereus | |
| TX 108 | B. cereus | |
| TX 87 | B. cereus | |
| E33L | B. cereus | |
| 97-27 | B. thuringiensis | |
| Al Hakam | B. thuringiensis | |
| HD 571 | B. thuringiensis | |
| BG 1 | B. atrophaeus | |
| Spore (Old Dugway) | B. atrophaeus |
The PFGE protocol was tailored from previous methods (3) specifically to facilitate lysis and macrorestriction digestion of spore-forming Bacillus species. Cultures were enriched for vegetative cells by diluting 5 μl of an overnight culture 1,000-fold in 5 ml of Trypticase soy broth medium and incubating for another 4 h at 37°C. Lysozyme (Sigma-Aldrich Co., St. Louis, MO; 250 mg/ml) was added to the diluted cells and molten agarose during plug casting to boost the standard lysis step, and a heat shock step (70°C for 30 min) was added after proteinase K treatment. The Bacillus genomic DNA was then digested with the NotI restriction enzyme (New England Biolabs, Inc., Beverly, MA), and the resulting macrorestriction DNA fragments were separated by PFGE. Each gel was loaded with 22 sample lanes and 8 reference lanes (Fig. 1). Thiourea (100 μM; Sigma-Aldrich Co.) was added to the PFGE running buffer, which improved the PFGE image quality of samples that contained residual nuclease activity (11). Three replicates were run on separate gels for each strain analyzed.
FIG. 1.
One example of a PFGE gel image of NotI restriction fragments. The four lambda ladder marker lanes were used for fragment size calibration. The low-range marker lanes were for reference only. Ba, B. anthracis; Bc, B. cereus strains; Bt, B. thuringiensis; Ba, B. atrophaeus.
BioNumerics (Applied Maths, Inc., Austin, TX) was used to analyze the PFGE patterns. PFGE gel images were normalized using lambda ladder markers (New England Biolabs) with the “snap-to-peak” algorithm. Fragment sizes of the three sequenced strains (4), B. anthracis Ames (A2084), B. cereus E33L, and B. thuringiensis 97-27, were highly correlated (R2 > 0.98) with those obtained from virtual enzyme digestion using Kodon (Applied Maths, Inc., Austin, TX), validating the high quality of fragment separation and normalization.
A dendrogram (Fig. 2) was produced from the PFGE profiles using band-based cluster analysis with the Dice coefficient and unweighted-pair group method with arithmetic mean algorithm. By using the “tolerance and optimization analysis” function, the optimal position tolerance was found at 0.72% and a 64.3% cluster cutoff value for the dendrogram was obtained. All B. anthracis isolates fell into the same cluster, with an overall similarity of 71.1%, and were well separated from B. cereus, B. thuringiensis, and B. atrophaeus isolates. Visual examination of the dendrogram showed that most of the B. anthracis strains shared the same profile, with similarities of over 94%, confirming previous reports of high homology (9).
FIG. 2.
Dendrogram resulting from cluster analysis of PFGE fingerprints from NotI digestion, including 25 B. anthracis (Ba) strains, 10 B. cereus (Bc) strains, 3 B. thuringiensis (Bt) strains, and 2 B. atrophaeus (Ba) samples. The symbol before the name of the strain represents the strain species.
Further improvements in the application of BioNumerics, along with the adaptation of a more standard PulseNet experimental design, e.g., the use of the PulseNet universal reference standard (Salmonella serotype Braenderup strain H9812) and fewer lanes per gel, will enhance the quality of the analysis for use in public health laboratories. However, the conditions and settings used in this study resulted in the accurate grouping of all B. anthracis isolates together and the clear differentiation of B. anthracis strains from B. cereus, B. thuringiensis, and B. atrophaeus strains.
B. cereus and B. thuringiensis species were not segregated by this method, confirming reports using other genotyping methods (1, 5, 6), and individual isolates showed greater molecular diversity than did B. anthracis. The two B. atrophaeus strains were clustered with 100% similarity and clearly differentiated from those of the group 1 Bacilli. Principal component analysis was performed on the band-matching matrix, and the score plot verified the grouping of B. anthracis strains and their separation from other samples by the first principal component. Other commonly used macrorestriction enzymes, SmaI, XbaI, and XmaI, were also tested in this study; however, none of these performed as well as NotI did in the differentiation of B. anthracis from B. cereus and B. thuringiensis. Only two to three fragments were generated by SmaI digestion, and more than 50 bands were created by XmaI and XbaI digestions, indicating greater difficulties in using PFGE profiles for differentiation. None of the enzymes tested resulted in PGFE profiles that could be used to differentiate the nine phylogenetic groups within B. anthracis as found by the multiple-locus variable-number tandem-repeat analysis system (10); moreover, neither could the composite data sets formed by profiles from any combinations of the four tested enzymes.
Our results show that B. anthracis can be distinguished from its closest relatives by using the PFGE profiles from NotI digestion, after a few modifications to the PFGE procedure to facilitate complete lysis. PFGE may be a useful technique for the initial genotyping of Bacillus species in a research or public health laboratory, where the equipment and methods used in this technique are familiar, readily standardized, and widely available. PFGE is a whole-genome characterization method that is unbiased relative to other genotyping methods that are more rapid but are based on specific target sequences. Such a capability added to the public health laboratory toolbox of diagnostic methods would enhance the response to food-borne outbreaks and suspected acts of bioterrorism.
Acknowledgments
We gratefully acknowledge Karen Hill for providing the strains of B. cereus and B. thuringiensis used in this study.
Funding was provided by the Department of Homeland Security, National Bioforensics Analysis Center, and the Federal Bureau of Investigation. W. Zhong was supported by the National Flow Cytometry Resource (NCRR grant RR-01315).
The statements and conclusions herein are those of the authors and do not necessarily represent the views of the FBI.
Footnotes
Published ahead of print on 30 March 2007.
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