Identification and Strain Differentiation of Bacillus Species using Automated rep-PCR DNA Fingerprinting 8080 North Stadium Drive, Suite 1200 Poster #177 Houston, TX 77054, USA R. Webb, M. Lising, K. Reece, and M. Healy P: 713-467-8500, F: 713-467-7766

March 9-12 2003, Baltimore, Maryland Bacterial BarCodes, Inc., Houston, Texas, USA www.bacterialbarcodes.com

assignments can be made for isolates with undetermined or partially determined species classification, such as Bacillus Table 1. Species and number Figure 3. DiversiLab dendrogram and corresponding gel-like images of BACKGROUND of isolates included in study. Bacillus isolates illustrating species and strain discrimination. spp., not anthracis or cereus (BNAC). For example, in Figure 3, BNAC 10 is clustering tightly with the B. subtilis isolates, indicating a close genotypic relationship between these five isolates, hence BNAC 10 can presumably be identified as B. Members of the genus Bacillus are aerobic, gram-positive, spore-forming, rod-shaped organisms. Some Bacillus species Species # subtilis. The curve overlay of these isolates is illustrated in Figure 2b. The same can be presumed for BNAC 3 and 4, are beneficial and are used in antibiotic or insecticide production; other species are implicated in food-poisoning or as “Bacillus cereus” group BNAC 1 BNAC 2 which is highly related to the B. licheniformis isolates. Interestingly, the B. anthracis (Ames strain) and a B. thuringiensis agents of bioterrorism. Due to the large number of species and often incomplete descriptions of newly reported Bacillus cereus 20 Bacillus licheniformis 1 1 Bacillus thuringiensis 2 isolate are clustering very closely. This is not unexpected as they are closely related and are both classified in the species, Bacillus species differentiation is difficult. Additionally, a limited number of assays are available to distinguish Bacillus licheniformis 2 3,6,7 Bacillus anthracis Ames 1 “Bacillus cereus” group. Other reports have also demonstrated the close relationship between these species . between members of the “Bacillus cereus” group, which includes B. cereus, B. thuringiensis, B. mycoides, and B. anthracis.2 BNAC 3 Bacillus circulans 2 BNAC 4 Although these two isolates appear to be highly similar in the dendrogram, slight differences can be seen in the banding Furthermore, studies have shown the only known difference between B. cereus and B. thuringiensis to be insecticidal toxin Bacillus pumilus 3 BNAC 5 3 patterns in the gel-like images. producing genes, which further complicates discrimination between these two species. Traditional identification has Bacillus subtilis 4 Bacillus lentimorbis 1 6 been based on Gram stain, colony morphology, motility, and biochemical testing, which are time-consuming, Bacillus megaterium 1 BNAC Bacillus lentimorbis 2 DISCUSSION somewhat subjective, and labor intensive procedures. Both traditional and automated identification systems have Bacillus licheniformis 6 BNAC 7 Bacillus lentimorbis 6 difficulty identifying some Bacillus species and do not differentiate between strains. As the reality of bioterrorism Bacillus pumilus 1 • A limited number of methods exist that can differentiate between the “Bacillus cereus” group of closely-related attacks increase, such as the anthrax incident beginning in 2001, there is a need to develop systems to rapidly identify BNAC 10 Bacillus pumilus ATCC 27142 species 2. These data indicate that the DiversiLab System can quickly and accurately discriminate between Bacillus and differentiate biothreat agents. One possible solution is a commercial assay applying automated repetitive sequence- Total 55 Bacillus thuringiensis 1 Bacillus circulans 1 cereus and Bacillus thuringiensis. 4 based PCR (rep-PCR), a method utilizing non-coding, interspersed repeat sequences as targets for oligonucleotide Bacillus circulans 2 • Due to limited access to B. anthracis DNA, only the Ames strain was included in this sample set. A recent study has primers, which can be used to discriminate between species. This study explores the use and applicability of automated Figure 1. Caliper® 1000 Analyzer with Bacillus pumilus ATCC 7061 DiversiLab DNA Fingerprinting Kit Bacillus cereus 1 shown, however, that B. anthracis is easily differentiated from other organisms in the “Bacillus cereus” group by rep- rep-PCR for the identification and strain differentiation of Bacillus species. 5 Bacillus cereus ATCC 14579 PCR. This same study also illustrates subspecies discrimination of B. anthracis, differentiating virulent from Bacillus cereus 2 avirulent strains using rep-PCR. Bacillus anthracis Ames Strain METHOD 8, Bacillus thuringiensis 2 • Previous studies and this report, demonstrate the utility of rep-PCR to identify Bacillus isolates to the subspecies Using the Mo Bio UltraClean™ Microbial DNA Isolation kit (Mo Bio Laboratories, Inc., CA), DNA was extracted Bacillus cereus 3 and strain levels. These results indicate that automated rep-PCR may also be a useful tool to identify Bacillus isolates Bacillus cereus 4 at the species level. from 55 Bacillus isolates of 10 different species including B. cereus, B. lentimorbis, B. licheniformis, B. thuringiensis, B. subtilis, B. Bacillus cereus 5 pumilus, B. megaterium, B. circulans, B. anthracis (Ames strain), and Bacillus spp., not anthracis or cereus (BNAC), previously Bacillus cereus 6 • Further studies should validate the use of the DiversiLab System as a diagnostic and/or outbreak tracking tool for characterized by traditional biochemical and automated analysis (Table 1). Fifty nanograms of DNA was amplified Bacillus cereus 7 Bacillus spp and other biothreat agents. The small footprint of the automated system would allow for portability, Bacillus cereus 8 Figure 2. a) rep-PCR fingerprint curve overlay of 4 while the web-based interface would allow for remote access and rapid data sharing. These are important features using the DiversiLab Bacillus Fingerprinting Kit (Bacterial BarCodes, Inc., TX). The various amplified fragments were B. cereus isolates from 2 different clusters. b) rep- Bacillus cereus 9 ® separated and detected using lab-on-a-chip technology and the Caliper 1000 Analyzer (Caliper Technologies, Corp., PCR fingerprint curve overlay of 3 B. subtilis Bacillus cereus 10 for biodefense applications. CA) (Figure 1). Rep-PCR fingerprint patterns are generated as electropherograms. Figures 2a and b illustrate isolates and an unclassified BNAC isolate. Bacillus cereus 11 Bacillus cereus 12 • The automated detection and standard algorithms for data analysis adds consistency to the discriminatory power of examples of curve overlays produced by the DiversiLab software. Results were analyzed using Pearson’s correlation Bacillus cereus 13 a. B. cereus 6 rep-PCR. Further studies with larger sample sets are needed to focus the application of this molecular method’s Bacillus cereus 14 coefficient and a dendrogram was generated using UPGMA. Using the DiversiLab software, a report was automatically B. cereus 7 usefulness and role in biodefense. generated that included the dendrogram and gel-like images reflecting each isolate’s rep-PCR fingerprint (Figure 3). B. cereus 16 Bacillus cereus 15 B. cereus 17 BNAC 8 BNAC 9 REFERENCES RESULTS Bacillus cereus 16

fluorescence Bacillus cereus 17 1. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST. 1994. Bergey’s Manual of Determinative Bacteriology. Endospore Forming Gram Positive The results showed multiple, visually distinct, fingerprint patterns of the 55 Bacillus isolates, which are illustrated by the Bacillus cereus 18 Rods and Cocci. 9:559. Bacillus cereus 19 2. Anderson GL, Simchock JM, Wilson KH. 1996. Identification of a region of genetic variability among Bacillus anthracis strains and related gel-like images in Figure 3. The software resolved many of the organisms into distinct species groups. Several groups Bacillus lentimorbis 3 species. J Bacteriol. 178(2):377-384. were seen for some species; this is presumably indicative of subtype differences within samples of the same species. Bacillus lentimorbis 4 3. Helgason E, Okstad PA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolsto AB. 2000. Bacillus anthracis, Bacillus cereus, Bacillus Some of the intense bands were similar within a species, while differences seen in the less intense bands differentiated Datapoints Bacillus lentimorbis 5 Bacillus subtilis 1 thuringiensis—One species on the basis of genetic evidence. Appl Env iron Microbiol. 66(6):2627-2630. the subspecies (for example, all the B. cereus isolates share at least one band, but other less intense bands are only seen b. B. subtilis 2 B. subtilis ATCC 23856 Bacillus subtilis 2 4. VersalovicJ, Schneider M, de Bruijn FJ, Lupski JR. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain within each B. cereus cluster). Some species, however, were not grouped in the same cluster (i.e. B. thuringiensis and B. B. subtilis 3 Bacillus subtilis ATCC 23856 reaction. Methods of Molecular and Cellular Biology. 5:25-40. BNAC 10 Bacillus subtilis pumilus). This is likely a result of inadequate representation of that specific subspecies or strain in this study. 3 5. Helgason E, Caugant DA, Lecadet MM, Chen Y, Mahilln J, Lovgren A, Higna I, Kvaloy K, Kolsto AB. 1998. Genetic diverisity of Bacillus BNAC 10 cereus/Bacillus thuringiensis isolates from natural sources. Curr. Microbiol. 37:80-87. Conceivably, multiple clusters for these isolates, as seen with B. cereus, might be present if more of the same, or closely Bacillus lentimorbis 6

related strains were included. The multiple groups within a species is indicative of the heterogeneity that is known to fluorescence Bacillus megaterium 1 6. Harrell LJ, Andersen GL, Wilson KH. 1995. Genetic variability of Bacillus anthracisand related species. J ClinMicr. 33(7):1847-1850. exist among some Bacillus species.5 Figure 2a illustrates curve overlays of four B. cereus isolates from two different Bacillus licheniformis 3 7. Shangkuan YH, Chang YH, Yang JF, Lin HC, Shaio MF. Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR Bacillus licheniformis 4 groups, presumed to be different strains. Another important observation is that although the B. thuringiensis isolates are and RAPD. 2001. Letters in Applied Microbiology. 32:139-145. Bacillus licheniformis 5 8. Keim P, Kalif A, Schupp J, Hill K, Travis SE, Richmond K, Adair DM, Jones M, Kuske CR, Jackson P. 1997. Molecular evolution and diversity not grouped together, they are well discriminated from the closely related B. cereus isolates. Presumptive speciation Bacillus licheniformis 6 in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J Bacteriol. 179(3):818-824. 55% 64% 73% 82% 91% 100% Datapoints The rep-PCR technology and rep-PCR primers are covered by U.S. patents (5,691,136 and 5,523,217) and by international patents for Canada and Europe. For Research Use Only. Not For Use In Diagnostic Procedures.