Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schumacher, W. C. Articles by Phipps, A. J. Search for Related Content PubMed PubMed Citation Articles by Schumacher, W. C. Articles by Phipps, A. J. Agricola Articles by Schumacher, W. C. Articles by Phipps, A. J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, August 2008, p. 5220-5223, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00369-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Bacillus anthracis Spores by Multiparameter Flow Cytometry

William C. Schumacher,¹ Craig A. Storozuk,^2,3 Prabir K. Dutta,¹ and Andrew J. Phipps^2,3*

Department of Chemistry,¹ Department of Veterinary Biosciences,² Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio 43210³

Received 13 February 2008/ Accepted 20 June 2008

Top ABSTRACT INTRODUCTION Bacterial stocks. Surface PA assay. Two-color assay. Characterization of Bacillus... REFERENCES

 
In response to the need for methods that can rapidly detect potentially virulent Bacillus anthracis spores, we developed a two-color flow cytometric assay capable of simultaneously identifying B. anthracis spores and the presence of spore-associated protective antigen, a virulence marker for strains harboring the pXO1 plasmid.

Top ABSTRACT INTRODUCTION Bacterial stocks. Surface PA assay. Two-color assay. Characterization of Bacillus... REFERENCES

 
Assays that can rapidly and accurately detect Bacillus anthracis, the etiologic agent of anthrax, are important tools for combating bioterrorism (5, 13, 16). This gram-positive spore-forming bacterium is a member of the Bacillus cereus group (along with B. cereus, B. thuringiensis, and B. mycoides) whose members exist ubiquitously in nature and are genetically related (7). One of the few distinguishing features among this group is plasmids that encode virulence factors (5). Fully virulent strains of B. anthracis harbor two virulence plasmids, pXO1 (1) and pXO2 (6), whereas minimally virulent strains lack one or both.

Common detection methods for B. anthracis include real-time PCR (11) and standard microbiological tests (14), but such assays require either the extraction of genetic material (PCR) or lengthy analysis times (microbiological tests). Comparatively, spore-based methods that analyze intact spores can be rapid because surface antigens are detected directly by using simple labeling procedures (5); however, many of these strategies are not selective for specific members of the B. cereus group (9, 17). Through a phage display screening process, short peptide fragments that exhibited species-specific binding to Bacillus spores were discovered (19, 21), and several investigators have used them successfully (2, 15). One such peptide, ATYPLPIRGGGC (abbreviated ATYP), was conjugated to R-phycoerythrin (RPE) and found by flow cytometry (FCM) to bind only to B. anthracis spores; however, the ATYP-RPE conjugate cannot differentiate spores from different strains of B. anthracis (21).

Recently, the protective antigen (PA) protein expressed by pXO1-harboring strains of B. anthracis has been identified as a spore surface antigen (4, 20). PA is present during the sporulation process and thought to be noncovalently entrapped in the spore coat and exosporium (4). Since the pXO1 plasmid encodes several virulence factors, surface PA can be considered a virulence marker for B. anthracis spores. Here, we report on a two-color FCM assay which couples a fluorescein isothiocyanate (FITC)-conjugated antibody-based PA assay to the B. anthracis spore-specific ATYP-RPE assay. We demonstrate the potential of this multiparameter assay by differentiating PA-positive (PA⁺) B. anthracis spores from PA-negative (PA^–) B. cereus and B. thuringiensis spores.

Top ABSTRACT INTRODUCTION Bacterial stocks. Surface PA assay. Two-color assay. Characterization of Bacillus... REFERENCES

 
The following Bacillus spp. were used in this study: B. anthracis Sterne 34F2 (pXO1⁺/pXO2^–, seed from the live-spore veterinary vaccine; Colorado Serum Company, Denver, CO), B. anthracis Ames (pXO1⁺/pXO2⁺), B. cereus (pXO1^–/pXO2^–), and B. thuringiensis (pXO1^–/pXO2^–). Bacterial strains were grown, sporulated, and purified according to previously described methods (10, 12, 18). Subsequent labeling assays were carried out in phosphate-buffered saline, pH 7.2.

Top ABSTRACT INTRODUCTION Bacterial stocks. Surface PA assay. Two-color assay. Characterization of Bacillus... REFERENCES

 
An amount of 80 µg/ml of a mouse monoclonal antibody against B. anthracis PA (BAP0101; Abcam) was mixed with approximately 10⁶ spores and incubated for 1 h at 37°C. Bound antibody was detected by incubation with 25 µl of a 1:100 dilution of FITC-conjugated goat anti-mouse immunoglobulin G (IgG) (heavy plus light chains) (IgG-FITC; Jackson ImmunoResearch) for an additional hour at 37°C. Wash/centrifuge steps were used before and after the addition of IgG-FITC to remove unbound reagents. Under these conditions, PA was detected on the surface of B. anthracis Sterne spores by using FCM (FACSCalibur instrument/CellQuest Pro software; BD Biosciences) (Fig. 1). As expected, PA was not detected on the surface of B. cereus or B. thuringiensis spores since they do not harbor the pXO1 virulence plasmid.

View larger version (40K): [in this window] [in a new window]

FIG. 1. Typical FCM data for the surface PA assay. The monoclonal antibody against PA was sensitive and selective for B. anthracis Sterne spores (BAS) as compared to B. cereus (BC) and B. thuringiensis spores (BT). In comparison to the intrinsic fluorescence of unlabeled spores, we did observe some nonspecific binding of IgG-FITC to Sterne spores. As such, the FITC cutoff marker (M1; M1 = positive event) was adjusted to minimize the contribution from nonspecifically bound IgG-FITC to the authentic surface PA signal. All FCM data are presented as the average of 10,000 events. , anti.

Top ABSTRACT INTRODUCTION Bacterial stocks. Surface PA assay. Two-color assay. Characterization of Bacillus... REFERENCES

 
The ATYP peptide (GenScript Corporation) was conjugated to RPE (Invitrogen) through the heterobifunctional cross-linker sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC; Pierce) (8). PA antibody-treated spores (10⁶) were prepared and labeled stepwise with 25 µl of 1:100 IgG-FITC and then 25 µl of 2 µM ATYP-RPE. Each reagent was incubated for 1 h at 37°C, and wash/centrifuge steps were used before and after the addition of ATYP-RPE. The FCM data showed that for one lot of B. anthracis Sterne spores (BAS7), the two-color assay double labeled 30% of the population (Table 1). An additional 26% of the spores were positive for ATYP-RPE and negative for surface PA, which reflects the heterogeneity of surface PA content. We also verified that double labeling occurred on individual Sterne spores by using confocal microscopy (TCS SP2 AOBS instrument; Leica Microsystems) (Fig. 2). Lower surface PA values were obtained for Sterne spores when the detection reagents were applied simultaneously or in opposite order (ATYP-RPE followed by IgG-FITC) (data not shown). This discrepancy may indicate that surface PA exists close to the ATYP peptide receptor(s) on the exosporium basal layer (3, 4) and can be blocked by binding of ATYP-RPE.

View this table: [in this window] [in a new window]

TABLE 1. Characterization of different spore lots by using the FCM two-color assay

View larger version (47K): [in this window] [in a new window]

FIG. 2. Phase-contrast (a) and fluorescence (b and c) images of double-labeled B. anthracis Sterne spores. The surface PA fluorescence image (b) and the ATYP-RPE fluorescence image (c) were used to produce the merged image (d).

Top ABSTRACT INTRODUCTION Bacterial stocks. Surface PA assay. Two-color assay. Characterization of Bacillus... REFERENCES

 
We next examined several spore lots to see if differences in preparation, purification, and storage conditions affected the outcome of the two-color assay (details presented in Table 1). Lots BAS3 through BAS8 were labeled similarly by ATYP-RPE (51 to 71% positive) and had comparable surface PA values (23 to 43% positive). Only lots BAS1 and BAS2 demonstrated lower ATYP-RPE binding (11 to 12% positive) and higher surface PA values (72 to 75% positive). Lots BAS1 and BAS2 were stored in double-distilled water at 4°C for a longer period of time (4 years) than any other lot used in this study. The examination of spores from lots BAS1 and BAS8 by using transmission electron microscopy revealed that a majority of lot BAS1 spores were missing the outermost exosporium (Fig. 3a and c). Though the reason is still unclear, we believe the structural damage to lot BAS1 spores was caused either by repeated handling during the prolonged storage or unknown protease activity (possibly through contamination). From the electron micrographs in Fig. 3 and data in Table 1, we concluded the following about the two-color assay: (i) B. anthracis spores only bind ATYP-RPE if they possess an exosporium and (ii) the detection of subexosporium PA (i.e., spore coat PA) is possible in the absence of an exosporium. As expected, B. cereus and B. thuringiensis spores produced double-negative results by the two-color assay.

View larger version (156K): [in this window] [in a new window]

FIG. 3. Transmission electron micrographs of spores from lots BAS1 (a and c) and BAS8 (b and d). Asterisks are placed on spores shown in the enlarged images. Arrowheads point to the exosporium (EX), spore coat (SC), and core (C). Image magnifications are x21,300 (a and b) and x60,000 (c and d).

Based on the two-color results, we have developed a set of prototypical FCM dot-plot patterns for predicting the species of spores belonging to the Bacillus genus and their potential virulence (Fig. 4). Pattern 1 (events occur in lower-left quadrant) corresponds to ATYP-RPE^–/PA^– non-B. anthracis spores, such as B. cereus or B. thuringiensis spores. Pattern 2 (events occur in lower-left, upper-left, and upper-right quadrants) is indicative of ATYP-RPE⁺/PA⁺ spores, such as B. anthracis Sterne or Ames spores, that are potentially virulent. Pattern 3 (events occur in lower-left and upper-left quadrants) is indicative of ATYP-RPE⁺/PA^– spores, such as B. anthracis Pasteur spores, that are likely to be minimally virulent. Like pattern 2, pattern 4 (events occur in lower-left, upper-left, upper-right, and lower-right quadrants) also occurs with ATYP-RPE⁺/PA⁺ B. anthracis spores that are potentially virulent; however, the population in the lower-right quadrant indicates that a significant fraction of the spores containing PA are nonreactive with the ATYP-RPE conjugate.

View larger version (55K): [in this window] [in a new window]

FIG. 4. Prototypical FCM dot-plot patterns for identification and characterization of unknown samples of bacterial spores. Bacillus species and potential virulence can be predicted by using one of the following patterns: pattern 1, ATYP-RPE–/PA–; pattern 2, ATYP-RPE+/PA+; pattern 3, ATYP-RPE+/PA–; and pattern 4, ATYP-RPE+/PA+ (no exosporium). Quadrant abbreviations are as follows: LL, lower left; UL, upper left; UR, upper right; and LR, lower right.

In conclusion, this two-color assay successfully differentiated pXO1⁺ strains of B. anthracis from pXO1^– strains in only a few hours. One drawback of this assay is that it cannot resolve B. anthracis Sterne spores from B. anthracis Ames spores since they both harbor the pXO1 plasmid. Preliminary data indicated that these two strains were labeled similarly by the two-color assay (data not shown). Work to identify another spore surface antigen capable of making that distinction is ongoing. Once discovered, we envision its use in a three-color assay. Nonetheless, this two-parameter detection assay marks an important step toward rapid and complete characterization of the dangerous pathogen known as B. anthracis.

 
This material is based upon work supported by the National Science Foundation under grant no. 0221678.

We are grateful to Mamoru Yamaguchi for performing the electron microscopy work. The B. anthracis Ames, B. cereus, and B. thuringiensis seed lots were generous gifts from Battelle, Columbus, OH.

 
* Corresponding author. Present address: Battelle, 505 King Avenue, Columbus, OH 43201. Phone: (410) 306-8526. Fax: (614) 458-6727. E-mail: PhippsA{at}battelle.org

Published ahead of print on 27 June 2008.

Top ABSTRACT INTRODUCTION Bacterial stocks. Surface PA assay. Two-color assay. Characterization of Bacillus... REFERENCES

 

1
1. Abrami, L., N. Reig, and F. G. van der Goot. 2005. Anthrax toxin: the long and winding road that leads to the kill. Trends Microbiol. 13:72-78.[CrossRef][Medline]
2
2. Acharya, G., D. D. Doorneweerd, C. Chang, W. A. Henne, P. S. Low, and C. A. Savran. 2007. Label-free optical detection of anthrax-causing spores. J. Am. Chem. Soc. 129:732-733.[CrossRef][Medline]
3
3. Boydston, J. A., L. Yue, J. F. Kearney, and C. L. Turnbough, Jr. 2006. The ExsY protein is required for complete formation of the exosporium of Bacillus anthracis. J. Bacteriol. 188:7440-7448.[Abstract/Free Full Text]
4
4. Cote, C. K., C. A. Rossi, A. S. Kang, P. R. Morrow, J. S. Lee, and S. L. Welkos. 2005. The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions. Microb. Pathog. 38:209-225.[CrossRef][Medline]
5
5. Edwards, K. A., H. A. Clancy, and A. J. Baeumner. 2006. Bacillus anthracis: toxicology, epidemiology and current rapid-detection methods. Anal. Bioanal. Chem. 384:73-84.[CrossRef][Medline]
6
6. Green, B. D., L. Battisti, T. M. Koehler, C. B. Thorne, and B. E. Ivins. 1985. Demonstration of a capsule plasmid in Bacillus anthracis. Infect. Immun. 49:291-297.[Abstract/Free Full Text]
7
7. Helgason, E., O. A. Okstad, D. A. Caugant, H. A. Johansen, A. Fouet, M. Mock, I. Hegna, and A. Kolsto. 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—one species on the basis of genetic evidence. Appl. Environ. Microbiol. 66:2627-2630.[Abstract/Free Full Text]
8
8. Hermanson, G. T. 1995. Bioconjugate techniques. Academic Press, San Diego, CA.
9
9. Kamboj, D. V., G. S. Agarwal, B. S. Dwarkanath, J. S. Adhikari, S. I. Alam, and L. Singh. 2006. Flow-cytometric analysis of Bacillus anthracis spores. Def. Sci. J. 56:769-774.
10
10. Kim, H. U., and J. M. Goepfert. 1974. A sporulation medium for Bacillus anthracis. J. Appl. Bacteriol. 37:265-267.[Medline]
11
11. Klee, S. R., H. Nattermann, S. Becker, M. Urban-Schriefer, T. Franz, D. Jacob, and B. Appel. 2006. Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group. J. Appl. Microbiol. 100:673-681.[CrossRef][Medline]
12
12. Leighton, T. J., and R. H. Doi. 1971. Stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J. Biol. Chem. 246:3189-3195.[Abstract/Free Full Text]
13
13. Lim, D. V., J. M. Simpson, E. A. Kearns, and M. F. Kramer. 2005. Current and developing technologies for monitoring agents of bioterrorism and biowarfare. Clin. Microbiol. Rev. 18:583-607.[Abstract/Free Full Text]
14
14. Marston, C. K., J. E. Gee, T. Popovic, and A. R. Hoffmaster. 2006. Molecular approaches to identify and differentiate Bacillus anthracis from phenotypically similar Bacillus species isolates. BMC Microbiol. 6:1-7.[CrossRef][Medline]
15
15. Pai, S., A. D. Ellington, and M. Levy. 2005. Proximity ligation assays with peptide conjugate "burrs" for the sensitive detection of spores. Nucleic Acids Res. 33:1-7.[Abstract/Free Full Text]
16
16. Petrenko, V. A., and I. B. Sorokulova. 2004. Detection of biological threats. A challenge for directed molecular evolution. J. Microbiol. Methods 58:147-168.[CrossRef][Medline]
17
17. Stopa, P. J. 2000. The flow cytometry of Bacillus anthracis spores revisited. Cytometry 41:237-244.[Medline]
18
18. Tamir, H., and C. Gilvarg. 1966. Density gradient centrifugation for the separation of sporulating forms of bacteria. J. Biol. Chem. 241:1085-1090.[Abstract/Free Full Text]
19
19. Turnbough, C. L., Jr. 2003. Discovery of phage display peptide ligands for species-specific detection of Bacillus spores. J. Microbiol. Methods 53:263-271.[CrossRef][Medline]
20
20. Welkos, S., S. Little, A. Friedlander, D. Fritz, and P. Fellows. 2001. The role of antibodies to Bacillus anthracis and anthrax toxin components in inhibiting the early stages of infection by anthrax spores. Microbiology 147:1677-1685.[Abstract/Free Full Text]
21
21. Williams, D. D., O. Benedek, and C. L. Turnbough, Jr. 2003. Species-specific peptide ligands for the detection of Bacillus anthracis spores. Appl. Environ. Microbiol. 69:6288-6293.[Abstract/Free Full Text]

---

Applied and Environmental Microbiology, August 2008, p. 5220-5223, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00369-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schumacher, W. C. Articles by Phipps, A. J. Search for Related Content PubMed PubMed Citation Articles by Schumacher, W. C. Articles by Phipps, A. J. Agricola Articles by Schumacher, W. C. Articles by Phipps, A. J.