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. 2003 Jan;41(1):524–525. doi: 10.1128/JCM.41.1.524-525.2003

Searching for Bacillus anthracis in Suspect Powders: a French Experience

Bernard La Scola 1, Pierre-Edouard Fournier 1, Didier Raoult 1,*
PMCID: PMC149647  PMID: 12517916

In a recent issue, we read the contributions of Kiratisin et al. (4) and Bell et al. (1), who reported large-scale screening of nasal swabs for Bacillus anthracis and detection of B. anthracis DNA by light cycler PCR, respectively. We report here our experience with large-scale screening of suspect powders by the light cycler PCR technique.

Following the appearance of press reports on inhaled anthrax in Florida on 4 October 2001 (2), “suspect powder fever” reached France. Some jokers began to send mail containing powder, mostly to administrative offices, in order to increase the terror. Any powder of unknown origin and any suspect mail coming from the United States were then analyzed for the detection of B. anthracis. We received samples from most parts of southern France from November 2001 to June 2002. Samples were opened and tested in a P3 laboratory. They were suspended in water, treated for 1 h at 65°C to destroy unsporulated bacteria, and then plated on Columbia 5% sheep blood agar (BioMérieux, Marcy l'Etoile, France). After 24 h of incubation at 37°C, nonhemolytic or weakly hemolytic colonies were sampled and DNA was extracted as previously reported (3) and submitted for DNA amplification by a procedure using primers obtained from the RNA polymerase (rpoB) gene of B. anthracis and a real-time PCR assay using light cycler technology (5). Positive amplicons were sequenced to discriminate among the Bacillus species amplified by this technique (5). We analyzed 757 samples, 668 of which were envelopes or mail packages. They apparently contained talc, flour, graphite, or coal. We obtained 222 isolates from 112 samples; 7 were amplified by our procedure. For these, the best BLAST matches were obtained with Bacillus subtilis (four isolates), B. mycoides (two isolates), and B. thuringiensis (one isolate) and none exhibited the sequence of B. anthracis. The suspect species isolated from powders in our study are different of those isolated from nasal swabs, most of which are related to B. megaterium (4). We believe that the technique used herein is useful as no real positive control is necessary and also only some strains of B. cereus (a hemolytic Bacillus species) can exhibit the same rpoB sequence as this amplicon. In the case of B. anthracis identification, a PCR assay using the technique described by Bell et al. (1) should be used as a rapid confirmatory test. Moreover, we believe that because of the rare occurrence of suspected colonies in the various samples tested in our study, as well as in nasal swabs (4), the use of large automated systems to test suspected bacteria is not necessary.

REFERENCES

  • 1.Bell, C. A., J. R. Uhl, T. L. Hadfield, J. C. David, R. F. Meyer, T. F. Smith, and F. R. Cockerill III. 2002. Detection of Bacillus anthracis DNA by light cycler PCR. J. Clin. Microbiol. 40:2897-2902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Centers for Disease Control and Prevention. 2001. Notice to readers: ongoing investigation of anthrax—Florida. Morb. Mortal. Wkly. Rep. 50:877. [Google Scholar]
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  • 4.Kiratisin, P., C. D. Fukuda, A. Wong, F. Stock, J. C. Preuss, L. Ediger, T. N. Brahmbhatt, S. H. Fisher, D. P. Fedorko, F. G. Witebsky, and V. J. Gill. 2002. Large-scale screening of nasal swabs for Bacillus anthracis: descriptive summary and discussion of the National Institutes of Health's experience. J. Clin. Microbiol. 40:3012-3016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Qi, Y., G. Patra, X. Liang, L. E. Williams, R. Rose, J. R. Redkar, and V. G. DelVecchio. 2001. Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Appl. Environ. Microbiol. 67:3720-3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
J Clin Microbiol. 2003 Jan;41(1):524–525.

Authors' Reply

Pattarachai Kiratisin 1,2, Caroline D Fukuda 1,2, Alexandra Wong 1,2, Frida Stock 1,2, Jeanne C Preuss 1,2, Laura Ediger 1,2, Steven H Fischer 1,2, Daniel P Fedorko 1,2, Frank G Witebsky 1,2, Vee J Gill 1,2, Trupti N Brahmbhatt 1,2

We thank La Scola et al. for their interest in our report and for contributing the account of their experience. They discuss an interesting application of the light cycler PCR technique to screening for B. anthracis in environmental samples, particularly those likely to contain considerable quantities of B. anthracis spores. The additional step of suspending samples in water and heating them at 65°C for 1 h would presumably eliminate most of the non-spore-forming bacteria that might also be present. Specimens sent to diagnostic laboratories may contain either vegetative cells (as, for example, in the case of blood specimens) or spores (as in the case of nasal swabs obtained for screening, although vegetative cells might also be present some time after initial exposure). In contrast, samples such as powders are likely to contain only spores, probably in much higher concentrations than would be likely in clinical material obtained for screening. Thus, it remains to be determined whether or not heating of specimens is a useful technique for the initial handling of clinical specimens. If La Scola et al. have not already done so, it would be important to determine the effect of their heating conditions on spore recovery. If the procedure produces a significant decrease in viability, it would probably not be suitable for use with clinical specimens such as nasal swabs. Our report (2) focused primarily on the practical methods that might be used in clinical laboratories for the initial screening of large numbers of specimens for B. anthracis in the event of a bioterrorism episode such as that with which we were presented. As is the case for the majority of clinical laboratories, we were not equipped to provide definitive identification of B. anthracis. Many clinical specimens contain a wide variety of normal flora. Initial plating of such specimens on sheep blood agar is required to detect possible B. anthracis strains on the basis of their colony morphology and hemolytic reaction. Particularly from a mixed culture, suspicious colonies need to be subcultured to a sheep blood agar plate and a motility test can be inoculated simultaneously. For clinical purposes, a nonhemolytic, nonmotile Bacillus sp. isolate with appropriate colonial morphology requires unambiguous determination of whether or not it is B. anthracis. Perhaps most importantly from a clinical perspective, as La Scola et al. mention, some strains of B. anthracis and B. cereus may exhibit indistinguishable rpoB gene sequences. Although a light cycler PCR technique has shown promising results as a rapid test (1), its use is still largely restricted to some reference laboratories; also, an initial direct plating step is still required. La Scola et al. noted that the Bacillus isolates obtained in their study were different from ours; this difference may well be due to different sources of specimens. We described in our report the biochemical reactions and 16S rRNA gene sequences of the organisms we isolated from nasal swabs that passed the screening criteria for B. anthracis but proved not to belong to that species; we thought this information might be useful for other laboratories that might be confronted with a situation similar to ours. Searches for more rapid, highly sensitive and specific methods that can be used as definitive tests would be useful for the detection of all potential agents of bioterrorism.

REFERENCES

  • 1.Bell, C. A., J. R. Uhl, T. L. Hadfield, J. C. David, R. F. Meyer, T. F. Smith, and F. R. Cockerill III. 2002. Detection of Bacillus anthracis DNA by light cycler PCR. J. Clin. Microbiol. 40:2897-2902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kiratisin, P., C. D. Fukuda, A. Wong, F. Stock, J. C. Preuss, L. Ediger, T. N. Brahmbhatt, S. H. Fisher, D. P. Fedorko, F. G. Witebsky, and V. J. Gill. 2002. Large-scale screening of nasal swabs for Bacillus anthracis: descriptive summary and discussion of the National Institutes of Health's experience. J. Clin. Microbiol. 40:3012-3016. [DOI] [PMC free article] [PubMed] [Google Scholar]

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