Aims: In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag.
Methods and Results: Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB, as well as the selective 16S rDNA sequence regions of B. anthracis, of the Bacillus cereus group and of Bacillus subtilis. Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected.
Conclusions: This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal (rpoB) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible.
Significance and Impact of the Study: The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.
The rapid identification of Bacillus anthracis in complex substrates such as dust and powder samples is a challenging diagnostic task and important for routine laboratories. Bacillus anthracis is difficult to differentiate phenotypically from members of the Bacillus cereus group by culture methods (Turnbull 1999). Further, B. anthracis has a nearly identical 16S rDNA sequence to B. cereus. Therefore, a molecular identification of B. anthracis can be achieved by PCRs targeting the plasmid-encoded virulence genes and specific chromosomal DNA sequences such as Ba813, vrrA and rpoB (Andersen et al. 1996; Jackson et al. 1997; Ramisse et al. 1999; Qi et al. 2001; Patra et al. 2002). This requires time-consuming tests susceptible to cross-contamination. Recently, a variety of molecular assays, e.g. real-time PCR, multiplex PCR and a combination of multiplex PCR with a DNA microarray, were developed to improve the diagnostics (Ellerbrok et al. 2002; Wang et al. 2004; Bavykin et al. 2008).
The objective of the study was to develop a DNA microarray to differentiate B. anthracis from environmental Bacillus isolates obtained from samples released as ‘white powders’ with regard to potential bioterrorism threats. Aforementioned microarrays are based on fluorescence-labelled PCR products. As the labelling reagents are expensive and the development of the techniques is time consuming, such systems are not ideal for routine use in diagnostic laboratories. In this study, the ArrayTube™ (AT) platform (Clondiag, Jena, Germany) and biotinylated whole bacterial DNA were used to diagnose B. anthracis. This platform is rapid and uses inexpensive reagents. Because of the hybridization with whole bacterial DNA instead of PCR products, the cross-contamination risk is minimized. All important genomic and plasmid markers can be analysed in parallel.
Materials and methods
Bacterial strains and growth conditions
Strains used are listed in Table 1. Environmental samples (1 g) were homogenized in 10 ml phosphate buffered saline and pasteurized (65°C, 10 min) to inactivate vegetative cells. Blood-TSA (blood-trypticase-soy-agar; Oxoid, Basel, Switzerland) was used for cultivation. Bacillus anthracis strains were handled under biosafety level 3 conditions.
Table 1. Strains used in this study. Characterization was performed by PCRs of the virulence genes, 16S gene sequencing and hybridizations to the array
Reference strains or isolates respectively
Highest homology with the 16S rRNA gene of
Sequence identity in percent
Result of ArrayTube hybridization
rpoB, gene encoding the subunit β of the RNA polymerase; pag, lef and cya, genes encoding the toxin components protective antigen, lethal factor and oedema factor respectively; capAC and capB, genes encoding the capsule proteins; +, a PCR product could be detected; −, no PCR product could be detected; NP, strain assignment by hybridization signals not predictable; ND, not done; Bold, hybridizations of these strains are shown in Fig. 2b-o.
*Prof. R. Böhm, University of Hohenheim, Germany.
†Prof. J. Frey, Institute of Veterinary Bacteriology, University of Berne, Switzerland.
Phenotypic characterization of ‘environmental’ bacillus strains
All environmental isolates were checked for the absence of B. anthracis virulence genes by PCR. Gram-staining characteristics (Romeis 1989), haemolysis, motility and penicillin susceptibility were evaluated.
An inoculation loop was tipped into a bacterial colony, and the attaching cells were lysed at 56°C for 2 h in 400 μl lysis buffer (0·1 mol l−1 Tris–HCl, pH 8·0, 0·05% Tween 20, 0·24 mg ml−1 proteinase K), denatured at 95°C for 10 min and filtrated [0·2 μm (Acrodisc®, Syringe filters; Pall Corporation, Ann Arbor, MI, USA); (Perreten et al. 2005)]. DNA concentrations were determined spectrophotometrically.
PCR and sequencing
16S rDNA, rpoB and B. anthracis virulence genes were amplified by PCR. Oligonucleotides and annealing temperatures are listed in Table 2. PCRs were performed using 100 ng template DNA, the HotStarTaq Master mix (Qiagen, Hombrechtikon, Switzerland) and 0·8 μmol l−1 primers.
Table 2. Oligonucleotides and annealing temperatures
Sequences of the 16S rDNA (MWG-Biotech, Ebersberg, Germany) were aligned with databank entries using fasta and the gcg package software (http://www.bio.uzh.ch/bioc/).
Twenty-two probes target the plasmid-encoded virulence genes and five probes cover the rpoB gene of B. anthracis. Fifteen16S rDNA targeting probes were designed on sites with higher variability between the members of the B. cereus group as well as of Bacillus subtilis compared with the corresponding B. anthracis sequence. Target genes of the microarray were selected from the GenBank database or from published primer/probe sequences (Table 3; Fig. 1). Common features are length between 22 and 30 nt, melting temperature between 67 and 69°C and GC content of 35–45%. Probes were custom-spotted by Clondiag onto a glass surface integrated into the AT system. Genetic markers and gene groups are represented by at least two different probes. All probes were spotted twice.
Table 3. Oligonucleotide probes spotted on the array
Bold: nucleotide differences from the sequence of B. anthracis Ames Ancestor NC_007530, as indicated in Fig. 1
Genomic DNA was biotinylated and hybridized to the array as described by Perreten et al. (2005). Instead of QMT hybridization buffer, 3× DNA Buffer (Clondiag) was used. Prehybridization (100 μl 1× BSA/1× SSPE; 10 mmol l−1 NaHPO4, 0·18 mol l−1 NaCl, 1 mmol l−1 EDTA, pH 7·4) was performed at 50°C for 15 min.
Results were obtained by reading the microarray in an AT Reader (ATR01; Clondiag) at 25°C taking a picture after 40 min. Analysis was performed by the ionoclust software provided by Clondiag. Hybridization signals were considered to be specific if they appeared in duplicates and if the mean intensity was 0·05 or above.
Phenotypic and genotypic characterization of the Bacillus isolates
A total of 158 environmental samples were submitted to our laboratory for B. anthracis diagnostic. Out of these samples, we obtained 43 Bacillus isolates that were characterized phenotypically. Based on the phenotype, the strains were assigned to seven groups. From each group, two isolates were chosen for array hybridization (group three consisted of only one member).
The 16S rDNA of the 13 environmental Bacillus isolates that were used for hybridization were sequenced (Table 1). Additionally, all isolates were examined using a B. anthracis-specific rpoB PCR (Qi et al. 2001). PCR products were obtained for three of the isolates (M100, M101/1 and M141/11). The PCRs targeting B. anthracis virulence genes were negative for all isolates.
To evaluate the specificity of the array probes, biotinylated PCR amplicons of the capAC, capB, pag, cya, lef, rpoB and the 16S rRNA gene region of B. anthracis A73 (capsule) and B. anthracis A15 (toxin genes) were hybridized to the AT. Specific hybridization signals were observed in all experiments (Fig. 2a). Hybridization with reference strains (Table 1) revealed the expected probe signals confirming their specificity (Fig. 2b–h). Four B. anthracis field isolates were also hybridized to the array (Fig. 2g). All virulent B. anthracis strains showed positive hybridization signals with spots 1–22. Only probe 18, detecting a region on the lef gene, was negative. Bacillus anthracis strains harbouring only one virulence plasmid could be recognized and differentiated by their hybridization patterns (positive for spots 1–12 and for 13–22 indicating the presence of pXO2 and pXO1 respectively). Strain A58 (cured of both plasmids) differed from B. cereus by its hybridization pattern of the genomic rpoB gene region (spots 23–27).
Hybridization with strains obtained from environmental samples
The discriminatory power of the array for B. anthracis from other members of the genus Bacillus was tested with DNA of strains isolated from environmental samples. Thirteen Bacillus isolates representing seven different groups were tested (Table 1).
All isolates could be differentiated from virulent B. anthracis because no positive hybridization results for any B. anthracis virulence gene probes were detected. Considering the rpoB and 16S rDNA probes, M101/1 and M141/11 could be typed as B. anthracis without plasmid-encoded genes. M63/3, M63/4_2, M71/1_4 and M125/2 were identified as B. subtilis and M100 as B. cereus. This result was confirmed by 16S rDNA sequencing. For the other isolates, no species identification was possible. Figure 2i–o illustrates arrays representing each phenotypic group.
State-of-the-art diagnosis of B. anthracis in environmental samples requires simultaneous detection of chromosomal markers as well as of plasmid-encoded virulence genes. An unambiguous routine diagnosis is time-consuming and sometimes uncertain because Bacillus strains with atypical characteristics have been described recently, i.e. penicillin-resistant B. anthracis strains (Bradaric and Punda-Polic 1992; Lalitha and Thomas 1997) or B. cereus isolates harbouring the virulence plasmids (Hoffmaster et al. 2004). In this study, we developed a microarray for the parallel detection of chromosomal markers (rpoB) and the virulence genes of B. anthracis. Furthermore, 16S rDNA-specific probes were included in the array to distinguish the B. cereus group from other Bacillus species.
Instead of fluorescence labelling of multiple PCR fragments, we biotinylated the entire bacterial DNA. The advantage is that it is not necessary to establish several PCRs or a multiplex PCR and multiple and expensive labelling reactions. This minimizes the required working steps, the total costs as well as the cross-contamination risk in routine application. Using our array, a definite diagnosis is achievable within 12 h.
Specificity of our microarray was proven by hybridization of biotinylated control PCR fragments and whole DNA of well-defined Bacillus strains. Thereby, all reference strains and field isolates of B. anthracis could be identified and unambiguously differentiated from other Bacillus strains. Only probes 18, 45 and 46 did not reveal the expected hybridization signals. However, the application of the whole bacterial DNA instead of specific PCR products as a template allows customizing the probe composition on newly produced microarray batches.
Interestingly, our results indicate that the reference strain B. cereus ATCC 14579 could be distinguished from the B. anthracis strains at the 16S rDNA level. Probe 30, originally designed to differ between Bacillus thuringiensis and B. anthracis, revealed a specific hybridization signal with B. cereus ATCC 14579. No signal was obtained with B. anthracis. It remains to be elucidated whether this is true for other B. cereus strains.
The hybridization results of the environmental Bacillus isolates confirmed the discriminatory power of our array, i.e. all isolates were clearly diagnosed as non-B. anthracis or avirulent B. anthracis. The 16S rDNA sequence of the strain M100 showed the highest identity with the B. anthracis strain Ames. The DNA did not hybridize with probes 24 and 26 on the array. Thus, it was identified as B. cereus; not as B. anthracis. The isolates M101/1 and M141/11 were assigned to B. cereus and the clusters B. cereus, B. thuringiensis and B. anthracis by 16S rDNA sequencing. By hybridization, both strains could be identified as B. anthracis without virulence genes because of high similarity to the rpoB hybridization pattern of B. anthracis A58. Therefore, the array has the potential to recognize B. anthracis strains cured of the virulence plasmids that would be overseen by 16S rDNA sequencing. Such strains are of epidemiological significance (Bode et al. 2004) and important to be recognized for bio-warfare prevention.
We conclude that our array is a reliable tool for the diagnosis of B. anthracis in environmental samples. It is possible to clearly distinguish all constitutions of B. anthracis (fully virulent, harbouring only one plasmid or without plasmids). Further, B. cereus harbouring the virulence plasmids would be detected. It is applicable in routine diagnostics because of its safety and simple handling. The cross-contamination risk and therefore false-positive results are minimized. Furthermore, the application of the entire bacterial DNA as a template enables the adaption of the array layout to novel findings in B. anthracis research.
This study was funded by the canton of Zurich, Amt für Abfall, Energie und Luft (AWEL), Switzerland. We thank Vincent Perreten, Institute of Veterinary Bacteriology, University of Berne, Switzerland, for the fruitful discussions concerning the biotinylation of the sample DNA. The strains B. anthracis A15 and B. anthracis A73 were kindly provided by Prof. R. Böhm, University of Hohenheim, Germany; the strains B. anthracis Sterne and B. anthracis A58 were obtained from Prof. J. Frey, Institute of Veterinary Bacteriology, University of Berne, Switzerland.