Kenneth D. Cole, Biochemical Science Division, Mailstop 8312, NIST, 100 Bureau Drive, Gaithersburg, MD 20899, USA. E-mail: email@example.com
Aim: To determine the stability and variability in concentration of spore suspensions of Bacillus anthracis (BA) spore suspensions by comparing different methods of enumeration and to detect changes, if any, under different storage conditions.
Methods and Results: Plate and microscope counts were compared to measuring the genomic equivalents based on DNA content BA spore suspensions. We developed chemical methods to extract spore DNA and extra-spore (ES) DNA. DNA mass was determined by gel electrophoresis and QPCR assays were developed using the markers on the chromosome (rpoB) and the pXO1 plasmid (pag). The plate counts and microscope counts were very stable (for up to 900 days). The effect of freezing and the presence of additives in samples were tested for up to 300 days, and the results indicated that the additives tested and freezing did not decrease the viability or microscope counts.
Conclusions: Bacillus anthracis spore suspensions can be stored for long periods of time without significant loss of viability or clumping. The content of ES DNA was variable and changed with time.
Significant and Impact of the Study: The study shows that BA spore suspensions can be developed for reference materials providing a uniform basis for comparing detection equipment and results from different laboratories.
Reference materials for Bacillus anthracis (BA) spores are needed to test detection devices and to calibrate laboratory instruments. The bioactivity, purity and concentration are essential properties of spore reference materials that need to be measured accurately and we are studying the best ways to determine these properties (Almeida et al. 2006). Uniform well-characterized reference materials for BA that are stable with time will facilitate the comparison of new detection devices and increase the confidence in the results obtained from existing monitoring devices and different laboratories.
Counting the bacterial colonies after growth on solid nutrient media is a classical microbiological method that yields valuable data on the biological activity of the samples, but only the viable cells are counted and plate counts underestimate the true cell number, if the cells are clumped. Another approach is to measure the genomic equivalents. The Mr of the BA chromosome and the plasmids (pXO1 and pXO2) can be accurately calculated based on the sequences (Okinaka et al. 1999a,b; Read et al. 2003). The Mr can then be used to calculate the genomic equivalents of the chromosome and plasmids. DNA markers for the chromosome (rpoB, present as a single copy) (Mollet et al. 1997) (Qi et al. 2001) and the two plasmids pXO1 (pag) and pXO2 (capC) were used to measure the genomic equivalents. Viable spores are not the only source of DNA; damaged (nonviable) spores and vegetative cells (or fragments) will contain DNA that may also be present. Extreme conditions must be employed to release the DNA from spores (Marmur 1961; Johns et al. 1994; Belgrader et al. 1999). Comparison of a real-time PCR instrument that incorporates automated spore lysis and DNA isolation steps showed the value of liberating the DNA for increased detection of BA spores (Ulrich et al. 2006). Physical disruption methods reduce the size of the DNA by shearing, which is satisfactory for assays that do not require high-molecular weight DNA, such as PCR.
We developed a procedure to extract the DNA from intact spores based on previous studies that used chemical and enzymatic dissolution treatments to disrupt spores to release and purify the DNA (Marmur 1961; Takahashi 1964; Tabatabai and Walker 1967; Miller et al. 1988). Using less stringent dissolutions conditions, we used an extraction method to recover DNA from vegetative cells, cell debris and damaged spores. This DNA fraction was referred to as the extra-spore (ES) DNA. Rupturing the spores by beating with beads was also compared in some cases. The bead beating method was the least selective in terms of removing ES DNA as the sample resulting from entire spore suspension is directly used for analysis.
The goals of this study are to determine the stability of the BA spore suspensions under different storage conditions and the best methods for their characterization for the development of reference materials. It is essential that the viability and the physical form (lack of clumping) of these materials remain consistent over their lifetime. Information about stability of BA spores with storage is incomplete and difficult to compare without the proper controls.
Materials and methods
Spore suspensions, plate counting, microscope counting and storage conditions
Spore suspensions of BA (Sterne strain) prepared at US Army Dugway Proving Ground (Dugway, UT, USA) were washed with water, and stored in sterile water at 4°C as concentrated stocks. A sample of gamma-irradiated BA (Ames, 39·3 kGy for 140 min on 24 February, 2005) was used as a positive control for the pXO2 plasmid DNA (prepared at U.S. Army Dugway Proving Ground, Dugway, UT, USA). The spore samples were diluted with phosphate-buffered saline (PBS, 10 mmol l−1 phosphate, 138 mmol l−1 NaCl, 2·7 mmol l−1 KCl, pH 7·4) containing 0·01% Triton X-100 (Luna et al. 2003), and vigorously mixed by vortexing. Samples were plated on LB plates. Disposable plastic cell-counting chambers were obtained from Nexcelom Bioscience (Lawrence, MA, USA)1 and phase bright spores counted at 400× magnification using phase contrast optics.
A portion of BA lot 3 was used to test the effect of different storage conditions on stability. The lot was diluted and dispensed into multiple sterile vials (4-ml borosilicate glass with a PTFE lined cap, Wheaton #W224582, Millville, NJ, USA). Concentrated additives were added for a final volume of 1. 2 ml. The following samples were stored at 4°C with the additives and final concentrations: ethanol 20% (v/v), ethylendediamainetetraacetic acid (EDTA) 10 mmol l–1 pH 8·0, phenol 1% (v/v), PBS containing 0·01% (v/v) Triton ×100, and controls in sterile water. In addition samples in sterile water were frozen at −20°C and −80°C. Samples to be frozen were mixed and then placed on the racks in the freezer. Samples were used only once for each time point.
DNA was extracted from spores using the following procedure.
A fresh solution of 8 mol l−1 urea, 10 g l−1 sodium dodecyl sulphate (SDS), 50 mmol l−1 dithiothreitol (DTT) was prepared by dissolving the components in TE buffer, pH 8·0 (10 mmol l−1 Tris and 1 mmol l−1 EDTA. A spore preparation (containing approx. 107 spores) was concentrated by centrifugation (5 min at 16 000 g). The supernatant was removed and the pellet was suspended in 200 μl of the urea/SDS/DTT solution by vortexing and the sample was incubated at 65°C for 90 min (vortexed each 30 min). The de-coated spores were recovered by centrifugation (5 min) and the supernatant removed. A solution of 200 μl of PBS was added to the side of the tube, being careful not to disturb the pellet and the tube was centrifuged (5 min) and the supernatant liquid removed. This PBS washing step was repeated. The pellet was suspended in 180 μl of lysozyme (20 mg ml−1 in TE) by vortexing and the sample was incubated at 37°C for 60 min. The samples were vortexed after 30 min of incubation during this step. Proteinase K digestion was done by adding 103 μl of TE buffer, 15 μl of 100 g l−1 SDS, and 2 μl of proteinase K (20 mg ml−1 in water) and the samples were incubated at 60°C for 60 min. TE buffer (280 μl) and solid NaCl (117 mg) were added to each sample to obtain a final concentration of 5 mol l−1 in a final volume of 400 μl. The samples were vortexed until fully dissolved. The samples were centrifuged (5 min) to remove insoluble proteins and carbohydrates. The supernatant liquid was carefully removed and transferred to a fresh microfuge tube. An equal volume (400 μl) of 10 mmol l−1 EDTA pH 8·0 was added to the samples, followed by 480 μl of isopropanol (0·6 volume) and the samples were mixed and incubated on ice for 45 min. The DNA was recovered by centrifugation (10 min), the pellet was air dried, suspended in 100 μl of TE and stored at 4°C.
DNA was also prepared by a bead beating method using a commercial apparatus (Mini Bead Beater 8, Biospec Products Inc., Bartlesville, OK, USA). Zirconia/silica beads (2·5 g from Biospec Products,#11079101Z, 0·1-mm diameter) were weighted into 2-ml plastic vials with screw caps. The beads were washed three times with 1·5 ml of TE buffer. The spore samples (0·1 ml containing approx. 8 × 106 spores) were added to 1·4 ml TE in the tubes for a final volume of 1·5 ml. The samples were sealed then shaken at maximum speed for 5 min. The beads were allowed to settle briefly and an aliquot of the supernatant removed and stored at −20°C prior to analysis by QPCR.
DNA measurement by gel electrophoresis and QPCR
The DNA samples were analysed by agarose gel electrophoresis using 45 mmol l−1 TRIS, 45 mmol l−1 boric acid and 1 mmol l−1 EDTA, buffer pH 8·3 using slab gels (0·8 gm agarose per 100 ml) run for 2 h at 6 V cm−1. The gels were stained with ethidium bromide (1 μg ml−1) for 1 h and rinsed twice with deionized water for 10 min.
We constructed plasmids containing the cloned PCR products to calibrate the QPCR assays. The PCR products from the gene markers listed in Table 1 were cloned into TOPO plasmids (Invitrogen, Carlsbad, CA, USA). We used a different reverse primer for the capC marker resulting in an amplicon length of 166 basepairs (compared to 291 basepairs in the original publication) and changed the annealing-polymerization temperature to 56°C. These changes resulted in increased fluorescence signals and improved the efficiency for the capC assay in our hands. The plasmids were purified using Qiagen plasmid purification kits (Qiagen, Valencia, CA, USA). Concentrations were determined by absorbance at 260 nm [assuming 1 optical density unit is equivalent to a DNA concentration of 50 μg ml−1 (Beaven et al. 1955)]. The identities of the PCR inserts in the plasmids were confirmed by DNA sequencing. The plasmid containing the capC PCR product (Table 1) had a single base pair difference when compared with the published sequence for BA at position 187 of the PCR product (substitution of a G for an A). This was not further investigated as it did not change the binding of the primers and the probe.
Table 1. DNA primers and probes used for the QPCR assays
Gene target (amplicon size)
The primers and probe sequences with the exception of Cap R-1 are from Ellerbrok et al. (Ellerbrok et al. 2002). GenBank accession numbers refer to Bacillus anthracis (BA) Sterne (AEO17225) for rpoB, BA plasmid pXO1 (AF065404) for pag and BA plasmid pXO2 (AF188935.1) for cap C marker.
The plasmids were linearized before use by digestion with EcoRI (1 h at 37°C followed by 65°C for 20 min). An ABI Prism 7000 (ABI, Foster City, CA, USA) instrument was used with TaqMan type probes. The probes were labelled with 5′ Reporter–6FAM (fluorescein) and 3′ Quencher-TAMRA. Mastermix was from Invitrogen (Carlsbad, CA, USA). The PCR conditions for rpoB and pag were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The capC reactions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 56°C for 1 min.
Stability under different storage conditions
Figure 1 shows the plate counts and microscope counts with time for the three lots of BA spores. These stocks were stored at 4°C in sterile water at high concentrations (shown in Fig. 1) in polypropylene tubes. The biological activity (viability) and microscope counts for the three lots did not significantly differ in the two methods and did not change during the time period examined. A portion of lot 3 was diluted and dispensed into multiple glass vials containing additives. The additives chosen are those commonly used to prevent the growth of contaminants or to explore different solution properties (low ionic strength vs high ionic strength vs the presence of an organic solvent). The results from these samples (Fig. 2) show that the additives or freezing the samples did not change the viability or result in cell clumping.
Measurement of genomic equivalents by QPCR
A spore DNA fraction was prepared by removal of the outer spore coat using urea, detergent and a reducing agent. The de-coated spores were recovered by centrifugation, so any DNA associated with the surface coats of spores or debris would be removed from the spore DNA fraction. A second, less stringent, extraction method was developed by omitting the spore coat dissolution step. The DNA prepared from BA (Sterne) spores using the chemical extraction method had a high molecular weight (approximately the size of the 48·5 kbp standard) and was sufficiently pure to be measured by gel electrophoresis (results not shown). The QPCR assays were calibrated by the use of plasmid DNA. Figure 3 shows examples of the standard curves (generated using the plasmid templates), run each time samples of spore DNA were analysed. The slope of the threshold cycle number (CT) plotted as a function of the log of the copy number (in this case dilution of the sample) should be close to the value of –3·32, corresponding to a doubling of the DNA product in each cycle of the PCR reaction (Bio-Rad 2005) and only runs that had values close to this were used in this study. A sample of virulent BA (Ames) spores inactivated by gamma irradiation was used as a positive control for the capC gene (marker for the pXO2 plasmid). The DNA extracted from this sample served as a positive control in the QPCR assays yielding slopes close to the –3·32 value (results not shown). As expected, the BA Sterne DNA was negative with the assay for the capC marker.
The genomic equivalents determined from the extracted DNA by mass and QPCR assays were compared to the plate and microscope counts for the three lots (Fig. 4). The QPCR data show a close correspondence between the copy number of the chromosomal marker (rpoB) and the pXO1 (pag) in BA (Sterne). The DNA extraction method gave higher genomic equivalent values compared to the plate counts by 2·2-, 2·2- and 1·5-fold for lots 1, 2, and 3, respectively. The differences between the chromosomal marker values and the plate counts for each of the three lots were significant as determined by an unpaired Student’s t-test (probability that they are significantly different of 0·997, 1·000, and 1·000 for lots 1, 2, and 3, respectively). The QPCR values obtained for the plasmid marker (pag) concentration was higher compared with the chromosomal marker (rpoB) concentration by 1·4-, 1·5- and 1·2-fold for lots 1, 2, and 3, respectively, using DNA extracted with the chemical method (Fig. 4).
The ES DNA contents of the lots were measured by chemical extraction and the QPCR assays. In this extraction method, the spores were collected by centrifugation and the extraction started at the lysozyme incubation step (the spore coat extraction step was omitted). The ES content of lot 3 was determined at beginning of the study and after 182 days, these values are shown in Fig. 5. The spore fraction of DNA determined by the chromosomal marker (rpoB) and the pXO1 (pag) marker did not change at 182 days, but the ES content as measured by both markers significantly decreased during this period. When the ES chromosomal DNA concentrations were expressed as a percentage of the spore chromosomal DNA concentrations, the values decreased from 18·0% (initial value) to 7·7% (182-day value).
We also used bead beating to extract DNA from diluted samples of lot 3 to compare to the results obtained with the chemical extraction method (Fig. 5). These results show that the initial DNA content obtained with bead beating was higher than the results obtained with the chemical extraction method; however, after storage at 4°C for 182 days, the values decreased to levels very similar to those obtained with the chemical extraction method (Fig. 5). The bead beating method is the least selective of the DNA extraction methods, as the concentrated spore suspensions was added directly to the beads and the resulting broken spores were used for the QPCR assays without any washing or purifications steps.
The additives or freezing did not significantly decrease the viability of the spores or result in clumping under the conditions tested. The use of additives or freezing will be especially helpful to prevent the growth of contaminants in reference materials that will be used frequently to calibrate instruments or used as positive controls in detectors. This study did not address the effect of repeated freeze-thaw cycles, as in this study each sample was frozen and thawed only once. The concentration of spores in the solution and the rate of freezing of the solution are important variables that may influence clumping of spores during storage of frozen samples. Additional studies are needed to determine the comprehensive effects of freezing conditions on stability.
A study examined the chromosome copy number per spore in different species of Bacillus by measuring the DNA content of spores (Hauser and Karamata 1992). Their data indicated that the spores of B. subtilis were monogenomic, while the spores of B. megaterium, B. cereus and B. thuringiensis contained two copies of the chromosome (based on the measurements of DNA content per spore). BA being a member of B. cereus family would be expected to behave in a similar manner to B. cereus and B. thuringiensis spores. A recent study measured the copy number of the plasmids and the chromosome in vegetative cells of different isolates of BA (Coker et al. 2003). They found using QPCR measurements that the copy number of the chromosome marker was six per vegetative cell, the copies of pXO1 plasmid ranged from 33 to 243 per cell, and the copies of pXO2 plasmid ranged from 1 to 32 per cell. Our data showed that the copy number of the pXO1 plasmid was similar (slightly higher) to the chromosome copy. Our data indicated that higher genomic DNA content compared to plate or microscope counts, but definitive measurements of the chromosome copy number per spore would best be done with measurements that directly measure the DNA content of individual spores and not large populations. The methods developed in this study for DNA extraction and calculations of genomic equivalents are a valuable adjunct to the classical methods for the characterization of spore reference materials. The genomic equivalents determined from spore DNA would reflect any additional contribution because of spore clumping and the presence of nonviable spores.
Other studies have shown that the ES DNA fraction is the source of the PCR products when intact spores are used in PCR assays (Johns et al. 1994). One study found that the ES DNA was not removed by simple washing (water and salt solutions), but enzymatic treatment, mechanical treatment and gradient purification of the spores reduced the amount of ES DNA, but did not entirely eliminate it (Belgrader et al. 1999). Our measurements show that the content of the ES DNA was not stable with time and cannot be relied upon as a reliable measurement characteristic.
Certain commercial equipment, instruments, or materials are identified in this paper to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.
We wish to thank Dr Bert Coursey (Department of Homeland Security) for the support of this project. We received helpful advice concerning QPCR from Joe Blasic, Marcia Holden and Margaret Kline (NIST).