Simple and rapid method for detection of bacterial spores in powder useful for first responders.

Introduction

In October of 2001, several letters containing Bacillus anthracis spores were sent through the U.S. Postal Service to recipients in government buildings in Washington, D.C., and in private office buildings (Atlas, 2002). Twenty-three cases of human anthrax infection occurred. Five of them were fatal. As a result of this terrorist act, several post offices, mailrooms in government buildings, and private office buildings were contaminated with B. anthracis spores.

The response to this incident highlighted the need for a reliable, rapid method of detecting Bacillus spores. Since it takes several days for laboratories to determine the existence of anthrax spores with typical culture-based analysis, anthrax hoaxes place a very heavy burden on public health laboratories. A simple and rapid screening procedure that could detect spores in an unidentified powder would reduce the workload of the laboratories significantly. Appropriate actions could be taken immediately upon detection of the spores.

B. anthracis is an aerobic, Gram-positive, spore-forming, nonmotile Bacillus species (Mock & Founet, 2001). Its spores germinate when they are in an environment rich in amino acids, nucleosides, and glucose, such as the blood or tissues of an animal or human host (Ireland & Hanna, 2002). By contrast, in harsh environmental conditions, such as boiling, freezing, desiccation, and nutrient exhaustion, vegetative Bacillus species will turn into spores; the spores can survive for decades in adverse ambient conditions (Titball, Turnball, & Hutson, 1991; Williams, 1986).

Current methods of bacterial-spore detection, such as colony counting or polymerase chain reaction (PCR), require more than a working day to provide results (Lester & Ponce, 2002). To prevent the spread of toxic spores from the environment to a human host, a test is needed that determines the existence of spores in a very short time so that corrective actions can be taken accordingly. An adenosine triphosphate (ATP) bioluminescence assay can satisfy the need to detect the presence of viable spores in near real time. The ATP bioluminescence assay allows estimation of viable bacterial cells within minutes (Deininger & Lee, 2001). Bacterial spores are, however, deficient in ATP and are almost undetectable by a regular bioluminescence technique. Therefore, they need to be germinated to a vegetative state by a nutrient addition (Stopa, Tieman, Coon, Milton, & Paterno, 1999). The purpose of this study was to find the best and fastest heat shock conditions that trigger the breakdown of endospore dormancy of Bacillus and give a higher luminescence signal. The variables include germination time, temperature, nutrient type, and nutrient concentration.

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Methods

The laboratory used in the study reported here was not allowed to have B. anthracis spores; therefore, the study focused on the use of brain heart infusion (BHI) (Becton Dickinson, Sparks, Maryland) and tryptic soy broth (TSB) (Becton Dickinson, Sparks, Maryland) as nutrients for the germination of Bt spores in the endospore stage. B. thuringiensis (Bt) is not pathogenic to human beings and is available in a powder form, so it can be used as a surrogate for B. anthracis. Before the nutrients were added to a powder sample, they were warmed and kept at a specific temperature (either 37[degrees]C or 55[degrees]C) to promote optimal germination. Dipel 150 Dust (Bonide Products, Yorkville, New York), which contains biologically active Bt spores (0.064 percent), was used in the experiments.

One milligram of powder that contained Bt spores was weighed and transferred into a sterile micro-centrifuge tube, and 1 mL of nutrient (preheated to 37[degrees]C or 55[degrees]C) was added to germinate the spores. Since many variables affect the germination rate of spores, different sets of conditions were tested: 1) temperature (37[degrees]C versus 55[degrees]C, with heating plates and heating blocks filled with sand); 2) germination time (2, 5, or 15 min); 3) nutrient type (BHI versus TSB); and 4) nutrient concentration (1/2X, 1X, or 2X strength).

The equipment needed for the ATP bioluminescence assay consists of a luminometer (Model 3560), Filtravette[TM], somatic cell-releasing agent (SRA), bacterial cell-releasing agent (BRA), and luciferine-luciferase (LL) (New Horizons Diagnostics, Columbia, Maryland). The Filtravette[TM] is a small cuvette with a filter membrane (0.45-[micro]m pore size) at the bottom; it enables filtration and measurements of light development at the same time. A luminometer measures the amount of light, which is produced by the reaction of ATP with Bacillus cells and LL, by counting the number of light flashes from the photo multiplier tube in the device. The light development is proportional to the number of Bacillus cells.

A certain volume (50-100 [micro]L) of the spore-containing solution was transferred to Filtravette, and the liquid was filtered through a gentle air pressure device. SRA (300 [micro]L) was added and was filtered so that nonbacterial cells would be removed while the bacteria remained intact (Lee & Deininger, 1999). The Filtravette, which retained the bacilli on the surface, was placed into the sample chamber of the luminometer. BRA (50 [micro]L) was added to lyse the bacterial cells, then 50 [micro]L of LL was added. The sample chamber was then closed, and the amount of light emission was measured. Relative-luminescence units (RLUs) were recorded from the luminometer proportional to the amount of extracellular ATP that originated from the bacteria in a sample (Lee & Deininger, 1999). ATP assay tests were conducted in triplicate for each germination condition. The results were converted to RLUs per milligram of powder after division by the amount used.

Germinated spores were also enumerated by pour plate count method with either BHI agar or TSA, depending on the type of germination nutrient used in the spore activation procedure. The plates were incubated at 30[degrees]C for 24 hours. Phosphatebuffered saline (pH 7.4, Sigma, St. Louis, Missouri) was used as a diluent. Pour plate count tests were done in triplicate for each germination condition.

L-alanine was used as a germinant during the study (data not shown) since it had been reported that L-alanine could initiate germination of B. anthracis (Ireland & Hanna, 2002). In the authors' tests, however, L-alanine did not germinate spores.

Results and Discussion

The results showed BHI to be more effective than TSB in germination of Bt spores at both temperatures, 37[degrees]C and 55[degrees]C, during the germination period of 2 to 15 minutes at regular concentration (IX) (Figure 1). At 37[degrees]C, as germination time increased, more Bt spores became vegetative cells with any type of germinant. The highest biological activity was obtained after 15 minutes. When the temperature was 55[degrees]C, however, this phenomenon was not observed. At 55[degrees]C, the highest biological activity of Bt spores was observed after 5 minutes, and further germination (15 min) decreased both the colony-forming-unit (CFU) and relative-light-unit (RLU) signals.

The effect of double-strength nutrients on the germination of Bt spores is delineated in Figure 2. The experiments aimed to examine whether a concentrated nutrient could effectively accelerate the germination of Bt. Compared with regular-strength nutrients, double-strength nutrients seemed to work rather ineffectively in inducing Bt spore germination. An evident increase of biological activity of Bt spores during the germination periods was not observed (Figure 2). From the results shown in Figure 1 and Figure 2, it is clear that at 37[degrees]C, Bt spore germination increased steadily with an increase of germination period up to 15 minutes, but at 55[degrees]C, the prolonged germination period did not necessarily support further germination. Thus, it is advisable to measure germinated Bt spores after 5 min when an elevated temperature is used. The results from the luminescence method and the pour plate count method agreed well in the number of spores found. The correlation coefficients between the two methods were 0.70 for both BHI and TSB.

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The detection limit of the method was investigated with liquid-phase Bt spores. Concentration started at 2,000 spores, and it was serially diluted until it reached less than 100 spores. The diluted spores were germinated with BHI at 37[degrees]C for 15 minutes. Concentrations of spores were determined with ATP assay and verified with the BHI agar method in triplicate. The control set was not added with the germinant. The result in Figure 3 indicates that the magnitude of the amplified RLU values, after the germination treatment, is proportional to the number of spores originally present in the sample. The RLU results were very low (<100) without the germination treatment, regardless of the number of spores present in the sample. Thus, a higher RLU increase after the germination treatment means that more viable spores are present. The result also shows that the heat shock and luminescence method can detect 15 bacterial spores in quantities as low as 15. This detection limit is a significant improvement over current test methods such as the PCR method, which have detection limits on the order of [10.sup.3] spores (Higgins et al., 2003). The ATP assay method can be a useful tool to screen for the presence of bacterial spores in a powder sample within minutes of sampling and to check the viability of spores in combination with other fast methods (e.g., PCR). All necessary equipment is portable, and the germinants can easily be warmed with a small portable incubator or a hand warmer.

Conclusion

The goal of the study reported here was to find the optimal germination conditions for the rapid detection of B. thuringiensis spores. Since the ATP test is rapid, sensitive, portable, and inexpensive, it can be used for on-site detection when a questionable powder is found. The optimum conditions for spore germination can be summarized as follows: 1) 37[degrees]C as a germination temperature, 2) 15 minutes as a germination time, and 3) single-strength solution of brain heart infusion as a nutrient. TSB can serve as a good alternative, however, and 5 minutes of germination time seems to be long enough to identify an increase in the biological activity of spores. The detection limit is less than 100 spores. Therefore, employing this rapid detection method will give a positive confirmation of viable spores in less time than traditional methods, at a higher level of sensitivity, and on site.

Acknowledgements: This study was partially funded by the Michigan Department of Community Health Bioterrorism Research Fund.

Corresponding Author: JiYoung Lee, Research Investigator, University of Michigan, Department of Environmental Health Sciences, 109 S. Observatory, Ann Arbor, MI 48109-2029. E-mail: [e-mail].

REFERENCES

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Jungwon Min, M.S.

JiYoung Lee, M.S., Ph.D.

Rolf A. Deininger, M.S., Ph.D.