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A Novel Surfactant Nanoemulsion with Broad-Spectrum Sporicidal Activity against Bacillus Species

  1. Tarek Hamouda1,
  2. Michael M. Hayes1,a,
  3. Zhengyi Cao1,
  4. Richard Tonda1,
  5. Kent Johnson2,
  6. D. Craig Wright3,
  7. Joan Brisker3 and
  8. James R. Baker Jr.1
  1. 1Center for Biologic Nanotechnology and Department of Medicine, University of Michigan Medical School, Ann Arbor
  2. 2Department of Pathology, University of Michigan Medical School, Ann Arbor
  3. 3NOVAVAX, Inc., Rockville, Maryland
  1. Reprints or correspondence: Dr. James R. Baker, Jr., University of Michigan Medical School, 9220 MSRB-III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0648 (jbakerjr{at}umich.edu).

  1. Presented in part: 98th general meeting of the American Society for Microbiology, Atlanta, May 1998 (poster A49); 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, September 1998 (late-breaker slide session II, LB-9); 99th general meeting of the American Society for Microbiology, Chicago, May 1999 (poster A300).

  • ↵a Present affiliation: Pulmonary/Critical Care Unit-Internal Medicine, University of Michigan, Ann Arbor.

 
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Abstract

Two nontoxic, antimicrobial nanoemulsions, BCTP and BCTP 401, have been developed. These emulsions are composed of detergents and oils in 80% water. BCTP diluted up to 1 : 1000 inactivated 190% of Bacillus anthracis spores in 4 h and was also sporicidal against three other Bacillus species. This sporicidal activity is due to disruption of the spore coat after initiation of germination without complete outgrowth. BCTP 401 diluted 1 : 1000 had greater activity than BCTP against Bacillus spores and had an onset of action of <30 min. Mixing BCTP or BCTP 401 with Bacillus cereus prior to subcutaneous injection in mice reduced the resulting skin lesion by 99%. Wound irrigation with BCTP 1 h after spore inoculation yielded a 98% reduction in skin lesion size, and mortality was reduced 3-fold. These nanoemulsion formulas are stable, easily dispersed, nonirritant, and nontoxic compared with other available sporicidal agents.

Bacteria of the Bacillus genus form stable spores that are resistant to harsh conditions and extreme temperatures. Contamination of farmlands with Bacillus anthracis leads to a fatal disease in domestic, agricultural, and wild animals [1]. Human infection by B. anthracis usually results from contact with infected animals or infected animal products [2]. Human clinical symptoms include a pulmonary form that has a rapid onset and is frequently fatal. The gastrointestinal and cutaneous forms of anthrax, although less rapid, can also result in fatalities unless treated aggressively [3, 4]. B. anthracis infection in humans is no longer common, because of effective animal control that includes vaccines, antibiotics, and appropriate disposal of infected livestock. However, animal anthrax still represents a significant problem because of contamination of farmland. Although a vaccine is available [5] and can be used for the prevention of anthrax, genetic mixing of different strains can render it ineffective [6]. The potential consequences of the use of B. anthracis spores as a biologic weapon were demonstrated by the accidental release of B. anthracis from a military microbiology laboratory in the former Soviet Union. Seventy-seven cases of human anthrax, including 66 deaths, were attributed to the accident. Some infections occurred as far as 4 km from the laboratory [7]. Genetic analysis of infected persons revealed the presence of either multiple strains or genetically altered B. anthracis [8].

Other members of the Bacillus genus are also reported to be etiologic agents for many human diseases. B. cereus is a common pathogen. It is involved in foodborne diseases because its spores can survive cooking procedures. Local sepsis and wound and systemic infections have also been attributed to B. cereus [9].

Disinfectants and biocides (e.g., sodium hypochlorite, formaldehyde, and phenols) that are highly effective against Bacillus spores are not well suited for decontamination of the environment, equipment, or exposed persons because of toxicity that leads to tissue necrosis and severe pulmonary injury after inhalation of volatile fumes. The corrosive nature of these compounds also renders them unsuitable for decontamination of sensitive equipment [10–15].

Concerns about these issues have stimulated interest in new types of biocidal agents that can safely decontaminate Bacillus spores. We have investigated the sporicidal properties of two antimicrobial lipid emulsions. Nanoemulsions are produced by mixing a lipid-oil “discontinuous” phase with an aqueous “continuous” phase under high shear forces. The result is an oil droplet of ∼400–800 µm in diameter that is able to fuse with and subsequently disrupt the membrane of a variety of different pathogens [16]. BCTP is a nanoemulsion made of soybean oil, Triton X-100 detergent, and tri-n-butyl phosphate in 20% water. BCTP 401 is a mixture of this emulsion and a liposome, P10. P10 is made of water, Tween 60, soybean oil, glycerol monooleate, refined soya sterols, and the cationic compound cetyl-pyridinium chloride. These two compounds have antimicrobial activity against enveloped viruses and bacteria through membrane disruption (unpublished data). In the current studies, we examined the ability of these emulsions to inactivate different Bacillus spores.

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Materials and Methods

Surfactant lipid preparations

BCTP is a water-in-oil nanoemulsion, in which the oil phase is made from soybean oil, tri-n-butyl phosphate, and Triton X-100. Stock solutions contained 80% lipid components and 20% water. Three different preparations of BCTP, 2, 8, and 16 months old, were tested for their stability. BCTP 401 was prepared by mixing equal volumes of BCTP with P10, the latter being a liposome-like compound. P10 is made of glycerol monosterate, refined soya sterols, Tween 60, soybean oil, a cationic ion halogen-containing cetylpyridinium chloride, and peppermint oil. The average size of these nanoemulsions is in the range of 400–800 nm, as determined by laser light scatter (LS230; Coulter, Hialeah, FL). These surfactant lipid preparations were stable after boiling for 1 h or exposure to 1 N nitric acid or 1 N sodium hydroxide for 2 h. This treatment resulted in a <20% reduction in the emulsion mean particle size [16]. These solutions were stored at room temperature and were diluted before each experiment to the working dilution. All dilutions herein are in reference to the stock solution.

Spore preparation

For induction of spore formation, B. cereus (ATCC 14579), B. circulans (ATCC 4513), B. megaterium (ATCC 14581), and B. subtilis (ATCC 11774) were grown for 1 week at 37°C on nutrient agar with 0.1% yeast extract and 5 mg/L MnSO4. The plates were scraped, and the bacteria and spores were suspended in sterile 50% ethanol and incubated at 22°C for 2 h with agitation to lyse the remaining vegetative bacteria. The suspension was centrifuged at 2500 g for 20 min, and the pellet was washed twice in cold distilled water. The spore pellet was resuspended in trypticase soy broth (TSB) and used immediately for experiments. B. anthracis spores, Ames and Vollum 1B strains, were supplied by Bruce Ivins (US Army Medical Research Institute of Infectious Diseases [USAMRIID], Fort Detrick, Frederick, MD) and were prepared as described elsewhere [5]. Four other strains of B. anthracis were provided by Martin Hugh-Jones (Louisiana State University, Baton Rouge). These strains (from South Africa; Mozambique; Bison, Canada; and Del Rio, TX) represent isolates with high allelic dissimilarity.

In vitro sporicidal assays

For assessment of sporicidal activity on solid medium, trypticase soy agar (TSA) was autoclaved and cooled to 55°C. BCTP was added to the TSA at a 1 : 100 final dilution and continuously stirred while the plates were poured. The spore preparations were serially diluted (10-fold), and 10-µL aliquots were plated in duplicate (highest inoculum, 105 spores/plate). Plates were incubated for 48 h aerobically at 37°C and evaluated for growth.

For assessment of sporicidal activity in liquid medium, spores were resuspended in TSB. Next, 1 mL of spore suspension containing 2 × 106 spores (final concentration, 106 spores/mL) was mixed with 1 mL of BCTP or BCTP 401 (at 2× final concentration in distilled water) in a test tube. The tubes were incubated in a tube rotator at 37°C for 4 h. Treatment of B. anthracis was done at 37°C, which promotes spore germination, and at 22°C, which does not promote spore germination [5]. After treatment, the suspensions were diluted 10-fold in distilled water. Duplicate aliquots from each dilution were then streaked on TSA and incubated overnight at 37°C; then colonies were counted. Sporicidal activity expressed as percentage of killing was calculated as follows: {[cfu(initial) —- cfu(posttreatment)]/[cfu(initial)]} × 100.

The experiments were repeated at least 3 times, and the mean and SE of the percentage of killing were calculated by use of StatView software (Abacus Concepts, Berkeley, CA). Analysis of variance tables and paired t test were used when applicable.

Electron microscopy

B. cereus spores were treated with BCTP at a final dilution of 1 : 100 in TSB by means of Erlenmeyer flasks in a 37°C shaker incubator. The spore-BCTP mixture was washed with saline and centrifuged at 2500 g for 20 min, and the supernatant was discarded. The pellet was fixed in 4% glutaraldehyde in 0.1 M cacodylate (pH 7.3). Spore pellets were processed for transmission electron microscopy, and thin sections were examined after staining with uranyl acetate and lead citrate.

Germination inhibitors or enhancers

B. cereus spores (final concentration, 106 spores/mL) were suspended in TSB with either the germination inhibitor D-alanine (final concentration, 10 mM) or the germination enhancer l-alanine (final concentration, 5 mM) [17–19]. This suspension was then immediately mixed with BCTP (final dilution, 1 : 100) and incubated for variable intervals. Then the mixtures were serially diluted, plated, and incubated overnight. The next day, growth on the plates was counted, and the percentage of sporicidal activity was calculated.

In vivo toxicity testing

Mice were exposed to various concentrations of the different emulsions by means of different routes of administration. The highest concentrations that produced no gross or histopathologic lesions in mice were reported. Exposures included subcutaneous or intramuscular injection of 100 µL, open wound irrigation with 2 mL of the emulsions, and intranasal instillation of 25 µL/naris. The emulsions are relatively viscous when not diluted, so toxicity testing in the nares was conducted at the highest concentration that would not suffocate the animals. Three to four mice were tested for each concentration of each compound, and the experiments were repeated on at least three occasions.

In vivo sporicidal activity

Two animal models were developed to confirm the sporicidal activity of the emulsions in vivo. In the first model, B. cereus spores (suspended in sterile saline) were mixed with an equal volume of BCTP to a final emulsion dilution of 1 : 10. As a control, the same B. cereus spore suspension was mixed with an equal volume of sterile saline. Next, 100 µL of each of the suspensions, containing 4 × 107 spores, was then immediately injected subcutaneously into CD-1 mice. Nine mice were inoculated in each group, and the experiment was repeated on three different occasions.

In the second model, a simulated wound was created by making an incision in the skin on the back of the mice. The skin was separated from the underlying muscle by blunt dissection. The pocket was inoculated with 200 µL of saline containing 2.5 × 107 spores and closed by use of wound clips. One hour later, the clips were removed, and the wound was irrigated either with 2 mL of sterile saline or with 2 mL of BCTP (1 : 10 in sterile saline). The wounds were then closed with wound clips. The animals were observed for clinical signs. Gross and histopathologic examination were done when the animals were euthanized 5 days later. The wound size was calculated by the following formula: 1/2a × 1/2b × π, where a and b are two perpendicular diameters of the wound. Five mice were used in each group, and the experiment was repeated on three different occasions. Both sets of animal studies were also conducted with BCTP 401 at identical dilutions.

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Results

In vitro sporicidal activity

To assess the sporicidal activity of BCTP, spores from four species of Bacillus genus (B. cereus, B. circulans, B. megaterium, and B. subtilis) were tested. BCTP at a 1 : 100 dilution showed 97% sporicidal activity against B. cereus and B. megaterium in 4 h (figure 1). B. circulans was less sensitive to BCTP, showing only an 83% reduction in spore count, whereas B. subtilis appeared resistant to BCTP in 4 h. The other nanoemulsion, BCTP 401, was more efficient in killing the Bacillus spores. At a 1 : 1000 dilution, it showed 99% killing of B. cereus spores in 4 h (compared with 50% with a 1 : 1000 dilution of BCTP). BCTP 401 at a 1 : 1000 dilution resulted in 96% killing of B. subtilis spores in 4 h, in contrast to its resistance to BCTP. Bleach diluted 1 : 100 (i.e., 0.0525% sodium hypochlorite) showed 98% sporicidal activity against B. cereus in 4 h. There was no significant difference in sporicidal activity against B. cereus between BCTP diluted 1 : 100, BCTP 401 diluted 1 : 1000, and bleach diluted 1 : 100 (P = .23).

Figure 1
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Figure 1

Sporicidal activity of BCTP against 4 different Bacillus species compared with that of BCTP 401 against 2 Bacillus species. BCTP showed significant sporicidal activity after 4 h of treatment against Bacillus cereus, B. circulans, and B. megaterium spores but not against B. subtilis spores. BCTP 401 showed more effective killing against B. cereus in 4 h and also had sporicidal activity against B. subtilis that was resistant to BCTP. Bleach diluted 1 : 100 was used as positive control and was comparable to BCTP or BCTP 401 at same dilutions.

Testing the stability of BCTP

Three different preparations of BCTP, stored for 2, 8, and 16 months at room temperature, were evaluated simultaneously for sporicidal activity against B. cereus spores to determine the stability of the emulsions. BCTP was diluted 1 : 10 and 1 : 100 for the experiments (figure 2), and there was no significant difference in the sporicidal activity of the preparations (P = .94 and .77).

Figure 2
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Figure 2

Comparison of sporicidal activity of 3 different preparations of BCTP aged 2, 8, and 16 months. Preparations have equivalent sporicidal activity, showing that BCTP is stable for up to 16 months.

B. cereus sporicidal time course. An 8-h experiment was done to analyze the time course of the sporicidal activity of BCTP (diluted 1 : 100) and BCTP 401 (diluted 1 : 1000) against B. cereus. Incubation of a 1 : 100 dilution of BCTP with B. cereus spores resulted in a 77% reduction in the number of viable spores at 1 h and a 95% reduction after 4 h. Again, BCTP 401 diluted 1 : 1000 was more effective than BCTP diluted 1 : 100 and resulted in an ∼95% reduction in count in 30 min (figure 3). The improvement in killing between BCTP 401 diluted 1 : 1000 and BCTP diluted 1 : 100 was statistically significant up to the 4-h time point (P < .05).

Figure 3
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Figure 3

Time course of nanoemulsion sporicidal activity against Bacillus cereus. Incubation with BCTP diluted 1 : 100 resulted in 95% killing in 4 h. Incubation with BCTP 401 diluted 1 : 1000 resulted in 95% killing in only 30 min. Difference in killing between BCTP diluted 1 : 100 and BCTP 401 diluted 1 : 1000 up to 4-h point was significant (P < .05).

Sporicidal activity of BCTP against

B. anthracis. After initial in vitro experiments, the sporicidal activity of BCTP was tested against two virulent strains of B. anthracis (Ames and Vollum 1B). We found that BCTP at a 1 : 100 final dilution incorporated into growth medium completely inhibited the growth of 1 × 105 B. anthracis spores. Sporicidal assays in fluid media, after 4 h of incubation with BCTP at dilutions up to 1 : 1000 with either the Ames or the Vollum 1B spores, resulted in 91% sporicidal activity when the mixtures were incubated at 22°C and 96% sporicidal activity when the mixtures were incubated at 37°C (table 1).

Figure 4
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Figure 4

Electron micrographs of Bacillus cereus spores before (top) and after (bottom) treatment with BCTP. Note uniform density in cortex and well-defined spore coat before treatment with BCTP. Spores after 4 h of BCTP treatment show disruption in both spore coat and cortex, with loss of core components.

Figure 5
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Figure 5

Effect of germination inhibition and stimulation on sporicidal activity of BCTP diluted 1 : 100 against Bacillus cereus spores. Sporicidal activity of BCTP was delayed in presence of 10 mM d-alanine (germination inhibitor) and accelerated in presence of 5 mM l-alanine (germination enhancer). All time points show significant difference between 3 treatments (P < .002).

Figure 6
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Figure 6

Gross and histologic photographs of animals injected subcutaneously with different combinations of BCTP and Bacillus cereus spores. A, B, animals injected with BCTP alone at dilution of 1 : 10. There was no gross tissue damage, and histology showed no inflammation. C, D, animals injected with 4 × 107 B. cereus spores alone subcutaneously. Large necrotic area resulted, with average area of 1.68 ± 0.35 cm2. Histologic examination of this area showed essentially complete tissue necrosis of epidermis and dermis, including subcutaneous fat and muscle. E, F, mice injected with 4 × 107 Bacillus spores that had been immediately premixed with BCTP nanoemulsion at final dilution of 1 : 10. These animals showed minimal skin lesions, with average area of 0.02 ± 0.01 cm2 (∼98% reduction from lesions resulting from untreated infection with spores; P < .002). Histology of F indicated some inflammation; however, most cellular structures in epidermis and dermis were intact. All histopathology is shown at ×4 magnification.

Figure 7
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Figure 7

Gross and histologic photographs of animals with experimental wounds infected with Bacillus cereus spores. A, B, mice with experimental wounds infected with 2.5 × 107 B. cereus spores but not treated. Histologic examination indicated extensive necrosis and marked inflammatory response. C, D, mice with wounds that were infected with 2.5 × 107 B. cereus spores and irrigated 1 h later with saline. By 48 h, large necrotic areas surrounded wounds, with average area of 4.86 ± 0.48 cm2. In addition, 60% of animals in this group died as result of infection. Histologic examination of these lesions indicated total necrosis of dermis and subdermis and large numbers of vegetative Bacillus organisms. E, F, mice with wounds infected with 2.5 × 107 B. cereus spores and irrigated 1 h later with 1 : 10 dilution of BCTP. There were small areas of necrosis adjacent to wounds (0.06 ± 0.03 cm2), 98% reduction compared with animals receiving spores and saline irrigation (P < .001). In addition, only 20% of animals died from these wounds. Histologic examination of these lesions showed no evidence of vegetative Bacillus organisms and minimal disruption of epidermis. All histopathology is shown at ×4 magnification.

Table 1
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Table 1

Sporicidal activity of BCTP against 2 different strains of Bacillus anthracis spores as determined by colony reduction assay (% killing).

Sporicidal activity of BCTP 401 against

B. anthracis. Because BCTP 401 was effective at higher dilutions and against more species of Bacillus spores than BCTP, it was tested against 4 different strains of B. anthracis at dilutions of up to 1 : 10,000 at 22°C to prevent germination. BCTP 401 showed peak sporicidal activity between ∼1 : 1000 and ∼1 : 5000 dilutions (table 2). It was less efficient at concentrations 11 : 100.

Table 2
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Table 2

Sporicidal activity of BCTP 401 against 4 different strains of Bacillus anthracis representing different clinical isolates.

Electron microscopic examination of the spores

We used B. cereus because it is the most closely related to B. anthracis. Transmission electron microscopic examination of B. cereus spores treated with BCTP diluted 1 : 100 in TSB for 4 h revealed physical damage to the B. cereus spores, including extensive disruption of the spore coat and cortex with distortion and loss of density in the core (figure 4).

Germination stimulation and inhibition

To investigate the effect of initiation of germination on the sporicidal effect of BCTP on Bacillus spores, the germination inhibitor, D-alanine [17, 18], and germination enhancer, l-alanine [19, 20], were incubated with the spores and BCTP for up to 1 h. Percentage of killing was calculated at different time points. The sporicidal effect of BCTP was delayed in the presence of 10 mM d-alanine and accelerated in the presence of 5 mM l-alanine (figure 5). All of the individual time points showed a significant difference in killing between the three treatments (P < .002).

In vivo toxicity testing

CD-1 mice injected with BCTP diluted 1 : 10 in saline did not exhibit signs of distress or inflammatory reaction, either grossly or histologically (figure 6A, 6B). Identical results were obtained when the toxicity of BCTP 401 was tested in mice subcutaneously. Intramuscular injection of the BCTP or BCTP 401 diluted 1 : 10 did not have any toxic effects in the form of inflammatory reaction, edema, or necrosis in mice. Open wound irrigation with 2 mL of the emulsions did not result in any pathologic damage. Intranasal instillation of the emulsion was less tolerable because of its viscosity; however, there was no injury from BCTP diluted 1 : 50 and BCTP 401 diluted 1 : 25. Oral administration of 10% BCTP (4 mL/kg of body weight daily) in rats for 1 week did not result in any gross or pathologic changes, and the rats maintained normal weight gain during this period (data not shown). In these tests, pathologic examination of local tissues and internal organs was done, and no abnormalities were detected.

In vivo sporicidal activity

B. cereus infection in experimental animals had been previously used as a model system for the study of anthrax and causes an illness similar to experimental anthrax [2, 9, 21–24]. Two animal models of cutaneous B. cereus disease were developed to assess the in vivo sporicidal activity of BCTP. A suspension of 4 × 107 B. cereus spores was mixed with saline or with BCTP at a final dilution of 1 : 10 and then immediately injected subcutaneously into the backs of CD-1 mice. Mice that were infected subcutaneously with B. cereus spores without BCTP developed severe edema in 6–8 h. This was followed by a gray, necrotic area surrounding the injection site at 18–24 h, with severe sloughing of the skin present by 48 h, leaving a dry, red-colored lesion (figure 6C, 6D). CD-1 mice injected with B. cereus spores premixed with BCTP never developed such a necrotic lesion, and edema and inflammation were minimal (figure 6E, 6F). The size of the necrotic lesion in BCTP-treated mice was ∼98% smaller than the necrotic lesion size in untreated mice (from 1.62 ± 0.35 cm2 to 0.02 ± 0.01 cm2; P < .002). Similar results were observed with BCTP 401 diluted 1 : 10.

In additional studies, a 1-cm skin wound was infected with 2.5 × 107 B. cereus spores and then closed (figure 7A, 7B). For some of the animals 1 h later, the wounds were irrigated with either BCTP diluted 1 : 10 or saline to simulate postexposure decontamination. Irrigation of experimentally infected wounds with saline did not result in any apparent benefit (figure 7C, 7D). BCTP irrigation of wounds infected with B. cereus spores showed substantial benefit, resulting in a consistent 98% reduction in the lesion size (from 4.84 ± 0.48 cm2 to 0.06 ±0.03 cm2; P < .001; figure 7E, 7F). This reduction in lesion size was accompanied by a 3-fold reduction in mortality (from 60% to 20%) compared with that in experimental animals receiving either no treatment or saline irrigation. Similar results were observed with BCTP 401 diluted 1:10.

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Discussion

In these studies, we demonstrated that BCTP and its derivative BCTP 401 have effective sporicidal activity against a variety of Bacillus spores, including B. anthracis. BCTP diluted 1 : 100 has a sporicidal activity against B. cereus, B. circulans, and B. megaterium, whereas 1 : 1000 is effective against B. anthracis in 4 h. BCTP 401, a BCTP-P10 mixture, appears to have a more rapid and broader sporicidal activity than BCTP. BCTP 401 diluted 1 : 1000 killed 95% of B. cereus spores in 30 min at 37°C, compared with a 70% reduction achieved by BCTP diluted 1 : 100. BCTP 401 diluted 1 : 1000 was also effective in 4 h against B. subtilis spores that were resistant to BCTP for up to 24 h. BCTP 401 did not show effective sporicidal activity against B. anthracis at dilutions of <1 : 100, contrary to the original BCTP, which showed killing at dilutions between 1 : 10 and 1 : 1000. The fact that BCTP 401 requires dilution to be effective against B. anthracis spores suggests that BCTP 401 needs dispersion by water to minimize its aggregation and to facilitate direct contact with spores.

Comparison of the sporicidal activity of BCTP against B. anthracis at 22°C, a temperature that does not promote spore germination, and at 37°C, at which germination occurs (as confirmed by microscopic examination), indicates that complete spore germination (i.e., outgrowth) is not necessary for the bactericidal activity of the emulsion. The small difference observed between the sporicidal activity at 37°C and 22°C may represent the killing of additional organisms from a few germinating spores. Sporicidal activity was also confirmed in water, a condition unsuitable for B. anthracis spore germination (data not shown). The sporicidal effect seems to start almost immediately and occurs within 30 min of incubation with the emulsion. Factors facilitating germination resulted in acceleration of the sporicidal activity of BCTP. Inhibition of the initiation of germination with d-alanine delayed BCTP's sporicidal activity. On the basis of these observations, we hypothesize that the sporicidal action of these emulsions occurs through initiation of germination before complete reversion to the vegetative form, leaving the spore susceptible to disruption by the emulsion. The initiation of germination could be mediated by the action of the emulsion or its components, but the emulsion appears necessary, as spores do not initiate germination in its absence. The results of the electron microscopy studies show disruption of the spore coat and cortex with disintegration of the core contents after BCTP treatment. However, the exact mechanism of killing is unclear and requires future investigation. Sporicidal activity appears to be mediated by both the Triton X-100 and tri-n-butyl phosphate components, because nanoemulsions lacking either component are inactive in vitro (data not shown). This unique sporicidal action of the emulsions, which is similar in efficiency to that of 1% bleach, is interesting because Bacillus spores are generally resistant to most disinfectants, including many commonly used detergents [15].

Animal studies demonstrated the protective and therapeutic effect of BCTP in vivo. B. cereus infection in experimental animals has been used previously as a model system for the study of anthrax [21, 22, 25]. The disease induced in animals experimentally infected with B. cereus is in many respects similar to anthrax [9, 23]. In this study, we demonstrated that mixing BCTP with B. cereus spores before injecting the spores into mice prevented the pathologic effect of B. cereus. We also demonstrated that BCTP treatment of simulated wounds contaminated with B. cereus spores markedly reduced the risk of infection and mortality in mice. Because the emulsion appeared to lose sporicidal activity when diluted past 1 : 100, higher concentrations of the emulsions (1:10) were used for the in vivo studies to make sure they remained effective after dilution with body fluids. Other experiments show that testing BCTP 401 in mice under similar conditions demonstrated similar effects. These results suggest that decontamination of spores prior to or after exposure can effectively reduce the morbidity and mortality from B. cereus infection. This appeared to be a valuable application, because unlike other sporicidal agents, BCTP or BCTP 401 did not demonstrate any toxic effects, grossly or by histopathologic examination of the mice [26]. Other tests in mice showed that these emulsions are nontoxic if administered intramuscularly, intranasally, or orally, providing other potential sites for treatment.

BCTP and its derivative BCTP 401 appear to have great potential as environmental decontamination agents or for treatment of exposed persons in either a military operation or a terrorist attack. The inactivation of a broad range of pathogens, including vegetative bacteria, enveloped viruses [27] (unpublished data), and bacterial spores, combined with low toxicity in experimental animals, seems to make it suitable for use as a general decontamination agent that can be deployed even before a specific pathogen is identified. The nanoemulsions can be rapidly produced in large quantities and are stable for many months unless frozen, which causes separation of the oil and lipid phases. Undiluted, they have the texture of a semisolid cream and can be applied topically by hand or mixed with water. Diluted, they have a consistency and appearance similar to skim milk and can be sprayed to decontaminate surfaces or potentially interact with aerosolized spores before inhalation. These properties provide a flexibility that will be useful for a broad range of decontamination applications. Further studies are warranted to determine the exact mechanism of the sporicidal effect of BCTP and its derivatives, and this may lead to further improvement in formulations.

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Acknowledgments

We thank Shaun B. Jones, Jane Alexander, and Lawrence DuBoise (Defense Science Office, Defense Advanced Research Project Agency) for their support; Bruce Ivins, Patricia Fellows, Mara Linscott, Arthur Friedlander, and the staff of USAMRIID for their technical support and helpful suggestions in the performance of the initial anthrax studies; Martin Hugh-Jones, Kimothy Smith, and Pamala Coker for supplying the characterized B. anthracis strains and the space at Louisiana State University (Baton Rouge); Robin Kunkel (Department of Pathology, University of Michigan) for her help with electron microscopy preparations; and G. Morris and A. Shih for their technical assistance with manuscript preparation.

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Footnotes

  • ↵D.C.W. and J.B. are employees of NOVAVAX, Inc., and have significant financial interest in the company. NOVAVAX, Inc., is the supplier of the emulsions. J.R.B., T.H., M.M.H., D.C.W., and J.B. have a patent application entitled: Methods of inactivating bacteria including bacterial spores.

  • The animal experiments were approved by and performed according to the guidelines of the Unit for Laboratory Animal Medicine, University of Michigan.

  • Financial support: Defense Advanced Research Project Agency (contract MDA 972-1-007 of the Unconventional Pathogen Countermeasures Program).

  • Received March 10, 1999.
  • Revision received June 30, 1999.
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  1. J Infect Dis. (1999) 180 (6): 1939-1949. doi: 10.1086/315124
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