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Ozone and Anthrax - Knowns and UnknownsRip G. Rice Ph.D. aa RICE International Consulting Enterprises 1331 Patuxent Drive , Ashton, MD, 20861 Phone:301-924-4224 Fax: 301-924-4224 E-mail:Published online: 05 Feb 2007.

To cite this article: Rip G. Rice Ph.D. (2002) Ozone and Anthrax - Knowns and Unknowns, Ozone: Science & Engineering: TheJournal of the International Ozone Association, 24:3, 151-158, DOI: 10.1080/01919510208901606

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OZONE SCIENCE & ENGINEERING Vol. 24. pp. 151-158 Printed in the U.S.A.

Ozone

0191-9512IO2 $3.00 + .00 International Ozone Association

Copyright 0 2002

and Anthrax - Knowns and Unknowns

Rip G. Rice, Ph.D.

RICE International Consulting Enterprises 1331 Patuxent Drive, Ashton, MD 20861

Tel: 301-924-4224; Fax: 301-774-4493; e-mail: ~p~ice40zone@cs.com

Received for Review: 16 April 2002 Accepted for Publication: 29 April 2002

Abstract

Mailings of envelopes containing anthrax spores (Bacillus anthracis) have caused serious disruptions of business operations in various parts of the United States, and several people tragically have lost their lives from resulting exposure to anthrax. These incidents have caused U.S. government agencies to investigate this material and to evaluate methods of neutralizing it. Ozone is one candidate countermeasure.

In this paper, what is known about ozone and its ability to destroy B. anthracis spores (and accepted surrogates) is discussed. High relative humidity is required to "soften up" the spore coating prior to addition of any disinfectant. Ozone clearly is a sufficiently powerful oxidant to destroy B. anthracis in relatively short exposure times. Of major concern, however, is ozone's equally clear ability to produce collateral damage in areas where it is used.

Research on ozone is needed to fill data gaps, in order to convince government authorities in charge of anti- terrorism activities that ozone should be included as a prime canhdate for combating anthrax contaminations. It is equally important that those in the ozone indusq be aware of the known facts and data gaps concerning ozone -- in order to minimize the number of (well-intentioned) overclaims for ozone that may be doomed to failure and give ozone a black eye in this field.

Key Words

Ozone; Anthrax; Bacillus anthracis; Bacillus subtilis; Bacillus globigii; Bacillus spores; Relative Humidity Effects on Inactivation; Hydrogen Peroxide;

Background

Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis, that occurs commonly in wild and domestic animals such as cattle, sheep, goats, camels, antelopes and other animals that eat grass. It can also occur in humans when they are exposed to the hides, wool, hair or meat of infected animals.

Three types of anthrax infections are known. Infections in humans most commonly develop on the skin (cutaneous). Thorough washing of the skin can

reduce the possibility of infection after exposure. Respiratory (inhaled) and gastrointestinal infections from naturally occurring anthrax are rare in humans. In order to develop respiratory anthrax, humans must inhale a minimum of 1000 to 2500 spores at one time.

What are the symptoms? Skin infections start with a raised, itchy bump that develops into a painless ulcer with a black center. Gastrointestinal infections cause nausea, loss of appetite, fever, vomiting,

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152 R. G. Rice

abdominal pain and diarrhea. Respiratory infections produce effects resembling those of a common cold, but then progress to breathing problems, shock and death. Direct person-to-person spread of anthrax from skin lesions is quite unlikely to occur. The inhaled or gastrointestinal infections are not transmitted from person to person.

Most infections caused by naturally occurring anthrax can be treated with penicillin. Infections caused by manufactured anthrax are treated with other types of antibiotics (Ciprol). Almost all cases of inhaled anthrax are fatal if left untreated. Cutaneous dosages of anthrax to humans are much less likely to cause death, because the likelihood of several thousand bacteria entering a lesion or open wound are much less likely than through inhalation.

Anthrax bacteria tend to clump together, thus lowering the probabilities of inhaling lethal dosages. Nevertheless, when properly prepared, so as to ensure a minimum of clumping, the bacteria can be aerosolized and then lethal dosages are more likely to be present when packages or envelopes containing the material are opened.

Thus it is desired to find processes or techniques to decontaminate rooms, dwellings, offices, buildings, etc. that may have been exposed to anthrax. Additionally, if the spores have settled onto desks, furniture, clothing, walls, rugs, floors, etc., it is desirable to be able to treat such items and enclosed spaces with some anthrax-destroying agent(s) so as to ensure decontamination while minimizing collateral damages.

Properties of Bacillus Spores

While most bacteria exist unprotected and are readily oxidized andlor disinfected by a variety of disinfectants, including ozone, Bacillus spp have a life cycle that includes a dormant phase during which a protective coating develops around the bacterium. This coating is very resistant even to strong chemical oxidants, including ozone. Further, coated spores also have a strong tendency to clump together, further protecting the surrounded spores from oxidantddisinfectants.

Many studies on the inactivation of Bacillus spp spores with a variety of disinfectants have made it clear that humidity plays a critical role in allowing an oxidantldisinfectant to breach the spore coating

and thereby inactivate (destroy) the encapsulated microorganism.

Antimicrobial Effectiveness of Ozone vs Bacterial Spores

In Aqueous Media

When compared to vegetative cells (absent the coatings), bacterial spores exhibit high resistance to ozone. Although only a few specific publications dealing with ozone inactivation of B. anthracis are available, there are many published studies of ozone inactivation of related Bacillus spp., such as B. subtilis, B. cereus, B. pumilum, B. globigii. Some of these bacteria are accepted surrogates for B. anthracis, in particular B. subtilis and B. globigii. Studies of ozone inactivation of various Bacillus spp in air and in water are summarized below.

Miller et al. (1959) at the U.S. Army laboratory in Ft. Detrick, MD studied the effects of ozone treatment of raw sewage spiked with B. anthracis, B. subtilis, a toxin of Clostridium botulinum and influenza virus. Ozone was generated by corona discharge and concentrations in the gas phase stream applied to aqueous samples varied from 1% to 5% by weight, the higher concentrations being obtained using high purity oxygen. Table I shows results obtained from ozonation of raw sewage spiked with B. anthracis spores.

A summary of data for ozone used to obtain sterility in samples spiked with B. anthracis, B. subtilis and botulinum toxin is presented in Table I1 (Miller et al., 1959).

These authors (Miller et al., 1959) concluded that under the conditions of these experiments, all the microorganisms inoculated into raw sewage could be completely sterilized or inactivated using ozone. as demonstrated by both cultural methods and animal inoculations. B. anthracis and B. subtilis var. niger spores were completely killed by 90 minutes of ozonation, and probably could be killed within 60 minutes. The results of ozone treatment of B. subtilis var. niger and B. anthracis spores in raw sewage confirm the suitability of B. subtilis var. niger spores as a simulant for pathogenic microorganisms with respect to ozone treatment.

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Ozone and Anthrax - Knowns and Unknowns 153

Table I.

In studies on other Bacillus spp organisms, Broadwater et al. (1973) reported that the lethal threshold ozone concentration for Bacillus cereus was 0.12 mgL, while that for E. coli and B. megaterium was 0.19 mg/L. Foegeding (1 985) found that acidic pH enhanced the lethality of ozone against Bacillus and Clostridium spores. This author also suggested that the spore coat is a primary protective barrier against ozone. Naitoh (1992b) found that the addition of metallozeolites (ascorbic

acid, and isoascorbic acid) improved the inactivation of B. subtilis spores by ozone treatment at 5 to 50 ppm for 1 to 6 h, suggesting that oxygen radicals are involved in the sporicidal activity. Naitoh (1992a) also investigated synergistic sporicidal activities of gaseous ozone and W irradiation (5-20 seconds at 80 mwlcm2). This author reported that the alternating treatment of W radiation followed by ozone at 80-95% relative humidity reduced the contact time required for inactivation.

Table I

Zhao and Cranston (1995) observed a 5-log decrease 6 Llmin. They also reported that S. aureus and B. for Staphylococcus aureus, B. cereus, E. coli, and cereus exhibited greater tolerance to ozone than did Salmonella in 10 to 20 min when they were sparged E. coli and Salmonella spp. in the water with 6.7 mg/L ozone at the flow rate of

I I I

3

a Initial count of B. subtilis var. niger spores in experiments at ARF was 1 to 2 x 10~lrnL; at Ft. Detrick 0.66 to 2.2 x 1 0 ~ l r n ~ . b Initial count of B. anthracis spores was 0.23 to 0.37 x 10~lrnL. c Initial concentration of botulinum toxin was 1 x 10' mouse minimum lethal level doselml.

Toxin ditto 30 4 250 210-290

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Inactivating spore-forming bacteria in water with ozone is one thing, but in air it is an entirely different matter, primarily because of the variations in relative humidities that can be made available.

Ishizaki et al. (1986) studied the inactivation of six strains of Bacillus spp [B. subtilis IF0 3134; B. subtilis (B. globigii) I F 0 13721; B. subtilis NCTC 10073; B. cereus AHU 1357; B. licheniformis AHU 1532; and B. megeterium AHU 13741 with gas phase ozone (0.5 - 3.0 mg/L) at varying relative humidities and found a lag phase during the initial exposure followed by an exponential decrease in the number of survivors with time. However, one strain, B. cereus, showed no lag phase. No inactivation was observed at relative humidities below 50%. Increasing relative humidity influenced the lag phase, but not the inactivation rate.

Whistler and Sheldon (1989b) evaluated ozone as an alternative hatchery disinfectant in air to replace formaldehyde. Cultures of Staphylococcus, Streptococcus, and Bacillus species previously isolated from poultry hatcheries and selected culture collections of Escherichia coli, Pseudomonas jluorescens, Salmonella typhimurium, Proteus species, and Aspergillus fumigatus were spread- plated on open petri plates and independently fumigated with ozone or formaldehyde in a prototype laboratory poultry setter. Ozone (1.52% to 1.65% by weight) reduced bacterial levels by >4 to 7 loglo and fungal levels by > 4 loglo.

Naito (1989) studied the microbiocidal effects of ozone in air in a Japanese candy factory. In the refrigeration room and cold room, large numbers of Bacillus, Micrococcus and yeast were found and the total microbial counts were 91-1 15 per 53-L, but that number decreased to 55-78/53 L after gas phase ozone treatment. The humidity in these rooms was low (5560% RH), which restrained ozone decomposition, leaving the remaining ozone concentration high in the range of 0.072-0.1 12 ppm. It was thought that the sterilizing effect of ozone in these rooms dropped somewhat in comparison with that of the refrigeration room and cold room on the first floor.

The most recent study on ozone for inactivation of anthrax spores in air phase was conducted by Currier et al. (2001) using Bacillus globigii var. niger as a

surrogate for Bacillus anthracis. Studies involved ozone (9,000 ppm, -1% ozone in air) deactivation of clumped and dirty spores of B. globigii. Rehydration of dry spores was found to greatly enhance the effectiveness of ozone. At high relative humidity (> 70%, samples preconditioned 15 hrs in air), capillary condensation of water within spore clumps was found to adversely affect the overall deactivation rate in clumped spores. However, the cleanliness of the spores did not significantly affect the overall deactivation rate of clumped spores.

Collateral Damages Caused by Ozone and High Relative Humidities

Currier et al. (2001) also studied the possible collateral damages that can be caused by applying ozone at 1% in air and 70% relative humidity for coping with anthrax in contaminated office buildings. Magnetic storage media, optical read- only disks, and a simple electronic calculator were placed in a small sealed chamber into which ozone- containing air (9,000 ppm) was fed. After 10 hours of exposure to ozone-laden air, all items operated properly. The magnetic media (Zip disk) failed first at about 16-20 hours. Failure was deemed to occur when bad blocks began to form on the diskette. Surface FTIR spectroscopy on damaged disks indicated a growth in C=O, OH, and C-H bonds, suggesting oxidation of organic constituents by ozone. Long-term exposure of the magnetic disk (36 hours at 9,000 pprn of ozone and 70% relative humidity) resulted in delamination of the magnetic material from the supporting substrate.

Subsequent tests verified that the components most likely to fail are the exposed "heads" used to read storage media. Disk drives exposed to 9,000 pprn of ozone-containing air typically failed at times comparable to failure of the magnetic storage media (16 hours). Relative humidity did not appear to influence the failure rate appreciably. With 36-hour exposure to 9,000 pprn ozone-containing air, the optical read-only disk and the calculator operated as designed.

Role of Relative Humidity on Spore Inactivation

Kim et al., (2002) conducted a detailed study of the effects of relative humidity on the inactivation of B. subtilis spores. They concluded that high humidity is needed for the inactivation of microorganisms by

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Hz02 Concentration (%)

0 5 10 15 20 25 30 Ozone Concentration (PPM)

Inactivation of spores of Bacillus subtilis OSU 494 when treated with aqueous ozone or hydrogen peroxide solution for I rnin at 22'C

Figure 2. Inactivation of B. subiilis spores by ozone and by hydrogen peroxide (Khadre and Yousef, 2002, unpublished information).

Figure 2 shows the inactivation of B. subtilis spores by ozone and by hydrogen peroxide.

With both antimicrobial agents, their effectiveness increases with increasing concentrations. However, the effectiveness of ozone is much higher at lower concentrations -which are practical to obtain in practice.

Recent NSF Research Project - Ozone vs Anthrax

Early in 2002, the prestigious National Science Foundation (Washington, DC) awarded a $100,000 (US.) grant to Dr. Mirat Gurol and her colleagues at San Diego State University to study the inactivation of bacterial spore surrogates for B. anthracis with gaseous ozone. This one-year research study will define parameters for ozone technology to combat bioterrorism through decontamination of enclosed spaces. Through this NSF grant, the inactivation rate of simulants of anthrax spores exposed to gaseous ozone will be investigated under various scenarios. The results will enable the industry to ready the technology within a short period of time for full-

scale applications in treatment of anthrax- contaminated buildings.

Dr. Gurol will investigate the inactivation of B. subtilis spores in a specially designed ozone chamber and on various different surfaces, including various types of carpeting and vinyl floor material, textile, wood, different types of plastics and plaster. Data will be gathered on the required ozone concentrations and contact times to produce desired levels of inactivation of the spores, the ozone demand of different type of surfaces, and the effect of air humidity, level of hydration of spores, and temperatures on the inactivation rates of the spores. The effect of ozone on surfaces for collateral damage will be investigated by inspecting the material exposed visually and microscopically.

This research was approved by the NSF under the SGER (Small Grant for Exploratory Research) Program due to the urgency of the subject matter. This program funds research when there is " . . . a severe urgency with regard to availability of, or access to data, facilities or specialized equipment,

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Ozone and Anthrax - Knowns and Unknowns 157

including quick-response research on natural disasters and similar unanticipated events". Availability of Gas-Phase Ozone Technology

Although gas-phase ozone has not yet been reported to have decontaminated rooms and buildings contaminated with anthrax spores, it is being used commercially to remediate rooms and buildings that have suffered fire or flood damages. At least in the USA, it is standard commercial practice for firelflood remediation firms to seal rooms/homes/buildings containing smoke odors (from fires) or molds and spores (from flood damages) and to expose the contaminated spaces with gaseous ozone for periods of time ranging from hours to days, depending on the extent of damages. Thus, the technology exists for gas phase ozone to be applied to rooms/offices/buildings to destroy B. anthracis spores, assuming that appropriate relative humidities can be created prior to ozonation.

Summary and Recommendations

B. anthracis spores inoculated into raw sewage can be inactivated by passing ozone (1-5 wt% in air) into this aqueous medium for 60 minutes. B. subtilis and other Bacillus spp can be used as surrogates for B. anthracis in air or aqueous media. A recent study (Cumer et al., 2001) of gas phase ozone to inactivate both clumped and dirty spores of B. globigii showed ozone inactivation at 9,000 ppm (-1 wt%) ozone during 15 hours of exposure. The optimum relative humidity was 75% - above this R.H., inactivation by ozone actually decreased. On the other hand, other recent studies (Kim and Yousef, 1999; Kim et al., 2002) using B. subtilis, showed that only when the water activity, a, (roughly equivalent to R.H.) is > 0.95 (-95% R.H.) can ozone inactivation of spores be attained by applying ozone in the gas phase. Minimum collateral damage to office equipment was caused by exposure to 9,000 ppm of ozone over 16 hours (time to inactivate B. globigii) at 70% R.H. (Currier et al., 2001). The exposed "heads" used to read storage media in data handling equipment failed under these conditions.

6. Gas-phase ozone technology is readily available from firms, who routinely provide equipment and services to restore fire andor flood damaged homes and offices. However, conditions for applying ozone to anthrax-contaminated areas need to be defined and optimized.

7. Definitive data need to be developed to optimize conditions for applying gas-phase ozone to anthrax-contaminated offices/rooms/buildings so as to obtain maximum decontamination while minimizing collateral damages. In particular, correlations need to be developed between ozone concentrations, exposure times, relative humidities (discrepancies need resolution) and collateral effects on specified office, home, data storage, etc. items.

Acknowledgments

The author is indebted to Dr. Ahmad Yousef, of the Ohio State University, for providing information dealing with studies by him and his colleagues on the effectiveness of ozone and hydrogen peroxide against B. subtilis spores (as surrogates for B. anthracis) and the critical role of relative humidity for the inactivation of bacterial spores in advance of formal publication of such data.

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