Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cross, J. B. Articles by Gladen, P. D. Search for Related Content PubMed PubMed Citation Articles by Cross, J. B. Articles by Gladen, P. D. Agricola Articles by Cross, J. B. Articles by Gladen, P. D.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, April 2003, p. 2245-2252, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2245-2252.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid, and Copper Ions

J. B. Cross,¹ R. P. Currier,^1* D. J. Torraco,¹ L. A. Vanderberg,¹ G. L. Wagner,² and P. D. Gladen²

Chemistry Division,¹ Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545²

Received 16 August 2002/ Accepted 26 December 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu²⁺) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O₂ is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Microbial life is abundant, tenacious, and is often very difficult to control. Organisms including viruses, bacteria, and fungi are often characterized by an ability to spread easily, reproduce rapidly, and thrive under conditions that can destroy higher life forms. Many of these organisms are completely harmless to humans and play an important role in many ecosystems and natural processes. Some organisms, though, cause human diseases, and exclusion or destruction of these organisms is important to prevent or block the spread of disease.

In addition to the problem of normal infections, the world is faced with the rapidly growing problem of "super bugs," or bacteria that have developed a resistance to one or more antibiotics or disinfectants. In its annual Report on Infectious Diseases, the World Health Organization (WHO) warned that many major infectious diseases are becoming resistant to the antibiotics used to treat them (press release WHO/41, 12 June 2000 [http://www.who.int/inf-pr-2000/en/pr2000-41.html]). As an example of this problem, WHO noted that in Estonia and Latvia, as well as parts of Russia and China, over 10% of those afflicted with tuberculosis have strains resistant to both of the most powerful antibiotics used to treat the disease.

Many of these drug-resistant diseases are acquired and spread in hospitals and, consequently, hospitals are being forced to take extraordinary measures to guard against patient infection. Hospitals and medical facilities commonly use sterilizing agents on patient contact surfaces, operating rooms, and medical instruments in order to combat growth and spread of disease. The sterilizing agents commonly used include formaldehyde and glutaraldehyde, which are cancer causing and put hospital personnel at risk. These agents, as well as commonly used oxidants, react directly with the outer membrane or coat of bacterial cells and spores. Though effective, these agents are also generally very highly reactive toward organic materials in general and some inorganic materials, thus causing corrosion or erosion. Because of the reactivity of these agents toward many different compounds, they could react with and thus be consumed by things other than their intended targets, thus becoming less efficient due to their tendency to cause collateral damage. Further, these substances often prove toxic, as has been mentioned. Use of these agents is thus conducted with a conscious understanding that such use will often eventually degrade the surfaces or materials sanitized and that extreme caution be employed to avoid contact with a patient, health care worker, or the environment. It would thus be a significant improvement to develop a sterilant that is relatively nonreactive to nontarget organic and inorganic materials and that is nonhazardous to humans or to the environment. The presently described method (modified Fenton chemistry) targets the interior of the spore, where copper ions are known to bind to the life-giving machinery such as DNA or other important molecules, enabling on-site hydroxyl radical generation that destroys the life-giving properties of that machinery. It should be noted that dissolved oxygen exterior to the spore is also converted to hydroxyl radicals, but their concentration is low because of the low concentration of dissolved oxygen, thus limiting corrosion of surfaces and reagent consumption by extracellular materials.

H. J. H. Fenton first studied the catalytic decomposition of hydrogen peroxide by transition metal ion (6) in 1894. This work led to over 100 years of studies on the Fenton reagent (12, 32) in which, in its simplest form, it was established that the hydroxyl radical (HO^·) was the species responsible for the reagent's high oxidation efficiency. The basic mechanism,

(1)

(2)

shows that the transition metal ion, in this case iron, is cycled between an upper and lower oxidation state by its interaction with hydrogen peroxide and that the net reaction,

(3)

employs the transition metal ion effectively as a liquid catalytic agent for forming hydroxyl and other reactive free radicals. Subsequent reactions of hydroxyl radical (12) with organic molecules include (1) hydrogen abstraction, (2) addition reactions to double bonds, and (3) oxidation reactions.

Fenton reactions can be operative in biological systems, especially where hydrogen peroxide is formed during the course of normal cell function. Low levels of transition metals such as iron and copper are normally present in biological systems where they aid in respiration and biological polymer conformation. For example, during cell respiration, the biomolecule NADPH is oxidized by dissolved oxygen to form the superoxide molecule O₂^-.

(4)

The superoxide is converted to hydrogen peroxide in a slightly acid medium by superoxide dismutase (SOD).

(5)

If transition metals ions are present, the Fenton reaction can form hydroxyl radicals that in many cases can damage the biological organism. In fact, there are numerous studies (33) indicating the toxic nature of iron and copper and their role in aging of biological systems.

Cells efficiently scavenge hydrogen peroxide and free radicals through the glutathione peroxidase-reductase and catalase (9) systems. In addition to these natural defense mechanisms, drugs such as erythromycin (10) and vitamins such as ascorbic acid (AA) (7) are employed as added defense mechanisms against free radical formation. AA (vitamin C) has long been recognized as an efficient free radical scavenger (11), but under certain conditions of high concentration and the presence of transition metals and dissolved oxygen, it can function as a prooxidant (7, 24). Copper ions are especially active (29) in forming hydroxyl radicals under aerobic conditions in the presence of AA (1). In biological systems, copper(II) chemically complexes with DNA or RNA, upon which a site-specific formation of hydroxyl radicals can occur causing DNA or RNA strand scission (23, 29). At high concentrations of AA, copper(II) is rapidly reduced to copper(I) with the formation of the oxidized form of AA, dehydroa(6)scorbic acid (DAA).

(6)

The large difference in solubilities between CuCl₂ (706 g/liter) and CuCl (0.062 g/liter) produces a white precipitate when Cu²⁺ (0.6 M) is reduced to Cu⁺, resulting in a 6 x 10^-4 M saturated solution of CuCl. Under aerobic conditions, however, copper(I) is spontaneously oxidized back to Cu^II with the subsequent formation of hydrogen peroxide through the superoxide intermediate (10),

(7)

(8)

the net reaction being:

(9)

Thus, under conditions of excess AA, where Cu^II is continually being reduced by AA to Cu^I, dissolved oxygen is converted to hydrogen peroxide. The peroxide is subsequently converted to hydroxyl radicals by the Fenton reaction:

(10)

Both copper and iron combined with hydrogen peroxide have been investigated (20, 21) as a substitute for conventional disinfectants (3, 4, 16-19, 22). Fenton-based disinfection using hydrogen peroxide can be more efficient with essentially no side effects compared to glutaraldehyde (21). The investigations reported here extend the concept of Fenton-based disinfection and show that hydrogen peroxide can be replaced with aqueous dissolved oxygen, AA, and a surfactant to effect a much higher kill effectiveness than either conventional or hydrogen peroxide-metal ion disinfectants.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organisms studied.
This study was limited to nonpathogenic biological surrogates. The surrogates were chosen to replicate specific properties of pathogens potentially found in natural environments. For example, the following were considered: spore-forming bacteria (e.g., anthrax), using Bacillus globigii as a surrogate; and vegetative bacteria (e.g., plague), using Erwinia herbicola as a surrogate.

B. globigii is a common simulant for Bacillus anthracis but may be somewhat easier to deactivate. B. globigii is a durable noninfectious gram-positive spore common in soils. Of course, final testing of decontaminants should take place on virulent strains of target organisms. E. herbicola is the standard simulant for plague and other non-spore-forming, gram-negative bacteria. It is noninfectious to animals and humans, but it is infectious to plant leaves. These surrogates are easily grown in culture, easily detected, and/or require only biosafety level 1 handling precautions. However, since spores are generally much harder to deactivate than vegetative bacteria, spores represent the most stringent test for a biocide. Vegetative bacteria such as E. herbicola were found to require less than 1/100 of the concentration of the Fenton reagent for complete kill compared to that for bacterial spores. Thus, this effort was concentrated on determining the effectiveness of a modified Fenton reagent against spores of B. globigii.

Imaging diagnostic technique.
Scanning electron micrographs (SEMs) were obtained using a JOEL 6300 instrument with spores mounted on an aluminum sample holder. The spores were deposited by drying an aqueous spore suspension with an ambient temperature desiccator. The mounted spores were gold coated to reduce charging. The SEM images showed the extent of spore coat damage.

Organism preparation.
The "B. globigii" strain used in these studies was Bacillus subtilis subspecies niger ATCC 9372 grown from stock obtained from Dugway Proving Grounds using the following protocol. A 2x SG-Schaeffer's sporulation medium (stock solution 1) was prepared from 16 g of Difco nutrient broth/liter, 0.5 g of MgSO₄ · 7H₂O/liter, and 2.0 g of KCl/liter. Additional stock solutions included 10% glucose, 1 M Ca(NO₃)₂, 0.1 M MnSO₄, and 0.001 M FeSO_4. The components of these stock solutions were dissolved one at a time, in the order indicated, in 1 liter of triple-distilled H₂O. Then, 100-ml aliquots were poured into media bottles. The caps were loosened and autoclaved in a pan of water for 20 min on slow exhaust. The bottles were allowed to cool completely, caps were tightened, and solutions were stored in the dark until use. As prepared, the Schaeffer's nutrient base lasts approximately 1 week. Stock solutions were filter sterilized using a 0.22-µm-pore-size Acrodisc filter.

The fermentation process used to produce spores consisted of the following steps. First, 2.8 liters of Schaeffer's medium was added to a 5-liter fermentor and inoculated with a 200-ml culture of B. globigii. Five days were allowed for growth and sporulation (30°C, moderate aeration). After harvesting, the culture was heat shocked at 65°C for 15 min to kill vegetative cells. The spores were then harvested by centrifugation (16,000 x g for 25 min) and washed with triple-distilled sterile water 10 times. The spores were then resuspend in 500 ml of sterile triple-distilled water. To enumerate the number of viable spores in the resulting preparation, a dilution series was performed on the suspension, followed by growth on nutrient agar medium deposited on petri dishes to determine the population of spores. The average of at least two plates was taken.

Testing procedures.
Test coupons were prepared on glass surfaces as follows. Sterile 2-cm² glass coupons were scored and cut from frosted glass microscope slides. The coupons were aligned with the smooth size exposed. The glass coupons were then inoculated with B. globigii stock solution, approximately 5 x 10⁷ spores/coupon. The pipette was fitted with a new tip for each coupon. The inoculated coupon(s) then was dried at room temperature overnight in a desiccator containing Drierite. Following exposure to Fenton reagents, coupons were placed in a cold (0°C) neutralization agent (sodium thiosulfate) and then sonicated for 60 min to remove spores from the substrate. Separate tests verified that the sonication procedure was capable of recovery from the coupons and that sonication did not induce spore deactivation. The spore population was then determined by performing triplicate serial dilutions of the resulting suspension. Serial dilutions were prepared for each sample, using sterile phosphate buffer as the diluent. Serial dilutions (generally over 5 to 7 logs of dilution) on both samples and controls were performed using nutrient agar growth medium plated on petri dishes. The dishes were incubated for 24 h minimum, 48 h maximum at 30°C. The magnitude of kill was then determined by comparing the treated colony count with that of untreated colonies. It was noted that the preparation techniques resulted in clumped spores on the impervious glass coupons.

For tests in aqueous solution (spore suspensions), the following steps were employed. Aliquots of 1 ml of spore culture were placed in test tubes. The tubes were spun for 10 min at 16,000 x g to pelletize the cells. The supernatant was discarded. Three tubes were resuspended in the reagent to be tested (the precise volume and tube size varied). A fourth sample tube was resuspended in sterile triple-distilled water as a control. The tubes were exposed to the Fenton reagents for the specified exposure times (30 min unless noted otherwise). Dilution with cold (0°C) liquid (sodium thiosulfate) quenched the reaction. The sample tubes and the control were then spun at 16,000 x g for 10 min to pelletize the cells. The supernatant was removed. Spores were rinsed two times in an appropriate diluent. Spores were then resuspended in 1 ml of fresh sterile phosphate buffer. As described above, serial dilutions were then prepared for each tube, using sterile phosphate buffer as the diluent. In all cases, a standard serial dilution of the stock spore culture was carried out to verify that the culture was still viable and that the number of CFU (CFU per milliliter) was constant over time. All spore-handling procedures used aseptic bacteriological techniques in laminar flow biohazard hoods.

Reagent-grade CuCl₂, AA, and sodium chloride were employed in the modified Fenton reagent. Cupric chloride and AA were dissolved in deionized, tap water, or salt water (2 M NaCl). A 1% surfactant (3 M FC-170 or FC-100 fluorinated surfactant) was also added in some experiments to reduce the surface tension of the aqueous solution. The exposure time of spores to the reagent was typically 30 min, and exposure was performed at ambient temperature (25°C).

Anaerobic test conditions were produced by bubbling N₂ gas through aqueous Fenton solutions and aqueous spore suspensions (2 h) to remove dissolved oxygen. A steady-state Clark-type polographic (voltammetric) dissolved oxygen sensor (electrode) having an accuracy of ±0.2 mg/liter was employed to confirm final dissolved oxygen concentrations. Dissolved oxygen concentration within the spores was not verified by independent means but was assumed to be lower than that under aerobic conditions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exposure (30 min) of B. globigii spores to various formulations of modified Fenton reagent produces a variety of results ranging from no kill to a 6-order-of-magnitude kill (Fig. 1). Copper ion (0.6 M) with 0.1 M hydrogen peroxide (Fig. 1B) shows very poor kill effectiveness with and without added AA (0.1 M). Other experiments with vegetative cells of E. herbicola show excellent kill with this formulation (Fig. 1B). Fenton formulations without hydrogen peroxide show excellent kill (4 to 6 orders of magnitude) with both an ionic strength and surface tension dependence. The ionic strength dependence can be seen in the results obtained with and without NaCl (Fig. 1C, E, and F), while the effects of a change in surface tension achieved by the use of a surfactant can be seen in Fig. 1G. The highest kill effectiveness (percent surviving = 5 x 10^-6) was obtained with 0.06 M CuCl₂, 0.1 M AA, 2 M NaCl, and 1% (by weight) surfactant (FC-100, a fluorinated surfactant [3M]). These results led to no growth in all serial dilutions. In presenting such results, we assume that at least 1 CFU was present on the lowest dilution; therefore, the data shown in Fig. 1G are an upper limit to the kill. Other experiments performed with AA, 2 M NaCl, and surfactant alone (no copper) showed no kill whatsoever.

View larger version (70K): [in this window] [in a new window]

FIG. 1. Comparison of various types of Fenton formulations on the disinfection (30-min exposure time) of B. globigii spores suspended in solution. The percent surviving spores is shown for control (A), 0.6 M Cu2+-0.1 M H2O2 (with and without 0.1 M AA) (B), 0.6 M Cu2+-0.1 M AA (no H2O2) (C), 0.06 M Cu2+-0.1 M AA (no H2O2) (D), 0.06 M Cu2+-0.1 M AA-2 M NaCl (no H2O2) (E), 0.06 M Cu2+-0.1 M AA-0.2 M NaCl (no H2O2) (F), and 0.06 M Cu2+-0.1 M AA-2 M NaCl-1% (by weight) surfactant (no H2O2) (G). Note that conventional Fenton reagent using H2O2 (B) produces very little kill of B. globigii spores. The variations between replicates used to calculate the average all fall within 1 log. Thus, in this figure (and those that follow) the mean value is shown and the error bars denote a 1-log variation.

The removal of dissolved oxygen from all aqueous solutions by bubbling nitrogen (2 h) through the deionized water to make anaerobic Fenton reagents, as well as the anaerobic B. globigii spore suspension solution, resulted in a dramatic lessening of kill effectiveness (Fig. 2C and D). The extent of oxygen removal from the interior of the spores is unknown, but the removal of aqueous oxygen in this case made a large difference (2 logs) in spore kill, indicating that the presence of dissolved oxygen is important in the kill mechanism. Over time, the AA ages (Fig. 3) by decomposing into diketo-L-gluonic acid, oxalic acid, and L-threonic acid (21) and therefore loses its reducing powers. CuCl₂ solution, which is blue-green to begin with (Cu²⁺ ion), converts to a colorless solution when AA is added, indicating the reduction of Cu²⁺ (CuCl₂) to Cu⁺ (CuCl). After a period of 14 days, the blue-green color slowly returned to the solution, indicating a back conversion of Cu⁺ to Cu²⁺, and the kill effectiveness was reduced by a factor of 100. When additional fresh AA was added, the colorless condition returned (conversion of Cu²⁺ to Cu⁺) and the kill effectiveness was reestablished. The copper remains active as long as it is in its reduced state; therefore, a disinfectant solution can be rejuvenated by the addition of fresh AA, which keeps the copper ion in its reduced state. Visible and UV absorption spectra measurements also confirmed that the copper ion was cycled between its upper and lower oxidation states by AA.

View larger version (63K): [in this window] [in a new window]

FIG. 2. Comparisons of aerobic and anaerobic conditions for kill effectiveness on B. globigii spores suspended in solution. Anaerobic and aerobic solutions were as follows: control (A), 0.6 M Cu2+-0.1 M hydrogen peroxide (no AA) (B), aerobic 0.6 M Cu2+-0.1 M AA (C), and anaerobic 0.6 M Cu2+-0.1 M AA (D). This set of data was obtained without the use of either salt or surfactant. Dissolved oxygen concentration exterior to the spores was measured using a steady-state Clark-type polarographic (voltammetric) dissolved oxygen sensor (electrode).

View larger version (54K): [in this window] [in a new window]

FIG. 3. Aging study of aerobic modified Fenton reagent on B. globigii spores suspended in solution. Note that after 14 days the kill effectiveness has decreased by 2 to 3 logs but that adding fresh AA on day 15 fully restores the kill effectiveness.

In order to gain additional information on the kill mechanism, experiments were performed with Cu⁺ (CuCl) versus Cu²⁺ (CuCl₂) and with AA versus DAA (Fig. 4). AA, as opposed to the dehydro form, is absolutely essential (Fig. 4, compare C and D) for good kill and the oxidized form of copper is also essential for effective kill (Fig. 4, compare C with E, F, and G). As employed, Cu⁺ exhibits much less kill (4 logs) than Cu²⁺ under the same conditions. This may be simply attributed to the fact that Cu⁺ has a much lower solubility than Cu²⁺. However, the speciation of the copper (Cu⁺ versus Cu²⁺) may be an important factor in transport through the spore coat.

View larger version (86K): [in this window] [in a new window]

FIG. 4. Comparisons of copper and AA oxidation states on spore kill effectiveness. (A) Control; (B) Cu2+, NaCl, surfactant; (C) Cu2+, AA, NaCl, surfactant; (D) Cu2+, DAA, NaCl, surfactant; (E) Cu+, AA, NaCl, surfactant; (F) Cu+, NaCl, surfactant; (G) Cu+, DAA, NaCl, surfactant. Concentrations are 0.6 mM (saturated solution) copper(I), 0.12 M copper(II), 0.1 M AA, and 0.1 M DAA, 0.5 M NaCl, 1% (by total weight) surfactant FC-100.

SEM analysis was employed to image the results of spore exposure to the modified Fenton reagent and aqueous solutions of ozone, a more indiscriminant hydroxyl radical generator (Fig. 5-7). Comparing the SEM of untreated B. globigii spores (Fig. 5) with the SEMs of 30-min (Fig. 6A) and 16-h (Fig. 6B) exposures of B. globigii spores to modified Fenton reagent shows that the spore coat appears intact even after 16 h of exposure. There is essentially no difference in appearance between 30 min and 16 h of exposure. The spore coat appears to be, for the most part, unaffected compared to live spores (Fig. 5) except for a few spores being broken open. These results are in stark contrast to the effect that other hydroxyl radical producers, such as ozonated water, have on B. globigii spores (21) (Fig. 7). Ozone attacks and dissolves the spore coat, and an overly long exposure time eliminates any trace of the spore. Clearly, the kill mechanisms for ozone and modified Fenton reagent are very different. Ozone kills by attacking the coat, while the copper-based modified Fenton reagent apparently attacks the inner workings of the spore, leaving the coat relatively untouched.

View larger version (164K): [in this window] [in a new window]

FIG. 5. SEM of dried viable B. globigii spores on glass coupon. An aqueous solution of spores was placed on the SEM sample holder and allowed to air dry.

View larger version (90K): [in this window] [in a new window]

FIG. 7. Left, SEM showing the results of exposing dried spores mounted on a glass slide to 9,000 ppm of ozone gas at 70% relative humidity. Right, SEM showing a spore after 1-h exposure to ozone-saturated aqueous solution (1% ozone gas bubbled through solution at 25°C). Spore coat appears to be attacked, whereas the coat is not appreciably affected by the modified Fenton reagent (see Fig. 6).

View larger version (80K): [in this window] [in a new window]

FIG. 6. SEMs showing exposure time results of solution-suspended B. globigii spores treated with modified Fenton reagent, 0.06 M CuCl2-0.1 M AA-2 M NaCl-1% surfactant mixture, for treatment times of 0.5 h (A) and 16 h (B).

In summary, the sterilant studied in this investigation, a modified Fenton reagent, appears to demonstrate an interior kill mechanism that does not require addition of a strong oxidizer such as hydrogen peroxide. After sterilization, the body of the spore is left relatively intact with no obvious degradation of the spore, which is in contrast to ozone, which clearly attacks the spore. The detailed mechanism by which the modified Fenton reagent operates is unclear, but a mechanism can be proposed that is consistent with the data presented here and those in the literature. In the absence of hydrogen peroxide, but in the presence of AA and dissolved oxygen, the reagent shows excellent kill, while the elimination of dissolved oxygen from the aqueous solutions reduces the kill by several logs. Note that it is unclear how much dissolved oxygen was actually removed from the spore interior by simply bubbling nitrogen through the spore suspension. Thus, the results shown in Fig. 2D may represent a less-than-fully anaerobic situation with respect to dissolved oxygen bound within the spore. It appears that hydroxyl radicals produced from the conventional Fenton reaction between copper ions and hydrogen peroxide (equations 1, 2, and 3, where iron is replaced by copper) are not particularly effective in killing when produced external to the spore. A comparison of experimental results shown in Fig. 1C, D, and E indicates that ionic strength is important. It was found that KCl produces results comparable to those with NaCl, but NaF did not give satisfactory kill. It is possible that the addition of a salt whose anion readily passes through the cell wall may result in more cations (i.e., copper) being driven into the spore in order to satisfy the electroneutrality and osmotic constraints. A comparison of Fig. 1E and G indicates that reduction of liquid surface tension also plays an important part in the mechanism, possibly providing more intimate contact of the reagent with either individual spores or clumps of spores. Results did not seem to be sensitive to the type of surfactant, as good kills were also obtained using a variety of common nonfluorocarbon surfactants. The SEMs in Fig. 6 indicated that spore coats are essentially intact, and the data in Fig. 1 show excellent kill. Thus, it appears that the kill mechanism is likely an interior one, with reactants diffusing through the spore coat and forming free radicals inside the spore via the Fenton reaction. However, relatively little is known about the details of water, small-molecular-weight species, and multivalent metal ion diffusion through the spore coat (26). The application of an in situ atomic force microscopy technique (J. J. Weimer [Laboratory of Materials and Surface Science, University of Alabama in Huntsville], personal communication), though, shows the extensive uptake of water (Fig. 8), and it is assumed that dissolved oxygen in the water would also be brought into the spore interior. Many studies (13-15, 31, 34) of AA entry into vegetative cells indicate that vitamin C is transported in its DAA form and subsequently reduced to AA. Since DAA is structurally similar to glucose, it has been proposed that glucose transporters (14, 31) mediate its mechanism of entry into cells. Our data (Fig. 4), though, do not support the idea that DAA is the ascorbate species transported. In some cases, transport is found to be Na⁺ ion dependent (5, 13), which may also explain the impact NaCl has on biocidal activity and our results. Although these studies indicate a possible path for AA entry, there have been no definitive studies of the transport mechanism of AA entry into spores. If AA is present in the spore interior along with Cu²⁺ ions, dissolved O₂ can ultimately be converted to hydroxyl radicals. After formation of hydroxyl radicals within the spore, these highly reactive species attack either the DNA in a site-specific manner (24) or attack enzymes necessary for spore conversion to a bacterial cell. Further investigations of ion transport into spores and oxidation-reductions within spores will be needed to determine precisely the mechanism of spore kill under these conditions.

View larger version (73K): [in this window] [in a new window]

FIG. 8. SEM of a dry B. globigii spore being converted to a hydrated spore, as shown in the atomic force micrograph (AFM) taken with spore immersed in deionized water (Weimer, personal communication). The spore was immersed in deionized water for several days before AFM was performed, so no rate data were obtained on water uptake. Note the swelling that occurs with water uptake.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Knowledge of the disinfection mechanism of pathogens is important for optimizing kill efficiency and minimizing undesired effects on surroundings and on higher-level multicellular organisms. Disinfectants can be classified into two groups according to whether the kill mechanism originates from inside the pathogen or outside. For example, ionizing radiation can form free radicals inside a cell (or spore) that then attack the interior life-giving machinery. Ionizing radiation kills by forming hydroxyl free radicals (from the decomposition of water) that then attack DNA or enzymes within the cell. By controlling the point of irradiation in an organism, discrimination can be achieved and serious side effects can be controlled. Alternatively, chemical sterilants can be employed that attack the cell or spore coat from the outside, either causing lysis or blocking nutrient or oxygen uptake. Exterior disinfectants are commonly used in hospitals (3, 4, 16-20, 22) and water treatment systems (ozone and chlorine compounds). These exterior disinfectants are usually powerful oxidizers that indiscriminately attack both inanimate organic matter and living organisms, are generally toxic to higher-level organisms (19), and may form toxic byproducts. The detailed disinfection mechanism for bacterial cells and viruses by metal ions (28) is well understood, but the mechanism operating in spore sterilization (25) is not as well developed. However, the environments within the spore, along with spore characteristics that provide long-term survival strategies, have been well elucidated (26).

A spore kill mechanism for the sterilant under investigation is proposed based on current knowledge of metal ion attack in cells. However, additional work is warranted to more fully and precisely elucidate the mechanistic details. For example, direct measurement of hydroxyl radical formation both within and exterior to the spore is needed. The use of hydroxyl traps or dyes may prove beneficial in such efforts. In a similar vein, direct measurement of mass fluxes would be beneficial in discriminating between intrinsic chemical kinetics and mass transfer rates. Such studies would verify and further refine our understanding of the underlying mechanism and provide a firm basis for optimizing Fenton-like formulations.

The development of this Fenton-dissolved oxygen-based sterilant for routine use will require future investigations into sensitive equipment corrosion, skin irritation and safety aspects of human exposure, and the optimization of transition metal ion composition and concentration, pH, temperature, and dissolved oxygen concentration. Binding and deactivation of Cu²⁺ on certain surfaces must also be assessed. Valid formulations of the sterilant might ultimately be made available in bar or liquid soap form. The one-time use of the sterilant under biological warfare (anthrax, etc.) conditions may be easier to develop. Victims exposed to anthrax could potentially be showered with the sterilant and/or the used wash water could be treated before release into the sewer system. Sterilization of corpses housing infectious agents before release to family members is another possible use of the Fenton-dissolved oxygen sterilant. However, before any such development, direct comparisons to competing sterilizing agents will be essential.

Due to the propensity of copper to form complexes with biological proteins, the modified Fenton reagent may prove capable of destroying prion proteins through the use of site-specific binding, subsequent reduction of copper, and the formation of HO^· species. It has been reported (27, 30) that Cu²⁺ binds strongly in a site-specific region of the prion protein, and one can expect the highly reactive hydroxyl to be capable of breaking the protein apart. Since prion proteins are involved in a number of neurodegenerative diseases and have been found to be resistant to normal disinfection methods, investigation into new methods of disinfection would be an important avenue for future study.

Conclusions.
Decontamination of a biological pathogen-simulant B. globigii was studied. Specifically, the use of an aqueous modified Fenton reagent was examined. A reagent formulation of cupric chloride and AA was shown an effective sporicide under aerobic conditions. Results were obtained for a variety of formulations. It was found that sodium chloride and a surfactant significantly enhanced the biocidal potency. The increase in ionic strength may promote mass transfer through the spore coat, while the surfactant may enhance reagent contact with individual spores or spore clumps by lowering the solution's surface tension. A chemical mechanism was proposed that is consistent with our experimental results and information found in the literature. This mechanism requires diffusion of relatively benign species through the spore coat. Once interior to the spore, dissolved O₂ can be converted into hydroxyl radicals. The free radicals then perform the killing action. A suggestion has been put forward that the modified Fenton reagent might be helpful in the destruction of prion proteins.

 
This work was supported in part by the DOE Chemical and Biological National Security Program.

We thank the anonymous referees for useful suggestions to improve the manuscript and insightful suggestions for future work.

 
* Corresponding author. Mailing address: Chemistry Division, Los Alamos National Laboratory, Los Alamos, NM 87545. Phone: (505) 665-3601. Fax: (505) 667-0440. E-mail: currier{at}lanl.gov.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Buettner, G. R., and B. A. Jurkiewiez. 1996. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat. Res. 145:532-541.[Medline]
2
2. Currier, R. P., J. B. Cross, D. T. Torraco, L. A. Vanderberg, G. L. Wagner, and P. D. Gladen. 2001. Deactivation of clumped and dirty spores of Bacillus globigii. Ozone Sci. Eng. 23:285-294.
3
3. Danielson, J. W., J. T. Peeler, and G. S. Oxborrow. 1994. Sporicidal activity of chemical germicides on Bacillus subtilis spores under various conditions. J. AOAC Int. 77:53-57.
4
4. Danielson, J. W., R. D. Thompson, and E. Bell. 1999. Sporicidal testing of commercial germicides using a chemical standard and a calibrated bioindicator. J. AOAC Int. 82:151-159.[Medline]
5
5. Diliberto, E. J., Jr., G. D. Heckmanm, and A. J. Daniels. 1983. Characterization of ascorbic acid transport by adrenomedullary chromaffin cells: evidence for Na⁺-dependent co-transport. J. Biol. Chem. 258:12886-12894.[Abstract/Free Full Text]
6
6. Fenton, H. J. H. 1894. Oxidation of tartaric acid in the presence of iron. J. Chem. Soc. 65:899-910.
7
7. Halliwell, B., and J. M. C. Gutteridge. 1999. Free radicals in biology and medicine, 3rd ed., p. 201-208. Oxford University Press, Oxford, England.
8
8. Ito, S., T. Yamamoto, and Y. Tokushige. 1980. Valence change of copper in catalytic-oxidation of L-ascorbid acid by molecular oxygen. Chem. Lett. 11:1411-1414.[CrossRef]
9
9. Mendiratta, S., Z. Qu, and J. M. May. 1998. Erythrocyte defenses against hydrogen peroxide: the role of ascorbic acid. Biochim. Biophys. Acta 1380:389-395.[Medline]
10
10. Muranaka, H., M. Suga, K. Sato, K. Nakagawa, T. Akaike, T. Okamoto, H. Maeda, and M. Ando. 1997. Superoxide scavenging activity of erythromycin-iron complex. Biochem. Biophys. Res. Commun. 232:183-187.[CrossRef][Medline]
11
11. Pauling, L. 1989. Vitamin C papers. Science 243:1535.[Free Full Text]
12
12. Prousek, J. 1995. Fenton reaction after a century. Chem. Lisy 89:11-21.
13
13. Rumsey, S. C., and M. Levine. 1998. Absorption, transport, and disposition of ascorbic acid in humans. J. Nutr. Biochem. 9:116-130.[CrossRef]
14
14. Rumsey, S. C., O. Kwon, G. W. Xu, C. R. Burnant, I. Simpson, and M. Levine. 1997. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J. Biol. Chem. 272:18982-18989.[Abstract/Free Full Text]
15
15. Rumsey, S. C., R. W. Welch, H. M. Garraffo, P. Ge, S. Lu, A. T. Crossman, K. L. Kirk, and M. Levine. 1999. Specificity of ascorbate analogs for ascorbate transport. J. Biol. Chem. 274:23215-23222.[Abstract/Free Full Text]
16
16. Russel, A. D. 1994. Glutaraldehyde: current status and uses. Infect. Control Hosp. Epidemiol. 15:724-733.[Medline]
17
17. Russell, A. D. 1998. Assessment of sporicidal efficacy. Int. Biodeterioration Biodegrad. 41:281-287.[CrossRef]
18
18. Rutala, W. A., M. F. Gergen, and D. J. Weber. 1993. Inactivation of Clostridium difficile spores by disinfectants. Infect. Control Hosp. Epidemiol. 14:36-39.[Medline]
19
19. Rutala, W. A., M. F. Gergen, and D. J. Weber. 1993. Sporicidal activity of chemical sterilants used in hospitals. Infect. Control Hosp. Epidemiol. 14:713-718.[Medline]
20
20. Sagripanti, J., and A. Bonifacino. 1996. Comparative sporicidal effect of liquid chemical germicides on three medical devices contaminated with spores of Bacillus subtilis. Am. J. Infect. Control 24:364-371.[CrossRef][Medline]
21
21. Sagripanti, J. 1992. Metal-based formulations with high microbicidal activity. Appl. Environ. Microbiol. 58:3157-3162.[Abstract/Free Full Text]
22
22. Sagripanti, J. L., and A. Bonifacino. 1997. Effects of salt and serum on the sporicidal activity of liquid disinfectants. J. AOAC Int. 80:1198-1207.[Medline]
23
23. Sagripanti, J. L. 1999. DNA damage mediated by metal ions with special reference to copper and iron, p. 179-209. In A. Sigel and H. Sigel (ed.), Metal ions in biological systems,vol. 36. Marcel Dekker, Inc., New York, N.Y.
24
24. Samuni, A., J. Aronovitch, D. Godinger, M. Chevion, and G. Czapski. 1983. On the cytotoxicity of vitamin C and metal ions: a site-specific Fenton mechanism. Eur. J. Biochem. 137:119-124.[Medline]
25
25. Setlow, P. 1995. Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Annu. Rev. Microbiol. 49:29-54.[CrossRef][Medline]
26
26. Setlow, P. 1994. Mechanisms which contribute to the long-term survival of spores of Bacillus species. J. Appl. Bacteriol. Symp. Suppl. 76:49-60.
27
27. Stockelo, J., J. Safar, A. C. Wallace, F. E. Cohen, and S. B. Prusiner. 1998. Prion protein selectively binds copper (II) ions. Biochemistry 37:7185-7193.[CrossRef][Medline]
28
28. Thurman, R. B., and C. P. Gerba. 1989. The molecular mechanisms of copper and silver ion disinfection of bacteria and viruses. CRC Crit. Rev. Environ. Control 18:295-315.
29
29. Ueda, J., M. Takai, Y. Shimazu, and T. Ozawa. 1998. Reactive oxygen species generated from the reaction of copper (II) complexes with biological reductants cause DNA strand scission. Arch. Biochem. Biophys. 357:231-239.[CrossRef][Medline]
30
30. Viles, J. H., F. E. Cohen, S. B. Prusiner, D. B. Goodin, P. E. Wright, and H. J. Dyson. 1999. Copper binding to the prion protein: structural implications of four identical cooperative binding sites. Proc. Natl. Acad. Sci. USA 96:2042-2047.[Abstract/Free Full Text]
31
31. Wang, Y., T. A. Russo, O. Kwon, S. Chanock, S. C. Rumsey, and M. Levine. 1997. Ascorbate recycling in human neutrophils: induction by bacteria. Proc. Natl. Acad. Sci. USA 94:13816-13819.[Abstract/Free Full Text]
32
32. Wardman, P., and L. P. Candeias. 1996. Fenton chemistry: an introduction. Radiat. Res. 145:523-531.[Medline]
33
33. Winterbourn, C. C. 1995. Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol. Lett. 82-83:969-974.
34
34. Zee, J. V., and P. J. A. Van den Broek. 1998. Determination of the ascorbate free radical concentration in mixtures of ascorbate and dehydroascorbate. Free Radic. Biol. Med. 25:282-286.[CrossRef][Medline]

---

Applied and Environmental Microbiology, April 2003, p. 2245-2252, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2245-2252.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Shapiro, M. P., Setlow, B., Setlow, P. (2004). Killing of Bacillus subtilis Spores by a Modified Fenton Reagent Containing CuCl2 and Ascorbic Acid. Appl. Environ. Microbiol. 70: 2535-2539 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cross, J. B. Articles by Gladen, P. D. Search for Related Content PubMed PubMed Citation Articles by Cross, J. B. Articles by Gladen, P. D. Agricola Articles by Cross, J. B. Articles by Gladen, P. D.