Killing of Bacillus subtilis Spores by a Modified Fenton Reagent Containing CuCl 2 and Ascorbic Acid

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2004, p Vol. 70, No /04/$ DOI: /AEM Copyright 2004, American Society for Microbiology. All Rights Reserved. Killing of Bacillus subtilis Spores by a Modified Fenton Reagent Containing CuCl 2 and Ascorbic Acid Michael P. Shapiro, Barbara Setlow, and Peter Setlow* Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut Received 29 August 2003/Accepted 21 December 2003 Bacillus subtilis spores were killed by CuCl 2 -ascorbic acid, chloride ions were essential for killing of spores, and spores with defective coats were killed more rapidly. CuCl 2 -ascorbic acid did not damage spore DNA, and spores killed by this reagent initiated germination. However, spores killed by CuCl 2 -ascorbic acid may have damage to their inner membrane. Spores of various Bacillus and Clostridium species are dormant and resistant to environmental stresses, including heat, radiation, and toxic chemicals (13, 31). This resistance, as well as the potential for a number of spore formers, including Bacillus anthracis, to cause disease has heightened interest in new sporicides. Further stimulating the development of new microbial biocides are the facts that some bacteria have become resistant to common sterilization agents and some currently used sterilization agents pose risks to health-care workers (11). One group of agents that have been tested as microbial biocides are Fenton reagents (34) that use various metal ions plus hydrogen peroxide or ascorbic acid to generate highly reactive hydroxyl radicals (2, 21, 22, 23). The reactivity of hydroxyl radicals means that they are most effective when they are generated in the immediate vicinity of their target, which is often DNA (8, 9, 19). Fenton reagent formulations with Cu 2 and hydrogen peroxide or ascorbic acid kill spores of Bacillus species (2, 21, 22, 23). Bacillus globigii spore killing by CuCl 2 - ascorbic acid requires oxygen, presumably for the generation of hydroxyl radicals, and killing is increased by the addition of NaCl or a surfactant (2). Since spores killed by Fenton reagent with Cu 2 and ascorbic acid were not visibly damaged, it was suggested that the Cu 2 and ascorbic acid had penetrated into the spore core and with dissolved oxygen had generated hydroxyl radicals that caused spore death by damage to DNA or enzymes (2). While this is a logical possibility, penetration of ascorbic acid into the spore core seems unlikely, since hydrophilic molecules such as monosaccharides do not normally penetrate the core of Bacillus cereus spores (6). Indeed, even a molecule as small as methylamine penetrates the spore core slowly (32). Since Cu 2 -ascorbic acid may be used to kill spores in applied settings, it seemed appropriate to determine how this reagent acts on spores. Of particular interest are the following issues: (i) whether this reagent kills spores by DNA damage; (ii) whether the reagent acts in the spore core or on some more external spore layer; and (iii) what spore features are important in protection against this reagent. Spore DNA is well protected against damage from toxic * Corresponding author. Mailing address: Department of Biochemistry, University of Connecticut Health Center, Farmington, CT Phone: (860) Fax: (860) setlow@nso2.uchc.edu. chemicals by the proteinaceous spore coats, the low permeability of the spore s inner membrane, the spore core s low water content, and the saturation of spore DNA with / -type small, acid-soluble spore proteins (SASP) (13, 31). The DNA protection afforded by these mechanisms is so great that many potentially genotoxic agents that kill spores, including many oxidizing agents, do not do so by causing DNA damage (5, 10, 13, 31, 37). To test whether Cu 2 -ascorbic acid kills spores by causing DNA damage, the killing of spores of B. subtilis strains lacking various DNA protective mechanisms was examined. The B. subtilis strains were isogenic derivatives of strain 168. PS533 is the wild-type strain and carries plasmid pub110, which contains a kanamycin resistance marker (30). Two genes (sspa and sspb) that encode the two major / -type SASP were deleted from strain 168 to create strain PS578, which carries pub110 (30). The reca gene was deleted from strain 168 and replaced with a gene providing resistance to macrolide antibiotics (MLS r ) to create strain PS2318 (30). The sspa and sspb genes were deleted from strain 168 and the reca gene was mutated to create strain PS2319 (30). Strain PS3394 carries pub110 and contains a tetracycline resistance cassette inactivating the cote gene essential for proper spore coat assembly (3, 37). Strain PS3379 contains the luxab genes from Vibrio harveyi under the control of the forespore-specific promoter of the sspb gene (10). Spores were prepared at 37 C on 2 SG medium plates without antibiotics and washed and cleaned as described previously (14, 15). Spore preparations used were free ( 98%) from growing cells, cell debris, or spores that had germinated, as determined by examination with a phase-contrast microscope. Spores were treated at an optical density at 600 nm (OD 600 )of 1.0 ( 10 8 CFU/ml) at room temperature (24 C) with 54 mm CuCl 2 and 90 mm ascorbic acid unless noted otherwise; the ph of this solution was 1.5. At various times, aliquots were diluted 100-fold in a solution containing 2.5 g of Na 2 S 2 O 3 per liter to neutralize the Cu 2 -ascorbic acid, and further diluted in water, and aliquots were spotted in duplicate on Luria-Bertani (LB) medium (15, 33, 37) plates. The plates were incubated for 24 h at 30 to 37 C, and colonies were counted to assess spore viability. Spores of all strains tested were not killed by being incubated at ph 1.5 for 2 h, as found previously (27). Spores of the wild-type B. subtilis strain (PS533) were killed 2535

2 2536 SHAPIRO ET AL. APPL. ENVIRON. MICROBIOL. FIG. 1. Killing of spores of B. subtilis strains with Cu 2 -ascorbic acid. (A) Spores of B. subtilis strains PS533 (wild type) (F), PS578 ( )(Œ), PS2318 (reca) ( ), and PS2319 ( reca) (}) were incubated with CuCl 2 -ascorbic acid. In each experiment shown, the viability of spores of all strains was determined in parallel and at least in duplicate as described in the text. (B) Spores of B. subtilis strains PS533 (wild type) (F and Œ), either intact (F) or decoated (Œ), and PS3394 (cote) ( ) were incubated with CuCl 2 -ascorbic acid, and spore viability was determined as described in the text. (C) Spores of B. subtilis strain PS533 (wild type) either intact (Œ,,, and }) or decoated (F) were incubated with CuCl 2 -ascorbic acid (Œ), 54 mm CuSO 4 90 mm ascorbic acid ( and F), 54 mm CuSO 4 90 mm ascorbic acid 108 mm NaCl ( ), or CuCl 2 -ascorbic acid 500 mm NaCl (}), and spore viability was determined as described in the text. The experiments shown in this figure were carried out two (B and C) or three (A) times with essentially identical results to those shown, and in these experiments, the confidence limits for the relative slopes of the killing curves were at least 11%. Consequently, the differences between the wild-type and spores shown in panel A are statistically significant, as are the differences seen in panels B and C, with the exception that the killing curves in panel C for intact spores killed with CuCl 2 -ascorbic acid or 54 mm CuSO 4 90 mm ascorbic acid 108 mm NaCl are not statistically different. by CuCl 2 -ascorbic acid (Fig. 1A), although more slowly than spores of B. globigii (2). Spores of strain PS578 that lack most of the DNA-protective / -type SASP (termed spores) exhibited slightly greater sensitivity to CuCl 2 -ascorbic acid, but in either a wild-type or background, loss of the reca gene responsible for much DNA repair in B. subtilis (36) did not reduce spore resistance (Fig. 1A). Previous work has shown that or reca spores are 3- to 10-fold more sensitive to agents that cause DNA damage than are wild-type spores (13, 33). Thus, the slight ( 1.5-fold) decrease in spore sensitivity to CuCl 2 -ascorbic acid upon loss of SASP- and SASP- may not be due to killing of the spores through DNA damage. Instead, this small difference is likely due to global changes in gene expression during sporulation of the strain, because of the absence of accumulation of most / -type SASP in the developing forespore (28). This alteration in gene expression causes slight changes in the properties of the spores, as seen previously in the decreased resistance of spores to other agents that do not cause DNA damage in spores (5, 28, 33, 37). In particular, the lack of synthesis of SASP- and - during sporulation has major effects on expression of genes coding for spore coat proteins (28), and as shown below, the spore coats are a major factor in spore resistance to CuCl 2 -ascorbic acid. To further examine whether spore killing by CuCl 2 -ascorbic acid is through DNA damage, wild-type and spores surviving CuCl 2 -ascorbic acid were tested for the presence of auxotrophic or asporogenous mutations by examining growth of survivors on plates containing rich, minimal, and sporulation media as described previously (4, 5, 10, 33, 37). In contrast to spores that survive treatments that kill by damaging DNA, 15% of which have obvious mutations (4, 33), wild-type spores surviving CuCl 2 -ascorbic acid (8% survival) had no more mutations (0 in 416 survivors) than the untreated spores (0 in 416 spores tested). The same finding was made for spores (83% killed) treated with CuCl 2 -ascorbic acid (6 mutants in 416 survivors; 6 mutants in 416 untreated spores examined). The significant level of mutations in untreated spores has been seen previously and ascribed to mutagenesis during spore preparation and storage (4, 5, 10, 33, 37). In contrast to the absence of DNA damage detected in spores killed by CuCl 2 -ascorbic acid, growing bacteria treated with a Fenton reagent do exhibit significant mutagenesis, and cell killing is accelerated by a reca mutation (35). The spore coat helps protect spores against many chemical agents (3, 5, 10, 31, 37). Indeed, B. subtilis spores with defective coats are 10- to 100-fold times more sensitive to many oxidizing agents than intact spores are (5, 10, 37). To assess the role of the coat in spore resistance to CuCl 2 -ascorbic acid, we examined wild-type spores that were chemically decoated (1) as well as cote spores (3). Not surprisingly, both the decoated and cote spores were much more sensitive to this reagent than were untreated wild-type spores (Fig. 1B). The protection against CuCl 2 -ascorbic acid by the spore coat may explain the observation that a surfactant sensitized B. globigii spores to this reagent (2). Perhaps the surfactant used in that study (2) removes some coat protein, allowing easier access to targets located inside the spore. It was reported that CuCl 2 -ascorbic acid killing of spores is increased significantly by the addition of NaCl (2), and we also found this to be the case (Fig. 1C). Why NaCl would increase spore killing by CuCl 2 -ascorbic acid if these chemicals were to react in the spore core to generate hydroxyl radicals is not clear. However, perhaps the agent killing the spores is not the hydroxyl radical but an oxidizing agent formed by reaction of hydroxyl radicals with chloride ions in free solution. A number

3 VOL. 70, 2004 SPORE KILLING WITH A FENTON REAGENT 2537 TABLE 1. Germination of B. subtilis spores with or without prior treatment with CuCl 2 -ascorbic acid a Treatment % Killing L-Alanine % Spore germination with LB medium plus L-alanine Ca 2 - DPA To assess the effect of CuCl 2 -ascorbic acid on spore germination, wild-type spores (94 to 99% of wild-type spores killed by CuCl 2 -ascorbic acid) were tested for spore germination with nutrients, with a 1:1 mixture of Ca 2 and dipicolinic acid (DPA), and with dodecylamine. Ca 2 -DPA and dodecylamine initiate germination of spores of many Bacillus species, including Bacillus subtilis, but are not nutrients (16, 26). The CuCl 2 - ascorbic acid-killed spores initiated germination normally with nutrients, judging from the conversion of the spores from the bright to dark phase using a phase-contrast microscope (Table 1). Spores killed 99% by CuCl 2 -ascorbic acid also germinated in LB medium plus L-alanine as rapidly as untreated spores, as assessed by the decrease in OD 600 of spore suspensions (data not shown), and after 90 min, these germinated spores appeared viable in an assay based on the inability of bacteria with a severely damaged plasma membrane to exclude the dye propidium iodide (Table 1). As judged by their ability to give rise to colonies, 95% of these spores were actually dead, indicating that the spores have indeed been damaged in some fashion. A similar discrepancy between these two viability assays was seen when chlorine dioxide or a superoxidized water, Sterilox, was used to kill spores, as a large fraction of spores killed by these reagents as assessed by colony formation appeared alive as measured by the exclusion of propidium iodide (10, 37). Presumably, the CuCl 2 -ascorbic acid-treated spores had accumulated sufficient damage such that they could not give rise to colonies but not so much damage that spores germinated 90 min in LB medium do not exclude propidium iodide. Previous work has shown that spores killed by a number of chlorine-containing oxidizing agents do not lose the core s large depot of DPA, but the killed spores do lose DPA upon a normally sublethal heat treatment (10, 37). The latter result has been interpreted as a lower resistance of the inner spore membrane to rupture at elevated temperatures due to membrane damage caused by the oxidizing agents (10, 37). Killing of wild-type spores to 99% with CuCl 2 -ascorbic acid also caused 5% release of spore DPA, determined as described previously (10, 37). However, incubation for 30 min at 80 or 85 C of wild-type untreated spores or spores killed 99% by CuCl 2 -ascorbic acid caused more release of DPA from the killed spores than from the untreated spores (8% from the killed spores and 3% from the untreated spores at 80 C; 15% from the killed spores and 8% from the untreated spores at 85 C; all values 15%), when DPA release was measured as described previously (5, 37). While the greater DPA release from the CuCl 2 -ascorbic acid-killed spores was significant, the amount was significantly lower than from spores killed by other oxidizing agents (10, 37), suggesting there is only a small amount of damage to the inner membrane in CuCl 2 -ascorbic acid-killed spores. Further evidence for damage to CuCl 2 -ascorbic acid-killed spores came from analysis of metabolism upon germination using spores that contain high levels of the V. harveyi LuxA and LuxB proteins. Untreated B. subtilis spores initiate metabolism soon after the initiation of germination and generate FMNH 2, and FMNH 2, LuxA, LuxB, and dodecanal result in light production by germinating spores (5, 7, 10). This was also observed in the current work (Fig. 2), when light production was measured as described previously (5, 10, 37). However, spores killed 99% by CuCl 2 -ascorbic acid and then germinated exhib- Dodecylamine None (97) b c ;37 d (97) e CuCl 2 -ascorbic acid (97) b 5 (5) f 98 c ;35 d (3) e CuCl 2 -ascorbic acid (95) b ND g (0.5) e a Spores of strain PS533 (wild type) were either not treated or treated with CuCl 2 -ascorbic acid, and percent killing was assessed on plates as described in the text. Spores at an OD 600 of1(l-alanine, LB medium plus L-alanine, and Ca 2 -DPA) or 2 (dodecylamine) were germinated by the following treatments: (i)3hat37 C with L-alanine (8 mm L-alanine, 25 mm Tris-HCl [ph 8.6]); (ii) 90 min at 37 C with LB medium with 4 mm L-alanine added to ensure as rapid germination as possible; (iii) 2hat24 C with Ca 2 -DPA (60 mm CaCl 2,60mM DPA, 20 mm Tris-HCl [ph 8.4]), followed by centrifugation and incubation in 20 mm Tris-HCl (ph 8.4) at 37 C for 1 h; and (iv) dodecylamine (1 mm dodecylamine, 20 mm Tris-HCl [ph 7.5]) at 37 Cfor3hor45 C for 4 h. Germination was assessed by examining the conversion of 200 spores from the bright to dark phase with a phase-contrast microscope (L-alanine, LB medium plus L-alanine, and Ca 2 -DPA) or measuring DPA release (dodecylamine) as described previously (25). b The values in parentheses are the viability of 200 spores that had germinated, as assessed by their staining either red (dead) or green (alive) with the BacLight viability stain (Molecular Probes, Eugene, Oreg.) as described previously (5, 12). However, note that this assay is different from the determination of percent killing as assessed on plates. c Value for spores incubated for 4hat45 C. d Value for spores incubated for 3hat37 C. e Values in parentheses are the maximum viability of spores that germinated after dodecylamine treatment compared to the viability of untreated spores determined without dodecylamine treatment. f The value in parentheses is the viability of the CuCl 2 -ascorbic acid-killed spores after incubation with Ca 2 -DPA compared to the viability of the untreated spores that had also been incubated with Ca 2 -DPA. g ND, not determined. of chlorine-containing species can be generated by the Fenton reaction in the presence of chloride ions, including chlorine atoms and hypochlorite (24, 25), and hypochlorite kills spores efficiently, especially as the ph is decreased (37). If the reaction of hydroxyl radicals with chloride ions does generate the killing species, then spores incubated with CuSO 4 -ascorbic acid should not be killed. This was indeed the case for both intact and decoated wild-type spores, and spore killing by CuSO 4 - ascorbic acid was restored by the addition of NaCl (Fig. 1C). The lack of spore killing by CuSO 4 -ascorbic acid further indicates that enhancement by NaCl of spore killing by Cu 2 - ascorbic acid was not an ionic strength effect but is more likely a specific effect of the Cl ion. Indeed, 0.5 M Na 2 SO 4 did not enhance spore killing by CuCl 2 -ascorbic acid (data not shown). The data noted above suggest that spore killing by CuCl 2 - ascorbic acid is through generation of oxidizing chlorine-containing species. In addition, since there is no evident DNA damage in spores killed by CuCl 2 -ascorbic acid, even in spores lacking the DNA protective / -type SASP, it seems likely that the killing agent does not act on the spore core but on some more exterior spore layer. Recent studies examining spore killing by hypochlorite and chlorine dioxide, as well as the new disinfectant Sterilox (which also contains a variety of oxidizing chlorine-containing compounds), have shown that these agents do not damage the DNA in the spore core but appear to kill spores by damaging the spore s inner membrane in some fashion (5, 10, 37). This damage, in particular by hypochlorite, can also greatly diminish the spore s ability to germinate (37).

4 2538 SHAPIRO ET AL. APPL. ENVIRON. MICROBIOL. FIG. 2. Light production during germination of untreated and CuCl 2 -ascorbic acid-killed spores carrying V. harveyi LuxA and LuxB proteins. Spores of strain PS3379 (PsspB::luxAB) either not treated (E) or treated with CuCl 2 -ascorbic acid giving 99% killing (F) were germinated at 37 C and an OD 600 of1in2 YT medium (5) containing 4 mm L-alanine. At various times, 0.5-ml samples were mixed with 0.5 ml of fresh medium, 10 l of 1% dodecanal in ethanol was added, and light production was measured as described in the text. More than 70% of both the treated and untreated spores had germinated by 60 min, as determined by observation with a phase-contrast microscope. ited 1% of the light production of germinated untreated spores (Fig. 2). Perhaps slight damage by CuCl 2 -ascorbic acid to the inner spore membrane that becomes the germinated spore s plasma membrane has compromised energy metabolism by these germinated spores. The CuCl 2 -ascorbic acid-killed spores also initiated germination normally with dodecylamine, an agent that triggers spore germination by causing release of spore DPA (26), although dodecylamine treatment did not revive the CuCl 2 - ascorbic acid-killed spores (Table 1). However, spores killed with CuCl 2 -ascorbic acid did not germinate with Ca 2 -DPA (Table 1). Germination of B. subtilis spores by Ca 2 -DPA requires the enzyme CwlJ, one of the spore s two redundant cortex lytic enzymes (15, 16). In spores, CwlJ is sensitive to exogenous chemicals, including at least one oxidizing agent (15, 37). Consequently, it seems likely that CwlJ is readily inactivated by CuCl 2 -ascorbic acid treatment. However, inactivation of all spore cortex lytic enzymes is not the reason for spore killing by CuCl 2 -ascorbic acid, since (i) spores killed by this reagent appear to initiate germination normally with nutrients and (ii) if spores were killed to 95% with CuCl 2 - ascorbic acid and decoated and treated with lysozyme in a hypertonic medium to degrade the spore s peptidoglycan and induce germination, the killed spores became phase dark (data not shown), an indicator of cortex lysis. However, the CuCl 2 - ascorbic acid-treated spores were not revived by this treatment (data not shown). In contrast, spores not treated with CuCl 2 - ascorbic acid remained viable after the same treatment (data not shown), as expected (20). The data presented in this communication allow three new conclusions. First, spore killing by Cu 2 -ascorbic acid largely if not completely requires Cl, suggesting that the killing agent is some oxidizing chlorine-containing species. Second, the spore coat provides major protection against spore killing by CuCl 2 - ascorbic acid, as they do against spore killing by a number of other oxidizing agents (5, 10, 37). Third, spore killing by CuCl 2 - ascorbic acid is not due to DNA damage, at least DNA damage that is either mutagenic, repaired in a RecA-dependent process, or prevented by / -type SASP. In addition, since wildtype and spores exhibit relatively similar sensitivities to CuCl 2 -ascorbic acid, it seems unlikely that this reagent generates hydroxyl radicals within the spore core. In contrast, hydrogen peroxide may generate hydroxyl radicals in the spore core, since spores are much more sensitive to hydrogen peroxide than wild-type spores and spores (but not wildtype spores) surviving treatment with hydrogen peroxide exhibit high levels of mutations (29). A remaining area of uncertainty about spore killing by CuCl 2 -ascorbic acid concerns the mechanism of killing. Previous work on spore killing by oxidizing chlorine-containing agents has shown that these agents do not kill spores by DNA damage but likely cause some damage to the spore s inner membrane that either prevents spore germination or results in loss of membrane integrity in the first minutes of spore germination (10, 37). However, there is no significant defect in the initiation of germination by CuCl 2 -ascorbic acid-killed spores, and these germinated spores appear to have retained their plasma membrane integrity, at least after 90 min of germination, although their ability to carry out energy metabolism is severely compromised. Consequently, the precise nature of the damage caused by CuCl 2 -ascorbic acid treatment that kills spores is not clear. Indeed, this damage may be different from that caused by other oxidizing chlorine-containing agents (10, 37), since these latter agents were used at ph values ranging from 3.5 to 11 and the CuCl 2 -ascorbic acid reagent has a ph of 1.5. 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