Optimizing acidified bleach solutions to improve sporicidal efficacy on building materials

Transcription

1 Letters in Applied Microbiology ISSN 0- ORIGINAL ARTICLE Optimizing acidified bleach solutions to improve sporicidal efficacy on building materials J.P. Wood, M.W. Calfee, M. Clayton, N. Griffin-Gatchalian and A. Touati United States Environmental Protection Agency, National Homeland Security Research Center, Research Triangle Park, NC, USA ARCADIS Geraghty & Miller, Inc., Durham, NC, USA Keywords Bacillus, bleach, building materials, decontamination, inactivate, spore, sporicide. Correspondence Joseph P. Wood, US EPA, National Homeland Security Research Center, Research Triangle Park, Mail Code E-0, NC 77, USA : received May 0, revised September 0 and accepted October 0 doi:0./j.7-7x.0.0.x Abstract Aims: We evaluated whether lowering ph (with acetic acid) and raising free available chlorine (FAC) levels in bleach solutions would improve efficacy in inactivating Bacillus spores on different materials. We also determined how varying ph and FAC levels affected bleach stability. Methods and Results: Acidified bleach solutions with ph levels of Æ, and 7Æ and FAC levels between 000 and ppm were evaluated for decontamination efficacy against Bacillus subtilis spores inoculated onto test coupons made from wood, ceramic and galvanized steel. Lowering the ph or increasing the FAC level improved efficacy in some of the tests, but depended on the material, which significantly affected decontamination efficacy. The acidified bleach at ph of 7Æ was significantly less effective than bleach at a ph of Æ or. The FAC levels in the bleach were the most stable at ph Æ, and stability at ph Æ was not significantly affected by the initial FAC level. Conclusions: It may be advisable to use bleach solutions with lower ph (rather than high FAC levels) in light of both the decontamination efficacy and bleach stability results. For wood materials, use of sporicides other than acidified bleach may be warranted. Significance and Impact of the Study: These results may be useful in preparing acidified bleach solutions for decontamination of materials contaminated with spores such as Bacillus anthracis. Introduction Bleach (sodium hypochlorite) has a long history of use for inactivation of vegetative bacteria and viruses, i.e. as a disinfectant (Rutala and Weber 997). Acidified bleach (also referred to as ph-amended) has also been demonstrated to have some effectiveness as a sporicide (sterilant) and has been used to decontaminate buildings contaminated with Bacillus anthracis spores (e.g. Nguyen et al. 00). In addition, interest in the use of sporicides, such as acidified bleach, is increasing because of emerging challenges with recalcitrant microorganisms such as Clostridium difficile spores (e.g. Perez et al. 00). When bleach is diluted with water, hypochlorite ion (OCl ) ) and hypochlorous acid (HOCl) are formed in equilibrium. When the ph is lowered (off-the-shelf chlorine bleach typically has a ph of ) via the addition of an acid (e.g. acetic), the equilibrium shifts: OCl ) levels decrease and levels of HOCl increase. Chlorine solutions are known to have stronger germicidal properties correlated with higher levels of HOCl, indicating that HOCl is a much stronger bactericide than hypochlorite ion (Dychdala 00). At a ph of, over 99% of the free available chlorine (FAC) in an acidified bleach solution is present as HOCl. As the ph is lowered below, however, the FAC equilibrium begins to shift from HOCl to formation of volatile Cl gas (Black and Veatch Corp. 00), and FAC may, therefore, be lost over time. Although several studies have demonstrated that lowering ph improves sporicidal activity of bleach solutions (Death and Coates 979; Kuroiwa et al. 00; Tomasino et al. 00), the indicated or suggested optimum ph level varied from to 9Æ, because of these authors competing criteria (sporicidal efficacy, FAC stability, corrosivity). In Letters in Applied Microbiology, 7 ª 0 The Society for Applied Microbiology

2 J.P. Wood et al. Acidified bleach sporicides an effort to optimize acidified bleach in terms of sporicidal efficacy for use as a building decontaminant against B. anthracis spores, we tested bleach solutions at ph levels lower than what has typically been used for B. anthracis spores (c. 7) and at FAC levels higher than typically used (c. 000 ppm). In addition, efficacy tests were conducted with spores inoculated onto various building materials, as efficacy of sporicidal chemicals is highly dependent on the material with which the spores are associated (e.g. Rastogi et al. 009), and efficacy studies often overlook the effect of carrier material. Lastly, we also measured bleach solution FAC levels over time to determine stability as a function of initial FAC and ph. Materials and methods Test organism Spores of Bacillus subtilis (ATCC strain 99) were used as a surrogate for B. anthracis (Tomasino et al. 00) and prepared and purified following the protocols described elsewhere (Carrera et al. 007). Stock solutions were stored at C. Test matrix The bleach formulations (required amounts of bleach, water and acetic acid) for each ph FAC combination tested were determined theoretically (see Table ) and confirmed experimentally prior to the decontamination tests. These bleach solutions were also tested for FAC stability over h. The FAC level was determined (method below) initially within a few minutes after each bleach solution was prepared and then four additional times over the course of approximately h. The bleach solutions were stored at c. C in sealed 0-ml plastic vials during the stability tests, and aliquots of ml were used for the FAC determination. Decontamination tests were conducted for each material (discussed later) at the different bleach ph FAC combinations shown in Table. Each material was tested in triplicate for each ph FAC combination, and five positive controls (inoculated with spores, but not exposed to acidified bleach) were used each day of testing. (We tested one material at one ph and all six FAC levels at each day of decontamination testing.) Inoculation procedures Approximately 0 7 colony forming units (CFU) of B. subtilis spores were inoculated onto test coupons in a biosafety cabinet (BSC-II). The inoculation was achieved with a micropipette, dispensing one droplet (0Æ0 ml) of the spore Table Required ratios of water, acetic acid and bleach to achieve test matrix ph and free available chlorine (FAC) levels ph FAC (ppm) stock suspension (c. 0 CFU ml ) ) onto the surface of each coupon. The actual titre of the inoculum was verified the day the coupons were inoculated through standard tenfold dilution using a solution of buffered peptone water (Becton, Dickinson, Franklin Lakes, NJ, USA) and Tween 0 (Fisher Scientific, Pittsburgh, PA, USA), and spread plating onto tryptic soy agar (TSA). After spiking, the coupons were left to dry overnight. Lastly, we note that although dry spore deposition techniques have been recently developed (e.g. Lee et al. 0), we used a liquid inoculation approach for ease of use and to maintain consistency with our previous decontamination research. Coupon materials Maple wood coupons were of mm diameter and -mmthick plugs (part SPM00; Woodworks Ltd, Haltom City, TX, USA). Coupons of spruce wood were prepared from commercially available (Lowe s, Mooresville, NC, USA) Number grade lumber and cut as -mm diameter discs, mm thick. Galvanized steel coupons were prepared using -gauge galvanized steel (Lowe s), mm discs. Unglazed porcelain square tiles were of Æ9 cm each side, mm thick (Item CO, Lyric; Mosaic Art Supply, Atlanta, GA, USA). Bleach solutions Ratio of water to bleach* (v v) Æ 0000 Æ7 Æ9 Æ 9000 Æ Æ Æ 000 Æ Æ Æ 7000 Æ Æ Æ 000 Æ7 Æ Æ 000 Æ Æ 0000 Æ7 0Æ Æ 0Æ9 000 Æ 0Æ Æ0 Æ Æ 0Æ Æ Æ0 7Æ 0000 Æ7 0Æ 7Æ 9000 Æ 0Æ 7Æ 000 Æ0 0Æ 7Æ 7000 Æ0 0Æ 7Æ 000 7Æ 0Æ 7Æ 000 Æ9 0Æ Ratio of % acetic acid to bleach* (v v) *Using Ultra Clorox Ò Germicidal bleach, containing Æ% sodium hypochlorite. The acidified bleach solutions were prepared immediately before both decontamination and stability testing using Letters in Applied Microbiology, 7 ª 0 The Society for Applied Microbiology 9

3 Acidified bleach sporicides J.P. Wood et al. % v v acetic acid (Ricca Chemical, Arlington, TX, USA), bleach (Ultra Clorox Ò Germicidal Bleach; The Clorox Ò Company, Oakland, CA, USA), and sterile deionized water. Within a few minutes of preparation, the ph of the acidified bleach solutions was determined using a ph probe (Oakton PC 0; Eutech Instruments Pte Ltd, Singapore, Singapore). The FAC of each decontamination solution was determined according to a standard method (ASTM International 00), with the following exceptions: A precision burette (±0Æ0 ml) was used, and sample size was adjusted to ml, so that c. ml of titrant would be used. Decontamination, neutralization and spore extraction procedures The morning of each decontamination test, each sporeinoculated coupon was aseptically transferred to a sterile 0-ml conical vial. Ten millilitre of acidified bleach was added to each vial, and the vial was then turned upside down and then righted. After 0 min, the stoichiometric amount of sodium thiosulfate (Æ0 N) was added to the vial to neutralize the sporicidal action of the bleach, and the vial was swirled to mix. The neutralized bleach was then decanted into a new sterile vial, and 0 ml of peptone-buffered water was added to the vial with the coupon. Each vial with the coupon was then sonicated for 0 min, vortexed for min and then re-vortexed c. 0 s immediately prior to plating. This extraction liquid was then serially diluted ( : 0) and 0Æ-ml aliquots of each dilution were spread plated onto TSA in triplicate. The CFU were enumerated following incubation at ± C for h. As a quality assurance measure, the neutralized bleach solutions were analysed for spores via filter plating (method below) to ensure that no or only minimal spores remained (which we confirmed was the case). For coupons in which no CFU were detected in undiluted extraction liquid, typically two volumes (e.g. 9 ml) of the remaining extraction liquid were filter plated. In this procedure, the extraction liquid was drawn through a sterile, cellulose nitrate 7 mm diameter filter with 0Æ um pore size (part number 0-00; Nalgene Labware, Rochester, NY, USA). The filter was then rinsed with 0 ml of sterile DI water and then placed directly on a TSA plate. The CFU were enumerated after incubation at ± C for h. Decontamination efficacy Decontamination efficacy for each material and ph FAC combination was calculated in terms of log reduction (LR). For each test and positive-control coupon, the CFU recovered after the test was transformed to its log 0 value. Then the mean of the log 0 values for each test coupon (t) was subtracted from the mean of the log 0 values from each positive control (pc), as follows: Efficacy ðlrþ ¼ðlog CFUpcÞ ðlog CFUtÞ Test coupons in which there were no CFU recovered were assigned a CFU count of, resulting in a log CFU of zero. The LR results are reported showing error bars based on the pooled standard error (SE), calculated as follows: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi S SE ¼ pc þ S t where S is the standard deviation of either the five positive controls or three test coupons for each combination of coupon material ph FAC tested. Statistical analysis of the data was performed using sas 9. (SAS Institute, Cary, NC, USA). The overall effect (combined dataset) of the FAC concentration on decontamination efficacy was estimated using a heterogeneity of slopes multiple regression approach. Significant differences in efficacy between the materials and ph levels were tested using the Tukey adjustment for multiple comparisons. For the bleach stability data, the non-parametric Mann trend test was used to examine the null hypothesis of no effect against the one-sided alternative that FAC loss increased with increasing ph or with increasing initial FAC level. Results The results of the decontamination tests for galvanized steel, maple wood and ceramic materials are shown in Figs, respectively. Overall, the FAC concentration was judged to have a significant effect on efficacy (P-value = 0Æ009). With respect to the effect of ph level on efficacy, ph levels of Æ and were not significantly different from each other, while ph 7Æ was significantly less effective compared with ph Æ and (both P-values <0Æ00). Overall, the ceramic material was the easiest to decontaminate (P-value <0Æ00), with over 90% of the test conditions showing a LR or higher. The overall efficacy for galvanized metal was significantly higher than maple (P-value <0Æ00). For maple and spruce (data not shown) wood, the mean LR for c. 90% of the tests was between Æ Æ and Æ0 Æ0, respectively. The acidified bleach stability results, in terms of percent FAC loss over time, are shown in Fig.. In general, there was a positive relationship between FAC loss and initial FAC level at ph and 7Æ, with the exception of 000 ppm FAC ph 7Æ, where the FAC loss rate was 70 Letters in Applied Microbiology, 7 ª 0 The Society for Applied Microbiology

4 J.P. Wood et al. Acidified bleach sporicides 7 Galvanized steel coupons 7 Ceramic coupons Bleach free available chorine ppm Bleach free available chlorine ppm Figure Effect of bleach free available chlorine level and ph on the inactivation of Bacillus subtilis spores on galvanized steel. Triangle with solid line = ph.; circle with dashed line = ph ; square with dotted line = ph 7.. Data are reported as mean ± standard error. Figure Effect of bleach free available chlorine level and ph on the inactivation of Bacillus subtilis spores on ceramic. Triangle with solid line = ph.; circle with dashed line = ph ; square with dotted line = ph 7.. Data are reported as mean ± standard error. 7 Maple wood coupons Bleach free available chlorine ppm Free available chlorine percent loss per minute Initial free available chlorine level ppm Figure Effect of bleach free available chlorine level and ph on the inactivation of Bacillus subtilis spores on maple wood. Triangle with solid line = ph.; circle with dashed line = ph ; square with dotted line = ph 7.. Data are reported as mean ± standard error. higher than all other conditions. Based on the results, the initial FAC level was judged to have a significant effect (P =0Æ0) only at a ph of. The ph had a significant effect (P < 0Æ00) on bleach stability at initial FAC levels of 7000, 000, 9000 and ppm. Discussion Decontamination efficacy clearly depends on the material being decontaminated, and improvements (or lack thereof) in efficacy by lowering ph or increasing FAC Figure Effect of initial bleach free available chlorine (FAC) level and ph on the stability of bleach FAC. Triangle with solid line = ph.; circle with dashed line = ph ; square with dotted line = ph 7.. level of the bleach solution also depend on the material. Lowering the ph and or increasing the FAC level of the bleach solution improved efficacy in some cases; at ph Æ, the ceramic and galvanized steel coupons were all completely decontaminated, and thus efficacy did not improve with increasing the FAC level. The FAC levels in the bleach were most stable at ph Æ (i.e. having the lowest average percent loss per minute), and at that ph, stability was not significantly affected by the initial FAC level. We expected the loss of FAC at the lower ph to be the highest, because Cl gas forms as ph levels are lowered. However, the loss of FAC Letters in Applied Microbiology, 7 ª 0 The Society for Applied Microbiology 7

5 Acidified bleach sporicides J.P. Wood et al. because of volatilization of Cl gas may not be an important stability mechanism until the ph is lowered below, because at that ph, the percentage of FAC owing to Cl gas is still only c. % (Black and Veatch Corporation 00). Additionally, although Cl is volatile, transfer to the gas phase may be minimal depending on how the solution is stored (e.g. headspace, agitation). In light of the results obtained in the present study, it may be advisable to use bleach solutions with a ph of or lower (rather than raise FAC levels), considering both efficacy and stability. However, for materials where corrosion may be a concern, raising the ph and FAC may be desirable. For wood materials that were ineffectively decontaminated at all ph and FAC levels, studies have shown that liquid sporicides, such as peracetic acid, are more effective (e.g. Wood et al. 0). In addition, longer contact times improve inactivation efficacy of spores in suspension (e.g. Kuroiwa et al. 00), but it is unclear whether this would improve efficacy against spores on wood. Acknowledgements The authors thank Luther Smith (of Alion Science and Technology, for statistical analyses), Shawn Ryan, Brian Attwood, Christina Slone, Jared Novak, Stella Payne, John Foley and Rob Delafield for their technical assistance in this study. Disclaimer The U.S. Environmental Protection Agency through its Office of Research and Development funded and directed the research described herein, with technical support provided by ARCADIS under contract EP-C It has been subject to an administrative review but does not necessarily reflect the views of the Agency. No official endorsement should be inferred. EPA does not endorse the purchase or sale of any commercial products or services. References ASTM International (00) ASTM Standard D0-9 Standard Test Methods of Sampling and Chemical Analysis of Chlorine-Containing Bleaches. doi: 0.0/D0-9R0. Available at: (last accessed 0 October 0). Black and Veatch Corporation (00) White s Handbook of Chlorination and Alternative Disinfectants, th edn. pp.. Hoboken, New Jersey, USA: John Wiley and Sons, Inc. Carrera, M., Zandomeni, R.O., Fitzgibbon, J. and Sagripanti, J.-L. (007) Difference between the spore sizes of Bacillus anthracis and other Bacillus species. J Appl Microbiol 0, 0. Death, J.E. and Coates, D. (979) Effect of ph on sporicidal and microbicidal activity of buffered mixtures of alcohol and sodium hypochlorite. J Clin Pathol,. Dychdala, G.R. (00) Chlorine and chlorine compounds. In Disinfection, Sterilization, and Preservation, th edn ed. Block, S.S. pp.. Philadelphia, PA: Lippincott Williams & Wilkins. Kuroiwa, K., Nakayama, H., Kuwahara, T., Tamagawa, K., Hattori, K., Murakami, K., Korai, H. and Ohnishi, Y. (00) Augmenting effect of acetic acid for acidification on bactericidal activity of hypochlorite solution. Lett Appl Microbiol, 9. Lee, S.D., Ryan, S.P. and Snyder, E.G. (0) Development of an aerosol surface inoculation method for Bacillus spores. Appl Environ Microbiol 77,. Nguyen, T.Q., Clark, N. and The 00 NYC Anthrax Working Group (00) Public health and environmental response to the first case of naturally acquired inhalational anthrax in the United States in 0 years: infection of a New York City resident who worked with dried animal hides. J Public Health Manag Pract, Perez, J., Springthorpe, V.S. and Sattar, S.A. (00) Activity of selected oxidizing microbicides against the spores of Clostridium difficile: relevance to environmental control. Am J Infect Control, 0. Rastogi, V.K., Wallace, L., Smith, L.S., Ryan, S.P. and Martin, G.B. (009) Quantitative method to determine sporicidal decontamination of building surfaces by gaseous fumigants, and issues related to laboratory-scale studies. Appl Environ Microbiol 7, 9. Rutala, W.A. and Weber, D.J. (997) Uses of inorganic hypochlorite (bleach) in health-care facilities. Clin Microbiol Rev 0, Tomasino, S.F., Pines, R.M., Cottrill, M.P. and Hamilton, M.A. (00) Determining the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard nonporous surface using the Quantitative Three Step method: collaborative study. J AOAC Int 9,. Wood, J.P., Choi, Y.W., Rogers, J.V., Kelly, T.J., Riggs, K.B. and Willenberg, J.Z. (0) Efficacy of liquid spray decontaminants for inactivation of Bacillus anthracis spores on building and outdoor materials. J Appl Microbiol 0, 7. 7 Letters in Applied Microbiology, 7 ª 0 The Society for Applied Microbiology