Takashi NAKANO 1, 2, Hideki HAYASHI 1, 2, 3, Hong WU 2, Chikao SHIMAMOTO 1, 4, and Kouichi SANO 1, 2 1

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1 Biomedical Research (Tokyo) 36 (2) , 2015 Disinfection potential of electrolyzed strongly acidic water against Mycobacteria: Conditions of disinfection and recovery of disinfection potential by reelectrolysis Takashi NAKANO 1, 2, Hideki HAYASHI 1, 2, 3, Hong WU 2, Chikao SHIMAMOTO 1, 4, and Kouichi SANO 1, 2 1 Project Team for Medical Application of Electrolysis, Central Research Center, Osaka Medical College, 2-7 Daigaku-machi, Takatsukishi, Osaka , Japan; 2 Department of Microbiology and Infection Control, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki-shi, Osaka , Japan; 3 Product Planning Department, Kaigen Pharma Co., Ltd., 5-14 Doshomachi 2-chome, Chuo-ku, Osaka-shi, Osaka , Japan; and 4 Laboratory of Pharmacotherapy II, Osaka University of Pharmaceutical Sciences, 20-1 Nasahara 4-chome, Takatsuki-shi, Osaka , Japan (Received 20 December 2014; and accepted 7 January 2015) ABSTRACT We attempted to clarify in detail the conditions of disinfection using electrolyzed strongly acidic water (ESW) against Mycobacteria, and the recovery of the disinfection potential of inactivated ESW by re-electrolysis. We mixed ESW containing 10, 20, and 30 ppm free chlorine with M. bovis cells ( CFU/mL) for 0 7 min. The disinfection potential of ESW positively correlated with free chlorine concentration, and negatively correlated with the initial density of bacterial cells. To clarify the recovery of the disinfection potential of inactivated ESW by re-electrolysis, we mixed ESW containing 10 ppm free chlorine with M. bovis cells (10 7 CFU/mL) for 1 min. The number of viable cells decreased to 1/10 3, but the cells were still detected. After re-electrolysis for 7 min, viable cells were not detected. Moreover, we confirmed by reusing the re-electrolyzed water against M. bovis cells that it regained its disinfection potential. These findings indicate that ESW once inactivated during disinfection can be re-activated by re-electrolysis. In conclusion, we were able to clarify in detail the conditions of ESW against Mycobacteria, and found the recovery of the disinfection potential of inactivated ESW by re-electrolysis. Electrolyzed strongly acidic water (ESW) has been widely used for infection control in clinical settings (7, 9) because it has been proved to be effective for the disinfection of disinfectant-resistant bacteria, such as Mycobacteria (15), Pseudomonas aeruginosa (6), and spore-forming bacteria (3, 4, 6), and viruses causing blood-borne infections such as hepatitis B virus (8, 11) and HIV (5). ESW is also expected to be used against pathogens of emerging diseases, such as the avian influenza virus H5N1 (12). Free Address correspondence to: Takashi Nakano, Department of Microbiology and Infection Control, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki-shi, Osaka , Japan Tel: , Fax: tnakano@art.osaka-med.ac.jp chlorine (residual chlorine), which is the active component of ESW for disinfection, is easily inactivated by organic molecules. This feature of ESW is advantageous in terms of reducing environmental load and safety for patients and healthcare workers; however, it may be a disadvantage due to its unstable disinfective effect. Considering the effectiveness and the stability of the disinfective effect of ESW, high free chlorine concentrations are better for infection control; however, ESW with high free chlorine concentrations is corrosive against metals. There are no data available regarding the relationship between the free chlorine concentration of ESW and the number of bacterial This article is dedicated to the memory of Mr. Keisuke Seto.

2 110 cells that can possibly be disinfected, particularly the cells of Mycobacteria, which is one of the disinfectant-resistant bacterial genus. If such data are available, we can determine the lowest possible free chlorine concentration at which the stability and reliability of the disinfective effect are maintained. Moreover, such data will be important, because a free-chlorine sensing method has recently been developed (10), and continuous, accurate measurement and control of free chlorine concentration will be available using an apparatus installable at clinical settings. Another method of decreasing free chlorine concentration with a minimum decrease in the disinfection potential is to circulate ESW during the disinfection process with continuous electrolysis (14). For the application of this method, we need to confirm whether inactivated ESW, which is the used ESW after disinfection and whose free chlorine is inactivated by organic molecules, can recover its disinfection potential by re-electrolysis; however, the recovery of disinfection potential has not been proved yet. In this study, we attempted to clarify in detail 1) the conditions of disinfection of Mycobacteria, which is one of the disinfectant-resistant bacterial genus, using ESW and 2) the recovery of the disinfection potential of inactivated ESW by re-electrolysis. We obtained Mycobacterium bovis, BCG Tokyo strain from the BCG vaccine provided by Japan BCG Laboratory (Tokyo, Japan). We obtained clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium kansasii from the Central Clinical Laboratory of Osaka Medical College Hospital. We cultured the bacteria in Mycobroth (Kyokuto Pharmaceutical Industrial, Tokyo, Japan) at 37 C for 1 week with agitation once a day and resuspended them in phosphate-buffered saline (PBS) at McFarland standard #4 [cell density, approximately 10 8 colony-forming units (CFU) /ml]. We then serially diluted the suspension at 10 7, 10 6, and 10 5 CFU/mL. To eliminate the effect of the solvent of bacterial suspensions, we centrifuged bacterial suspensions, discarded the supernatant and resuspended the resulting pellets in ESW. We kept the suspension at room temperature for a designated duration of contact between bacterial cells and ESW. For recovery culture and measurement of CFU, we neutralized free chlorine of ESW with the bacterial suspension with 1% bovine serum albumin (BSA). For recovery culture (qualitative and semiquantitative assays), we inoculated bacterial suspensions on agar slants of 1% Ogawa medium (Nissui Pharmaceutical, Tokyo, T. Nakano et al. Japan) and incubated them at 37 C for days. We measured CFU (quantitative assay) by seeding 100 μl of each serially diluted suspension on the modified medium of 7H11 (7H11C agar plates; Kyokuto Pharmaceutical Industrial) and incubating it at 37 C for days, after which we counted the number of colonies. All experiments were conducted in triplicate. ESW was produced using an apparatus with a 2,000-mL well separated by a cationic membrane (Nafion 450; Dupont, NY, USA). Positive and negative platinum-coated titanium electrodes (11 19 cm each; Tanaka Kikinzoku Kogyo, Tokyo, Japan) were installed in each well. Then, ml of 0.1% NaCl in distilled water was electrolyzed for a designated duration at room temperature and a constant voltage of 24 V. ESW with the designated free chlorine concentration and ph was obtained by adjusting electrolysis time (7 15 min) and the amount of solution in each well. Inactivated ESW was reactivated using a smaller apparatus with a 200-mL well separated by the same cationic membrane and using platinum-iridium-coated titanium electrodes (4 6.5 cm each; Tanaka Kikinzoku Kogyo). Free chlorine concentration was measured with a chlorine meter (RC- 2Z; Kasahara Chemical Instruments, Saitama, Japan). The ph of ESW was measured using a ph meter (HM-31P; DKK-TOA, Tokyo, Japan) equipped with a ph electrode (GST-2729C; DKK-TOA). The ph of ESW samples used in the experiments was adjusted to 2.60 ± ESW samples containing ± 0.49, ± 0.35, and ± 0.86 ppm free chlorine were designated as ESW (10 ppm), ESW (20 ppm), and ESW (30 ppm), respectively. To analyze the bactericidal activities of ESW containing different concentrations of free chlorine, ESW (10 ppm), ESW (20 ppm), and ESW (30 ppm) were mixed with M. bovis cells of different densities. The initial densities of bacterial cells were determined to be approximately 10 5, 10 6, 10 7, and 10 8 /ml by the turbidity method. ESW (10 ppm) showed its complete bactericidal effect against 10 5 and 10 6 /ml of bacterial suspension within 1 and 3 min, respectively, but not against 10 7 and 10 8 /ml of bacterial suspension within 7 min (Fig. 1-a). Fig. 1-b shows the complete bactericidal effect of ESW (20 ppm) against 10 7 /ml of bacterial suspension within 3 min. The number of viable cells decreased to less than 1/10 5 for 10 8 /ml of bacterial suspension; however, the complete bactericidal effect was not obtained within 7 min of contact. Fig. 1-c shows the complete bactericidal effect of ESW (30 ppm) against bacterial suspensions of up to 10 7 /ml within 1 min. The

3 ESW against Mycobacteria 111 Fig. 1 Disinfection potential of electrolyzed strongly acidic water (ESW) containing various concentrations of free chlorine against M. bovis (BCG strain) cells at different densities. M. bovis (BCG strain) suspensions at 10 8, 10 7, 10 6, and 10 5 CFU/mL were mixed with ESW containing 10 ppm (a), 20 ppm (b) and 30 ppm (c) free chlorine for designated durations, and the number of viable cells was counted. All experiments were conducted in triplicate. Detection limit: <10 CFU /ml. number of viable cells decreased to less than 1/10 5 ; however, the complete bactericidal effect was not obtained within 7 min of contact. The disinfection potential of ESW correlated with free chlorine concentration, and inversely correlated with the initial density of bacterial cells. One of the reasons why disinfection potential reversely correlated with the initial density of bacterial cells is considered that organic materials, which are contained in bacterial cells, can inactivate free chlorine in ESW in a relatively short duration. Recently, Yamamoto et al. (15) have shown the disinfection potential of the electrolyzed acidic product of sodium chloride solution containing 30 ppm free chlorine (ph 3.5) against disinfectant-resistant bacteria, that is, Mycobacterium species. However, neither various densities of bacterial cells nor ESW samples containing different concentrations of free chlorine were used in their study. In our study, we analyzed the relationship between the free chlorine concentration of ESW and the density of Mycobacterium cells with regard to the disinfection potential of ESW. The improvement of sensors for measuring free chlorine concentration accurately and continuously (10) makes these data more important. In the quantitative analysis using the BCG strain, ESW (10 ppm) showed the potential to disinfect 10 6 /ml of bacterial suspension within 3 min. Therefore, we examined the disinfection potential of ESW against other Mycobacterium species using clinical strains by a qualitative or semiquantitative method using agar slants of Ogawa medium. We mixed bacterial cells with ESW for a designated duration and detected viable cells by culturing them on agar slants of 1% Ogawa medium. ESW showed the complete bactericidal effect against all the six clinically isolated strains of M. tuberculosis within 1 min of contact (the initial density of bacterial cells was estimated as 10 6 CFU/mL). Against non-tuberculous Mycobacteria (NTM), one strain each of M. avium, M. intracellulare, and M. kansasii, we evaluated the disinfection potential of ESW (10 ppm). The initial densities of cells of M. avium, M. intracellulare, and M. kansasii were determined as 6.51, 6.63, and 6.45 log CFU/mL, respectively. Against all the species/strains of NTM used, ESW (10 ppm) decreased the number of viable cells by less than 1/10 5 within 1 min of contact. ESW showed a sufficient disinfection potential against clinical, pathogenic strains of Mycobacterium species including M. tuberculosis. ESW can be applied to clinical settings with sufficient evidence from the data obtained in this study. As shown in Fig. 1, in the case when the complete bactericidal effect was not achieved, the number of viable cells decreased within 1 min, after which, it seemed that the ESW mixed with bacterial cells showed almost no disinfection potential. When this inactivated ESW was re-electrolyzed, its disinfection potential may be recovered. To confirm this reactivation effect, we performed the following experiment. ESW (10 ppm) was mixed with 10 7 CFU/mL of M. bovis (BCG strain) suspension for 1 min. The mixture was then transferred to the well of the

4 112 T. Nakano et al. Fig. 2 Recovery of disinfection potential of inactivated ESW (10 ppm) by re-electrolysis. An M. bovis (BCG strain) suspension at 10 7 /ml was mixed with ESW (10 ppm) for 1 min. The mixture was then transferred to the well of the smaller apparatus and re-electrolyzed at a constant voltage of 24 V for 7 min. At 0, 1, 3, 5, and 7 min of re-electrolysis, the mixture was sampled to determine free chlorine concentration and viable cell count. Experiments were conducted in triplicate. Detection limit of viable cells: <10 CFU/mL. smaller apparatus with the positive electrode and then re-electrolyzed at a constant voltage of 24 V for 7 min. It was sampled at 1, 3, 5, and 7 min to determine the number of viable cells and free chlorine concentration. Results are shown in Fig. 2. After 1 min of contact with ESW (10 ppm), the number of viable cells decreased to less than 1/10 3 ; however, the complete bactericidal effect was not achieved. At 1 min of contact, the residual free chlorine concentration was 1.7 ± 0.8 ppm, suggesting that free chlorine molecules were nearly completely consumed and inactivated. After re-electrolysis, the free chlorine concentration increased, and the bactericidal effect was recovered. After 7 min of re-electrolysis, viable cells were not detected, and the concentration of free chlorine was 9.2 ± 3.3 ppm, suggesting that the disinfection potential of the solution was recovered. Actually, when bacterial cells (2.82 ± CFU/mL) were again placed in 7-min-re-electrolyzed ESW, viable cells were below the detection limit within 1 min (result of triplicate experiments); which means that the re-electrolyzed ESW truly recovered its disinfection potential. These findings suggest that even when ESW is inactivated by disinfection, its disinfection potential can be recovered by re-electrolysis. One of the methods of decreasing free chlorine concentration when ESW is used for the disinfection of clinical devices is to circulate ESW during disinfection with continuous electrolysis. A disinfector for endoscopy devices has been developed on the basis of this principle and widely used in clinics and hospitals (14). In this study, it is proved that the disinfection potential of inactivated ESW can be recovered by re-electrolysis. This finding is considered to be important for applying ESW to disinfection method in clinical settings. High-level disinfectants, such as glutaraldehyde (GA), have disinfection potentials against any microorganisms; however, it has been reported that it may be harmful to patients and healthcare workers (1, 2). ESW is considered less toxic than GA (13), and it has been reported to be effective against disinfectantresistant bacteria, such as Pseudomonas aeruginosa (6), spore-forming bacteria (3, 4, 6), and bloodborne infectious pathogens such as hepatitis B virus (8, 11) and HIV (5) depending on the condition. Further study is needed to clarify both the disinfection spectrum of ESW and the optimum conditions for disinfection, particularly the relationship between

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