Hospital Infection Control Bactericidal Effect of Medilox, an super oxidized water generated by Medilox against Bacteria, Fungi and Mycobacteria Tae Youn Choi, Bui Young Jang Feb 5, 2009 Departement of Clinical Pathology, College of medicine, Soonchunhyang University 657, Hanna: BL: a2) dong, Yongsan~gu, Seoul, Korea 709-9425 FAX : 82-2~790"-5820Journal of Soonchunhyang Medical Science 14(3) p.785~794 Feburary 2009 1 Bactericidal Effect of Medilox’, an super oxidized water generated by Medilox against Bacteria, Fungi and Mycobacteria Tae Youn Choi, Bui Young Jang Department of Laboratory Medicine, Soonchunhyang University Hospital, Seoul, Korea = Abstract = Background : Disinfection is essential for the prevention of hospital infection. Medilox, an super oxidized water generated by Medilox (SOO SANE&C CO., LTD. Yongin, Korea) was developed as a disinfectant in Korea. This is not costly and does not cause any clinical problems and environmental pollution. We evaluated bactericidal activity of Medilox against several clinical isolates and standard strains, Method : Clinical isolates and reference ATCC strains were exposed to Medilox, an super oxidized water (80 ppm of HOCI) generated by Medilox (SOO SAN E&CCO., LTD. Yongin, Korea) for the various periods (0.5, 1, 2, 5, and 10 minutes). After the exposure mixture of microorganisms and Medilox solution was inoculated into tryptic soy broth and onto tryptic soy agar, Sabouraud dextrose agar or Ogawa medium and cultured at 35°C. Results : All strains of bacteria, yeasts, mycobacteria and vegetative form of Bacillus subtilis were killed within 30 seconds after an exposure to Medilox (80 ppm of HOCI) under clean and dirty conditions. But, spore form of Bacillus subtilis was killed within 5 minutes. Conclusion: It may be recommended that Medilox can be used for the effective disinfectant for hospital environments and high-level disinfectant for hospital infection control. Key Words : Disinfectant, Medilox, Hospital infection control, super oxidized water Introduction There are lots of methods used to prevent an infection by pathogenic or opportunistic Author : Tae Yoon, Choi Address : Laboratory Medicine dept, Soonchunhyang University Hospital, 657, Hannam-dong, Yongsan-gu, Korea, 140-743, Tel :02-709-9425, / Fax : 02-790-5820 E-mail :choity@hosp.sch.ac.kr Date of Receipt : Oct 29, 2008 / Publish Confirmed Date: Feb 5,2009 (M) (D) (¥) soul, infection in the hospital, including washing, disinfection, and sterilization, Disinfectants are defined as the agents that fully kill the fungi and vegetative bacteria causing an infection within 10 minutes, not ones that exterminate the clostridial spores.2. Journal of Soonchunhyang Medical Science Vol. 4 No.3 Feburary 2009 Disinfectants are divided into a low level that can kill bacteria, virus or some fungi but not the myco- bacteria or clostridial spores; a mid level that actually kills the mycobacteria or fungi but not the clostridial spores; and a high level one that with enough time of exposition can kill even the clostridial spores, Disinfection involves different medicines and methods depending on the subject to be disinfected ; and nevertheless, selecting of proper disinfectants is important in the economic aspect and critical in terms of the effect of disinfection, Of the disinfectants used in the hospital, chlorine compounds are can apply to a wide range of treatment, work fast and cost lower than other compounds but the hypochlorous acid (HOCH indicates a big gapin antiseptic effects with ° With the brisk their different pH densities.* activities of infection management undertaken by hospitals, the super oxidized water containing the HOCI with a great antiseptic effect with water and clorine components electrolyzed is used as a disinfectant; its range of use is also expanding" HOCI is a disinfecting water that is generated at the point when the electrolyte containing is electr- lyzed around the neutral part. The mechanism in which this disinfectant works are not known explicitly but it seems to work in the way an activated oxygen is oxidized inside a cell to mitigate the enzyme functions while the HOCT creates a combination of nitrogen and chlorine, denatures the protein and inactivates the nucleic acid at the cell membrane and in a cell to introduce a electric shock to microorganisms with higher pH and transposition of oxidation-restoration.”” A common low-acid HOCT is colorless, odorless, and non-stimulus so poses no harm to human bodies and used in its liquefied state, In addition, it works so fast that itis used in a wide range of areas from washing of fruits and vegetables, disinfection of instruments under the Food Safety Law to disinfection of non- hazardous instruments used in hospitals and pseudo- hazardous ones such as an endoscope.) Since the Bac jokiller system (TRE, Japan) was first developed in Japan as @ running water type disinfecting device generating HOC, the Medilox (SOO SANE&C CO., LTD. Korea) and WATRIX-10G(MicroBank, Korea) were also developed in Korea as well as Sterilox (St- erilox medical UK Ltd., Abingdon, Oxon, UK) and the Cleantop (Ka- igen Co., Japan), an endoscope dedicated disinfecting device utilizing the HOCI. The authors evaluated the disinfecting capacity of the HOCI, Medilox, generated by 2 HOCI generating device, Medilox (SOO SAN E&C CO.,LTD. Yongin, Korea) developed in the nation with pathogenic and standard strains collected from the patients in Soon- chunhyang University Hospital, based on the time they are exposed to the disinfecting agent. Subjects and Methods of the Study 1. The Subject Bacteria Types and Strains A total of 6 strains of gram-positive bacteria sepa- rated from the specimens of inpatients at Soonchun- yang University Hospital including methicillin. susceptible Staphylococcus aureus (I strain),Journal of Soonchunhyang Medical Science 14(3) p.785~794 Feburary 2009 3 methicillin - resistant Staphylococcus aureus (1 strain), methicillin - susceptible Staphylococcus epidermidis (1 strain), methicillin-resistant Stap- hylococcus epidermidis (1 strain), vacomycin- resistant Enterococcus faecium (1 strain), and Enterococcus faecalis (1 strain) ; a total of 12 strains of Gram Negative Bacillus including Sal- monella Typhi (1 strain), Salmonella Enteritidis (1 strain), Shigella sonnei (1 strain), Pseudomo- nas aeruginosa (1 strain), Acinetobacter bauma- nnii (strain), Escherichia coli (1 strain), Serratia marcescens (1 strain), Klebsiella pneumoniae (1 strain), Enterobacter cloacae (1 strain), Citrobae- ter freundii (1 strain), Stenotrophomonas malto- philia (1 strain), and Proteus mirabilis (1 strain); a total of 2 strains of fungi including Candida albicans (1 strain) and Candida glabrata (1 strain); a total 5 strains of mycobacteria including Myc- obactetium tuberculosis (5 strains), Mycobacter~ ium avium (1 strain) and Mycobacterium absce- ssus (1 strain), The standard strains of bacteria include Staphylococcus aureus ATCC 29213, E. coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, and Bacillus subtilis ATCC 6633. 2. Subject Disinfectants Employed as a disinfectant into the study is Medilox (pH 5.7, HOC! 80 ppm) generated by Medilox (SOO SAN E&C CO., LTD. Yongin, Korea), a HOCI generating device. 3. Evaluation of Disinfecting Capacity Candida albicans was cultured in the tryptic soy broth (TSB) for 48 hours; others for 18 hours. ‘The mycobacteria were cultured in Ogawa culturing room for 6 weeks. The bacteria were disinfected at 3,000 rpm for 15 minutes with the upper layers discarded; 10 mL. of phosphate buffered soluiton(pH 7.2) put into the precipitated bacteria and wafted evenly with a mixer so that the turbidity be fixed to McFarland No. 3. Each of 0.5 mL bacteria solutions were put in 4.5 mL physiological saline solution and diluted in TSB containing 0.75% of lecithin from 10 to 10” times step by step to produce the final 10’~10° CFU/mL. of bacteria, Each 0.5 mL bacteria solution were added to Medilox 4.5 mL to be kept in a room temperature; the exposition time was set to 0.5, 1,2, 5, and 10 minutes; with the bacteria and fungi were inoculated with the disinfectant of 0.1 mL in 5 mL TSB broth containing lecithin 0.75%; the mycobacteria were cultured after inoculated to MGIT culturing solution (MGIT system, Be- cton Dickinson, USA) for the observation of whether the bacteria develop. Besides, to calculate the viable cell numbers and estimate the develo- pment of bacteria, a phased dilution was applied to the solution; 0.01 mL of standard platinum loop was used to culture the bacteria in tryptic soy agar (TSA), Candida species in Sabouraud dextrose agar, and Mycobacter- ium tuberculosis in Ogawa culture ground. To identify the antiseptic effect when the or ganic matters exist, the bacteria solution was added to the standard bacteria strain until the serum density reaches up to 3%; the test was repeated in the same way.4. Journal of Soonchunhyang Medical Science Vol.14 No.3 Feburary 2009 Results 1. Bactericidal effect against Gram - Positive Bacteria The number of all bacteria strains including Methicillin - susceptible Staphylococcus aureus, methicillin - resistant Staphylococcus aureus, methicillin - susceptible Staphylococcus epider - midis, methicillin - resistant Staphylococcus epi - dermidis, vacomyein - resistant enterococcus fa- ecium, Enterococcus feacalis, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis 29212 decreased more than 5 log10 per 30 second of exposition time at 80 ppm of HOCI (the rem- aining bacteria less than 1 x 10'/mL) (Table 1). 2. Bactericidal effect against Gram - Negative Bacteria The number of all bacteria strains including Salmonella Typhi, Salmonella Enteritidis, Shige- lla sonnei, Pseudomonas aeruginosa, Acinetoba- cter baummanii, E. coli, Serratia marcescens, Klebsiella pneumoniae, Entero - bacter cloacae, Citrobacter freundii, Stenotro - phomonas malto- philia, Proteus mirabilis, E. coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 dro- pped more than 5 log10 per 30 second of exposi- tion time at 80 ppm of HOCI (the remaining ba teria less than 1x 10°/mL) (Table 1), 3. Bactericidal effect against Yeast Fungi The number of one strain of Candida albicans and Candida glabrata, all declined more than 5 logl0 per 30 second of exposition time at 80 ppm of HOCI (the remaining bacteria less than 1 x 10° /mL) (Table 1), 4, Bactericidal effect against mycobacteria (Table 1).Journal of Soonchunhyang Medical Science 14(3) p.785~794 Feburary 2009.5 Table 1. Biocidal activity of Medilox (80pm of HOCI) against various microorganisms according to exposure time Initial Colonies remaining (C Test microorganism count | (CFU/mL) 95min Imin 2min 5 min 10min U/mL) o <10° <10° <10° <10° Methicillin-susceptible S.aureus 82x10? < Methicillin-resistant S. aureus 58x10 <10° <10° <10° <10° <10° Methicillin-susceptible S. epidermidis. 7.010" <10° <10° <10° <10° <10° Methicillin-resistant S. epidermidis. 8.9x10" << 10" <10° <10° <10° Vancomycin-resistant E. faecium 7.7x107 <10 <10 <10° <10 Enterococcus faecalis 91x10" <10° <10° <10° <10° <10° Escherichia coli 88x10" <10° <10° <10° <10° <10° Klebsiella pneumoniae 75x10’ <10° <10° <10° <10° <10° Serratia marcescens 65x10’ <10° <10° <10° <10° <10° Enterobacter cloacae 74x10’ <10° <10° <10° <10° <10° Salmonella Typhi 69x10? <10° <10° <10° <10° <10° Salmonella Enteritidis 71x10’ <10° <10° <10° <10° <10° Shigella sonnei 69x10? < 10° <10° <10° <10° Citrobacter freundii 6.2x107 <10° <10° <10° <10° Proteus mirabilis 9.1x107 <10° <10° <10° <10° <10° Pseudomonas aeruginosa 66x10) <10° <10 <10 <10° <10° Acinetobacter baumannii 6.2x107 <10° <10° <10° <10° Stenotrophomonas maltophilia 5.9x107 <10° <10° <10° <10° <10° Candida albicans 54x10? <10° <10° <10° <10° <10° Candida glabrata 52x10? <10' <10° <10° <10° <10° Bacillus subtilis, vegetative form 58x10? <107 < <10° <10° <10° Bacillus subtilis, spore form 62x10" <10° 2.0x10' 4.5x10°<10° <= 10°6 Journal of Soonchunhyang Medical Science 14(3) p.785~794 Feburary 2009 ‘Table 2. Biocidal a according to exposure time tivity of Medilox (80ppm of HOC!) against various Colonies remaining (CFU/mL) Test microorganism Culture medium Control 0.5 min min 2min Smin 10min Mycobacterium tuberculosis 1 fet + - ee Mycobacterium tuberculosis2 NE + - oe ee ‘Mycobacterium tuberculosis 3 ae + - See Mycobacterium tuberculosis 5 a + - ee ee Mycobacterium avium i Mycobacterium abscessus aes th + - - oe ee Five strains of Mycobactehum tuberculosis, ones train of Mycobacterium avium, and one strain of Mycobacterium abscessus all declined more than 5 log 10 per 30 second of exposition time at 80 ppm of HOCI (the remaining bacteria less than 1 x 10°/mL) (Table 2).Journal of Soonchunhyang Medical Science 14(3) p.785~794 Feburary 2009 7 5. Bactericidal effect against spore-forming bacteria, Bacillus subti ‘The number of Bacillus subtilis ATCC 6633 strain dropped more than 5 log 10 per 30 second of ex- position time at 80 ppm of HOCI (the remaining bacteria less than 1x 10'/mL) ; while the number of spore type decreased more than 5 logl0 per 5 minute exposition time(the remaining bacteria less than 1x10 /mL) (Table 1). 6, Bactericidal effect with the Organic Matters Contaminated In the contamination of the organic matters the numbers of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and E. coli ATCC 25922 strains dropped more than 5 log 10 per 30 second of exposition time at 80 ppm of HOCI (the remaining bacteria less than 1 x10" mL). Pseudomonas aeruginosa ATCC 27853 de- clined less than 5 log10 per 30 second of exposi- tion time at 80 ppm of HOCI (to less than 5 x10 {mL) and no growth was observed during the 1 minute of exposition (the he remaining bacteria less than 1x10"/mL) (Table 3). Table 3. Bactericidal activity of Medilox (80 ppm of HOC!) under clean and dirty conditions Initial Test microorganism count lean condition (CFU/mL) (CFU/mL) 05min Imin 2min 5 min Dirty condition (CFU/mL) 05min Imin 2min 5 min Staphylococcus aureus ATCC 29213 Toxo Enterococcus faecalis 9 919° <0" <10° ‘ATCC 29212 E.coli : ATCC 25922 62x10, < 10" = 10 Pseudomonas aenusi- ¢ +19 nosa ATCC 27853 <10° <10° <10° <10° <10° <10° <10° <10 <10° <10° <10° <10° <10° <10 <10 <108 Journal of Soonchunhyang Medical Science Vol.14 No.3 Feburary 2009 Discussion The Antiseptic effect of the HOCI natrium (NaOCl, lox) was derived from the ion (OCL-) of HOCK, The HOCT ion killed the ‘vegetative bacteria at 5 ppm, the mycobacteria at 1,000 ppm and of the bacterial spores, espe- cially the Bacillus subtilis at 100 ppm within 5 minutes up to 99.9%, In addition, the virus was killed at 200 ppm within10 minutes, the yeast fungi at 100 ppm within 1 hour, the spawn fungi at 200 ppm with 1 hour and the fungus spores at 500 ppm.’ However, the HOCT which has a antiseptic effect of more than 80 times higher than that of HOCI Jon showed a formi- dable performance at a low density equal to 30-60 ppm. The chlorine compounds predomi- nantly used so far include HOCI natrium and HOCI calcium products with a higher density have posed contamination related environ- mental problems. As an effort to address this issue and escalate the proportion of HOCI which has a strong disinfecting capacity and causes little environmental problems, super oxidized water (HOCI) generated from elec- trolysis of water and chlorine components is under development as a disinfectant. Super oxidized water is divided into higher, "middle and low acid ones depending on its pH super oxidized (bigh acid water), super oxidized water (middle acid water) and super oxidized water (lowacid water), developed higher acid superoxidized water has pH level of 2.3 - 2.7, which has a good perfor- ‘mance in killing bacteria but produces chlorine gas harmful to the human body, involves a strong metal decay and short time period for storage, a series of managerial problems Developed to complement these short comings water the earliest are middle acid super oxidized water with pH level of 2.8 - 5.0 and low acid super oxidized water with pH of 5.0 - 7.0. The low acid super oxidized water is manufactured in the way the 100% HOC, Provides enough antiseptic effects even at a low density, Bacterio killer (BK) system (Japan), which was first developed by means of the method to compound the super oxidized water is chlorine residues becomes a running water type disinfecting instrument that let out the mixture of high acid super oxidized ‘water and tap water could be applied to common hand washings or disinfecting and cleaning of various devices, with its chlorine residues density of pH 6.28-6,38 at 20-25 ppm!” After- wards, Medilox, a low acid super oxidized water producing device and WATRIX, a middle acid super oxidized water compounding tool were domestically developed.” In U:K., a low acid super oxidized water producing system, Sterilox ‘was introduced.’ Medilox is currently produc- ing a super oxidized water with pH level of 4.5- 7.0, oxidation-restoration transposition of 800- 1,000 mV, and HOCI density of 60-80 ppm: Sterilox produces a super oxidized water with pH level of 5.0-6.5, oxidation-restoration transposi« tion of above 950 mV, and HOCT density of around 144 ppm!" Developed in Japan, an endo- scope disinfecting device using super oxidized ‘water, Clentop produces a super oxidized water with pH level of 2.5.0.2, oxidation-restoration transposition of higher than 1,120 mV, and the chlorine residues’s density of 5.0+0.2 ppm.” Super oxidized water, which has no color, smell and is not stimulus, does not raise any side effects to the skin from its repeated use and is _espe- cially useful in washing the hands, the primaryJournal of Soonchunhyang Medical Science 14(3) p.785~794 Feburary 2009 9 culprit of infection. The infectants commonly used in hand washing before an operation or invasive procedure include alcohol, chlothex- idine, povidone - iodine, etc; however, using these agents in the hospital by the medical st- aff for daily hand washing can cause a poor adaptability including damages to the skin. The best solution for this problem has been washing the hand with flowing tap water and soap for a long time; super oxidized water may possibly replace this method since it poses little harms to the skin with its repeated use and is relatively outstanding in its disinfecting performance. As a result of the test on the disinfection capacity of super oxidized water (the density of chlorine residues at 20-25 ppm, pH 6.28-6. 38), its antiseptic effect was strong enough to kill both the common germs and fungi within 30 seconds; some of Bacillus species died within 30 seconds but some were not killed even until the elapse of 4, 8 or 128 minutes.” Domestically developed super oxidized water, Hicro-S and WATRIX-10G showed its disinf- ecting capacity to kill both the common bact- ceria and fungi within 30 seconds at the HOCI density of 30-50 ppm.*® A spore forming germ, vegetative form of Bacillus subtilis in 10 min at 30 ppm, 4 min at 40 ppm and 2 min at 50 ppm; spore form of Bacillus subtilis within 30 min at 30 ppm, 15 min at 40 ppm and 10 min at 50 ppm.5® ‘The results of the test for Me- dilox with the HOCI density of 80 ppm reve- aled that the vegetative type of Bacillus subt- ilis was killed with the exposition time of 30; spore type of Bacillus subtilis when exposed for 5 min. Sterilox killed the spore-forming bacteria within 2 min at the density level of 144 ppm, which indicates a higher density of HOCI means a shorter disinfecting time.!!"”) Sterilox, a super oxidized water developed in U.K. has the HOCI density of 144 ppm ; it could kill not only the common bacteria but also Helicobacter pylori, vancomycin-resistant Enterococcus species (VRE), Candida albicans, Mycobacterium species, Clostridium difficile spore, Bacillus subtilis var niger spore, Can- dida albicans, poliovirus, and HIV (human immunodeficiency virus), showing its disin- fection capacity to kill more than 5 logl0 germs within 2 min. Besides, Sterilox, in an environment where the serum is contaminated by 5%, killed Helico -bacter pylori, vancom- ycin-resistant Enteroco-ccus species (VRE), Candida albicans, Myco -bacterium species, etc up to 5 logl0 within 2 min, Not decayed by a metal and untoxic to the organization of human body, any Sterilox generated within 24 hours will be suitable for the disinfection of endoscopes.'"") However, in a case a large number of organic matters, which inactivate the disinfection are contaminated, the disinf- ecting capacity of Sterilox for the Clostri- dium difficile spore can be prominently dete- riorated so it is indispensible to wash the en- doscope.' It has been indicated that the super oxidized water sterilizes most of bacteria and fungi except spore type germs at the HOCI density of 20-40 ppm in 30 sec, which is a disinfecting capacity equivalent to Wydex, Vipon, Tego-51, 70% ethanol, Biospot, etc.) With regard to the disinfecting capacity for Bacillus subtilis in a nourished state, Hicro-S and WATRIX super oxidized water except Bacteriokiller system killed the bacteria within 4 min at the HOCI density of 30-40 ppm de~ pending on different strains of bacteria; while10. Journal of Soonchunhyang Medical Science Vol.14 No.3 Feburary 2009 the spore - forming type Bacillus subtilis generally was not killed until 15 min at 30 ppm but some strains were disinfected in 15 to 30 min at 40 ppm; and in 10 min at 50 ppm.>% As a result of this study, the low acid Medilox showed a antiseptic effect to kill all of the common bacteria as well as fungi and mycobacteria (inclu- ding the mycobacteria) up to more than 5 logl0 in 30 sec of exposition time at 80 ppm; and killed the Bacillus subtilis spore up to than 5 log10 in 5 min. In addition, the same performance to kill up to more than 5 log10 in 30 sec of exposition time was maintained even in the environment that the serum was contaminated by 3% When the floor and the walls were disinfected with super oxidized water, Bactericidal effect against methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii was equal to killing up to 4 log10 or 6 log10 with only one time of aerosol; two times of aerosol resulted in disin- fecting both germs up to around 7 log10, which reportedly can be useful also for disinfections in a hospital environment." The endoscope which contacts the membranes of human body and belongs to a semi- critical item requires a high class disinfectant if all the bacteria can be removed. What recommended so far was to dip it in 2% glutaraldehyde for about 20 min) but now it has been started to import the super oxidized water from Japan and Europe to reduce the time taken for disinfecting the endos- cope.) The endoscope dedicated disinfection system developed in Japan, Cleantop (Kaigen Co., Japan) uses a high acid super oxidized water and disinfects the endoscope for 7 min." One study estimating this system's disinfecting perfo- rmance reported the bacteria and yeast fungi am- ounting to 3-5 log 10 CFU/ mL before the disinfection were almost gone after the disinfection using the Cleantop.”” And also, one of domestic studies employing the Cleantop WM-1 revealed its excellent efficiency of disinfection." ‘The result of a study that compared the antiseptic effects of HOCI between in a clean state and ina contamination by organic matters showed the effective chlorine density performing more than 99.99% was 50-65 ppm for E. colia in a clean state; while in a contamination by organic matters, 120-250 ppm; for the Staphylococcus aureus, the figure further jumped to 320-385 ppm from 80-100 ppm, which indicates a disinfectant in the inorganic chlorine class has lower disinfecting capacity in a contaminated state.!® Any compounds categ- orized into chlorine class show a rapidly deterio- rating performance due to organic matters or metals, which means a contaminated state requires an extra amount of disinfecting agents as well as a thorough washing of matters that can potentially exist on the surface of a certain instruments compared with other disinfectants not affected by organic matters. With the existence of organic matters, the disinf- ecting performance of super oxidized water dec!- ines so washing a device contaminated with blood or organic fluids prerequisites a thorough washing of the same device. To put the above observations of other literatures and the results of this study, the super oxidized water Medilox as a high-level disinfectant, has a good performance in disinfec- ting not only the ordinary bacteria, fungi and mycobacteria but also spore - forming bacteria, presumably useful from hand washing, disinfection of skin and cleanings in hospital environments to infection management including the disinfection of non-critical and semi-critical medical items.Journal of Soonchunhyang Medical Science 14(3) p.785~794 Feburary 2009 II References 1. Nosocomial Infection Control 3rd edition, pp. 53-97, Korean Society for Nosocomial Infection Control, Hanmi Publish, Seoul, 2006, 2. Rutala WA. Disinfection, sterilization and waste disposal. 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