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BRIEF COMMUNICATION Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 4  |  Page : 757-759
Evaluation of microbiocidal activity of superoxidized water on hospital isolates


1 Department of Microbiology, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India
2 Department of TB and Respiratory Medicine, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India

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Date of Web Publication27-Oct-2010
 

   Abstract 

Background: Prevention of nosocomial infections, pre-operative and post-operative complications is directly linked with effective disinfection and decontamination. Microbial decontamination is the most serious challenge to the today's health care practice despite the abundance of disinfectants and chemicals as there are increasing reports of emergence of resistance to the action of commonly used disinfectants. There is a need to evaluate the efficacy of newer methods of asepsis for better patient management. Aim: This study was designed to evaluate the microbiocidal activity of superoxidized water (SOW) on common clinical isolates, ATCC strains, vegetative cells and spores of Bacillus subtilis. Materials and Methods: Bacterial suspensions were treated with SOW and deionized water (control). All the tubes were incubated at 37°C for 0.5, 2.5 and 5.0 min. The number of viable cells was counted. Results: All the clinical isolates and ATCC strains were killed within 0.5 min of exposure to the SOW. Vegetative cells and spores of B. subtilis were killed after 5.0 min. Conclusion: We conclude that SOW is an effective microbiocidal agent for routine hospital use.

Keywords: ATCC strains, spores, superoxidized water, vegetative cells

How to cite this article:
Aggarwal R, Goel N, Chaudhary U, Kumar V, Ranjan K P. Evaluation of microbiocidal activity of superoxidized water on hospital isolates. Indian J Pathol Microbiol 2010;53:757-9

How to cite this URL:
Aggarwal R, Goel N, Chaudhary U, Kumar V, Ranjan K P. Evaluation of microbiocidal activity of superoxidized water on hospital isolates. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Dec 9];53:757-9. Available from: http://www.ijpmonline.org/text.asp?2010/53/4/757/72076



   Introduction Top


In today's health care, a burning issue is prevention of nosocomial infections, affecting almost 20-30% of the hospitalized patients. Prevention of pre-operative and post-operative complications is directly linked with effective disinfection of surgical instruments, equipments and microbial decontamination of environmental and inanimate objects. Notorious microbial species that are prevalent in the hospital environment are Pseudomonas aeruginosa, Acinetobacter, Escherichia coli, Enterobacter and Klebsiella. Microbial decontamination is the most serious challenge to the practical health care despite the abundance of disinfectants and chemicals. [1] There are increasing reports of emergence of resistance to the action of commonly used disinfectants. [2] Recently, newer chemical methods of asepsis, such as superoxidized water (SOW), are gaining importance as a hospital disinfectant. There is a need to evaluate the efficacy of newer methods of asepsis for better patient management. Thus, this preliminary study was designed to evaluate the microbiocidal effect of SOW on the common hospital bacterial strain under in vitro conditions.


   Materials and Methods Top


Test Solution (SOW)

SOW was produced on site using a disinfectant generation system. The raw material was tap water and electrolyte solution. The solution was fed into a special unit, which activated the water using an electric current. Specific gravity of the SOW was 1.02-1.06 g/ml, the oxidation reduction potential was +900 mV ± 100 mV and the pH was 7.0 ± 0.5. Shelf-life of the SOW was 120-144 h.

Microbial Isolates, Culture and Treatment with SOW

The test organisms S. aureus, P. aeruginosa, Acinetobacter, E. coli, Enterobacter, Klebsiella and Candida were isolated from hospitalized patients. Five different strains from each genera were included in the study. Bacillus subtilis was isolated as an environmental contaminant using standard microbiological techniques. [3] Standard control ATCC organisms S. aureus (ATCC 25923), E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) were also included in the study. All the isolates were subcultured on nutrient agar to obtain a single pure growth. Pure growth of the test organism was inoculated into nutrient broth and was incubated at 37 o C aerobically for 24h. Following incubation, organisms of each strain were collected, washed twice with 0.85% sodium chloride solution and resuspended in 1 ml of sodium chloride solution. Optical density of the suspension was adjusted to 0.5 McFarland (1.5 Χ 10 8 bacteria/ml) standards. Each suspension was divided into control and experimental parts. The control part was treated with 4.5 ml of deionized water and the experimental part was treated with SOW. The control and experiment tubes were incubated at 37ºC for 0.5, 2.5 and 5.0 min. After incubation, the number of viable cells was determined by the semi-quantitative culture technique. [3] Colonies of the inoculated organisms were counted on the plates after incubation at 37ºC for 24 h. All the organisms were inoculated in triplicate and the mean colony count was interpreted.

Preparation of Spores


To prepare a suspension of spores of B. subtilis, the organism was grown on agar slants for 7-10 days at 37ºC. Microscopic examination of the growth was performed daily after the 7 days till the microscopy showed profuse sporulation. After that, growth was harvested and washed thrice in distilled water. The spores were resuspended in 2 ml of distilled water and the suspension from this tube was divided into four equal parts. In this way, four tubes of 0.5 ml spore suspension were prepared. [4]

Treatment of Spores with SOW

Of the four tubes, to one control tube 4.5 ml of deionized water was added and to three experimental tubes, 4.5 ml of SOW was added. Three experimental tubes were incubated at 37ºC for 0.5, 2.5 and 5.0 min. After incubation, the tubes were centrifuged at 5,000 rpm for 5.0 min. The supernatant was discarded and to the sediment, 5 ml of glucose broth was added. All the three tubes were incubated for 24 h at 37ºC. After the incubation, the number of viable spores that had turned into vegetative cells was determined by the semi-quantitative culture technique. Colonies of the inoculated bacteria were counted on the plates after incubation at 37ºC for 24 h. The control tube was incubated at 37ºC for 24 h. After that, the tube was centrifuged at 5,000 rpm for 5.0 min and, to the sediment, 5 ml of glucose broth was added. This tube was incubated for 24 h at 37ºC. After incubation, the number of viable spores that might have turned into vegetative cells was determined by the semi-quantitative culture technique. [3]


   Results Top


Colony counts of all the hospital isolates and standard control ATCC organisms were reduced to zero after 0.5 min of treatment with SOW, whereas the counts of vegetative cells and spores of B. subtilis were reduced to zero after 5.0 min of exposure to SOW [Table 1]. Control tubes for all the organisms showed growth. Colony counts for the control are depicted in [Table 2].
Table 1 :Mean viable counts of organisms treated with SOW


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Table 2 :Mean viable counts of organisms treated with deionized water


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   Discussion Top


SOW is a high-level disinfectant. The active ingredients of this neutral anolyte are hydrogen peroxide, hypochlorous acid, hypochlorite ion, ozone, chlorine dioxide, chloric acid and chlorous acid. [1] SOW works by creating oxidative stress on the cell membrane and cell walls thus causing total destruction of the chromosomal and plasmid DNA, RNA and proteins. It is highly microbiocidal and is considered harmless to human tissue. [5] The present study shows that SOW generated in our conditions renders a strong microbiocidal action to Gram-positive, Gram-negative, Yeast cells as well to the vegetative cells and spores of B. subtilis.

At the exposure time of 0.5 min, all the potential pathogenic bacterial strains, Candida cells and standard ATCC strains were killed by treatment with SOW, whereas the vegetative cells and spores of Bacillus were killed after 5.0 min of treatment with SOW. Emergence of antimicrobial resistance and adaptation to other disinfectants have been reported in the literature. [2] Because of this resistance, it is difficult to contain the environmental dissemination of such microbial strains. Chances of adaptation of the microorganism to the SOW are suggested to be negligible. Also, SOW has been reported to increase a microorganism's susceptibility to antibiotics. [6] One of the potential limitations of our study is that the mycobactericidal action was not evaluated.

The SOW was used initially for sanitation, water disinfection and regeneration, but, considering its high biocidal activity, unique combination of detergent and antimicrobial properties and good compatibility with human tissues, made it a potential candidate for all types of microbial decontamination, including instrument and glassware disinfection, aerial fumigation, floor mopping and hand and feet disinfection. Our study shows that SOW is an effective microbiocidal against clinical isolates, control ATCC strains and vegetative cells and spores of B. subtilis and it can be a used as an effective disinfectant in hospitals and laboratories.

 
   References Top

1.Mikhailov SN, Mistryukov VV, Chuyeva IM. Disinfecting military hospital surgery unit with neutral anolyte. Available from: http://www.bakhir.com/publications/08-VoennoMedZhurnal.htm [last accessed on 2009 May 5].  Back to cited text no. 1
    
2.Windmer AF, Frei R. Decontamination, disinfection and sterilization. In: Murray PR, Baron EJ, Landry ML, Jorgensen JH, Pfaller MA, editors. Manual of clinical microbiology. 9 th ed. Washington: ASM Press; 2007. p. 65-96.  Back to cited text no. 2
    
3.Collee JG, Miles RS. Tests for identification of bacteria. In: Collee JG, Duguid JP, Fraser AG, Marmion BP, editors. Mackie and McCartney Practical medical microbiology. 13 th ed. Edinburgh: Churchill Livingstone; 1989. p. 141-61.  Back to cited text no. 3
    
4.Scott AC. Laboratory control of antimicrobial therapy. In: Collee JG, Duguid JP, Fraser AG, Marmion BP, editors. Mackie and McCartney Practical medical microbiology. 13 th ed. Edinburgh: Churchill Livingstone; 1989. p. 161-82.  Back to cited text no. 4
    
5.Marais J, Brozel V. Electrochemically activated water in dental unit water line. Br Dent J 1999;187:154-8.  Back to cited text no. 5
    
6.Menikova VM, Belikov GP, Lotionova NV, Bakhir VM, Sukhova OI. Prevention and therapy of nosocomial infections using electrochemically activated solutions. Available from: http://www.bakhir.com/publications/09-Melnikova-K o.htm [last accessed on 2009 Jul 5].  Back to cited text no. 6
    

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Correspondence Address:
Ritu Aggarwal
House No. 717, Sector 1, HUDA, Rohtak - 124 001, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.72076

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    Tables

  [Table 1], [Table 2]

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