Comparative evaluation of disinfection mechanism of sodium hypochlorite, chlorine dioxide and electroactivated water on Enterococcus faecalis

Highlights

• The mode of action of NaOCl, ClO₂ and electroactivated water (EAW) was investigated.

• Enterococcus faecalis was used as an indicator microorganism.

• The EAW was the most effective disinfectant with more damage in less time.

• The bacterium was inactivated due to cell membrane damage at first.

• The leakage of intracellular molecules was the main reason of bacterial death.

Abstract

The aim of this study is to compare the efficiency of chlorine-based disinfectants, namely, sodium hypochlorite (NaOCl), chlorine dioxide (ClO₂), and electroactivated water (EAW), and to investigate their inactivation mechanisms on a Gram positive, opportunistic pathogen Enterococcus faecalis. Cell viability, damages on membrane integrity and cellular components were examined to reveal underlying mechanisms of the disinfection process. Among three disinfectants, EAW was the most effective one to inactivate E. faecalis in less time with more damage. According to flow cytometry results, EAW destroyed 95.4% of bacteria within 1 min whereas NaOCl and ClO₂ provided 73.9% and 72.6% bacterial inactivation after 20 min of treatment. The rapid increment at conductivity and lipid peroxidation within 30 s of treatment indicates that EAW causes membrane damage much more expeditiously compared to other disinfectants. DNA and protein leakage increased gradually during the treatment with EAW and reached 44.9 and 5.8 μg/ml, respectively. All disinfectants were found as inactivating the bacteria by disrupting cell membrane integrity, unbalancing the ion concentration and damaging the cellular components such as DNA, protein, lipid molecules. It is considered that EAW is more effective due to its superior content including chlorine dioxide, free radicals and ozone besides hypochlorous acid.

Introduction

Disinfection is a process of deactivation of pathogenic or non-pathogenic microorganisms and chemical disinfection is mainly used for this purpose. Chlorine-based technologies are favorable disinfection methods; chlorine gas, hypochlorite solution and other chlorine compounds in liquid and solid form are available as chemical agents (EPA, 1999). Sodium hypochlorite (NaOCl), which is the major compound of bleach, is an effective antiseptic substance against microorganisms (McDonnell & Russell, 1999). Chlorine dioxide (ClO₂) includes both oxygen and chlorine as oxidizing agents; thus, it is highly effective against bacteria, virus (Morino, Fukuda, Miura, & Shibata, 2011), fungi (Burton, Adhikari, Iossifova, Grinshpun, & Reponen, 2008) and even against spores (Young & Setlow, 2003). Chlorine dioxide has a wide range of application areas such as wastewater and/or drinking water treatment, food and beverage processing (Jonnalagadda & Nadupalli, 2014).

Electro activated water (EAW) is produced by electrolysis of a saline solution passing through the electrolysis chamber. When electric current is applied to the solution, electron exchange takes place between electrodes and water by means of a unipolar electrochemical process. Physical-chemical character of water changes due to the formation of polarization energy on electrodes during the electrolysis process. Inside the electrolysis chamber, negative and positive ions are separated; positive charges are accumulated at the anode surface while negative charges gather at the cathode surface. Consequently, two new products called catholyte and anolyte (namely EAW) are formed in the aqueous solution. Electro activated solution of anolyte has pH values from 7 to 1 and ORP from 0 to +1250 mV, while the catholyte solution has pH values from 7 units to 14 units and ORP from 0 to −950 mV. Anolyte is an aqueous solution with anomalously enhanced electro-acceptor properties. Thus, under unipolar electroprocessing of liquid, reduction products with alkaline reaction are being formed in a diaphragm cell in the cathode zone, and in the anodic zone, there are highly oxidative products with acid reaction.

Electro activated water is composed of oxygen gas and many active species such as chlorine, chlorine dioxide and free radicals, while catholyte contains hydrogen gas and sodium hydroxide (Koseki & Itoh, 2000; Kumon, 1997). Due to the presence of a sufficient number of strong oxidants and free radicals, EAW is a solution that has strongly biocidal properties (Hao et al., 2012). Several studies have presented inhibition capacity of EAW on vegetative and spore forms of various bacteria (Hao, Wu, Li, & Liu, 2016; Huang, Hung, Hsu, Huang, & Hwang, 2008; Rahman, Ding, & Oh, 2010; Udompijitkul, Daeschel, & Zhao, 2007). The mechanism of inhibition by EAW has also been evaluated on different bacterial cells so far (Table 1).

As stated before, the EAW contains more than one active specie, unlike chlorine and chlorine dioxide. The reason for these three disinfectants to cause different effects may be the diversity of components in the EAW content and their mode of action in the bacterial inactivation process. Therefore, the cell inactivation mechanism of EAW is thought to be different from other disinfectants. Although HOCl is the most abundant in the content of EAW, there are also ozone and free radicals increasing its ORP, which are responsible for the cell membrane degradation (Ding et al., 2016). Hypochlorous acid is present in both NaOCl and EAW as an oxidant, and it is responsible for the protein deterioration. Hypochlorous acid destroys certain enzymes participating in carbohydrate metabolism by oxidizing their sulfhydryl groups (Kim, Hung, & Brackett, 2000; Rahman et al., 2010).

The fact that the EAW has different physicochemical properties than chlorine, an interest has arisen in determining the differences in the inactivation mechanism. E. faecalis was selected as a test organism in this study to assess the disinfection mechanisms of three chlorine-based disinfectants since E. faecalis is an opportunistic pathogen and it can easily become dangerous and infectious for the human body (Rashid et al., 2017). Our previous study (Turkay et al., 2017) on E. faecalis inactivation presented only preliminarily results of inactivation mechanism of EAW. Indeed, E. faecalis was inactivated by the release of protein and intracellular components following cell membrane degradation. However, in order to propose the use of EAW as an alternative to presently used NaOCl and ClO₂, it is more appropriate to assess the inactivation effects comparatively and to discuss the results based on the differences in mechanisms of action. Therefore, in this study, sublethal concentrations of chlorine-based disinfectants (i.e. EAW, NaOCl and ClO₂) were applied to the pure culture of E. faecalis and a deeper investigation on the inactivation mechanism of EAW was conducted by using various methods. Cell viability determination by flow cytometry and TTC-dehydrogenase enzyme activity assay, investigation of cell membrane integrity by measurement of DNA and protein leakage, conductivity and lipid peroxidation levels, and examination of cellular components by the SDS-PAGE and FTIR analysis were conducted to clarify this effect.