In Vitro Study Of The Bactericidal Efficiency Of Electrolyzed Water On Enterococcus Faecalis Biofilm

In the endodontic clinical, pulpal and periapical diseases are frequently-occurring.Many factors like microbial infection, physical and chemical stimuli, and immuneresponse are the cause of pulpal and periapical diseases. And microorganism is the maincause. Many clinical studies and animal experiments have shown that they could isolatebacteria from diseased pulp and periapical tissue. Microorganisms that caused pulpal andperiapical diseases are Black-pigmented bacteria (BPB), Fusobacteria, Actinomyces,Spirochetes, Enterococcus faecalis (E.faecalis，E.f), and so on.Up to now, root canal therapy (RCT) is the most commonly used and the mosteffectively method to treat endodontic disease. RCT prepared root canal by mechanicaldebridement and chemical disinfection. The source of irritation and the diseased pulp canbe eliminated through the preparation, disinfection and obturation. And it can also avoid the secondary infection of the root canal. But the clinical, some root canal treatment isfailed because of the secondary infection of some microorganism, and E.faecalis is themain one.E.faecalis belongs to streptococcus and is the representative of the genusenterococcus. It is the resident bacteria of upper respiratory tract, oral cavity and intestinaltract. Many studies have shown that they could isolate E.faecalis from diseased pulp andtissue. It is hardly isolated in the first infected root canals, but there are many bacteria inthe secondary infected root canal. Usually, E.faecalis caused endodontic infections byitself or as the main pathogenic bacteria of various bacteria mixed infection. E.faecalis cannot only adhesion on the root canal wall and form biofilm, but also intrude into thedentinal tubule and survive and reproduce in very hard conditions. Biofilm is a populationof cells which encases in the extracellular matrix, and attaches on various biotic andabiotic surfaces. Bacteria in the biofilm could live without adverse factor and haveenhanced virulence. RCT could not eliminate it thoroughly by preparation, disinfectionand obturation, it also need effective irrigation. The irrigant which is used commonly inclinic is not effective and has virulence. So, we need a safe and effective irrigant toeliminate E.faecalis in the root canal, thoroughly.In recent years, electrolyzed water has been used in clinic as a new irrigant, whichwas produced by electrolyzed water generator. SAEW and AEW are both electrolyzedwater. SAEW was first used in the disinfection of environment, food and agriculture, andhas been used as irrigant since1996, in Japan. SAEW has many advantages such assterilization, wide antibacterial spectrum, safe to tissue, and it can change into water afterbe used. AEW has strong cleaning and decontamination ability, can dissolve the necrotictissue safely and effectively, eliminate smear layer and is harmless to the environment.The aim of the research was to observe the bactericidal efficiency of electrolyzed water toE.faecalis biofilm through different experiments. The research has three parts.1. Observe the bactericidal efficiency of electrolyzed water to E.faecalis biofilmby scanning electron microscopy The24h E. faecalis biofilm on coverslips were separatedly treated by SAEW, AEW,5.25%NaOCl, and NS for1min,5min, and10min. And then, they were fxed overnightwith2.5%glutaraldehyde in4℃, dehydrated in an ascending cetonitrile series (50%,70%,80%and90%for20min each and100%twice for20min), dried in a lyophilizer, sputtercoated with platinum, and observed by scanning electron microscopy. After treatment withSAEW, AEW and5.25%NaOCl, the biofilm decreased greatly, and the bacterial cellsbecame rough, shrunken, and even lysed; the cell damage became more obvious as thetreating time increased. Our findings suggest electrolyzed water has bactericidal effect toE.faecalis biofilm.2. The bactericidal efficiency of electrolyzed water on E.faecalis biofilm in thebovine dentin tubules in vitroAfter the in vitro model of E. faecalis infected bovine dentin tubule was prepared,they were separated treated by SAEW, AEW,5.25%NaOCl, and NS for1min,5min, and10min. Dentin powder of different depths inside the dentinal tubules was collected toextract bacterial gene DNA which was used for real-time quantitative PCR. The resultshowed that the bactericidal efficiency of SAEW and AEW were both enhanced as thetreating time increased, and the bactericidal efficiency of SAEW was equal to that of5.25%NaOCl; AEW also had a favorable bactericidal efficiency, although it was not aseffective as SAEW or5.25%NaOCl. Our findings determine the bactericidal effect ofelectrolyzed water to the E.faecalis biofilm again, and suggest that SAEW could be usedas irrigant in the root canal therapy.3. The bactericidal efficiency of electrolyzed water on E.faecalis biofilm undershear forceThe BioFlux system, which provides constant and controlled shear forces, was usedfor bacterial biofilm formation. After E.faecalis biofilms under shear force were formed,they were separated treated by SAEW, AEW,5.25%NaOCl, and NS for1min,5min, and10min. And then, the treated E.faecalis bioflms were stained with a LIVE/DEAD BacLightTMBacterial Viability Kit and observed by CLSM. The live and dead cells wereseparated stained into green and red fuorescence. The fuorescence intensity of the imageswas relatively quantifed using the NIH Freeware ImageJ. Fluorescence images wereintegrated and used to calculate the ratio of green fuorescence intensity to that of greenand red fuorescence. The ratios represented the relative viability of E.faecalis afterdifferent treatments. The results were the same as previous. The E.faecalis biofilms oncoverslips, which were stained with a LIVE/DEAD BacLightTMBacterial Viability Kitand viable cell counts, were used as control. We got the same result. SAEW appears to bean ideal root canal irrigant.