Research ArticlePlasma Sterilization Effectively Reduces BacterialContamination in Dental Unit Waterlines

Sarocha Noopan, Phattranit Unchui, Supitcha Techotinnakorn,and Ruchanee Salingcarnboriboon Ampornaramveth

Research Unit on Oral Microbiology and Immunology, Department of Microbiology, Faculty of Dentistry,Chulalongkorn University, Bangkok, !ailand

Correspondence should be addressed to Ruchanee Salingcarnboriboon Ampornaramveth; ruchanee.a@chula.ac.th

Received 22 March 2019; Revised 1 July 2019; Accepted 14 July 2019; Published 30 July 2019

Academic Editor: Andrea Scribante

Copyright © 2019 Sarocha Noopan et al. &is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Objective. To investigate the effectiveness of plasma sterilization in reducing bacterial contamination and controlling biofilms indental unit waterlines.Materials and Methods. Ten identical dental chair units (DCUs) were used. Five DCUs were installed withan automated plasma sterilization system (PSS) and the other five were kept as nontreated controls (CTL). Water flushed from theairotor line served as the output water of the dental unit waterlines (DUWLs). Water samples were collected at the beginning andon a weekly basis for 4months. Water was analyzed for bacterial contamination (CFU/mL). Scanning electron microscopy (SEM)was used to investigate the amount of biofilm in the waterlines. Biofilm viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. All statistical analyses were performed using the Mann–WhitneyU test. A valueof p< 0.05 was considered significant. Results. &e DCU output water was found to be heavily contaminated with bacteria. Plasmasterilization effectively reduced bacterial contamination from an average of 212CFU/mL to 8CFU/mL. During the entire period of4months, the level remained below 500CFU/mL, the standard level recommended by the Centers for Disease Control andPrevention (CDC) of the USA. &e reduction in the bacterial count was significant compared with the CTL group (p< 0.05).Plasma sterilization could not eradicate the existing biofilms in the waterlines, and it did reduce biofilm mass and viability.Moreover, treatment with plasma sterilization did not induce a change in the composition of microorganisms, as analyzed byGram staining. Conclusion. Plasma sterilization, which is part of electrochemically activated water, effectively reduces bacterialcontamination and reduces biofilms in dental unit waterlines.

1. Introduction

Dental chair units make use of water as a coolant for high-speed rotary dental instruments and for washing the mouthduring the dental procedure. &e input water of a dentalchair unit can be derived from different sources: eitherdirectly from municipal water or from a separate waterreservoir. Once it enters the dental chair unit, the water willbe flushed through the complex small tubing system tosupply the airotor handpiece and air-water syringe. &edental chair unit output water has been reported to be highlycontaminated by microorganisms [1]. Predominant bacteriaspecies found in dental unit waterlines (DUWLs) are Gram-negative aerobic heterotrophic environmental species

including Pseudomonas species, particularly Pseudomonasaeruginosa, and Legionella species, particularly Legionellapneumophila, and nontuberculosis mycobacterial species[2–4]. &e microorganisms in the tubing system formbiofilms which continuously release microorganisms thatcontaminate output water. Contamination of DUWLs thusposes a serious concern in infection control in dentalpractice.

&e main obstacle to eliminate effectively microorgan-isms in DUWLs is the formation of biofilm that aggravatesresistance to a wide range of disinfection methods. Cur-rently, several methods are proposed to reduce bacterialcontamination in DUWLs. &e most popular method isdisinfecting with chemical disinfectants. Commonly used

HindawiInternational Journal of DentistryVolume 2019, Article ID 5720204, 6 pageshttps://doi.org/10.1155/2019/5720204

products are peracetic acid, ethylenediaminetetraacetic acid,tetrasodium salt, sodium hypochlorite, chlorhexidine glu-conate, povidone iodine, hydrogen peroxide, and chlorinedioxide [5]. Previous studies indicated that chemical dis-infectants including peracetic acid [6], hydrogen peroxide[7],N-halamine [8], D-amino acid [9], povidone iodine [10],and nanosilver [11] significantly reduced the bacterial countin DUWLs. However, most of them could not totallyeradicate biofilm in long-term use [12]. Some disinfectants(e.g., chlorine compounds and acetic acid) even causeddamage to the water pipes [13, 14]. Most of these disin-fectants could only be used as shock treatment due to theirharmfulness to the patients and need more compliance fromstaff in dental clinic.

An alternative approach using plasma sterilizationtechnology for disinfecting DUWLs is widely used in theKorean dental profession. &e machine produces positiveand negative charges, so-called plasma ions, and this dis-infects the water system. &e plasma particles are generatedby high-voltage electricity that split passing water into thereactive oxygen species OH−, H2O2, and O3. &e plasmasystem utilized in this study is a platinum catalyst andproduces plasma with low temperature in water, and as aresult, H2O is resolved into H+, O−, and O3 and form OH−,HOCl, and H2O2. &ese plasma particles have a bactericidaleffect due to their strong oxidizing properties. Despite theeffectiveness claimed by the company, very few studies re-port the efficacy of plasma sterilization in decontaminatingbiofilms in dental unit water lines [15].

&e purpose of this study was to investigate the effec-tiveness of plasma sterilization on reducing bacterial con-tamination and controlling biofilms in dental unit waterlines.

2. Materials and Methods

2.1. Sample Collection. Ten identical dental chair units(DCUs) at the Faculty of Dentistry, Chulalongkorn Uni-versity, were selected for the study. &e DCUs were dividedinto 2 groups: (1) plasma sterilization system (PSS) and (2)nontreatment control (CTL). &e chairs of the PSS groupwere equipped with a plasma sterilization system (DPS-110bi, Dentozone, Seoul) which resulted in a continuousgeneration of plasma ions into the DUWLs.

At the start of the experiment, water from 2 sources wascollected from each unit to assess baseline bacterial con-tamination. &ese two sources were as follows: the cup-filledwater was kept as input water which was obtained directlyfrom the main water inlet of the building and water flushedfrom the airotor line served as DUWL output water. &ewater samples were collected weekly for 4months to analyzebacterial contamination of the dental unit output water.

2.2. Laboratory Investigation. After collection, the watersamples were transferred directly to the laboratory and keptat 4°C until plate count was done. In order to break upclumped bacterial biofilms, the water samples were soni-cated for 10 seconds before serial dilution and they weresubsequently plated onto R2A agar (HiMedia, Mumbai,

India) and incubated at 36°C for 3–7 days. &e number ofbacterial colonies was counted and expressed as coloniesforming unit per mL (CFU/mL). Colony formation andGram staining were performed to characterize bacteriafound in the water before and after treatment with PSS.Gram staining was performed as follows: the colonies weresmeared onto a glass slide and fixed with heating using aflame.&e smear was then stained with crystal violet, iodine,washed with alcohol, and counterstained with safranin.

Scanning electron microscopy was used to investigatewhether plasma sterilization could eliminate biofilm for-mation on the waterlines. At the end of the experimentalperiod (4months), the tubes of the DUWLs were collected,fixed in 2.5% glutaraldehyde for 24 hours, and washed with1% PBS before sectioning into small pieces. &e specimenswere serially dehydrated, critical point dried, gold sputtercoated, and examined by using scanning electron micros-copy (SEM) (FEI Quanta 250, OR).

MTTassays were used to determine the biofilm viability.&e tubes from DUWLs were soaked in 100 μL of 0.5mg/mLMTTsolution (Affymetrix, CA) and incubated for 3 hours at37°C. &e MTT solution was then removed, and 100 μLDMSO was added and incubated for 15minutes at roomtemperature. &e OD value was measured at a wavelength of570 nm.

All data were analyzed and compared using theMann–Whitney U test. A p value of 0.05 was consideredstatistically significant. &e Ethics Committee of the Facultyof Dentistry, Chulalongkorn University, &ailand, has ap-proved the study to be carried out according to the protocoland informed permission dated and/or amended as followsin compliance with the ICH/GCP (HREC-DCU 2018-006).

3. Results

Dental chair unit input water was heavily contaminated withbacteria ranging from an average of 4,800 to 30,440CFUs/mL. &ere was not a significant difference in the bacterialcount of input water between the PSS and the nontreatmentcontrol group (Figures 1(a) and 1(b)). &roughout the entireperiod of 4months, the dental unit output water was found tobe heavily contaminated with bacteria. &e maximum bac-terial counts were as high as an average of 35,400CFUs/mL.

After the first week of plasma sterilization, the number ofbacteria was dramatically reduced and maintained at thesame level until the end of the experiment at week 16(Figures 2(a) and 2(b)). &e average CFU/mL of bacteriarecovered from airotor line of PSS group was 212CFU/mL,which was significantly lower than the control group(p< 0.05) (Figure 2(c)).

Scanning electron microscopy revealed the presence ofbacterial biofilms covering the inner surface of the waterlinesin the control group (Figure 3). &e biofilm mass wasstrongly reduced in the PSS group (Figure 3). In line with thereduced amount of biofilm, MTT analysis revealed a sig-nificantly reduced viability of these samples (Figure 4).

According to the results obtained from the Gramstaining, the PSS did not induce a selective shift in types ofmicroorganisms. &e colonies found in output water at

2 International Journal of Dentistry

baseline and 16weeks after PSS installation still have thesame Gram staining profiles. &e ratio of Gram positive,Gram negative bacteria, and fungi were not differentcompared to the baseline from week 16 (Figure 5).

4. Discussion

Our results demonstrated that plasma sterilization signifi-cantly reduced bacterial contamination in dental unit wa-terline output water when applied as continuous treatment.&e average CFUs/mL of bacteria recovered from DUWL ofthe plasma sterilization group was less than 500CFU/mL.&is value is within the standard level recommended by theAmerican Dental Association (ADA). SEM analysis revealedthat after 4months of PSS treatment the bacterial biofilmsinside the waterlines were reduced. &e MTT assay alsoindicated that the system effectively decreased biofilm massas reflected by a reduction in viability. According to previousstudies reported by Marais and Brozel [12], the efficacy ofelectrochemically activated (ECA) water was demonstrated;ECA markedly reduced the number of bacterial colonies(<1CFU/mL) and also eliminated the biofilm more

completely than a conventional disinfectant (0.5% sodiumhypochlorite solution). Treatment with ECA resulted in asmooth and clean surface of the waterlines. Meanwhile, thebiofilm in the sodium hypochlorite-treated group proved tobe cracked. Our study proposes a clinical application of theplasma sterilization system for the reduction of bacterialcontamination in dental unit waterlines.

&ere are several approaches to control bacterial con-tamination in dental unit waterlines including both non-chemical and chemical methods. However, most of thesemethods provide uncertain effects on controlling bacterialbiofilms in the complex tubing system of DUWLs. &ere-fore, the effectiveness of the plasma sterilization system inreducing both bacterial count and biofilmmass appears to bean attractive way to control biofilms inside the complextubing system of dental chair units.

Currently, for decontamination processes, plasma ionsare used as an energy reservoir to activate a variety ofchemicals present in or added to the solutions. Likewise, inour study, by using the electrochemically activated principle,the plasma particles are positively and negatively chargedand produced by high-voltage electricity. &ese charges will

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Figure 1: (a) Total bacterial count from dental chair unit input water. Each line indicates the average CFU/mL at different time points. &eblue line represents the average CFU/mL from input water of the PSS group.&e red line represents the average CFU/mL from input water ofthe control group. &e average number of the bacterial count in CFU/mL is presented in the table below the chart. (b) &e average CFU/mLof the entire period of the experiment. &e bacterial count of input water of the two groups is not significantly different.

International Journal of Dentistry 3

split water molecules into the reactive oxygen species (ROS)OH−, H2O2, and O−3 . In this way, PSS generates strongoxidants which provide bactericidal effects so that the

exopolymer matrix that coats the biofilms collapses due tohydroxyl radicals. &en, the bacterial colonies inside areexposed to other oxidative agents and subsequently killed

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Figure 2: (a) Total bacterial count from dental chair unit output water (from airotor line). Each line indicates the average CFU/mL at thedifferent time points. &e blue line represents the average CFU/mL from output water of the PSS group. &e orange line represents outputwater of the control group. &e number of bacterial counts (CFU/mL) is presented in the table below the chart. (b) Magnified view of thearea shown in the red box of (a). PSS effectively reduced the bacterial number and was found to be within the standard level recommendedby CDC of less than 500CFU/mL (the red dotted line). (c)&e average CFU/mL throughout the period of experiment.&e average bacterialcount observed in the PSS group is significantly lower than that in the controlled one. ∗Statistically significant difference at p≤ 0.05.

4 International Journal of Dentistry

[12]. &is could explain the marked reduction in bacterialcounts and the elimination of the biofilm as noted in ourstudy. &e plasma technology, and especially hydrogenperoxide gas plasma sterilization, has been developed toprovide a broad-spectrum bactericidal effect. Due to their

instability, plasma ions tend to release an extra amount offree energy which drives it toward an equilibrium stage.&us, it is difficult to measure the amount of plasma ionquantitatively. In this study, we qualitatively confirmed thepresence of hydroxyl radical in output water by the meth-ylene blue dye test as reported by Satoh et al. [16]. Briefly,PSS water generated from the machine (at the machineoutlet tube) was immediately dropped on the methylene bluedye test trip. Bleaching of methylene blue dye, due to thepresence of hydroxyl radicals in a sample, was indicated by adiscoloration from a dark blue color to an almost whitecolor. A lack of bleaching indicated the absence of hydroxylradicals in the sample. &e result confirmed the presence ofOH− in the PSS water.

According to the results obtained with the Gramstaining, treatment with PSS did not induce a change in thecomposition of the microorganisms. However, this ap-proach does not give information on the presence and/orabsence of specific groups of bacteria. Further studies areneeded to analyze the exact composition of microorganismsfound before and after treatment with PSS.

A sincere concern of the use of the PSS system is itssafety. Plasma ions are very strong oxidizing agents that canoxidize biological molecules found in living organisms.Although there is no evidence that confirms or disclaimssafety of the system, the system is likely to be safe for thefollowing reasons. First, the plasma particles are producedby an electrical charge applied to water and do not consist ofany toxic chemical compound. Second, the patients are onlyexposed to a very small volume of water from DUWLs. &us,the limited exposure of small quantities of free radicals shouldnot be hazardous. &ere are some experiments that supportsafety issue of electrochemically activated solution whichshares the same principle as our PSS. A single-dose and a 28-day repeated oral dose of super oxidized water in rats did notproduce any serious adverse effects [17]. Furthermore, a testwith human cells demonstrated no oxidative damage on

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Figure 3: Scanning electron microscopy revealed the presence of biofilms that covered the inner surface of the waterlines. Bacterial biofilmswere reduced in the PSS group (right panel) compared with the control group (left panel). ∗Bacterial biofilm.&e lower panels show a highermagnification (×2000).

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Figure 4: MTT assay indicated the viability of biofilm in thewatertubes. PSS reduced the amount of viable biofilms in thewaterlines. ∗Statistically significant difference at p≤ 0.05.

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Figure 5: Gram staining of bacterial colonies recovered fromoutput water before and after PSS treatment. Gram positive (bluebar); Gram negative (red bar); and fungi (orange bar). PSS did notinduce a shift in types of microorganisms.

International Journal of Dentistry 5

nucleic acids or the cells themselves [18]. Nevertheless, ex-periments with animals and cell cultures are still needed toprove the safe use of the plasma sterilization system.

5. Conclusions

&e plasma sterilization system, which is part of electro-chemically activated water, effectively reduced bacterialcontamination and biofilms in dental unit waterlines. &isstudy, therefore, proposes PSS as a treatment to de-contaminate dental unit waterlines. In addition, it may beapplied to control bacterial biofilms in other medical devices.

Data Availability

&e datasets used in this study were supplied by Ruchanee SAmpornaramveth, DDS, PhD. Requests for access to thesedata should be made to Ruchanee S Ampornaramveth(ruchanee.a@chula.ac.th).

Conflicts of Interest

&e authors declare that there are no conflicts of interestregarding the publication of this paper.

Acknowledgments

&e plasma sterilization system (PSS) was donated byDentozone Corporation (Seoul, Korea). Special thanks aredue to J.E.B. Millennium Company (&ailand) for theirenthusiastic coordination. We would like to express oursincere gratitude to Prof. Vincent Everts for his valuablecomments and correcting English grammar. &is work wassupported by the Dental Research Fund, Dental ResearchProject 3200502#5/2016, Faculty of Dentistry, Chula-longkorn University.

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