Bull Tokyo Dent Coll (2014) 55(2): 77–86

Original Article

Antiseptic Effect of Slightly Acidic Electrolyzed Water

on Dental Unit Water Systems

Miho Komachiya1), Akira Yamaguchi2), Kaname Hirai3), Yuichiro Kikuchi4),

Shinya Mizoue1), Nao Takeda1), Michio Ito5), Tetsuo Kato6),
Kazuyuki Ishihara4), Shuichiro Yamashita7) and Akihiro Kuroiwa1)
1)
Department of Removable Prosthodontics, School of Dentistry,
Matsumoto Dental University,
1780 Gobara, Hirooka, Shiojiri, Nagano 399-0781, Japan
2)
Clover Dental Clinic,
2-14-9 Matsushiro, Tsukuba, Ibaraki 305-0035, Japan
3)
Hirai Dental Clinic,
3316 Mikkaichi, Kurobe, Toyama 938-0031, Japan
4)
Department of Microbiology, Tokyo Dental College,
2-1-14 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan
5)
Institute for Biomaterials Co., Ltd.,
2265 Seba, Shiojiri, Nagano 399-6462, Japan
6)
Laboratory of Chemistry, Tokyo Dental College,
2-9-7 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
7)
Department of Clinical Oral Health Science, Tokyo Dental College,
2-9-18 Misaki-cho, Chiyoda-ku, Tokyo 101-0061, Japan

Received 14 December, 2012/Accepted for publication 11 December, 2013

Abstract
Biofilm formation in dental unit water systems (DUWSs) can contaminate water
from three-in-one syringes, air rotors, and low-speed handpieces. This may serve as a
potential source of infection for dentists, dental staff, and patients, so these systems
must be sterilized. Because slightly acidic electrolyzed water (SAEW) is often used as a
disinfectant for food, the aim of this study was to investigate the possibility of using SAEW
as a DUWS disinfectant. Slightly acidic electrolyzed water was injected into a dental
unit and its effects evaluated. Chemical properties such as chlorine ion and potential
hydrogen in the SAEW were measured. Detection of both ordinary and heterotrophic
bacteria from the DUWS was performed by culture, and biofilm formation of the bacteria
in the DUWS evaluated. Polymerase chain reaction (PCR) was used to detected contami-
nation by nosocomial pathogens. Almost all the chlorine ions in the SAEW were exhausted
during the two-day trials, and the pH value of the SAEW fell from 5 to 4. No viable cells
were detected in the SAEW collected. Biofilm formation in the water from the DUWS
with SAEW was almost at a baseline level, whereas that without SAEW was 4 times higher.
The PCR analysis showed that no nosocomial infecting pathogens were detected in the
SAEW. The present study demonstrated the antiseptic effect of SAEW in DUWS.
Key words: Dental unit water system (DUWS) — 
Slightly acidic electrolyzed water (SAEW) — Nosocomial infection

77
78 Komachiya M et al.

Introduction Welfare41). Among these different types of

EOW, the strongly acidic type has one disad-
Water obtained from dental units via three- vantage in that metallic material can be easily
in-one syringes, air rotors, and low-speed corroded, with Co-Cr alloys, in particular,
handpieces may be heavily contaminated with showing a greater degree of corrosion with
microorganisms, and thus constitute a poten- the strong type than with the slightly acidic
tial source of infection for dentists, dental type12). This suggests that the slightly acidic
staff, and patients6,46). Several studies have type of EOW would be more suitable for
indicated that rates of respiratory infections preventing bacterial contamination of dental
are higher in dentists and dental staff than instruments and dental units.
in the general public8,9), and contaminated In the present study, we evaluated the
handpieces are believed to be at least partially ability of slightly acidic electrolyzed water
­
responsible for this phenomenon26). Elderly (SAEW) to prevent microbial contaminations
people, as compromised hosts, are particu- in DUWSs.
larly susceptible to infection with respiratory
pathogens such as Candida albicans, MRSA,
and Pseudomonas aeruginosa via the oral cavity1). Materials and Methods
In addition to contamination via a dental
unit water system (DUWS), other instruments 1. Properties of SAEW in dental unit
commonly used in dental practice such as The outline of the tested system is shown
air rotors and ultrasonic scalers can also lead in Fig. 1. The SAEW was obtained by the
to the creation of aerosols involving micro­ ­electrolysis of municipal tap water after the
organisms. The range of microorganisms addition of 2% HCl solution (Purestar Mate3,
isolated from DUWSs includes both harmless Morinaga Engineering, Tokyo, Japan) in a
environmental organisms and nosocomial commercial EOW generator (Purestar Mp240-
pathogens such as P. aeruginosa, Legionella B, Morinaga Engineering). For testing, the
pneumophila, and Staphylococcus species2,5,7,15,48). Exceed Flora Over Arm type dental unit was
Electrolyzed oxidizing water (EOW) has
recently been developed as a new type of
disinfectant agent. One strong version of
this type of agent is produced through the
electrolysis of water containing a low concen-
tration of sodium chloride in an electrolysis
chamber in which anode and cathode elec-
trodes are separated by a diaphragm, which
imparts strong bactericidal and virucidal
properties to the water collected from the
anode43). Strongly acidic water obtained via
this method has been used to disinfect den-
tal material, dental instruments and clinical
specimens16,29,30,47). There is a slightly or weakly
acidic type, which is produced by electrolysis
of water containing a low concentration of
hydrochloride in an electrolysis chamber
where the anode and cathode electrodes are
not separated, which imparts strong bacte­
Fig. 1 Tested DUWS incorporating electrolysis chamber
ricidal properties to the water28). Both types for SAEW
of EOW are permitted as food additives by Circles indicate valves. Valves were opened after office
the Japanese Ministry of Health, Labour and hours and shut before office hours.
 Electrolyzed Water Helps Keep Dental Units Clean 79

used (Yoshida Dental MFG, Tokyo, Japan). pollution by waste water, was also evaluated
The generator was connected to the DUWS in the same way as described above, but using
by tubes. All tested SAEW was obtained via Endo agar (Eiken Chemical, Tokyo, Japan).
three-in-one syringes. A control DUWS was
connected to the dental unit and standard 3. Biofilm formation
municipal tap. Water samples from both types Two hundred-microliter aliquots of water
of dental unit were collected after 64 hrs rest from the dental units were inoculated into a
from dental use. 96-well tissue culture plate (Greiner Bio-One,
Levels of free and total chlorine ions Frickenhausen, Germany) at 377C. Then,
were measured by diethyl-p-phenylenediamine 100 μl was added to the same samples every
(DPD) colorimetric and potassium iodide 3 days for 2 weeks to supplement loss by
(KI) colorimetric method using a commercial ­transpiration. Semi-quantification of biofilm
kit (ATK100DA, ATD100DA; Advantec Toyo formation using toluidine blue was performed
Kaisha, Tokyo, Japan). The pH value and by the method of Tanaka with modifications39).
temperature were measured with the pH The biofilm in the wells of the tissue cultures
tester 30 (Eutech Instrument, Ayer Rajah was incubated with 200 μl of 0.1% toluidine
Crescent, Singapore). blue for staining at room temperature. The
dye was decanted 30 min later and excess
2. Microbiological assessment by culture stain removed by washing twice with distilled
method water. Next, 100 μl ethanol was added and
Heterotrophic bacteria and ordinary bacte- left for 30 min to extract the stain. The optical
ria were detected by culture from both SAEW density of each sample was determined at
and municipal water obtained via the dental 590 nm.
units. Total numbers of contaminated cells
were determined according to the America 4. DNA extraction from water samples
Method 9215A, as set forth in the Standard A total of 500 ml each water sample was
Methods for the Examination of Water and filtered through a polycarbonate filter (0.45-
Wastewater3). Water samples collected from μm pore size; HVLP; Millipore, Billerica, MA,
the DUWSs via a three-way syringe after USA) under sterile conditions. The filters
64 hrs rest from dental use were employed. were cut into eighths and each filter piece
An aliquot of 100 μl water sample was imme- placed in 10 ml phosphate buffered saline
diately transferred into 900 μl soybean-casein (pH 7.2, PBS).
broth containing lecithin and polysorbate 80 A 10-ml portion of the water sample was
(SCDLP; Nihon Seiyaku, Tokyo, Japan) and centrifuged at 20,000×g for 30 min. The sedi-
diluted in a 10-fold series down to 1013. The ment was then transferred to a sterile 1.5-ml
SCDLP used as the dilution solution was tube and centrifuged at 20,000×g for 10 min,
prepared by the addition of 0.8 mm glass
­ and the resulting segment re-suspended in
beads. Each 100 μl SCDLP sample was plated 300 μl PBS. The suspension was then divided
onto an appropriate plate, spread evenly with into 3 tubes. After another centrifugation step
a Conrage stick, and cultured at 377C for at 20,000×g for 10 min, the resulting pellet
48 hrs. This procedure was carried out in 3 was used for DNA extraction, which was
separate assays performed in triplicate. After carried out with a commercially available
­
culture, the number of colony forming units kit (QiAamp DNA Mini Kit; Qiagen, Hilden,
(CFUs) was counted. Bacteria were grown Germany) according to the manufacturer’s
on R2A agar (Merck, Darmstadt, Germany) instructions. First, bacterial cells were lysed by
for heterotrophic bacteria, or standard agar enzymatic treatment. For Gram-negative rods,
(Nissui Pharmaceutical, Tokyo, Japan) for a pellet was resuspended in ATL buffer and
ordinary bacteria. Also, evaluation of Entero- treated with 10 μg/ml lysozyme (Sigma-Aldrich,
bacteriaceae contamination, which indicates St. Louis, MO, USA)31). For Staphylococcus
80 Komachiya M et al.

aureus, a pellet was resuspended in TEN of a denaturation step at 957C for 1 min, a
buffer and treated with 20 μg/ml lysostaphin primer annealing step at 557C for 1 min, an
(Sigma-Aldrich)18). For C. albicans, a pellet was extension step at 727C, and a final step at
resuspended in Zymolyase buffer and treated 727C for 5 min. The PCR temperature profile
with 10 μg/ml lyticase (Sigma-Aldrich)25). All for P. aeruginosa included an initial denatur-
samples that were treated with each enzyme ation step at 957C for 3 min, followed by 40
were incubated at 377C for 60 min. Then, cycles of a denaturation step at 957C for 1 min,
200 μl kit buffer AL and 20 μl proteinase a primer annealing step at 607C for 1 min, an
K were added and incubated at 567C for extension step at 727C, and a final step at
10 min, after which 200 μl ethanol was added 727C for 5 min. A negative control (i.e., sterile
to each tube. After brief centrifugation, the water without bacteria) was included in each
supernatant was transferred to the column batch of samples for DNA preparation and
and washed twice with AW1 and AW2 by spin PCR performed to exclude contamination of
down. Isolated DNA was then eluted in 50 μl the buffer solutions.
elution buffer AE (supplied in the kit) and Five microliters of each of the amplified
stored at 1207C until analysis by PCR. products was analyzed by electrophoresis in
3% agarose gel (NuSieve 3:1 Agarose; Cambrex
5. Detection of nosocomial pathogens by Bio Science, ME, USA) in 1×TBE buffer
PCR (90 mM Tris-borate, 2 mM EDTA; pH 8.3) at
PCR was used to detect P. aeruginosa10), 100 V for 40 min using the Mupid system
L. pneumophila45), S. aureus32), MRSA32), and (Advance, Tokyo, Japan). A low-DNA mass
C. albicans17). The oligonucleotides used as ladder (TaKaRa Bio) was used as the molecular
primers for PCR are shown in Table 1. Oligo- size standard. The gel was stained with ethid-
nucleotide primers and probes were synthe- ium bromide (0.5 μg/liter) and photographed
sized at GE Healthcare, UK Ltd. (Amersham under ultraviolet illumination with Polaroid
Place, England, UK). The PCR reagent used film (Polaroid, St. Albans, England, UK) and
was from the Ready-to-go PCR kit (GE Health- Tri-X film (Eastman Kodak, Rochester, NY,
care, UK). PCR amplification was performed USA).
in the DNA thermal cycler Dice (TaKaRa
Bio, Shiga, Japan). The PCR temperature 6. Statistical analysis
profile for L. pneumophila, S. aureus, MRSA and Viable cell numbers in biofilm formation
C. albicans included an initial denaturation were analyzed using a one-factor ANOVA
step at 957C for 3 min, followed by 40 cycles (StatView Program, SAS Institute Inc., San

Table  1  Primers for PCR detection of nosocomial pathogens in waters

Annealing
Strain Direction Sequence (58–38) Product
temperature
F TTC CCT CGC AGA GAA AAC ATC
Pseudomonas aeruginosa 520 bp 607C
R CCT GGT TGA TCA GGT CGA TCT
F AGG GTT GAT AGG TTA AGA GC
Legionella pneumophila 386 bp 557C
R CAA ACA GCT AGT TGA CAT CG
F AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG
Staphylococcus aureus 422 bp 557C
R CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA
F AAA ATC GAT GGT AAA GGT TGG C
MRSA 533 bp 557C
R AGT TCT GCA GTA CCG GAT TTG C
F CCT GAA CCA CAA GAT GGA ACC ATT A
Candida albicans 490 bp 557C
R CGC AGT TTT CTA CTA CCA CCA TCG
 Electrolyzed Water Helps Keep Dental Units Clean 81

Francisco, CA, USA), and Fisher’s PLSD as a film formation in the micro-wells under static
post hoc test at a 5% level of significance. conditions. The results of staining revealed
biofilm formation was very low, almost at
­baseline level with SAEW. Biofilm formation
Results with municipal water, on the other hand, was
approximately 4 times greater than that with
1. Properties of SAEW from dental unit SAEW at one week, indicating that SAEW
The amount of chlorine ions, pH value, inhibited growth more strongly than the
and temperature are shown in Table 2. Almost control (Fig. 3).
all chlorine ions in the SAEW were exhausted
during the 64 hrs trials, and the pH value 4. PCR detection
of the SAEW fell from 5 to 4. These results No nosocomial pathogens were detected
indicate that the SAEW was resolved to 2% in SAEW (Fig. 4), whereas S. aureus and
HCl solution. The amount of chlorine ions P. aeruginosa were detected in municipal water.
and the pH value of the municipal water P. aeruginosa was detected only in the morning,
showed no change over the 64 hrs period. whereas S. aureus was detected both in the
evening and the morning. Both bands of
2. Microbiological assessment by culture S. aureus and P. aeruginosa were faint. This
method finding, that template DNAs obtained from
No viable cells were detected in the SAEW municipal water via dental units were pres-
collected from the dental units after 64 hrs ent in quite small amounts, indicates that
(Fig. 2). In municipal water, bacteria grown on minute amounts of P. aeruginosa or S. aureus
standard agar showed a significant increase had contaminated the municipal water.
to approximately 300 CFU/ml (Fig. 2A) after
64 hrs; heterotrophic bacteria grown on R2A
agar, however, showed no significant increase Discussion
after 64 hrs (Fig. 2B). This indicated a large
number of heterotrophic bacteria in the In 2004 the American Dental Association
municipal water prior to testing. No entero- (ADA) issued a statement on DUWSs recom-
bacteriaceae were detected in either of the mending that water delivered during non-
two dental unit systems (data not shown). surgical dental procedures consistently contain
no more than 200 CFU/ml aerobic meso-
3. Biofilm formation philic heterotrophic bacteria in an unfiltered
Figure 3 shows the effect of SAEW on bio- dental unit output4). This level is equivalent to

Table  2  Properties of tested waters via dental unit

Slightly acidic electrolyzed water Municipal water

Start After 64 hrs Start After 64 hrs
Free chlorine ion (mg/liter) 3.26±1.80 0.09±0.04 0.05±0.04 0.03±0.01

Total chlorine ion (mg/liter) 3.14±1.69 0.20±0.08 0.06±0.03 0.04±0.03

pH 4.99±0.32 3.96±0.18 7.32±0.11 7.37±0.31

Oxidation Reduction
857±110 503±71 555±105 391±76
Potential (ORP, mV)
Temperature (7C) 23.1±1.9 21.2±1.9 23.0±0.9 21.5±1.7
Results are expressed as mean±standard deviation from 5 separate assays. n=15.
82 Komachiya M et al.

the assurance standard for dialysis fluids. On number of bacteria in water supplied through
the other hand, the American Public Health a dental unit should be the same as for drink-
Association, the American Water Works Asso- ing water, which should contain less than
ciation, and the Environmental Protection 500 CFU/ml of aerobic mesophilic hetero­
Agency have all set the standard for drinking trophic bacteria19). Palenik et al. reported that
water at less than 500 CFU/ml of aerobic only one waterline specimen contained more
mesophilic heterotrophic bacteria. Water than 500 CFU/ml among 10 tested specimens35).
supplied from dental units should also abide In Europe, while the guidelines for potable
by this standard. The Centers for Disease water standards have recommended a bac­
Control and Prevention consider that the terial contamination level of <100 CFU/ml,

CFU/ml

CFU/ml

Fig.  2  Microbiological assessment by culture method

Bar indicates standard deviation from 3 separate assays. n=9.
* Significantly different (p<0.05) by one-way ANOVA.
 Electrolyzed Water Helps Keep Dental Units Clean 83

there are as yet no guidelines in the European Health, Labour and Welfare requires that
Union for the control of microbial con­ municipal water have less than 100 CFU/ml21),
tamination in DUWSs40). Several investigators yet there are presently no guidelines in Japan
reported that most units tested were unsatis- on the control of microbial contamination
factory according to the recommendations in DUWSs. Some investigators have reported
set by the ADA44). In Japan, the Ministry of that DUWSs in Japan also failed to meet
the recommendations set by the ADA, and
sometimes over 1,000 viable bacterial cells
OD
were obtained from 1  ml of dental unit
system water3,20). In the present study, over
200 CFU/ml planktonic bacteria were found
in the control DUWS. In the DUWS employ-
ing SAEW, however, no viable heterotrophic
or ordinary bacteria were detected. The use
of SAEW allowed the DUWS to meet the
water quality standards set for prevention of
bacterial contamination in the USA, EU, and
Japan.
Chlorine ions produced by the electro-
chemical hydrolysis are believed to be the
main agent responsible for disinfection in
Fig.  3 Biofilm formation by microorganisms in water EOW 23). The antimicrobial effects of electro-
from DUWS
lyzed water are similar to those of hypochlo-
Bar indicates standard deviation from 3 separate assays.
n=9. * Significantly different (p<0.05) by one-way rite solution. Diluted sodium hypochlorite is
ANOVA. effective in improving the quality of effluent

Fig.  4 Detected nosocomial pathogens by PCR

Representative of 5 separate assays. n=15.
84 Komachiya M et al.

water and reducing the amount of biofilm in temperatures were used in the PCR, with PCR
DUWSs11). With EOW, it has been reported for each strain carried out separately. How-
that approximately 40% of the chlorine was ever, no significant difference was observed
lost under closed dark storage conditions for in the amplification efficiency of the positive
2 months22). Chlorine dioxide also inhibits the control, which is shown in Fig. 4. No amount
formation of natural biofilms37). Our results of pollution of a DUWS with nosocomial
showed that SAEW which had passed through infection-inducing agents is permissible, so
the DUWS inhibited biofilms formation to every effort must be made to eradicate them
within almost baseline levels. completely. In the present study, injection
The type of chlorine ions often used in the of SAEW into a DUWS for 64 hrs resulted
decontamination of water, ordinary sodium in no detection of nosocomial pathogens,
hypochlorite, is usually referred to as bleach. indicating that SAEW offers an effective
Disinfection with chlorine has been the single means of disinfecting such units.
most important measure in ensuring the
microbiologic safety of potable water supplies.
Since the institution of the routine chlorina- Acknowledgements
tion of water supplies, waterborne outbreaks
of infectious agents have been exceedingly Part of this study was supported by a Scien-
rare. Most waterborne outbreaks are believed tific Research Special Grant from Matsumoto
to result from the use of untreated water, Dental University to Miho Komachiya. We
systems receiving inadequate treatment, or would like to thank Professor David M. Carlson
contamination after treatment36). The Japanese of Matsumoto Dental University for his assis-
Ministry of Health, Labour and Welfare recom- tance with the English of this manuscript.
mends the removal of chlorate ions to below
0.6 mg/ml in the concentration of municipal
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