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Reduction in bacterial contamination of toothbrushes using the Violight
ultraviolet light activated toothbrush sanitizer
ROBERT BOYLAN, PHD,YIHONG LI, DDS, DRPH,LIDIA SIMEONOVA, DDS,GENE SHERWIN,DDS,JUDITH KREISMANN, RDH,
RONALD G. CRAIG, DMD, PHD,JONATHAN A. SHIP, DMD &JANE A. MCCUTCHEON,DDS, PHD
ABSTRACT: Purpose: This two armed, self-controlled, investigator blinded, clinical study tested the efficacy of an
ultraviolet (UV) light toothbrush holder (Violight) to decrease toothbrush bacterial contamination. Methods: 25 subjects
were randomly assigned to control or experimental groups and received two toothbrushes for home use on either even or
odd days. The control group rinsed both toothbrushes after use in cold tap water with no mechanical manipulation. The
experimental group rinsed one toothbrush in cold running water while storing the other toothbrush in the Violight
toothbrush holder after use. The toothbrushes were returned after 2 weeks use in sealed plastic bags and were analyzed for
the number of colony forming units (CFU) of S. mutans,S. salivarius, lactobacilli, E. coli, and other coliforms, and total
bacterial counts by culture. An additional analysis of the total bacterial profile was performed using denaturing gradient gel
electrophoresis (DGGE). Results: The Violight toothbrush holder reduced total CFU by an average of 86% (ANCOVA,
P= 0.037). In addition, a tendency was noted for a reduction in total bacterial population as detected by DGGE. (Am J Dent
CLINICAL SIGNIFICANCE: These results suggest that the Violight toothbrush holder can decrease bacterial contamination
of toothbrushes between uses.
: Dr. Jane McCutcheon, Department of Basic Sciences and Craniofacial Biology, New York University College of
Dentistry, Mail Code 9436, 345 East 24th Street, New York, New York 10010-4086, USA. E-: firstname.lastname@example.org
The uninterrupted accumulation of oral bacterial plaque is
strongly associated with an increased risk for dental caries, the
development of gingivitis and the promotion of oral malodor.1-3
Mechanical disruption of bacterial plaque through the use of
toothbrushes, or a combination of toothbrushing, interproximal
hygiene procedures and use of oral mouth rinses has been
regarded as an optimal means of preventing plaque accumula-
tion and the promotion of overall oral health. However, bacteria
remain on the toothbrush after use and may provide a potential
source for recontamination of the oral cavity on the next use.
One study4 has reported a bacteremia due to a viridans strepto-
cocci (S. oralis) strain that was cultured from the patient’s oral
cavity and from the subject’s toothbrush suggesting that
colonization of toothbrushes with oral microflora may be
associated with bacteremia in some patients. Environmental
enteric bacterial contamination of toothbrushes resulting from
bathroom aerosols has also been reported5,6 which may also
contaminate faucets, combs and hairbrushes.7
Several bactericidal agents have been promoted to reduce
the possibility of toothbrush bacterial contamination between
uses. These include the use of chlorhexidine,8 Brushtox,9 and
several dentifrices.10-12 While all of the above have shown
varying degrees of efficacy, none are widely used as a home
based application. A possible reason for non-compliance with
these methods is they are time consuming and may result in
unwanted product residues.
Ultraviolet (UV) light is bactericidal to a wide array of
bacterial species including antibiotic resistant species13-15 and
has been extensively used in water purification systems.
Violighta (Fig. 1) is a toothbrush holder that uses UV light for
bacterial decontamination. The toothbrush is inserted into the
device and a button is pressed to deliver a 10-minute UV
irradiation dose. The design of the Violight toothbrush holder
prevents exposure to UV light when in use. Due to its ease of
use, Violight may increase compliance in toothbrush bacterial
decontamination. However, the extent of bacterial decon-
tamination using the Violight toothbrush holder has not been
determined in a clinical setting. Therefore, the objective of the
present investigator-blinded, self-controlled, clinical study was
to determine the efficacy of Violight in decreasing toothbrush
Materials and Methods
Subject recruitment and study design - The study design was
reviewed and approved by the New York University School of
Medicine Institutional Board of Research Associates. Subjects
were recruited from the patient pool seeking dental care at the
New York University College of Dentistry and from advertise-
ments in local media. Inclusion criteria included subjects be ≥
18 years of age, in good general health, able to give informed
consent and comply with the study protocol, have at least 10
natural teeth per arch, and brush their teeth twice daily.
Exclusion criteria included the clinical evidence of gross caries
or periodontal disease, the presence of systemic diseases or
conditions that would affect the oral cavity such as uncontrolled
diabetes mellitus, use of any medications associated with
xerostomia or any antibiotic therapy within 7 days prior to the
start of the study protocol.
Subjects were randomly assigned to either the control or the
experimental groups. Each subject was provided with two
toothbrushes of different color (green and silver) for home use
on either odd or even days. The control group was instructed to
rinse both brushes after use in cold running water without any
mechanical manipulation. The experimental group was instruct-
ed to rinse one brush (green) in running water without mechani-
cal manipulation and treat the second toothbrush (silver) with
314 Boylan et al
Fig. 1. Violight toothbrush holder storing toothbrushes. The device can be used
store toothbrushes after the treatment cycle is completed. to
the Violight toothbrush holder (Fig. 1) after every use. The
treated brush was stored in the Violight toothbrush holder
between uses in the subject’s room at home where they
typically brush their teeth (i.e. typically their bathroom).
Subjects were instructed to start the study the evening after
enrollment and to follow their regular oral hygiene regimen.
Subjects returned the first toothbrush in a pre-labeled sealed
plastic bag on day 13 of the study and the second toothbrush in
a pre-labeled sealed plastic bag on day 14 of the study.
Toothbrushes were returned to the Bluestone Center for
Clinical Research within 1 hour after use in the morning. The
toothbrushes were promptly delivered to the laboratory for
bacterial extraction and cultivation (see below). After
xtraction, the toothbrushes were stored at -20 ºC. e
Bacterial culture - Methods used for bacterial culture followed
standard techniques.16 Media used for bacterial culture were
purchased from Difcob unless otherwise noted. Media included
Trypticase Soy Agarc for total counts, Mitis salivarius agarc for
total streptococci, Mitis salivarius agar with 2 IU/ml of
bacitracinc for mutans streptococci, MacConkey agar with 1%
lactosec for E. coli and other coliforms, and Rogosa SL agarc
or lactobacilli. f
For bacterial extraction, the toothbrushes were individually
placed in pre-labeled, sterile 50 ml centrifuge tubes containing 10
ml of trypticase soy brothc (TSB) to immerse the bristles, then
vortexed vigorously for 1 minute, squeezed against the side of
the tube to drain, rinsed with 5 ml TSB and drained again. A
series of undiluted and 10-fold dilutions of each sample were
prepared and plated onto the surface of selective and non-
selective media. A duplicate series of plates was then incubated
aerobically or anaerobically at 37°C for 2-4 days, until colony
formation was visible. The number of colonies, measured as
olony forming units (CFU), was counted using a colony counter. c
Polymerase chain reaction (PCR) and denaturing gradient gel
American Journal of Dentistry, Vol. 21, No . 5, October, 2008
electrophoresis (DGGE) assays - PCR and DGGE assays were
used to identify both cultivable and non-cultivable bacteria
present on four pairs of randomly selected toothbrushes from
the 25 pairs of clinical samples. The toothbrushes were washed
by agitation in 5.0 mL of phosphate-buffered saline (PBS) and
centrifuged at 126,800 x g. for 30 minutes. The supernatant was
discarded and the cell pellet was re-suspended in 0.5 ml TE
buffer (10 mM Tris-Cl, pH 7.5, and 1 mM EDTA). The total
bacterial genomic DNA was isolated by means of a DNA
purification kit (MasterPured) with modifications as previously
A nested-PCR approach was used to first amplify the entire
1500-bp 16S-rRNA locus for all extracted bacterial DNA sam-
ples with a set of universal 16S rRNA PCR primers19 followed
by a second amplification of a hyper-variable region (~300 bp)
of the 16S-rRNA locus.19 In the first PCR, each 50 μL reaction
mixture contained a standardized 100 ng of the total genomic
DNA, 200 μM of each dNTP, 50 pmole of universal primers
16S-8f and 16S-1492r (19), 1.5 mM MgCl2, 5 L of 10X PCR
buffer II, and 2.5 U of Taq DNA polymerase.e In the second
PCR, a specific set of universal bacterial 16S rRNA primers
(prbac1 and prbac2)20 was used with a 40-nucleotide GC-clamp
to facilitate the DGGE analysis.21-23 All PCR procedures were
performed with the GeneAmp PCR System 9700.e PCR condi-
ions and reagents were as described elsewhere.17,18 t
A standardized 20 μL of each PCR-amplified product was
separated on gradient gels as previously described.17,18 A 40% to
60% linear DNA denaturing gradient was formed in an 8%
(w/v) polyacrylamide gel. PCR products were directly loaded in
each lane and were run along with known species-specific
DGGE reference markers.17 After electrophoresis, the gels were
rinsed and stained for 15 minutes in 0.5 g/mL ethidium bro-
mide. The DGGE images were digitally captured and recorded.f
The DNA fingerprints of the DGGE were compared and
analyzed by means of Fingerprinting II Informatixg software as
reviously described.17,18 p
Statistical analysis - Data were entered and checked into a
password-protected data set and analyzed using SPSS for
Windowsh (Version 13). All bacterial counts were log
transformed to normalize their distributions prior to analysis.
An ANCOVA was performed to compare the differences in
bacterial levels between the control and treatment groups using
as a covariate the log of the total bacterial count on the first
(cold water rinsed) toothbrush. The ANCOVA was adjusted for
the individual specific CFU counts in the untreated situation
and compared the second untreated toothbrush in the control
group to the treated (Violight) toothbrush in the treatment
group. The differences in total and each specific bacterial
counts were also expressed as a percent reduction in CFUs
comparing the Violight treated brush to the untreated, cold
water rinse brush. Group characteristics were compared by
Mann-Whitney-Wilcoxon test for continuous variables and
Fisher’s exact test for categorical measures. A value of P< 0.05
as accepted for statistical significance. w
Twenty five subjects (20 females, five males) aged 21-65
years (28 ± 10; mean ± standard deviation of the mean) were en-
rolled in this study consisting of seven Caucasians, 10 African-
American Journal of Dentsitry, Vol. 21, No . 5, October, 2008
Fig. 2. Changes in CFU of bacterial levels on the paired toothbrushes for each
subject. Each pair of circles connected by a line represents one subject. The left
hand group represents the control subjects and the right hand group represents the
treatment subjects whose second day toothbrush received Violight treatment.
Seven of the 10 treatment subjects show reductions in CFU for the toothbrush
eated with the Violight as compared to toothbrushes treated with cold water. tr
Violight & toothbrush bacteria 315
Table 1. Descriptive statistics for log CFU.
Toothbrush #1 Toothbrush #2 Toothbrush #2
__________________ ___________________ _____________________
Group N Mean SD Mean SD Mean SD
ontrol 12 4.99 1.25 4.88 0.85 4.89 0.75
Treatment 10 5.02 1.18 4.17 0.86 4.17 0.75
Statistics for the two toothbrushes in each treatment group and for toothbrush 2
adjusted for the log CFU values on Toothbrush 1 based on an ANCOVA.
Toothbrush 1 was always a cold water rinse brush (regardless of group).
Toothbrush 2 was cold water rinsed in the control group and Violight treated in the
treatment group. The data are for log transformed CFU’s. Visually looking at the
difference in mean values, there was little difference in brush 1 and 2 in the control
roup but a reduction in the treatment group. g
Table 2. ANCOVA. Total CFU.
Type III sum Mean
Source of squares df square F P value
Baseline 4.008 1 4.008 7.155 0.015
roup 2.822 1 2.822 5.038 0.037
Error 10.643 19 0.560
The dependent variable was the log CFU on Toothbrush 2. The independent factor
oup and the covariate was the log CFU on Toothbrush 1 (a baseline value). was gr
Fig. 3. Comparison of DGGE profiles between the controls (A) and experimental toothbrushes (B). The pair-
wise similarity was 95% for the controls, suggesting no changes in bacterial population between the two
samples. The similarity was 60.0% for the experimental pair, indicating a 40% reduction in the bacterial
population after Violight treatment.
Americans, four Hispanics, two Asians and one other ethnic
group. One subject failed to return and was excluded from the
final data analysis. The control and treatment groups were
balanced for age (P= 0.295), ethnicity (P= 0.805), and gender
Twenty-four of the 25 subjects were able to return their
toothbrushes on day 13 and 14 in sealed labeled bags as
instructed. Bacteria were extracted from the toothbrushes and
used to determine CFU or for DGGE analysis. Streptococcus
mutans was cultured from only two subject’s toothbrushes and
lactobacilli were cultured from only one subject’s toothbrush,
therefore no further analyses were performed for these two
species. Log transformation of E. coli and S. salivarius and
subsequent ANCOVA analysis revealed no statistically
significant differences between the experimental and control
groups (F= 1.05, df= 1, P= 0.32). However, an 86% reduction
in the CFU of the total bacterial counts was observed between
the Violight and control groups. An approximate 10-fold mean
reduction from 104,713 to 14,791 CFU was observed in the
Violight treated group (Table 1). ANCOVA was used to adjust
for the individual specific CFU counts in the untreated situation
compared between the second untreated toothbrush in the
control group to the treated (Violight) toothbrush in the experi-
mental group. As reported in Table 2, there was a statistically
significant difference (F= 5.04, df= 1, P= 0.037) in the log CFU
of the total bacterial counts between the control and experi-
316 Boylan et al
mental groups. Finally, a comparison of the difference between
the toothbrushes for both the control and experimental groups
was determined for each individual. The results in Fig. 2 show
that the number of bacterial counts on the treated toothbrushes
decreased in the experimental group compared to that in the
To further determine the efficiency of the above bacterial
extraction method and the Violight treatment, a pair of
toothbrushes were analyzed using DGGE. No bacterial DNA
samples were detectable by PCR after extraction for culture,
suggesting the extraction procedure was highly efficient. A
second two pairs of toothbrushes from two subjects in the
control group were directly examined for bacterial population
profile using DGGE without cultivation. The comparison was
conducted between the toothbrushes before and after regular
water rinsing, and the two bacterial profiles obtained were
found to be highly similar (94.6% concordance) (Fig. 3). A
third pair of toothbrushes was obtained from a subject in the
experimental group. The bacterial profile analysis showed that
the similarity was only 60% (Fig. 3) suggesting a reduction in
bacterial contamination may occur after using the Violight
The paired toothbrush design used in this study was
developed to determine if the Violight toothbrush holder can
reduce the bacterial load remaining on toothbrushes after use.
This study, using both conventional bacterial culture methods
and more sensitive PCR-based non-culture methods, demon-
strates that the Violight group had an average reduction of 86%
in the bacterial load (Fig. 2, left side, Fig. 3 bottom). This
finding was further supported by an approximate 40% decrease
n bacterial population by the DGGE analysis. i
Decreasing the total bacterial load in the oral cavity, as well
as decreasing specific bacterial species, is a fundamental
therapeutic approach designed to decrease the incidence and
severity of gingival inflammation, caries and oral malodor.
Although few studies have examined whether bacteria trans-
ferred to a toothbrush can be a source for oral re-colonization,
studies have shown that bacteria from the oral cavity and other
sources can be found on toothbrushes and use of various
chemical products can decrease the bacterial load on the
toothbrush.9-12 Despite evidence demonstrating that chemical
rinses and dentifrices can reduce the total bacteria load on a
toothbrush, these methods are not widely in use. Possible
reasons for poor compliance include ease of use, the need for
additional procedures after brushing or unwanted product
residues. The results of this study suggest that a UV light
toothbrush holder can effectively reduce by an average of 86%
total cultivatable bacteria on a toothbrush. This result was
supported by the DNA-based DGGE technique. Since the
device requires no additional input from the subject other than
pushing a button to activate the device, it is possible that the
ease of use of the Violight toothbrush holder will increase
patient compliance while reducing between use bacterial load
n toothbrushes. o
It is important to note the limitations of the present study.
First, bacterial culture demonstrated that one subject had an
American Journal of Dentsitry, Vol. 21, No . 5, October, 2008
increase in the total cultivatable bacteria with the Violight
treated toothbrush compared to the untreated toothbrush (Fig.
2). One possibility for this anomalous result could be that the
subject had poor compliance in treating the toothbrush with
Violight toothbrush holder prior to delivering it to the
laboratory. Although subjects were asked if there were any
problems using the Violight toothbrush holder, the study design
did not include a questionnaire or any other means to evaluate
that the treated toothbrush was indeed treated before being
returned for bacterial analysis. Second, as S. mutans and
lactobacilli are associated with caries initiation and progression,
we were interested to determine the effect of the Violight
toothbrush holder on these species. However, this study
observed low detection rates for both S. mutans and lactobacilli.
However, both species are known to have low retention rates on
E. coli is not generally considered to be a common resident
of the oral cavity, yet it and other common enteric bacteria have
been found in the oral cavity by other studies.26,27 The presence
of E. coli is more common in patients with periodontal disease,
but not exclusively so.26 E. coli and other enteric bacteria are
found as pathogens in elderly subjects with hospital acquired
pneumonia.27 Despite some association with disease, it is
important to remember that many strains of E. coli are
avirulent, so that the mere presence of these bacteria may not
ecessarily be an indicator of disease. n
Finally, reducing the bacterial load and population in the
oral cavity is generally thought to be beneficial to oral health
by reducing caries, gingivitis and oral malodor, as well as
promoting general health. Yet, data in support of this
contention are limited.4 While this study demonstrates that the
Violight toothbrush holder reduces both the bacterial load and
overall population on toothbrushes, this result should not be
extrapolated to conclusions about the effect of the Violight
toothbrush holder on oral or general health. Further clinical
studies will be required to determine the effect of reduction of
toothbrush bacterial load and diversity on oral and general
a. Violight Inc., Elmsford, NY, USA.
b. Difco, Detroit, Michigan, USA.
c. Sigma, St. Louis, MO, USA.
d. Epicenter, Madison, WI, USA.
e. PE Applied Biosystems, Foster, CA, USA.
f. Alpha Innotech Corporation, San Leandro, CA, USA.
g. Bio-Rad Laboratories Inc., Hercules, CA, USA.
h. SPSS, Chicago, IL, USA.
Acknowledgements: To Dr. Robert Norman (Director of Biostatistics and Data
Management, Bluestone Center for Clinical Research, NYU) for
statistical analysis, Dr. Zhou Chen for technical support, the research
coordinators of the Bluestone Center for Clinical Research and the research
subjects, all of whom helped make this study possible. This study was funded in
part by Violight Inc., Elmsford, NY, USA.
Dr. Boylan is Associate Professor, and Dr. Li is Associate Professor,
Department of Basic Sciences and Craniofacial Biology, Dr. Simeonova is
researcher/trainer, Department of Reconstructive and Comprehensive Care, and
Dr. Sherwin is Clinical Assistant Professor, Department of Cariology and
Comprehensive Care; Ms. Kreismann is Clinical Associate Professor, Dental
Hygiene Program, Dr. Craig is Associate Professor, Department of Basic
Sciences and Craniofacial Biology and Department of Periodontology and
Implant Dentistry, Dr. Ship (deceased) was Professor, Department of Oral &
Maxillofacial Pathology, Radiology, and Medicine and Director, Bluestone
American Journal of Dentsitry, Vol. 21, No . 5, October, 2008
Center for Clinical Research; Dr. McCutcheon is Associate Professor,
Department of Basic Sciences and Craniofacial Biology and Bluestone Center
for Clinical Research, New York University College of Dentistry, New York,
ew York, USA. N
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