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The Effectiveness Of A U-V Toothbrush Sanitizing device In
reducing The Number Of Bacteria, Yeasts And Viruses On
Toothbrushes
In the early 1980's, a systematic study
of toothbrushes revealed that toothbrushes from both patients with
oral disease and those with normal mouths had a substantial number
of different types of microorganisms. many of these microorganisms
had the ability or potential ability to produce disease. Further
studies revealed that Herpes Simplex Virus, Type 1, could
survive on a moist toothbrush for seven days or more. More recent
studies using an animal model (kennel dogs) have shown a definite
correlation between toothbrushes contaminated by daily use and at
least oral disease.
Studies were also conducted to determine the effectiveness of an
experimental ultra-violet light device in sanitization of the
toothbrush (not the Pollenex unit). Thirty-one toothbrushes of
various designs, colors, and from a variety of manufacturers were
sterilized (using ethylene oxide gas) and divided into two groups.
Ten brushes were contaminated with Candida albicans. The
brushes were exposed to an experimental ultra-violet germicidal
light device for different time intervals and then cultured at three
different bristle levels. Ultra-violet light was found often to
decontaminate the toothbrush bristle surface, however, midbristle
and bristle depth remained contaminated. The effectiveness of
ultra-violet light decontamination was in part dependent upon the
opacity and ornateness of the toothbrush. In addition, prolonged
ultra-violet light exposure with the experimental unit appeared to
harden the bristles (unpublished data by funding manufacturer's
request).
An ultra-violet light device for toothbrushes has also been
developed by Pollenex: the DS60 Daily Dental Sanitizer. In order to
test the decontamination effectiveness of this ultra-violet light,
toothbrushes exposed to Streptococcus mutans and Candida
albicans were contaminated at varying time intervals (simulating
human toothbrushing experience) and placed either in an ultra-violet
light or in a humidified environment (simulating a bathroom
environment) for 24 hours. The brushes were then cultured to
determine:(1)whether the Pollenex ultra-violet light device
decontaminates toothbrushes exposed to Streptococcus mutans
and Candida albicans as compared to natural light, and
(2)whether toothbrush design, color, or opacity alter
decontamination by the Pollenex unit. The Pollenex DS) Daily Dental
Sanitizer demonstrated both a qualitative and a quantitative
reduction in Streptococcus mutans and Candida albicans
compared to placing toothbrushes on a shelf (unpublished pilot data,
superseded by the following studies).
In order to further study the effectiveness of the DS60, six
microorganisms were chosen for quantitative analysis (as suggested
by the U.S. Food and Drug Administration as being their indicator
microorganisms of sanitization): 1.Staphylococcus aureus, 2.
Pseudomonas aeruginosa 3.Escherichia coli, 4. Bacillis subtilis, 5.
Serratia marcescens, 6. bakers yeast. Similarly, in order to
test the effectiveness of the DS60 on viruses; Herpes Simplex
Virus, Type 1 (HSV), a virus responsible for recurrent fever
blisters and Parainfluenza Virus, Type III (PV), a common
cause of upper respiratory disease and colds in children, were
chosen.
Materials and Methods
Brain heart infusion (BHI) broth (100 ml) was seperately
inoculated with Staphylococcus aureus (ATCC 25923), Pseudomonas
aeruginosa (ATCC 25922), Escherichia coli (ATCC 27853), Bacillis
subtilis (Difco spore suspension), Serratia marcescens (ATCC 8100),
and Saccharomyces sp.(solated from commercially obtained "Baker's
yeast").
The Broth solutions were incubated at 37C for hours.
Each organism was absorbed on to 11Oral B toothbrushes, previously
gas sterilized in 25x200mm screw cap test tubes, by adding 7ml of
the 24 hour inoculum to the tube for 10 minutes. The brushes were
then removed and (see Flow Sheet 1): a. 3 brushes/organism were
placed in sterile 20x 150mm test tubes( inoculum control); b. 4
brushes/organism were placed in the Pollenex DS60 Daily Dental
Sanitizer (U,V.) for 24 hours (all sanitizers were found to have 3
minute+/- 2second exposure/30 minute) and c. 4 brushes/organism were
placed in a test tube rack in a bacteriological hood at room
temperture for 24 hours (shelf).
Four milliliters of BHI broth were added to each tube of the
inoculated control brushes and the brushes were individually
vortexed for 30-45 seconds. Dilutions of the control were made by
removing 0.5 ml of the vortexed toothbrush washing and adding it to
4.5 ml of sterile BHI broth. Ten-fold serial dilutions were made
through 6 to 8 dilutions. Each dilution was quantitated by removing
0.1 ml and placing it on a trypticase soy agarplate (the Baker's
yeast were placed on Sabouraud's media). The inoculum was spread by
placing the inoculated plate on a revolving, the liquid was spread
via a sterilized bent glass rod. Duplicate plates were used for each
delution. Plactes were incubated at 37C (yeast plates were incubated
at 30C ) for 24 to 48 hours prior to counting bacterial colonies.
After 24 hours, the inoculated brushes from the "UV" and the
"shelf" brushes were placed into sterile 20x150 mm tubes. BHI broth
(4ml) was added to each test tube and processed as decribed abovefor
the "control" brushes. Bacterial colonies were counted after 24-48
hours incubation. Counts from duplicate plateswere averaged for the
final results.
A Total of seventy two toothbrushes (twenty-four each od Oral-B,
Reach, Clogate) were placed in a glass test tube and sterilized by
ethylene oxide gas. Equal numbers of colored, opaque, or clear
toothbrushes were used. All seventy-two toothbrushes were exposed to
Herpes Simplex Virus, Type1, approximately 105 TCID
50/0.1ml (50% Tissue Culture Infective Dose) in hanks
Ballance Salt Solution(HBSS) for ten minutes to allow viral
absorption. In a seperate trial, seventy-two brushes were exposed to
Parainfluenza Virus, Type III, approximately 106.5 TCID
50/0.1 ml in HBSS, for ten minutes. To determin the
numbers of virus particles attacheding to the toothbrush prior to
exposure to UV light or drying conditions, 12 toothbrushes (4 of
each brand) were immersed in HBSS immediately after incubation with
virus. The toothbrushes were triturated (vortexed) for 45 seconds
and the virsus contained fluids subsequently titrated for viral
quantitations as describer below. Thirty viral contaminated brushes
were exposed to the Pollenex DS60 unit and thirty viral contaminated
brushes were exposed to natural light. The exposure time for both
groups was twenty-four hours (see Flow Sheet 2) .
After a 24 hour exposed period each brush was placed in HBSS and
triturated for 45 seconds. This allowed the virus particles to
disloge from the brush bristles and accumulate in the fluid. The
viral particals were quantitated by performing 10-fold dilutions of
the virus containing fluids and adding.0.1ml of each didution,
inquadruplicate, to 96-well tissue culture plates containing a
monolayer of appropriate cells. The tisue cultures plates were
monitored dayily for CPE (cytopathic effort) of the cell monolayer
in the case of HSV ande hemadsorption using guinea pig erythrocytes
in the case of PV infected cells. After approximately 5 days the
plates were fixed with formalin, stained, and observed
microscopically. The TCID50 was calculated and the
results tabulated. All tests were performed using coded brushes to
prevent observer bias.
Results
The results of the bacterial and yeast study are summarized in
Table 1. The initial inoculum of the first three microorganisms was
at 10 8-9/ml level. The initial inoculum of the last
three microorganisms was reduced to 107/ml due to a
difference in growth curves. From the results, it is clear that all
microorganisms could be transfereed to the toothbrush with only a
101-2/ml reduction from the initial inoculum. further,
storage of the contaminated toothbrushes on the shelf of a low
humidity microbiology hood reduced the microorganism count by only
101/ml. Most important, following Pollenex recommended
procedures, the pollenex DS60 Daily Dental Sanitizer was found to
consistently reduce the number of microoganisms by at least a factor
103-5/ml. In the case of Pseudomonas and Escherichia, two
toothbrushes per microorganism were completely negative.
Seventy-two toothbrushes were exposed to Herpes Simplex Virus,
Type I, for a period of 10 minutes at a concentration of
105 TCID50/ ml. The Toothbrushes retained an
average of 104 TCID50/ml after the ten minute
(range 103.75 - 104.25 TCID 50/ml).
No HSV was found on any of the 30 toothbrushes placed in the
Pollenex DS60 Unit for 24 hours. The ten reach toothbrushes placed
on a shelf had the highest return of virus particals (ave.
102.35 TCID 50/ml ) from a retained initial
concentration of virus particals: ave 104.125 TCID
50/ml. The ten colgate toothbrushes (shelf stored)
returned an average of 102.23 TCID 50/ml from
a retained initial concentration of virus particles: ave.
103.3875 TCID 50/ml. The ten Oral B
toothbrushes (shelf stored) had the lowest return of virus particles
(ave.104.0 TCID 50/ml. One Colgate and one
Oral B toothbrush (shelf stored) had no evidence of virus particles.
The overall average of the 30 toothbrushes placed on the shelf was
102.06 TCID 50/ml. In general, the lighter
colored or clear toothbrushes appeared to retain or to return fewer
virus particles. The results are summerarized in Tables 2 and 3.
Seventy-two toothbrushes were exposed to Parainfluenza Virus,
Type III, for a period of 10 minutes at a concentration of
106.5 TCID 50/ml. The toothbrushes retained an
average of 103.71 TCID 50/ml virus particles
after the 10 minute (range 103.25-104.0 TCID
50/ml). No PV was found on any of the 30 toothbrushes
placed in the Pollenex DS60 unit for 24 hours. The ten Colgate
toothbrushes placed on a shelf had the highest return of virus
particles (ave. 102.0 TCID 50/ml) from a
retained intiial contration of virus particles: ave.
104.0 TCID 50/ml. The ten Reach toothbrushes
(shelf stored) returned an average of 101.77 TCID
50/ml from a retained initial concentration of virus
particles: ave. 103.625 TCID 50/ml. The ten
Oral B toothbrushes (shelf Stored) hade the lowest return of virus
particles (ave. 101.41 TCID 50/ml) from a
retained initial concentration of virus particles:ave
103.5 TCID 50/ml. Two Oral B toothbrushes
(shelf stored) had no evidence of virus particles. The overall
average of the 30 toothbrushes placed on the shelf was
101.75 TCID 50/ml. In general, the lighter
colored or clear toothbrushes appeared to retain or to return fewer
virus particles.
The results are summarized in Tables 2 and 3.
Discussion
With the findings that toothbrushes from humans were
contamintated with a wide range of microorganisms, it seemed
imperative that some type of instrument or proces be developed to
lessen the problem of toothbrush contamination.1
Certainly, high risk patients could profit from use of a toothbrush
bacteriocidal device, 4,5,6 but so too could the general
population.7 Even though some of the initial levels of
microbial contaminants were somewhat higher than what would be exp
ected in normal saliva (ca. 108 total microorganisms per
ml of saliva), the finding that substantial reduction in numbers of
microorganisms identified by the U.S. Food and Drug Administration
as target microorganisms of decontamination by the Pollenex DS60
speaks well for its use.
With additional studies demonstrating viral retention on
toothbrushes2 and the harm caused by repeated brushing
with the same toothbrush3, and effective, low-cost
solution to the problem was critical. The imperative was intensified
by the finding that 4.5% of healthy individiuals, 20% of the
patients undergoing oral surgery and 38% of immunocompromised
patients, regularly shed HSV in their saliva.8 The
finding that the Pollenex DS60 unit completely kills viruses on all
toothbrush types and colors is certainly sufficient reason to
advocate its use.
Similarly, it seemed important to evaluate other virus retention
on toothbrushes. The Parainfluenze Virus, Type III was chosen
because it represented a prototype of the common cold viruses in
children and upper respiratory viruses in adults. The findings that
the PV could be retained on toothbrushes and completely killed by
the Pollenex unit again advocates the use of the device. Goh et al
have implicated by statistical means, the toothbrush in the
transmission of the Hepatitis B Virus in families.9
Goodman et al have also shown transmission of a wide range of viral
systemic diseases by oral means.10 Finally, if the
statistics on waterborne disease outbreaks in the United States,
1986-1988, are considered, one third of the 25, 846 cases had either
a definite viral or possible viral etiology.11
Multiple techniques to decontaminate toothbrushes have been
employed, including trials with several of the ultraviolet
toothbrush sanitizers (unpublished data). While simple toothbrush
rinsing reduces some of the toothbrush microbial burden, the only
device which was found to consistently reduce the number of a wide
range of microorganisms was the Pollenex DS60 Daily Dental
Sanitizer.
Finally, all experiments performed, using whatever method, were
found to be toothbruse dependent. Toothbrush color, toothbrush
opacity, numbers of bristles and bristle arrangement appeared to be
the major factors in determining the ability of a toothbrush to
become contaminated and to retain microorganisms.2 Sharp
edges on the toothbrush heads also appeared to be a factor in
microorganism retention (clinical observation). Silverstone and
Featherstone, in an excellent scanning electron microscopic study of
toothbrushes, found that toothbrush bristle rounding in new, unused
toothbrushes varied from 88% to 22% rounding/brush.12 A
recent experiment demonstrated a substantial decrease in the number
of rounded bristles after two weeks of use (unpublished data).
Previous studies also demonstrated a propensity of microorganisms to
acumulate and adhere to roughened toothbrush bristles. It is
important, therefore, to develop new toothbrushes which can retain
rounded bristles and to continue to regard the toothbrush as an
hygienic device, to be discarded every two weeks.3 Based
upon this study and previous studies, it appears that the most
biologically sound toothbrush available at this time is a clear, two
row soft toothbrush .
Conclusions
1. The Pollenex DS60 Daily Dental Sanitizer was found effective
at sanitizing toothbrushes contaminated with the cited
microorganisms. 2. A new toothbrush needs to be developed; one which
will take into consideration microbial contamination.
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