Transcript Document

Toothbrush Bristle Wear
and Adherence of
Streptococcus mutans
Robert Goldsmith, DMD
UMDNJ – Department of Pediatric Dentistry
May 12, 2006
Purpose of the Study
To determine if toothbrush bristle wear:
•impacts the adherence of S. mutans
•affects the extent of adherence at 0, 8 and 24 hours
after air-drying
Review of Literature
Microbial Contamination of Toothbrushes
Svanberg M (1978). Contamination of toothpaste and toothbrush by Streptococcus
mutans. Scand. J Dent. Res. 86:412-414.
Purpose: Examined the contamination of toothbrushes by S. mutans
Methods: Two Swedish adult subjects with a salivary S. mutans
concentration of 106 colony forming units (CFU) were
each given three new toothbrushes. After three consecutive days of use
toothbrushes were air dried at room temperature for 15 minutes, 12
hours, and 24 hours and CFU were counted.
Results: Greater than 106 CFU of S. mutans remained on toothbrush bristles up to
15 minutes after use and that approximately 104 CFU of S. mutans
remained after 24 hours of air-drying.
Kozai K, Iwai T, Miura K. Residual contamination of toothbrushes by
microorganisms. J Dent Child 56:201-204, 1989.
Purpose: Investigated the microbial contamination of toothbrushes
after regular use and rinsing by children.
Methods: 150 Japanese children, 6 years of age, were given a new toothbrush
without toothpaste and asked to brush and rinse the toothbrush three
different ways - rinsed slightly in running tap-water, rinsed in running
tap-water with a finger used to manipulate the bristles slightly, and rinsed
well in running tap-water with vigorous manipulation of bristles.
Toothbrushes were then collected and allowed to air-dry for 0, 6 and 24
hours.
Results: Toothbrushes harbored approximately 4.47 x 104 S. mutans CFU
immediately after rinsing with the number decreasing to 2.55 x 104 CFU
after 6 hours and to 1.35 x 103 CFU after 24 hours.
Conclusions: The extent to which the toothbrush was rinsed affected the degree of
contamination, with well-rinsed brushes containing fewer microorganisms.
Malmberg E, Birkhed D, Norvenious G, Noren JG, Dahlen G. Microorganism
on toothbrushes from day-care centers. Acta Odontol Scand 52:93-98, 1994.
Purpose: Examined microbial adherence on 44 toothbrushes used by
children 4-to-6 years old at Swedish day-care centers.
Methods: Unsupervised tooth brushing without toothpaste after breakfast and
lunch using toothbrushes provided by their parents. Toothbrushes
were air-dried for two hours after each use. Vigorous hand shaking
was performed for two minutes to remove bacteria from
toothbrushes. Bacteria were plated on agar media and CFU were
counted
Results: Over 50% of the time the most frequent bacteria found on the
toothbrush two hours after use was Streptococci, predominantly S.
salivarius, S. sanguis, and S. mitis. Forty-one percent of the
toothbrushes contained Lactobacilli and none showed beta-hemolytic
Streptococci.
Conclusion: After two hours of air-drying toothbrushes become heavily
contaminated with microorganisms and that the level and viability of
the microorganisms vary based on the extent of dryness.
Wetzel WE, Schaumburg C, Ansari F, Kroeger T, Sziegoleit A. Microbial
contamination of toothbrushes with different principles of filament anchoring.
JADA, 136:June 2005:758-765.
Purpose: Examined the microbial contamination of toothbrushes with
different filament anchoring using 45 German children ages 6to-13 years old.
Methods: Toothbrushes were divided into three groups by type of anchoring
construction: staple-set tufting, in-mold tufting, and individual
in-mold placement of filaments. Subjects used two toothbrushes with a
pea-sized amount of toothpaste, cleaning the maxillary and mandibular
teeth on one side with one toothbrush and those on the opposite side
with the other toothbrush. The brushes were examined immediately
after brushing, two hours later and eight hours later.
Results: Anchoring systems and the drying intervals both had a significant effect
on the microbial contamination of the brushes with individual in-mold
filament placement retaining the least amount of microorganisms
compared with tufting.
Review of Literature
Toothbrush Bristle Wear
McKendrick AJW, McHugh WD, Barbenel LMH. Toothbrush age and wear: An
analysis. Br Dent J 130:66-68, 1971.
Purpose: Examined toothbrush wear over two years using 103 adults with a
mean age of 20.7 years.
Methods: 50 subjects were issued electric toothbrushes and 53 subjects
were issued hand-held toothbrushes. They were instructed to return their
used toothbrushes when they thought the brush was worn out.
Results: The average brush age at replacement was 10.5 weeks.
Conclusions: The researchers concluded that the way in which a toothbrush is used
for cleaning teeth is more important than length of time in use.
Glaze PM, Wade AB. Toothbrush age and wear as it relates to plaque control. J
Clin Periodontol 13:52-56, 1986.
Purpose: Examined toothbrush wear and plaque control.
Methods: 40 British dental students, ages 19-to-26 years old. One group used a
single toothbrush for 10 weeks while the other group were given new
toothbrushes every two weeks for ten weeks. At biweekly visits, plaque
and calculus were measured. The degree of toothbrush wear was
assessed subjectively and placed into one of three categories: good, fair,
and poor condition. Brush head surface area was assessed using calipers
at different locations on the trim of the toothbrushes and by multiplying
the greatest measurements in each direction.
Results: Subjects using the same toothbrush for the ten week period had
significantly more plaque on their teeth than subjects who replaced their
brushes. However, it was only when the mean brush-head surface area
increased to 68% above that of unused brushes that plaque scores
significantly increased.
Rawls HR, Mkwayi-Tulloch NJ, Casella R, Cosgrove R. The measurement of
toothbrush wear. J Dent Research 68(12):1781-1785, 1989.
Purpose: Develop a quantitative measure of toothbrush wear based on bristle
splaying
Methods: Toothbrushes were damaged using a toothbrush wear machine. Bristle
wear was measured subjectively and scored as follows:
“0” A brush that a person could not be sure if
it had been used or not (0-25% wear)
“1” bristles that spread apart in many of the tufts (2549% wear)
“2” all tufts were spread apart and many bristles
were curled and/or matted (50-75% wear)
“3” most tufts overlap and were matted and many
curled and bent bristles were seen (76-100% wear)
Results: Bristle splaying was strongly influenced by the length of time a toothbrush
was used and that wear rating provided a quick and effective way of
determining bristle deterioration.
Summary
Microbial Contamination:
• Toothbrushes become heavily contaminated with many microorganisms
after regular use.
• Different amounts of bacteria adhere to toothbrushes at different time
points.
• Level and viability of the microorganisms varies based on the amount of
toothbrush bristle rinsing and dryness after use.
Toothbrush Bristle Wear:
• Bristles wear out over time and can affect plaque removal
• Bristle wear can be created and assessed through different means
Hypotheses
1.
Toothbrush group will affect adherence of S. mutans to new
and worn toothbrushes as measured by the number of
recoverable microorganisms.
2.
Worn toothbrush bristles will harbor more S. mutans than
new toothbrush bristles immediately after contamination
with bacteria as measured by the number of recoverable
microorganisms.
3.
After 8 hours of air-drying, worn toothbrush bristles will
harbor more S. mutans than new toothbrush bristles as
measured by the number of recoverable microorganisms.
4
After 24 hours of air-drying, worn toothbrush bristles will
harbor more S. mutans than new toothbrush bristles as
measured by the number of recoverable microorganisms.
Methods
Creating Toothbrush Bristle Wear
An orthodontic typodont from a front and side view with metal bands and
brackets on the teeth with four rubber bands placed around the typodont to
hold it closed and to assure constant pressure of the toothbrushes against the
bracketed teeth.
Standardization of Toothbrush
Bristle Wear
Goal: To identify worn toothbrushes that meet the criteria for a
category “3” toothbrush classification as specified by Rawls et al
(1989): most tufts overlap and are matted together or many bristles
are bent and curled.
Method:
•4 independent observers were given a verbal description
and visual representations of new and worn toothbrushes
by group (labeled A, B and C). They were then given 30
worn toothbrushes, 10 from each group and asked to
rate whether the worn toothbrushes met the appropriate
criteria for a category “3” toothbrush.
•Two training sessions produced 100% reliability.
Visual Representation of a New and Worn Toothbrush
New
Worn
Examining New and Worn toothbrush bristles under a
dissecting microscope
New toothbrush bristles are tightly packed
Worn toothbrush bristles are splayed
Examining New and Worn toothbrush bristles
under a scanning electron microscope
New Bristle (360X)
Worn Bristle (370X)
•Round
•Jagged
•Smooth
•Irregular
Measurement of Toothbrush
Bristle Splaying
Goal:
•To compare bristle splaying between new and
worn toothbrushes by group
Method:
•10 toothbrushes from each group, 5 new and 5
worn were used.
•Three randomly selected tufts, magnifying loops
of 2X magnification, a millimeter caliper and
an adequate light source.
•The degree of bristle splaying was measured
within a tuft from one edge to the most
splayed bristle at the other edge of the tuft.
Toothbrush bristle splaying for all toothbrush
groups both new and worn N=45
4.5
Mean Bristle Splaying (mm)
4
N=45
3.5
3
2.5
2
N=45

1.5
1
0.5
0
New
Worn
Toothbrush Status
A statistically significant difference in mean bristle splaying of 1.7 mm +/- 0.74 mm was seen between all
toothbrushes, both new and worn (t = 172.7; P = 0.0001).
Toothbrush bristle splaying between new and worn
toothbrushes by group
6
Mean Bristle Splaying (mm)
5
4
New
3
2
Worn



1
0
A
B
C
Toothrush Group
Toothbrush group A had a mean difference in bristle splaying of 2.21 mm +/- 0.99 (t = 67.8; P = 0.0001).
Toothbrush group B had a mean difference in bristle splaying of 0.68 mm +/- 0.13 (t = 271.0; P = 0.0001).
Toothbrush group C had a mean difference in bristle splaying of 2.10 mm +/- 0.27 (t = 270.1; P = 0.0001).
Methodology Flow Chart
for Bacteria
30 adult toothbrushes
Group A (10)
Group B (10)
Submitted to bristle wear
(15 - 5 per group)
Group C (10)
Not submitted to bristle wear
(15 - 5 per group)
Toothbrushes dipped into a test tube containing
S. mutans
Four random tufts were removed from
the toothbrush heads with a hemostat
and placed into a test tube containing
3ml of phosphate buffered saline
Rinsed for five seconds by dipping into
non-sterile tap water
100 microliters of this
bacterial solution (sample)
and 100 microliters of Brian
Heart Infusion media
(control) were pipetted into
two separate wells of a 96well microplate
Serial dilutions 10-1 to 10-5
were performed and plated
on Mitis Salivarius Agar
Vortexed for 30 seconds to
remove bacteria
Serial dilutions (10-1 to 10-3) and 100
microliters of each was plated on Mitis
Salivarius Agar and incubated at 37° C for two
days
Colony forming units of S. mutans
were visually counted
Optical reading at
620nm was used to
measure the
amount of S.
mutans in the
sample compared
to the control
Plates were grown
aerobically in a
humidity chamber at
37°C for two days
until colony forming
units were large
enough to be
visually counted
Statistical Analysis
Dependent Variable
• Bacterial adherence to toothbrush bristles
Independent Variables
• Toothbrush status (new vs. worn) and toothbrush group (A, B and C)
Stat View program; Super ANOVA program
• Means and Standard Deviations - Adherence of S. mutans to new and
worn toothbrushes at different time points
• Bacterial data was transformed to log10.
• Independent t-tests were used to compare (new vs. worn)
• A one-factor ANOVA was used to compare (toothbrush groups A, B
and C). Post Hoc Scheffe’s test for significance
•  = 0.05, Power = 80%
Adherence of S. mutans at different time points by
toothbrush group
7
6

Mean log CFU/ml
5

4
A
B

3
C
2

1
0
0
8
24
Time (hours)
At 0 hours, the results of the analysis of variance (F = 8.2; df = 2) comparing the three toothbrush groups
indicated that there were significant differences (P = 0.0008). At 8 hours, the results of the analysis of
variance (F = 28.2; df = 2) comparing the three toothbrush groups indicated that there were significant
differences (P = 0.0001). At 24 hours, the results of the analysis of variance (F = 15.46; df = 2) comparing the
three toothbrush groups indicated that there were significant differences (P = 0.0001).
Adherence of S. mutans at different time points by
toothbrush status
7
6

Mean log CFU/ml
5
4
New
Worn
3
2
1
0
0
8
24
Time (hours)
At 0 hours, the results of the t-test comparison (t = 4.21; df = 1) indicated that S. mutans adherence to new
toothbrushes were significantly (P = 0.0448) more than S. mutans adherence to worn toothbrushes. At 8 and 24
hours respectively, the results of the t-test comparison (t = 1.44 and t = 2.13 respectively) indicated that S.
mutans adherence to new and worn toothbrushes were not significantly different (P = 0.2358 and P = 0.1496
respectively).
Adherence of S. mutans to new toothbrushes by group at
different time points
7
6

Mean log CFU/ml
5
4
A

B
3
C
2
1

0
0
8
24
Time (hours)
At 0 hours, the results of the analysis of variance (F = 8.81; df = 2) comparing the three toothbrush groups
indicated that there were significant differences (P = 0.0011).  At 8 hours, the results of the analysis of variance
(F = 19.48; df = 2) comparing the three toothbrush groups indicated that there were significant differences (P =
0.0001).  At 24 hours, the results of the analysis of variance (F = 16.88; df = 2) comparing the three toothbrush
groups indicated that there were significant differences (P = 0.0001).
Adherence of S. mutans on worn toothbrushes by
group at different time points
7
6

Mean log CFU/ml
5
4
A
B
C
3
2

1
0
0
8
24
Time (hours)
At 0 hours, the results of the analysis of variance (F = 2.99; df = 2) comparing the three toothbrush groups
indicated no significant differences (P = 0.0670).  At 8 hours, the results of the analysis of variance (F = 12.88; df
= 2) comparing the three toothbrush groups indicated that there were significant differences (P = 0.0001).  At 24
hours, the results of the analysis of variance (F = 4.44; df = 2) comparing the three toothbrush groups indicated that
there were significant differences (P = 0.0215).
Adherence of S. mutans on New and Worn
Group C Toothbrushes at different time points
6
Mean log CFU/ml
5
4
New

3
Worn
2
1
0
0
8
24
Time
No significant differences were seen between adherence of S. mutans on new and worn group C toothbrushes at 0
hours (t = 3.43; df = 1; P = 0.0805) and 8 hours (t = 3.68; df = 1; P = 0.0711).  At 24 hours (t = 21.58; df = 1;
P = 0.0002), a significant difference in mean bacterial adherence was seen between new and worn group C
brushes.
Conclusions
1. Toothbrush group affects adherence of S. mutans to both
new and worn toothbrushes.
2. New toothbrushes tend to harbor more S. mutans than worn
toothbrushes at 0, 8 and 24 hours after air-drying, but was
only significant at 0 hours.
3. S. mutans adheres to toothbrushes immediately after
inoculation and can be recovered on toothbrushes 8 and 24
hours later.
Explanation of Results
• Capillary action of the liquid bacterium on the toothbrush bristles
• Different toothbrush head shapes/bristle surface area
• Different lengths of toothbrush bristles
• Different number of bristles per tuft
• Different means of tufting
Limitations of the Study
1. The use of a one-time application of a liquid bacterial
culture of S. mutans to measure adherence does not
replicate the oral environment in the mouth.
Toothbrushes were only inoculated once and then
measured over time.
2. Times points chosen were 0, 8 and 24 hours, to facilitate
the conduct of the study when the laboratory was
available. Even though this was practical, it may have
simulated life more closely if 0, 12 and 24 hours where
chosen because people brush their teeth in the morning
and then 12 hours later when they go to bed.
Future Studies
1) Repeated inoculations of toothbrushes with S. mutans
over time
2) Another study could look at specific sites on the
bristles where bacteria adhere and colonize over time
3) Different bacteria
Acknowledgements
1) Ms. Marie McKiernan for her help, time, knowledge and support in the laboratory. Without her, this project
could not have been completed.
2) Dr. David Furgang for his help and guidance with all aspects of this project, especially the statistical analysis.
3) Dr. Kabilan Velliyagounder for his help with the use of the scanning electron microscope.
4) Ms. Weijuan Han for her help with the statistical analyses.
5) My fellow post-graduate students, especially Dr. Natalie Sanche and Dr. Shylon Mathew for their help in the
laboratory.
6) The third year dental students, Ms. Janna Kohout, Ms. Heather Wolen, Ms. Maryam Shariff, Mr. Mark Danbe
and Mr. Reza Movahed who helped in evaluating toothbrushes.
7) The Pediatric Dental faculty for sharing their knowledge of pediatric dentistry: Dr. Jack Budnick, Dr. Mary
Burke, Dr. Jorge Caceda, Dr. Jerry Guzzy, Dr. Mahdu Mohan, Dr. Melvyn Oppenheim, Dr. R. Glenn Rosivack
and Dr. Nanci Tofsky.
8) Ms. Debra Goldsmith and Ms. Maria Navarro for supplying toothbrushes for this study.
9) Ms. Carmen Logatto and Ms. Debra Bereski for their help in scheduling committee meetings.
10) My family, especially my mother Dr. Rachelle Goldsmith for her support and guidance throughout this
project.
11) My thesis advisory committee: Dr. Zia Shey, Dr. Daniel Fine, Dr. Helen Schreiner, Dr. Barbara Greenberg,
and Dr. Milton Houpt for their advice and encouragement throughout this project.