Toothbrush Bristle Wear and Adherence of Streptococcus mutans

1
Toothbrush Bristle Wear and Adherence of
Streptococcus mutans

• Robert Goldsmith, DMD
• UMDNJ Department of Pediatric Dentistry
• May 12, 2006

2
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
3
Review of Literature Microbial Contamination of
Toothbrushes
Svanberg M (1978). Contamination of toothpaste
and toothbrush by Streptococcus mutans. Scand. J
Dent. Res. 86412-414. 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.
4
Kozai K, Iwai T, Miura K. Residual contamination
of toothbrushes by microorganisms. J Dent Child
56201-204, 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.
5
Malmberg E, Birkhed D, Norvenious G, Noren JG,
Dahlen G. Microorganism on toothbrushes from
day-care centers. Acta Odontol Scand 5293-98,
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.
6
Wetzel WE, Schaumburg C, Ansari F, Kroeger T,
Sziegoleit A. Microbial contamination of
toothbrushes with different principles of
filament anchoring. JADA, 136June
2005758-765. Purpose Examined the microbial
contamination of toothbrushes with different
filament anchoring using 45 German children ages
6- to-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.
7
Review of Literature Toothbrush Bristle Wear
McKendrick AJW, McHugh WD, Barbenel LMH.
Toothbrush age and wear An analysis. Br Dent J
13066-68, 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.
8
Glaze PM, Wade AB. Toothbrush age and wear as it
relates to plaque control. J Clin Periodontol
1352-56, 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.
9
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 (25-
49 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.
10
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
11
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.
12
MethodsCreating 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.
13
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.
14
Visual Representation of a New and Worn Toothbrush
New
Worn
15
Examining New and Worn toothbrush bristles under
a dissecting microscope
New toothbrush bristles are tightly packed
Worn toothbrush bristles are splayed
16
Examining New and Worn toothbrush bristles under
a scanning electron microscope
New Bristle (360X) Round Smooth
Worn Bristle (370X) Jagged Irregular
17
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.
18
Toothbrush bristle splaying for all toothbrush
groups both new and worn
N45
N45
N45
?
?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).
19
Toothbrush bristle splaying between new and worn
toothbrushes by 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).

20
Methodology Flow Chart for Bacteria
30 adult toothbrushes
Group A (10)
Group B (10)
Group C (10)
Submitted to bristle wear (15 - 5 per group)
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
Serial dilutions 10-1 to 10-5 were performed and
plated on Mitis Salivarius Agar
100 microliters of this bacterial solution
(sample) and 100 microliters of Brian Heart
Infusion media (control) were pipetted into two
separate wells of a 96-well microplate
Rinsed for five seconds by dipping into
non-sterile tap water
Vortexed for 30 seconds to remove bacteria
Plates were grown aerobically in a humidity
chamber at 37C for two days until colony forming
units were large enough to be visually counted
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
Optical reading at 620nm was used to measure the
amount of S. mutans in the sample compared to the
control
Colony forming units of S. mutans were visually
counted
21
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
Scheffes test for significance ? 0.05,
Power 80
22
Adherence of S. mutans at different time points
by toothbrush group
?
?
?
?
?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).  
23
Adherence of S. mutans at different time points
by toothbrush status
?
?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).  

24
Adherence of S. mutans to new toothbrushes by
group at different time points
?
?
?
?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).
25
Adherence of S. mutans on worn toothbrushes by
group at different time points
?
?
?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).
26
Adherence of S. mutans on New and Worn Group C
Toothbrushes at different time points
?
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.
27
Conclusions

• Toothbrush group affects adherence of S. mutans
  to both new and worn toothbrushes.
• 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.
  
• S. mutans adheres to toothbrushes immediately
  after inoculation and can be recovered on
  toothbrushes 8 and 24 hours later.

28
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
29
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.
30
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
31
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.