Clinical Studies
The effects of toothpastes on the residual microbial contamination of toothbrushes.
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Donna P. Warren, R.D.H., M.Ed.; Millicent C. Goldschmidt, Ph.D.
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Mathew B. Thompson; Karen Adler-Storthz, Ph.D.; Harris J. Keene, D.D.S.
Introduction:
As early as 1920, Cobb reported that toothbrushes could be a source of repeated oral infections. Others have reported a more serious consideration-bacterial endocarditis-resulting from bacteremia caused by toothbrushing. Studies by Glass and colleagues have shown that contaminated toothbrushes not only harbor, but also transmit, both viruses and bacteria that cause systematic, localized and oral inflammatory diseases. Toothbrushes kept in a moist environment like that of a bathroom retained up to 50 percent of herpes simplex virus Type I for a week. And in vivo study involving 59 patients who had oral inflammatory disease found that 34 percent required no additional therapy after they changed their toothbrushes biweekly. Of the remaining 39 patients in the study who were treated with antimicrobials, 78 percent had no recurrent symptoms after initial therapy combined with a toothbrush change. In another study using dogs, repeated brushing with the same toothbrush for a month resulted in a more intense infection of Candida albicans,Staphylococcus aureus and Prevotella melaninogenica than occurred in the group whose brushes has been changed.
Other studies have reported that Streptococcus mutans was found on toothbrushes after six hours of drying time, thus increasing the risk of dental plaque that adheres to and can remain on the toothbrushes. Transparent toothbrush heads with the minimal amount of bristles per tuft were found to harbor the least amount of S. mutans Loesche and colleagues found that S. mutans could be transmitted by a dental explorer from one tooth to another, while Kohler and Bratthall found that the bacteria could be spread by using eating utensils contaminated by saliva.
Barnett and colleagues used scanning electronic microscopy to determine that periodontal pathogens from an infected site could adhere to probes and be transmitted to other sites. Christersson and colleagues studied patients who had juvenile periodontitis and reported that Actinobacillus actinomysetemcomitans, or Aa, could be transmitted with a probe with infected sites to noninfected sites within the same mouth. Several studies have shown that Aa and Porphyromonas gingivalis, or Pg, have been transmitted between spouses and or between siblings. Preus and Olsen reported two cases of Aa transmission from dogs to children. Muller and colleagues demonstrated that Aa could be cultured from the toothbrushes of subjects who had advanced periodontitis.
In response to these reports, several studies have focused on methods of toothbrush disinfection. Caudry and colleagues found that soaking toothbrushes for 20 mins in a mouth rinse containing essential oils killed 100 percent of the bacteria present. The use of a UV toothbrush-sanitizing device also has been shown to be effective. Meier and colleagues tested the use of a cetylpyridinium chloride spray with toothbrushes and found it to be bactericidal.
Recently, antimicrobial toothpastes that contain triclosan have been introduced; triclosan is a compound commonly used for disinfection. To date, these toothpastes have not been studied for any effect on the residual microbial contamination of toothbrushes. We conducted an in vivo pilot study to evaluate the effect of a triclosan containing toothpaste on the residual microbial.
MATERIALS AND METHODS
We obtained approval to conduct this study from the
Committee for the Protection of Human Subjects at the University of
Texas-Houston Health Science Center. We recruited 20 subjects
from the dental hygiene patient clinic, and they agreed to
participate in the study and signed the appropriate consent
forms. To participate in the study they had to meet the
following inclusion criteria:
-Must have Type III or Type
IV periodontitis;
-Must not be pregnant, as hormones can
affect gingival health;
-Must not be taking medications that
affect periodontal pathogens (for example, antimicrobial
agents)
-Must not have systemic conditions associated with
periodontal flora (for example, juvenile periodontitis,
diabetes);
-Must not be using a tricolson-containing
toothpaste;
-Must be 25 to 79 years of age.
-Must have a
minimum of three posterior teeth in each quadrant.
This
preliminary pilot study comprised 20 subjects, or 40
half-mouths. We used a cross-arch study design, in which each
subject served as both a control and a test subject. One-half
of each subject?s mouth served as a control, as the teeth in it were
not brushed with toothpaste. The other side?s teeth were
brushed with regular toothpaste or a triclosan-containing
toothpaste. We randomly placed each of the 20 subjects into
one of four groups (box, ?Subjects? Group Assignments?)
To control for any differences in bacterial growth caused by translucency, we autoclaved and used Crest Complete (Procter & Gamble) toothbrushes that had white heads and handles. We also used Colgate Regular (Colgate-Palmolive) toothpaste, which contains sodium monofluorophosphate, 0.15 percent weight per volume and triclosan 0.30 percent.
To control for variations in brushing technique,
one dental hygienist (D.P.W.) performed all of the toothbrushings
using Bass? method. A ?pea-sized? amount of toothpaste was
used in the experiments involving toothpaste. The teeth on
each side of the mouth were brushed for 30 seconds, and then the
toothbrush was rinsed for 10 seconds under a stream of sterile
bottled water, tapped three time to remove excess moisture and
placed in an individual labeled sterile bag to be transported to the
laboratory. In the laboratory, the brushes were air-dried in a
rack at room temperature (25 C) for four hours. We selected a
four-hour drying period as Aa, Pg, and Prevotella species, or Ps,
are considered to be sensitive to oxygen, and a previous study also
had used this interval.
After the drying period, we aseptically
removed toothbrush heads from the handles placed them in 10
milliliters of prereduced peptone-saline diluent and pulse agitated
them using a vortex mixer for one minute. We prepared suitable
serial dilutions in this reduced diluent and plated them on various
perreduced anaerobically sterilized, or PRAS Brucella agar with 5
percent sheep blood (AS-141, Anaerobe Systems). We used selective
media to detect Pg (AS-6422, Anaerobe Systems) and Aa (tryptic
soy-serum-bacitracin-vancomycin agar AS-648, Anaerobe
Systems). We placed the petri plates containing PRAS in
standard Brewer jars. Anaerobiosis was obtained by placing a
BBL Gas Pak Plus (Becton Dickinson) anaerobic system envelope
containing a palladium catalyst in each jar before firmly scaling
it. We anaerobically incubated the jars containing the plates at 35
C for one week in a standard laboratory incubator and examined them
under long-wave (366 nanometer) UV light to differentiate Ps
colonies that fluoresced brick red. We also determined the
presence or absence of the three test microorganisms by examining
the petri plates under a low-power colony counter. WE recorded
group isolation frequencies and used X ? analysis to test for
significance of inter-group differences. We compared the
control group and regular-toothpaste group, the control group and
the triclosan-containing-toothpaste group, and the
triclosan-containing-toothpaste group and the regular toothpaste
group. We considered P values equal to or less than .05 to be
statistically significant.
Results
Isolation frequencies for the microorganisms
observed in each of the groups are shown in the table. We
found Aa on 85.o percent of the control toothbrushes, on 87.5
percent of the regular-toothpaste toothbrushes and on 66.7 percent
of the triclosan-containing-toothpaste toothbrushes. We found
Pg on 80.0 percent of the control, toothbrushes and on 83.3 percent
of the triclosan-containing-toothpaste toothbrushes. We found
Ps on 20.0 percent of the control toothbrushes and on 8.3 percent of
the triclosan-containing-toothpaste toothbrushes. We did not,
however, find Ps on the regular-toothpaste toothbrushes.
We
performed a series of nine X ? tests to determine the significance
of the different isolation frequencies for each of the three
microorganisms in the three treatment groups. None of the
intergroup frequency companions was significant by X ? analysis
(P>.05). In the control group, however, the lower isolation
frequency of Ps (20.0 percent) was statistically significant (X ? =
22.14; P<.001) compared with the frequency of Aa (85.0 percent)
and Pg (80.0 percent).
Discussion
Since Aa, Pg and Ps are anaerobic bacteria commonly
found in periodontal disease, we chose them to represent periodontal
pathogens that are able to survive on toothbrushes exposed to air
fro four hours. Pg is a strictly anaerobic, gram-negative rod
that forms black colonies on blood agar. Ps also is a strictly
anaerobic, gram-negative rod, and it forms dark colonies that
fluoresce red when exposed to long-wave UV light. It is
associated strongly with necrotizing ulcerative gingivitis and
hormonal gingivitis, which occurs in women during puberty and
pregnancy. Aa is a facultative anaerobe that is associated
consistently with aggressive periodontitis and chronic
periodontitis.
Although we detected all three bacteria on
most of the toothbrushes used in this preliminary study, Ps occurred
at a much lower frequency than did Pg or Aa. We do not know if these
different isolation frequencies actually reflect their relative
prevalence in the subjects? oral cavities. In a more
comprehensive study, it would be important to determine these
interrelationships. The low-isolation frequency of Ps observed
in this study could be an artifact, or it could be related to its
anaerobic properties.
Nearly all of the clinically
significant obligate anaerobes isolated from the oral cavity are
moderate or aerotolerant anaerobes; they can tolerate being exposed
to oxygen?s toxic reduction products. The three bacteria we
studied in this article have been classified as either moderate or
aerotolerant anaerobes. For example, Aa is catalase positive
Oral specimens that are cultured immediately can have very high
counts of bacteria. There is a much lower survival rate when
the organisms on the toothbrushes air exposed to air at four hours
and a still lower rate at 24 hours before they are cultured
anaerobically. What is valuable from this study is the fact
that enough of these bacteria are surviving and can become
reestablished when they are grown in an anaerobic environment,
including the oral cavity. Thus, they may be capable of
reinfecting patients from toothbrushes that have been left in air to
dry.
All oral organisms, including facultative anaerobes
that can grow aerobically or anaerobically, usually grow well on the
nonselective Brucella blood agar plates. We made no attempt to
isolate or identify other periodontal pathogens or facultative
anaerobes in this study.
This study supports the findings
of previous studies of the residual microbial contamination of
toothbrushes. In the regular-toothpaste group, we found Aa on
87.5j percent of the toothbrushes. The control group has
slightly lower Pg isolation frequencies than did the
regular-toothpaste group. We found no Ps on the toothbrushes
from the regular-toothpaste group, while we found Ps on 8.3 percent
of the toothbrushes from the triclosan-containing-toothpaste
group. This was much less than the 20 percent frequency rate
we observed for the control toothbrushes. While the intergroup
differences were not statistically significant, this last
observation could be clinically important for patients who have
periodontal disease associated with Ps infection.
A more
definitive study is necessary, however, to determine if the presence
of periodontal pathogens on toothbrushes is inhibited by
toothpaste. Long-term use of a toothbrush with a toothpaste
may result in findings different form those of our study, which
involved one-time brushing with a new toothbrush. Also,
researchers may want to culture patients? periodontal pockets before
the study begins to determine the existence of certain bacteria,
thereby ensuring that specific pathogens are present and
establishing baseline levels. A crossover design could be used
to enable reduction of certain threats to validity, as well as to
increase the population sample size.
Conclusions
We isolated periodontal pathogens from
toothbrushes that were air-dried for four hours after
toothbrushing. Neither regular toothpaste nor
trilosan-containing toothpastes appear to inhibit the presence of
periodontal pathogens. We detected Aa, Pg, and Ps on
toothbrushes from each treatment group, except for in the
regular-toothpaste group, in which we did not find Ps.
This
study supports other studies that suggest that dental professionals
should advise patients who have periodontal disease to disinfect or
change their toothbrushes between brushings to prevent
self-infection.
Background: Contaminated toothbrushes have been shown to harbor and transmit viruses and bacteria. The authors conducted a study to evaluate the effect of triclosan-containing toothpaste on the residual anaerobic microbial contamination of toothbrushes.
Methods: Twenty patients who had Type III or Type IV periodontitis participated in this study. One side of each of their mouths served as a control (no toothpaste). The teeth on the other side were brushed with a regular toothpaste or a triclosan-containing toothpaste. After the toothbrushes were allowed to dry in air for four hours, the authors placed the toothbrush heads in solution, dislodged the microbes form the brushes by vortexing and plated them in culture dishes. The authors anerobically incubated the culture dishes and determined the presence or absence of Prevotella species or Ps; Porphyromonas gingivalis, or Pg; and Actinobacillus actinomycetemcomitans, or Aa.
Results: The authors detected Aa and Pg on the control toothbrushes more frequently than they did Ps. This variation in isolation frequency was statistically significant by x? analysis (P<.001). The authors compared the isolation frequency of the three test organisms between the control and regular-toothpaste groups, between the control and triclosan-containing-toothpaste groups, and between the triclosan-containing-toothpaste and regular-toothpaste groups. They found no significant intergroup differences in the isolation frequencies after using x? analysis.
Conclusions: Toothpaste use reduced the
residual microbial contamination for two of three test organisms,
but the lower isolation frequencies were not statistically
significant. Further study in this area is
indicated.
Clinical Implications: Dental professionals should advise patients who have systemic, localized or oral inflammatory diseases to disinfect or frequently replace their toothbrushes.
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