Clinical Studies

The effects of toothpastes on the residual microbial contamination of toothbrushes.

  • Donna P. Warren, R.D.H., M.Ed.; Millicent C. Goldschmidt, Ph.D.

  • Mathew B. Thompson; Karen Adler-Storthz, Ph.D.; Harris J. Keene, D.D.S.


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.


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.


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).


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.

 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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