Other studies have reported that Streptococcus mutans was found on toothbrushes after six hours of drying time, thus increasing the risk of dental caries.^9,10 S. mutans cells exist in moist dental plaque that adheres to and can remain on toothbrushes.⁹ Transparent toothbrush heads with the minimal amount of bristles per tuft were found to harbor the least amount of S. mutans.⁷ Loesche and colleagues¹¹ found that S. mutans could be transmitted by a dental explorer from one tooth to another, while Kohler and Bratthall¹² found that the bacteria could be spread by using eating utensils contaminated by saliva.

Barnett and colleagues¹³ used scanning electron microscopy to determine that periodontal pathogens from an infected site could adhere to probes and be transmitted to other sites. Christersson and colleagues¹⁴ studied patients who had juvenile periodontitis and reported that Actinobacillus actinomycetemcomitans, or Aa, could be transmitted with a probe from 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.^15–21 Preus and Olsen²² reported two cases of Aa transmission from dogs to children. Muller and colleagues²³ 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 colleagues²⁴ found that soaking toothbrushes for 20 minutes in a mouthrinse 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.²⁵ Meier and colleagues²⁶ 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 contamination of toothbrushes.

	MATERIALS AND METHODS

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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 not have had scaling or root planing within the last six months;

– 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 triclosan-containing toothpaste;

– must be 25 to 70 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 a 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 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 times 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.²⁴

After the drying period, we aseptically removed toothbrushes’ 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 prereduced anaerobically sterilized, or PRAS, selective media and nonselective 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 sealing 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 ² analysis to test for significance of intergroup 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

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Isolation frequencies for the microorganisms observed in each of the groups are shown in the table. We found Aa on 85.0 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, on 87.5 percent of the regular-toothpaste 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.

View this table: [in this window] [in a new window]   TABLE ISOLATION FREQUENCIES OF TOOTHBRUSH CONTAMINATION BY Aa, Pg AND Ps AFTER DIFFERENT TREATMENTS.*

Long-term use of a toothbrush with a toothpaste may result in findings different from those of this study, which involved one-time brushing with a new toothbrush.

	DISCUSSION

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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 for four hours.^27,28 Pg is a strictly anaerobic, gram-negative rod that forms black colonies on blood agar.²⁸ 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.²⁸ Aa is a facultative anaerobe that is associated consistently with aggressive periodontitis and chronic periodontitis.²⁸

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 for various periods, and they have developed protective agents to fight against oxygen’s toxic reduction products. The three bacteria we studied in this article have been classified as either moderate or aerotolerant anaerobes.²⁹ 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 are exposed to air at four hours and a still lower rate at 24 hours before they are cultured anaerobically.²⁹ 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^4–7 of the residual microbial contamination of toothbrushes. In the regular-toothpaste group, we found Aa on 87.5 percent of the toothbrushes. The control group had 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 from 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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
We isolated periodontal pathogens from toothbrushes that were air-dried for four hours after toothbrushing. Neither regular toothpaste nor triclosan-containing toothpastes appeared 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.^{1,4–10,23–26}