Infection of Dental Implements and Appliances Part 1: The Toothbrush

Studies by Glass et al in the 1980s indicated that toothbrushes harbored microorganisms that could play a role in transmission of disease.1-3 The same investigators examined dentures and more recently have evaluated other dental devices such as protective athletic mouth guards for their potential to harbor and transmit microorganisms. Dentures will be the focus of Part 2 of this article. The purpose of this first part is to review the evidence concerning the potential role of the toothbrush in transmission of infection and also to provide suggestions regarding the selection, use, and care of these devices.

Initial Studies of the Potential Role of the Toothbrush in Microbial Transmission

The initial study involved 2 populations of patients: 10 clinically normal individuals (control group) and 10 patients who had either mucosal or periodontal disease (study group).1 Each patient was asked to wrap his or her toothbrush after the last use and bring it to the clinic. As negative controls, 5 toothbrushes from 2 leading brands were cultured directly from the package. All toothbrushes were handled in an aseptic manner and cultured using an enriched microbial media (thioglycollate). The 2 predominating microorganisms found on the initial plating were isolated and identified for each toothbrush to determine if the microorganisms might be considered either pathogenic or opportunistic to humans. The results showed a wide array of microorganisms found on the toothbrushes used by the control group, many of which were the same microorganisms cultured from toothbrushes used by the study group (see Table).

Table. Toothbrush Contamination: This table is a compilation of the predominating microorganisms that were cultured from toothbrushes of individuals who had normal-appearing mucosa and gingiva (N001 to N0010; positive controls), from toothbrushes of patients who had either periodontal disease (P001 to P002; study group) or mucosal lesions (M001 to M008; study group). The third groups of toothbrushes were from 2 different companies and were cultured directly from the package (negative controls). Note the similarity of microorganisms in the positive controls as compared to the study population. Also note that 80% of the negative control toothbrushes were contaminated in the package.
ID # Microorganisms ID # Microorganisms ID # Microorganisms
N001 Candida albicans P001 Staphylococcus epidermidis A001 No Growth
N002 Bacteroides melaninogenicus
a hemolytic Streptococcus
P002 Enterobacter cloacae A002 No Growth
N003 Klebsiella pneumoniae
Bacteroides oralis
M001 Staphylococcus epidermidis A003 No Growth
N004 Clostridium ramosum Staphylococcus epidermidis M002 Enterobacter cloacae Staphylococcus epidermidis A004 No Growth
N005 Enterobacter cloacae M003 Enterobacter cloacae Clostridium clostridiforme A005 No Growth
N006 Clostridium ramosum M004 Serratia sp. Staphylococcus epidermidis B001 Staphylococcus epidermidis
N007 Staphylococcus epidermidis M005 Staphylococcus epidermidis Propionibacterium sp. B002 No Growth
N008 Staphylococcus epidermidis Bacteroides sp. M006 Staphylococcus epidermidis Bacteroides sp. B003 Staphylococcus epidermidis
N009 Staphylococcus epidermidis M007 Staphylococcus epidermidis a hemolytic Streptococcus B004 Staphylococcus epidermidis
N010 Staphylococcus epidermidis M008 Enterobacter cloacae B005 Staphylococcus epidermidis



The difference between toothbrushes from the control group and toothbrushes from the study group was that the concentration of microorganisms was higher in the study group. This study also showed that many of the microorganisms could be opportunistic or pathogenic for the respiratory and gastrointestinal tracts. What was unexpected was the finding that all 5 unused toothbrushes from one manufacturer had no microbial growth, while 4 of the 5 unused toothbrushes from a second manufacturer were contaminated with Staphylococcus epidermidis prior to use.

Once the concept of toothbrush contamination by use was established, the next study focused on where the contamination actually occurred on the toothbrush.2 Toothbrushes were contaminated in vitro with Candida albicans by immersing the toothbrushes in 106 microorganism/mL for 24 hours. The ends of the toothbrush bristles were streaked across Sabouraud's dextrose agar (specific for C albicans). The toothbrush bristles were then sectioned at mid-bristle in an aseptic manner and were again streaked across the media. Finally, using sterile end-cutting nippers, the toothbrush heads were sectioned and touched to the media.

The data from this study indicated that microorganisms were not just on the bristle ends but were also in the mid-portion of the toothbrush and in the head. Qualitatively, the concentration of microorganisms appeared to be equally dense in all 3 areas. One additional but unpublished finding from this study was that when toothbrushes were immersed in 106 microorganisms/mL for 3 minutes, vortexing the toothbrushes yielded initial concentrations of 104 to 106 microorganisms/mL. When these same toothbrushes were allowed to stand in the open air (similar to patient use) for 24 hours, vortexing the same toothbrushes yielded a concentration of 108 or 109 microorganisms/mL. Thus, the study indicated that yeasts could not only contaminate the entire toothbrush, but could actually increase in concentration over a 24-hour period.

Figure 1. This is a photomicrograph of bristles that were stained with a vital dye for viruses. (Original magnification: 100x.) Note that the arrow points to a black dot at the tip of the bristle, which would be consistent with a very large aggregate of viral particles. Then note the overall sharpness of not only the viral-contaminated bristle but also the surrounding bristles.

After finding that both bacteria and yeasts could adhere to toothbrushes, the next step was to look at the possibility of viral contamination of toothbrushes. The first virus that was studied was the herpes simplex virus type-1 (HSV-1).3 HSV-1 is a DNA virus that is known to cause not only a generalized stomatitis in infants and children, but recurring lip lesions, conjunctivitis, en-cephalitis, herpetic whitlow, esophagitis, pneumonia, and disseminated infections in the adult.4 Twelve ethylene oxide-sterilized toothbrushes were immersed in 3 mL of 105.5 tissue culture infective dose (TCID50) of HSV-1, with some toothbrushes being subsequently placed in a moist environment simulating a bathroom. This study demonstrated that toothbrushes could be contaminated by viruses. It also determined that both the number of bristles/tuft and the number of tufts/toothbrush were important in the pattern of contamination by the virus. Higher concentrations of virus were found in those toothbrushes with the highest number of bristles/tuft and the highest number of tufts/brush as compared to those toothbrushes with a lower number of both bristles/tuft and tufts/brush. Additionally, the study showed that clear toothbrushes retained fewer viruses than opaque toothbrushes, underscoring the role of light in inhibiting microbial growth. Even more notable was the finding that when toothbrushes were contaminated at a level of 105.5 TCID50 and subsequently stored in a moist environment (simulating the bathroom) for 168 hours (7 days), they retained 102.3 TCID50 viable viruses (Figure 1). The only reasonable conclusion from this study was that toothbrushes contaminated with HSV-1 could transmit the disease more than 7 days after initial contamination. This same study was repeated using a number of other pathogenic viruses, with essentially the same results.5

In regard to viral infections, it is important to note a small study that looked for toothbrush contamination by HIV.6 Because HIV is an RNA virus, in order to detect the presence of the virus the toothbrushes were examined for the presence of the HIV precursor (HIV proviral DNA). Ten toothbrushes (5 from HIV-positive individuals and 5 from HIV-negative controls) were examined for HIV proviral DNA. One of the 5 toothbrushes from the HIV-positive individuals was positive for HIV proviral DNA. This observation could relate to increased occurrence of periodontal disease in HIV-positive individuals and therefore toothbrush contamination by gingival hemorrhage.7 A search of the literature revealed 2 reports of suggested transmission of HIV from an infected brother to a noninfected brother occurring after sharing a toothbrush.8,9

FULFILLING KOCHS POSTULATES FOR DISEASE TRANSMISSION


Koch established a paradigm for microbial disease transmission that is now known as Koch's postulates.10 Koch stated that (1 ) the microorganism must be present in every case of the disease; (2) the microorganism must be isolated and grown in pure culture; (3) the microorganism must be introduced into a susceptible animal and produce the disease; and (4) the original microorganism must be recovered and identified.

In order to fulfill Koch's postulates as applied to disease transmission by a toothbrush, Glass et al used a canine model.11 The study employed a triple crossover design where each animal received all 3 experimental treatments. Toothbrushes were initially sterilized using ethylene oxide. The 3 arms of the experimental design were (1) brushing each day for a month with a new sterile toothbrush each time; (2) brushing each day for a month with a sterile toothbrush that had been subsequently contaminated with a known number of microorganisms; and (3) brushing each day for a month with the same toothbrush (sterilized only prior to the first brushing). This latter arm simulates the daily brushing pattern of most humans. Local disease in the oral mucosa was scored by daily counting of the number of discrete oral inflammatory lesions (ulcerations and erythema) on both the mucosa and gingiva. Blood was also drawn daily for microbial culturing to evaluate the vascular-dissemination of microorganisms.

This in vivo study demonstrated that daily brushing, even with a new sterile toothbrush, can produce both discrete ulcers and areas of erythema, even when performed carefully (arm 1). The data indicated that if the toothbrush was contaminated with a pathogenic microorganism, it not only increased the number of intraoral lesions, but the same microorganisms were cultured from the bloodstream after brushing (arm 2). However, the most striking finding of this study was that the repeated use of the same toothbrush (arm 3) was associated with the development of a greater number of intraoral lesions and more positive blood cultures. These findings are similar to the observations reported for patients by other investigators.12,13

ADDITIONAL TOOTHBRUSH STUDIES

Figure 2. This scanning electron photomicrograph shows the rough, jagged surface with the finger-like extension of the tip of a toothbrush that had been contaminated with Candida albicans. The round structures are consistent with adherent yeasts. (Original magnification: 5,000x)

Additional studies were performed to examine the effect of toothbrush tuft end-rounding on retention of microorganisms. Using a dissecting microscope, tufts on new toothbrushes from 3 manufacturers were examined. The bristles were scored as to whether they were end-rounded or sharp. The toothbrushes were then used for 1-week or 2-week intervals and were again examined and scored for end-rounding. At the beginning of the study, 98% to 100% of the bristles from all 3 toothbrush brands were rounded. After a week of use, one third of the bristles from 2 of the toothbrush manufacturers became sharp and jagged; after 2 weeks of use, two thirds of the bristles from the same 2 toothbrush manufacturers were sharp and jagged. The toothbrushes produced by the third manufacturer did not change over the 2-week period. Compared to the bristles of the first 2 toothbrush groups, these latter bristles were 3 to 4 times larger in diameter. As part of this study, bristles were also studied using a scanning electron microscope. At the ultrastructural level, all 3 toothbrush brands showed rough and jagged edges after use for 2 weeks (Figure 2). A similar study was performed by Silverstone and Feather-stone,14 who found that end-rounding on 8 brands of brushes varied from 22% to 88%. The importance of ab-sence or loss of end-rounding is that the sharper bristles can penetrate the mucosa, injecting microorganisms into the submucosa and its vessels.

Presently, studies are being conducted on electronic toothbrushes. The potential effect of the motorized action on persistent infection of the bristles and dissemination of that infection are being examined.

POSSIBLE SOLUTIONS TO THE PROBLEM

While the persistent contamination of toothbrush bristles was described, studies were also being conducted on a number of disinfection techniques. Soaking used toothbrushes overnight in commercially available mouthrinses, cleaning disinfectants, hydrogen peroxide, or even alcoholic beverages was not an effective means of reducing microbial infection of the bristles. Placing the toothbrush in a dishwasher was also ineffective in reducing the numbers of microorganisms. Sterilization of the toothbrushes could be achieved by either autoclaving or microwaving. However, these techniques caused the toothbrush heads to melt and the bristles to splay, rendering the toothbrush ineffective. As noted earlier, toothbrushes can be sterilized without distortion using an ethylene oxide sterilizer, but ethylene oxide is not practical for home use.

Two studies have examined the use of toothbrush sanitizing devices. The first study was conducted in vitro using an ultraviolet light device manufactured by Associated Millin.5 This device was effective at reducing the concentration of microorganisms used as test subjects by the FDA; however, it did not eliminate the microorganisms completely. It was also found to inhibit completely the contamination of the toothbrush by viruses. In vitro studies were conducted on a second ultraviolet toothbrush sanitizer, the Purebrush (Murdock Laboratories). In this study, toothbrushes were contaminated with Candida albicans, Beta-Hemolytic streptococci, and Capno-cytophaga species. This study demonstrated that the Purebrush unit effectively killed not only bacteria but also yeasts.15 Unpublished studies with the Purebrush revealed that it also inhibited toothbrush contamination by viruses.

CONCLUSION

Based on the data presented here, the following suggestions are offered: First, the toothbrush is a hygienic device and removes microorganisms from the body. While it certainly may be used more than once, it should be replaced regularly, perhaps every 2 weeks due to both contamination and changes to bristle end-rounding. Second, it would seem reasonable that a toothbrush needs to be discarded at the beginning of an upper respiratory or other illness, which can directly or indirectly involve the oral cavity; when the patient is first feeling better; and when the patient is completely well. This regime would be expected to break the reinfection cycle so often seen in patients. Third, people with either chronic oral diseases (periodontal disease, dental caries, or mucosal infections) or chronic systemic diseases that may be affected by the oral microflora (cardiovascular, cerebrovascular, pulmonary, or gastrointestinal disorders) should change their toothbrushes even more frequently. Fourth, the smallest manual toothbrush with the fewest number of bristles/tuft, the fewest number of tufts/toothbrush, and a clear head would be the best toothbrush selection. Finally, care should be taken to disinfect toothbrushes, for example by use of an effective ultraviolet toothbrush sanitizing device such as the Purebrush.


References

1. Glass RT, Lare MM. Toothbrush contamination: a potential health risk? Quintessence Int. 1986;17:39-42.
2. Glass RT. Other factors in infections: the transmission of disease. Gerodontics. 1986;2:119-120.
3. Glass RT, Jensen HG. More on the contaminated toothbrush: the viral story. Quintessence Int. 1988;19:713-716.
4. Rubin E, Farber JL, eds. Pathology. 3rd ed. Philadelphia, Pa: Lippincott-Raven; 1999:368-370.
5. Glass RT, Jensen HG. The effectiveness of a u-v toothbrush sanitizing device in reducing the number of bacteria, yeasts and viruses on toothbrushes. J Okla Dent Assoc. Spring 1994;84:24-28.
6. Glass RT, Carson SR, Barker RL, et al. Detection of HIV proviral DNA on toothbrushes: a preliminary study. J Okla Dental Assoc. Winter 1994;84:17-20.
7. Lamster IB, Begg MD, Mitchell-Lewis D, et al. Oral manifestations of HIV infection in homosexual men and intravenous drug users. Study design and relationship of epidemiologic, clinical, and immunologic parameters to oral lesions. Oral Surg Oral Med Oral Pathol. 1994;78:163-174.
8. HIV infection in two brothers receiving intravenous therapy for hemophilia. MMWR Morb Mortal Wkly Rep. Apr 10, 1992;41:228-231.
9. Fitzgibbon JE, Gaur S, Frenkel LD, et al. Transmission from one child to another of human immunodeficiency virus type 1 with a zidovudine-resistance mutation. N Engl J Med. 1993;329:1835-1841.
10. Smith AL. Microbiology and Pathology. 12th ed. St Louis, Mo: Elsevier Science Health Science Div; 1980:97.
11. Glass RT, Martin ME, Peters LJ. Transmission of disease in dogs by toothbrushing. Quintessence Int. 1989;20:819-824.
12. Malmberg E, Birkhed D, Norvenius G, et al. Microorganisms on toothbrushes at day-care centers. Acta Odontol Scand. 1994;52:93-98.
13. Warren DP, Goldschmidt MC, Thompson MB, et al. The effects of toothpastes on the residual microbial contamination of toothbrushes. J Am Dent Assoc. 2001;132:1241-1245.
14. Silverstone LM, Featherstone MJ. Examination of the end rounding pattern of toothbrush bristles using scanning electron microscopy: a comparison of eight toothbrush types. Gerodontics. 1988;4:45-62.
15. Glass R, Furgason M, Moody J. In vivo study of the efficacy of an ultra-violet toothbrush sanitizer [abstract]. IADR. 1997; 76:385 (abstract 2972).


Dr. Glass is professor of forensic sciences, pathology, and dental medicine; adjunct professor of microbiology; chairman, department of forensic sciences; and director of the Forensic Sciences Graduate Program at Oklahoma State University Center for Health Sciences. He can be reached at (918) 561-8240.

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