The Effectiveness Of A U-V Toothbrush Sanitizing device In reducing The Number Of Bacteria, Yeasts And Viruses On Toothbrushes
In the early 1980's, a systematic study of toothbrushes revealed that toothbrushes from both patients with oral disease and those with normal mouths had a substantial number of different types of microorganisms. many of these microorganisms had the ability or potential ability to produce disease. Further studies revealed that Herpes Simplex Virus, Type 1, could survive on a moist toothbrush for seven days or more. More recent studies using an animal model (kennel dogs) have shown a definite correlation between toothbrushes contaminated by daily use and at least oral disease.
Studies were also conducted to determine the effectiveness of an experimental ultra-violet light device in sanitization of the toothbrush (not the Pollenex unit). Thirty-one toothbrushes of various designs, colors, and from a variety of manufacturers were sterilized (using ethylene oxide gas) and divided into two groups. Ten brushes were contaminated with Candida albicans. The brushes were exposed to an experimental ultra-violet germicidal light device for different time intervals and then cultured at three different bristle levels. Ultra-violet light was found often to decontaminate the toothbrush bristle surface, however, midbristle and bristle depth remained contaminated. The effectiveness of ultra-violet light decontamination was in part dependent upon the opacity and ornateness of the toothbrush. In addition, prolonged ultra-violet light exposure with the experimental unit appeared to harden the bristles (unpublished data by funding manufacturer's request).
An ultra-violet light device for toothbrushes has also been developed by Pollenex: the DS60 Daily Dental Sanitizer. In order to test the decontamination effectiveness of this ultra-violet light, toothbrushes exposed to Streptococcus mutans and Candida albicans were contaminated at varying time intervals (simulating human toothbrushing experience) and placed either in an ultra-violet light or in a humidified environment (simulating a bathroom environment) for 24 hours. The brushes were then cultured to determine:(1)whether the Pollenex ultra-violet light device decontaminates toothbrushes exposed to Streptococcus mutans and Candida albicans as compared to natural light, and (2)whether toothbrush design, color, or opacity alter decontamination by the Pollenex unit. The Pollenex DS) Daily Dental Sanitizer demonstrated both a qualitative and a quantitative reduction in Streptococcus mutans and Candida albicans compared to placing toothbrushes on a shelf (unpublished pilot data, superseded by the following studies).
In order to further study the effectiveness of the DS60, six microorganisms were chosen for quantitative analysis (as suggested by the U.S. Food and Drug Administration as being their indicator microorganisms of sanitization): 1.Staphylococcus aureus, 2. Pseudomonas aeruginosa 3.Escherichia coli, 4. Bacillis subtilis, 5. Serratia marcescens, 6. bakers yeast. Similarly, in order to test the effectiveness of the DS60 on viruses; Herpes Simplex Virus, Type 1 (HSV), a virus responsible for recurrent fever blisters and Parainfluenza Virus, Type III (PV), a common cause of upper respiratory disease and colds in children, were chosen.
Materials and Methods
Brain heart infusion (BHI) broth (100 ml) was seperately inoculated with Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 25922), Escherichia coli (ATCC 27853), Bacillis subtilis (Difco spore suspension), Serratia marcescens (ATCC 8100), and Saccharomyces sp.(solated from commercially obtained "Baker's yeast").
The Broth solutions were incubated at 37C for hours. Each organism was absorbed on to 11Oral B toothbrushes, previously gas sterilized in 25x200mm screw cap test tubes, by adding 7ml of the 24 hour inoculum to the tube for 10 minutes. The brushes were then removed and (see Flow Sheet 1): a. 3 brushes/organism were placed in sterile 20x 150mm test tubes( inoculum control); b. 4 brushes/organism were placed in the Pollenex DS60 Daily Dental Sanitizer (U,V.) for 24 hours (all sanitizers were found to have 3 minute+/- 2second exposure/30 minute) and c. 4 brushes/organism were placed in a test tube rack in a bacteriological hood at room temperture for 24 hours (shelf).
Four milliliters of BHI broth were added to each tube of the inoculated control brushes and the brushes were individually vortexed for 30-45 seconds. Dilutions of the control were made by removing 0.5 ml of the vortexed toothbrush washing and adding it to 4.5 ml of sterile BHI broth. Ten-fold serial dilutions were made through 6 to 8 dilutions. Each dilution was quantitated by removing 0.1 ml and placing it on a trypticase soy agarplate (the Baker's yeast were placed on Sabouraud's media). The inoculum was spread by placing the inoculated plate on a revolving, the liquid was spread via a sterilized bent glass rod. Duplicate plates were used for each delution. Plactes were incubated at 37C (yeast plates were incubated at 30C ) for 24 to 48 hours prior to counting bacterial colonies.
After 24 hours, the inoculated brushes from the "UV" and the "shelf" brushes were placed into sterile 20x150 mm tubes. BHI broth (4ml) was added to each test tube and processed as decribed abovefor the "control" brushes. Bacterial colonies were counted after 24-48 hours incubation. Counts from duplicate plateswere averaged for the final results.
A Total of seventy two toothbrushes (twenty-four each od Oral-B, Reach, Clogate) were placed in a glass test tube and sterilized by ethylene oxide gas. Equal numbers of colored, opaque, or clear toothbrushes were used. All seventy-two toothbrushes were exposed to Herpes Simplex Virus, Type1, approximately 105 TCID 50/0.1ml (50% Tissue Culture Infective Dose) in hanks Ballance Salt Solution(HBSS) for ten minutes to allow viral absorption. In a seperate trial, seventy-two brushes were exposed to Parainfluenza Virus, Type III, approximately 106.5 TCID 50/0.1 ml in HBSS, for ten minutes. To determin the numbers of virus particles attacheding to the toothbrush prior to exposure to UV light or drying conditions, 12 toothbrushes (4 of each brand) were immersed in HBSS immediately after incubation with virus. The toothbrushes were triturated (vortexed) for 45 seconds and the virsus contained fluids subsequently titrated for viral quantitations as describer below. Thirty viral contaminated brushes were exposed to the Pollenex DS60 unit and thirty viral contaminated brushes were exposed to natural light. The exposure time for both groups was twenty-four hours (see Flow Sheet 2) .
After a 24 hour exposed period each brush was placed in HBSS and triturated for 45 seconds. This allowed the virus particles to disloge from the brush bristles and accumulate in the fluid. The viral particals were quantitated by performing 10-fold dilutions of the virus containing fluids and adding.0.1ml of each didution, inquadruplicate, to 96-well tissue culture plates containing a monolayer of appropriate cells. The tisue cultures plates were monitored dayily for CPE (cytopathic effort) of the cell monolayer in the case of HSV ande hemadsorption using guinea pig erythrocytes in the case of PV infected cells. After approximately 5 days the plates were fixed with formalin, stained, and observed microscopically. The TCID50 was calculated and the results tabulated. All tests were performed using coded brushes to prevent observer bias.
The results of the bacterial and yeast study are summarized in Table 1. The initial inoculum of the first three microorganisms was at 10 8-9/ml level. The initial inoculum of the last three microorganisms was reduced to 107/ml due to a difference in growth curves. From the results, it is clear that all microorganisms could be transfereed to the toothbrush with only a 101-2/ml reduction from the initial inoculum. further, storage of the contaminated toothbrushes on the shelf of a low humidity microbiology hood reduced the microorganism count by only 101/ml. Most important, following Pollenex recommended procedures, the pollenex DS60 Daily Dental Sanitizer was found to consistently reduce the number of microoganisms by at least a factor 103-5/ml. In the case of Pseudomonas and Escherichia, two toothbrushes per microorganism were completely negative.
Seventy-two toothbrushes were exposed to Herpes Simplex Virus, Type I, for a period of 10 minutes at a concentration of 105 TCID50/ ml. The Toothbrushes retained an average of 104 TCID50/ml after the ten minute (range 103.75 - 104.25 TCID 50/ml). No HSV was found on any of the 30 toothbrushes placed in the Pollenex DS60 Unit for 24 hours. The ten reach toothbrushes placed on a shelf had the highest return of virus particals (ave. 102.35 TCID 50/ml ) from a retained initial concentration of virus particals: ave 104.125 TCID 50/ml. The ten colgate toothbrushes (shelf stored) returned an average of 102.23 TCID 50/ml from a retained initial concentration of virus particles: ave. 103.3875 TCID 50/ml. The ten Oral B toothbrushes (shelf stored) had the lowest return of virus particles (ave.104.0 TCID 50/ml. One Colgate and one Oral B toothbrush (shelf stored) had no evidence of virus particles. The overall average of the 30 toothbrushes placed on the shelf was 102.06 TCID 50/ml. In general, the lighter colored or clear toothbrushes appeared to retain or to return fewer virus particles. The results are summerarized in Tables 2 and 3.
Seventy-two toothbrushes were exposed to Parainfluenza Virus, Type III, for a period of 10 minutes at a concentration of 106.5 TCID 50/ml. The toothbrushes retained an average of 103.71 TCID 50/ml virus particles after the 10 minute (range 103.25-104.0 TCID 50/ml). No PV was found on any of the 30 toothbrushes placed in the Pollenex DS60 unit for 24 hours. The ten Colgate toothbrushes placed on a shelf had the highest return of virus particles (ave. 102.0 TCID 50/ml) from a retained intiial contration of virus particles: ave. 104.0 TCID 50/ml. The ten Reach toothbrushes (shelf stored) returned an average of 101.77 TCID 50/ml from a retained initial concentration of virus particles: ave. 103.625 TCID 50/ml. The ten Oral B toothbrushes (shelf Stored) hade the lowest return of virus particles (ave. 101.41 TCID 50/ml) from a retained initial concentration of virus particles:ave 103.5 TCID 50/ml. Two Oral B toothbrushes (shelf stored) had no evidence of virus particles. The overall average of the 30 toothbrushes placed on the shelf was 101.75 TCID 50/ml. In general, the lighter colored or clear toothbrushes appeared to retain or to return fewer virus particles.
The results are summarized in Tables 2 and 3.
With the findings that toothbrushes from humans were contamintated with a wide range of microorganisms, it seemed imperative that some type of instrument or proces be developed to lessen the problem of toothbrush contamination.1 Certainly, high risk patients could profit from use of a toothbrush bacteriocidal device, 4,5,6 but so too could the general population.7 Even though some of the initial levels of microbial contaminants were somewhat higher than what would be exp ected in normal saliva (ca. 108 total microorganisms per ml of saliva), the finding that substantial reduction in numbers of microorganisms identified by the U.S. Food and Drug Administration as target microorganisms of decontamination by the Pollenex DS60 speaks well for its use.
With additional studies demonstrating viral retention on toothbrushes2 and the harm caused by repeated brushing with the same toothbrush3, and effective, low-cost solution to the problem was critical. The imperative was intensified by the finding that 4.5% of healthy individiuals, 20% of the patients undergoing oral surgery and 38% of immunocompromised patients, regularly shed HSV in their saliva.8 The finding that the Pollenex DS60 unit completely kills viruses on all toothbrush types and colors is certainly sufficient reason to advocate its use.
Similarly, it seemed important to evaluate other virus retention on toothbrushes. The Parainfluenze Virus, Type III was chosen because it represented a prototype of the common cold viruses in children and upper respiratory viruses in adults. The findings that the PV could be retained on toothbrushes and completely killed by the Pollenex unit again advocates the use of the device. Goh et al have implicated by statistical means, the toothbrush in the transmission of the Hepatitis B Virus in families.9 Goodman et al have also shown transmission of a wide range of viral systemic diseases by oral means.10 Finally, if the statistics on waterborne disease outbreaks in the United States, 1986-1988, are considered, one third of the 25, 846 cases had either a definite viral or possible viral etiology.11
Multiple techniques to decontaminate toothbrushes have been employed, including trials with several of the ultraviolet toothbrush sanitizers (unpublished data). While simple toothbrush rinsing reduces some of the toothbrush microbial burden, the only device which was found to consistently reduce the number of a wide range of microorganisms was the Pollenex DS60 Daily Dental Sanitizer.
Finally, all experiments performed, using whatever method, were found to be toothbruse dependent. Toothbrush color, toothbrush opacity, numbers of bristles and bristle arrangement appeared to be the major factors in determining the ability of a toothbrush to become contaminated and to retain microorganisms.2 Sharp edges on the toothbrush heads also appeared to be a factor in microorganism retention (clinical observation). Silverstone and Featherstone, in an excellent scanning electron microscopic study of toothbrushes, found that toothbrush bristle rounding in new, unused toothbrushes varied from 88% to 22% rounding/brush.12 A recent experiment demonstrated a substantial decrease in the number of rounded bristles after two weeks of use (unpublished data). Previous studies also demonstrated a propensity of microorganisms to acumulate and adhere to roughened toothbrush bristles. It is important, therefore, to develop new toothbrushes which can retain rounded bristles and to continue to regard the toothbrush as an hygienic device, to be discarded every two weeks.3 Based upon this study and previous studies, it appears that the most biologically sound toothbrush available at this time is a clear, two row soft toothbrush .
1. The Pollenex DS60 Daily Dental Sanitizer was found effective at sanitizing toothbrushes contaminated with the cited microorganisms. 2. A new toothbrush needs to be developed; one which will take into consideration microbial contamination.