2024 Volume 66 Issue 1 Pages 5-8
Purpose: This study investigated the efficacy of various antiseptics for disinfection of rubber dams used during endodontic treatment, the duration of disinfection effectiveness, and the disinfection protocol employed by dental schools in Thailand.
Methods: The efficacy of 10% povidone-iodine, 1.5% tincture iodine and 70% ethyl alcohol in eliminating Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans) on the rubber dam was investigated. Time duration of disinfection was evaluated at 0, 30, 60, and 120 min. The two-step disinfection method adopted at Thai dental school was examined. Independent t-test or Kruskal-Wallis followed by a Dunnett’s test was used for statistical analysis.
Results: Among the three antiseptics, 10% povidone and 1.5% tincture iodine eradicated the microorganisms completely, whereas 70% ethyl alcohol did not achieve a statistically significant decrease. The duration of sterilization effectiveness was 120 min for 10% povidone-iodine, but bacteria were eliminated only at 0 min by 1.5% tincture iodine. The results also indicated that the two-step protocol scarcely eliminated the microorganisms.
Conclusion: The best antiseptic for rubber dam disinfection is 10% povidone-iodine, which remains effective for 120 min. The two-step protocol typically practiced in Thai dental schools needs to be updated. The use of 10% povidone-iodine alone is adequate for complete eradication of E. faecalis and C. albicans.
A rubber dam has many benefits when used during root canal treatment, as it reduces the risk of adverse events such as swallowing of objects or materials, protects the patient’s mucosa from contact with disinfectant compounds, reduces the operating time, maintains aseptic control, and decreases the risk of aerosol contamination, reinfection and cross-infection [1,2]. Kwak et al. [3] have reported that the use of a rubber dam increases the survival rate of endodontically treated teeth. The European Society of Endodontology and the American Association of Endodontists have recommended the use of a rubber dam during endodontic treatment [4] in situations where the equipment and work area need to be free of any microorganisms to prevent reinfection [2,4]. For this purpose, the rubber dam and operating field should be decontaminated before initiating treatment using a variety of compounds, including 3% sodium hypochlorite, 1.5% hydrogen peroxide, 70% ethyl alcohol, tincture iodine, and povidone-iodine [2,5]. In medicine, povidone- iodine or tincture iodine has been used in conjunction with ethyl alcohol to disinfect the skin before surgery, as the two compounds have different bactericidal pathways and the efficacy is increased when iodine preparations and ethyl alcohol are used together [6]. Conventionally, although rubber dam sheets are sterilized by the manufacturer, contamination of sheets remaining in the box may occur once the package has been opened.
In Thailand, various dental schools teach students to eliminate bacteria on rubber dams placed around teeth before treatment by scrubbing the area with iodine and ethyl alcohol. However, rubber dams can be contaminated by, for instance, aerosols generated during tooth preparation or through contact with equipment during root canal treatment [3]. Bahador et al. [7] have reported that pulpectomy and nonsurgical root canal therapy release aerosols along with bacteria, the number of bacteria being higher in aerosols near the mouth and during long treatments. Failure of endodontic treatment may be due to bacteria entering the root canal after treatment (secondary infection) or resistance of bacteria to treatment (persistent infection). Most of the bacteria involved are Gram-positive anaerobes and facultative anaerobes, both of which occur at similar levels in the root canal [8,9].
Persistence of intraradicular and extraradicular bacteria is one of the most important causes of endodontic treatment failure [10]. Many types of microorganism have been identified in the root canal after treatment, including Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans) [11]. E. faecalis is an aero-tolerant bacterium that has been widely discussed in the context of persistent apical periodontitis [9,12,13]. E. faecalis shows a high degree of resistance to various antimicrobials such as vancomycin, gentamicin, ampicillin, teicoplanin, and linezolid [14], as it has several characteristics that aid its survival throughout root canal treatment procedures and alterations in the root canal environment after the completion of treatment. For example, E. faecalis is able to form biofilms [15], enter and grow within dentinal tubules [16], and survive in conditions that are usually unfavorable for bacterial growth such as very basic pH [17] and lack of nutrients. Once conditions become favorable, E. faecalis resumes growth [18,19]. Furthermore, E. faecalis has the ability to thrive alone without support from other species [20].
C. albicans is normally present in small numbers in the oral cavity, being present in saliva, and the oral and buccal mucosa. However, C. albicans can cause opportunistic fungal infection when changes occur in the host microenvironment and the balance of the oral flora is disrupted. For instance, long-term use of antibiotics or immunodeficiency enables C. albicans to multiply and replace much of the normal flora, leading to infection and development of lesions. It has been shown that C. albicans can often be isolated from the root canal system after failure of endodontic treatment, together with E. faecalis, Gram-negative facultative anaerobic bacilli, and Pseudomonas. Accordingly, C. albicans is believed to be one of the major causes of endodontic treatment failure [21]. These findings are in line with other studies of bacteria in the root canal system of patients with persistent apical periodontitis, which have demonstrated a variety of morphological types, mostly facultative bacteria, including E. faecalis, Streptococcus spp., and Lactobacillus spp. Additionally, anaerobic bacteria can also be present. Therefore, persistent apical periodontitis is more likely to be associated with mixed, rather than single-species, infection [22].
The present study was designed to investigate the efficacy of two types of iodine preparation and ethyl alcohol for disinfection of E. faecalis and C. albicans on rubber dams and for prevention of reinfection during dental procedures. As it has been established that experienced dentists require less time than inexperienced dentists to perform any given dental procedure [23], differences in the period for which rubber dams remain sterile were also investigated, together with the efficacy of disinfection methods currently applied at dental schools in Thailand.
E. faecalis (ATCC 29212) and C. albicans (ATCC 10231) were used in this study. E. faecalis was grown in brain heart infusion (BHI) medium (Difco, Sparks, MD, USA) at 37°C overnight. The bacteria were then diluted to an OD600 of 0.29 (i.e., 108 cells/mL). C. albicans was cultured in BHI broth at 37°C for 40 h and then diluted to an OD600 of 1.00 (106-107 cells/mL).
Efficacy of iodine and ethyl alcohol for disinfection of E. faecalis and C. albicansThis study investigated the disinfection efficacy of three antiseptics: 10% povidone-iodine (Betadine solution, Pathum Thani, Thailand), 15% tincture iodine (M-dent, Nakhon Pathom Thailand), and 70% ethanol (GPO, Pathum Thani, Thailand). One hundred microliters of E. faecalis or C. albicans was dropped onto a separ rubber dam measuring 1.5 × 1.5 inches. Then a sterile cotton swab soaked with 500 μL of each disinfectant, or 0.85% sterile normal saline (NSS) as a control, was rubbed over the rubber dam for 40 s. Then 5 min later, the rubber dam was placed in a 50-mL centrifuge tube with 10 mL of NSS, followed by a series of ten-fold dilutions. One hundred microliters of each concentration was spread onto BHI agar and then cultured at 37°C for 24 h and 40 h for E. faecalis and C. albicans, respectively. The number of colony-forming units per milliliter (CFU/mL) was then determined and compared between the treated and control samples.
Time duration for efficient disinfectionThis experiment was performed to determine the duration of effectiveness of disinfection for 10% povidone-iodine and 1.5% tincture iodine applied individual to separate rubber dams. One hundred microliters of either E. faecalis or C. albicans was dropped onto a previously sterile rubber dam at time intervals of 0, 30, 60, and 120 min to mimic the spread of aerosolized microorganisms that might possibly occur during endodontic treatment. These time intervals were chosen to match the actual timing of a dental operation by dentists with different levels of experience ranging from highly experienced to practicing dental students. Each rubber dam sheet was then washed and handled using the same method as that in the previous experiment. An untreated group was subjected to an identical process but without application of antimicrobial compounds.
Duration of antimicrobial efficacy using the protocol practiced at Thai dental schoolsFollowing the protocol practiced at Thai dental clinics, a rubber dam sheet was rubbed with 10% povidone-iodine or 1.5% tincture iodine and then wiped with 70% ethyl alcohol. Then, 100 μL of either E. faecalis or C. albicans was dropped onto the previously disinfected rubber dam sheet after disinfection with the previously described protocol for 0, 30, and 60 min. A rubber sheet subjected to no antimicrobial treatment was also included as a control group.
Statistical analysisLog reduction of the bacterial count was calculated as log10(mean untreated CFU/mL) - log10(mean treated CFU/mL) [24]. Percentage reduction was calculated as (mean untreated CFU/mL – mean treated)/mean untreated CFU/mL × 100. All experiments were performed at least three times independently. The results were expressed as mean ± SD and log (mean ± SD) of CFU/mL. SPSS program version 18 was used to analyze the collected data. Normally distributed data were analyzed with independent sample t-test, while non-normally distributed data were analyzed by Kruskal-Wallis test followed by Dunnett’s test.
Table 1 summarizes the average counts of remaining bacteria after application of the various antiseptics. In the control group, the average counts of E. faecalis and C. albicans after rubbing with 0.85% NSS were 1.38 × 108 ± 3.48 × 107 and 7.20 × 106 ± 6.93 × 105 CFU/mL, respectively. However, E. faecalis and C. albicans were completely eradicated after rubbing with 10% povidone-iodine and 1.5% tincture iodine. This resulted in 100% reduction of both species as illustrated in Fig. 1. The use of 70% ethyl alcohol resulted in residual E. faecalis and C. albicans counts of 4.80 × 107 ± 6.08 × 106 and 2.33 × 106 ± 8.96 × 105 CFU/mL, respectively. The average counts of E. faecalis and C. albicans after treatment with 70% ethyl alcohol were significantly lower than those for the control group (0.85% NSS) with P-values of 0.008 and 0.002, respectively. As seen in Fig. 1, 70% ethyl alcohol was able to reduce the E. faecalis and C. albicans counts by 65.1% and 67.6%, respectively.
| Antiseptic | Mean ± SD (CFU/mL) | |
|---|---|---|
| E. faecalis | C. albicans | |
| 0.85% NSS (control) | 1.38 × 108 ± 3.48 × 107 | 7.20 × 106 ± 6.93 × 105 |
| 10% Povidone-iodine | 0.00 | 0.00 |
| 1.5% Tincture iodine | 0.00 | 0.00 |
| 70% Ethyl alcohol | 4.80 × 107 ± 6.08 × 106 | 2.33 × 106 ± 8.96 × 105 |
This experiment focused only on 10% povidone-iodine and 1.5% tincture iodine since only both were able to completely eradicate E. faecalis and C. albicans, as described in the previous section (Table 1). Initially, the mean counts of E. faecalis and C. albicans were 1.85 × 108 ± 3.16 × 107 and 6.05 × 106 ± 9.50 × 105 CFU/mL, respectively, and remained constant at all time points when untreated with any antimicrobial agent. When 10% povidone-iodine was applied to the rubber dam, it completely eradicated both species immediately and was still effective for up to 120 min (Table 2). Figure 2A shows the percentage reduction in the counts of E. faecalis and C. albicans relative to the initial counts (untreated rows in Table 2). Before application of 1.5% tincture iodine, the mean counts of E. faecalis and C. albicans were 1.85 × 108 ± 3.16 × 107 and 6.05 × 106 ± 9.50 × 105 CFU/mL, respectively, but after application both species were totally eradicated at 0 min. Thereafter, the counts had recovered to 1.68 × 108 ± 6.40 × 107 and 6.00 × 106 ± 3.08 × 106 CFU/mL, respectively, by 30 min and then remained roughly constant (Table 2). The counts of E. faecalis at 30, 60, and 120 min after treatment with 1.5% tincture iodine were in agreement with those in the untreated group with P-values of 0.664, 0.720, and 0.677, respectively. Likewise, the corresponding mean counts of C. albicans at the same time points were comparable to those in the untreated group, with P-values of 0.980, 0.594, and 0.624, respectively. The counts of both bacterial species at 30, 60, and 120 min were less than 10% of the initial count (Fig. 2B).
| Strain | Time (min) | Mean ± SD (CFU/mL) | Log reduction |
|---|---|---|---|
| 10% Povidone iodine | |||
| E. faecalis | untreated | 1.85 × 108 ± 3.16 × 107 | NA |
| 0 | 0.00 | - | |
| 30 | 0.00 | - | |
| 60 | 0.00 | - | |
| 120 | 0.00 | - | |
| C. albicans | untreated | 6.05 × 106 ± 9.50 × 105 | |
| 0 | 0.00 | - | |
| 30 | 0.00 | - | |
| 60 | 0.00 | - | |
| 120 | 0.00 | - | |
| 1.5% Tincture iodine | |||
| E. faecalis | untreated | 1.85 × 108 ± 3.16 × 107 | NA |
| 0 | 0.00 | - | |
| 30 | 1.68 × 108 ± 6.40 × 107 | 0.041 | |
| 60 | 1.70 × 108 ± 7.00 × 107 | 0.036 | |
| 120 | 1.70 × 108 ± 5.85 × 107 | 0.037 | |
| C. albicans | untreated | 6.05 × 106 ± 9.50 × 105 | NA |
| 0 | 0.00 | - | |
| 30 | 6.00 × 106 ± 3.08 × 106 | 0.004 | |
| 60 | 5.60 × 106 ± 1.51 × 106 | 0.034 | |
| 120 | 5.67 × 106 ± 1.29 × 106 | 0.028 | |
NA, not applicable
The disinfection protocol typically practiced at dental schools in Thailand is a two-step process of scrubbing the rubber dam with iodine followed by 70% ethyl alcohol in sequential order. The two groups tested in this study were rubbed with two different iodine preparations, i.e., 10% povidone-iodine or 1.5% tincture iodine, followed by 70% ethyl alcohol. Regardless of the type of iodine preparation, this two-step process was unable to eliminate E. faecalis and C. albicans across all time intervals (0, 30, and 60 min), as shown in Table 3. The initial mean counts of E. faecalis and C. albicans were 1.73 × 108 ± 8.34 × 107 and 7.18 × 106 ± 1.51 × 106 CFU/mL, respectively. At most of these time points, the percentage reduction of both species became negative, meaning that the counts of E. faecalis and C. albicans even increased after the two-step process (Fig. 3). However, no statistically significant difference was evident between the untreated and treated groups. Comparison of the E. faecalis count between the two groups using 10% povidone-iodine with 70% ethyl alcohol showed P-values of 0.262, 0.372 and 0.495 at 0, 30, and 60 min, respectively, and the corresponding P-values for C. albicans were 0.325, 0.554, and 0.674, respectively. Comparison of the E. faecalis count between the two groups using 1.5% tincture iodine with 70% ethyl alcohol showed P-values of 0.745, 0.416 and 0.664 at 0, 30, and 60 min, respectively. The corresponding P-values for C. albicans were 0.964, 0.792, and 0.828, respectively. It appeared that C. albicans was more resistant than E. faecalis to the two-step process involving both types of iodine preparation (Fig. 3A and 3B).
| Strain | Time (min) | Mean ± SD (CFU/mL) | Log reduction |
|---|---|---|---|
| 10% Povidone-iodine followed by 70% ethyl alcohol | |||
| E. faecalis | untreated | 1.73 × 108 ± 8.34 × 107 | NA |
| 0 | 1.74 × 108 ± 5.62 × 107 | −0.002 | |
| 30 | 9.27 × 107 ± 7.66 × 107 | 0.272 | |
| 60 | 1.84 × 108 ± 2.31 × 107 | −0.025 | |
| C. albicans | untreated | 7.18 × 106 ± 1.51 × 106 | NA |
| 0 | 8.33 × 106 ± 1.62 × 106 | −0.064 | |
| 30 | 8.07 × 106 ± 2.91 × 106 | −0.050 | |
| 60 | 7.70 × 106 ± 2.01 × 106 | −0.030 | |
| 1.5% Tincture iodine followed by 70% ethyl alcohol | |||
| E. faecalis | untreated | 1.73 × 108 ± 8.34 × 107 | NA |
| 0 | 6.18 × 108 ± 6.72 × 108 | −0.065 | |
| 30 | 1.28 × 108 ± 4.33 × 107 | 0.132 | |
| 60 | 2.85 × 108 ± 7.14 × 107 | −0.216 | |
| C. albicans | untreated | 7.18 × 106 ± 1.51 × 106 | NA |
| 0 | 7.23 × 106 ± 1.55 × 106 | −0.003 | |
| 30 | 7.53 × 106 ± 2.40 × 106 | −0.021 | |
| 60 | 7.50 × 106 ± 2.86 × 106 | −0.019 | |
NA, not applicable
Panel (A) shows the sample scrubbed with 10% povidone-iodine and 70% ethyl alcohol, while panel (B) is scrubbed with 1.5% tincture iodine and 70% ethyl alcohol. The untreated groups were not applied any compound.
Disinfection is undeniably necessary to reduce the possibility of endodontic treatment failure due to re-infection or new infection from the involved area. Alcohol and iodophors are disinfectants commonly used for this purpose prior to any medical/dental procedure [Centers for Disease Control and Prevention; 2017]. This study showed that among 3 types of compound tested, 10% povidone-iodine and 1.5% tincture iodine were able to eradicate E. faecalis and C. albicans completely. This was consistent with the findings of Tan and Johari [25], who reported that povidone-iodine was able to kill 99.99% of bacteria and fungi in vitro. On the other hand, 70% ethyl alcohol was able to kill only an insignificant number of E. faecalis and C. albicans. Pidot et al. [26] found that E. faecium, which is in the same genus as E. faecalis, was resistant to 70% ethyl alcohol and 70% isopropyl alcohol. This inefficient disinfection by 70% ethyl alcohol might have been due to the rubbing technique used. As the compounds were spread all over the rubber dam as a thin film, 70% ethyl alcohol might have evaporated rapidly, leaving no antimicrobial ability against E. faecalis or C. albicans [27,28,29]. In fact, 70% ethyl alcohol generally requires an exposure time of more than 5 min for bactericidal activity [30]. Furthermore, 70% ethyl alcohol was mixed with the microorganisms in BHI, thus decreasing its concentration and antimicrobial efficacy and hindering its disinfection properties. The ideal concentration of ethyl alcohol for exerting a bactericidal effect is 60-85% with an exposure time of 0.5-5 min [30]. In a systematic review of the effectiveness of 70% ethyl alcohol for disinfection of semi-critical medical instruments, Ribeiro et al. [31] found that 36.9% of studies that had used alcohol for disinfection found surviving microorganisms. Therefore, the use of 70% ethyl alcohol alone is not enough to disinfect medical instruments thoroughly [31].
In this study, 10% povidone-iodine and 1.5% tincture iodine were able to disinfect the two investigated microorganisms. Further experiments were conducted to clarify the time required for both types of iodine preparation to maintain aseptic conditions, as the longer the time needed for root canal treatment the greater the number of microorganisms generated from aerosols during the procedure [7]. Root canal treatment typically requires 35-88 min per visit depending on the complexity [32]. Therefore, clarification of the disinfection time is important for maintaining a microbial-free working area. The present study demonstrated that 10% povidone-iodine was able to kill both bacterial species at all time points up to 120 min, similar to a previous study using the same antiseptic to prepare skin for surgical operations [33]. On the other hand, although 1.5% tincture iodine was able to kill E. faecalis and C. albicans immediately after rubbing it onto the rubber dam, it was unable to do so thereafter. This may have been due to its less complex molecular structure in comparison with povidone iodine, which is more stable. Thus, povidone-iodine was able to keep the treated area aseptic for a longer time [34]. Furthermore, the povidone iodine used in this study was an aqueous solution, whereas tincture iodine was dissolved in alcohol [35], which evaporates faster, thus possibly affecting antiseptic efficacy.
Dental schools in Thailand teach students to sterilize rubber dams using a two-step procedure. First, an iodine preparation – 10% povidone iodine or 1.5% tincture iodine – is rubbed onto the rubber dam followed by 70% ethyl alcohol. The present results indicated that this two-step process was unable to kill either E. faecalis or C. albicans completely, regardless of the iodine preparation used in the first step. This may be because povidone iodine and tincture iodine dissolve when ethyl alcohol is applied [34,35], thus removing the active antimicrobial component. Clearly, therefore, the two-step protocol typically used in Thai dental schools is not effective for disinfection of important microorganisms related to endodontic treatment failure.
Among the three compounds tested in this study, 10% povidone-iodine was the best for eliminating E. faecalis and C. albicans and maintained its antimicrobial ability for 120 min, which is a sufficient amount of time for root canal treatment. On the other hand, 1.5% tincture iodine was able to kill E. faecalis and C. albicans but unable to maintain its antiseptic ability for an extended period. The third option, 70% ethyl alcohol, was unable to kill or control either of the two microorganisms.
The two-step disinfection protocol for rubber dams used in Thai dental clinics was shown not to be effective for elimination of bacterial contamination. Therefore, 10% povidone-iodine without additional 70% ethyl alcohol is recommended for clinical practices. This is because alcohol may inadvertently remove the iodine through evaporation, thus decreasing the efficacy of bacterial killing.
It is suggested that dental schools should revise the existing protocol to a single step before endodontic treatment. This may help to reduce chair time, save costs, and efficiently sterilize the operating area. Finally, the use of disinfectants produced by different companies may result in varying effects.
The authors have no conflicts of interest to declare in relation to this study.
