Evaluation of the Antibacterial Effects of Single and Combined use of Different Irrigation Solutions Against Intracanal Enterococcus Faecalis.

OBJECTIVES: This study assessed the antibacterial activity of both separate and combined uses of 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 17% ethylenediaminetetraacetic acid (EDTA), 3% hydrogen peroxide (H2O2), MTAD, SmearClear (SC) and 13.8% chlorine dioxide (ClO2) irrigation solutions against Enterococcus faecalis.
MATERIALS AND METHODS: Two hundred eighty single rooted human premolars were randomly grouped into 26 test and 2 control (negative and positive) groups and were incubated for 24 h with E. faecalis, except for the negative control group. The tested solutions were as follow: NaOCl; CHX; ClO2; MTAD; SC; EDTA; H2O2; NaOCl + CHX; NaOCl + MTAD; SC + NaOCl; EDTA + NaOCl; H2O2 + NaOCl; ClO2 + CHX; CHX + MTAD; SC + CHX; EDTA + CHX; CHX + H2O2; ClO2 + MTAD; SC + ClO2; EDTA + ClO2; ClO2 + H2O2; SC+MTAD; EDTA+MTAD; MTAD + H2O2; SC + H2O2; and EDTA + H2O2. Optic density values were recorded at 0, 6, 12, 18, 24, 30, 36, 42 and 48 h and bacterial growth curve created for each solution.
RESULTS: The CHX, MTAD and ClO2 showed a high potential for the elimination of E. faecalis, both alone and in all combinations. The EDTA, H2O2, H2O2+ EDTA, H2O2 + NaOCl and SC + NaOCl groups showed less antibacterial activity than the other groups. The SC + CHX group showed the best antibacterial effect against E. faecalis.
CONCLUSION: The SC + CHX combination can be recommended as the most effective irrigation regimen against E. faecalis in persistent endodontic infections.

Introduction

Microorganisms are one of the significant etiological factors in the pathogenesis of periapical tissue diseases. For this reason, it is very important to completely remove microorganisms from the infected root canals during a root canal treatment. Some ex vivo and clinical studies have reported that there are some untouched areas in the root canal walls during mechanical preparation. Therefore, to combine mechanical instrumentation with chemical irrigation is extremely important (-) in elimination of the microorganisms known to be extremely resistant against antimicrobial agents, such as Enterococcus (E.) faecalis ().

The relationship of E. faecalis with different forms of periradicular infections, including primary and permanent infections, is well known because of its ability to grow in the presence or absence of oxygen. E. faecalis has been reported to be associated more with asymptomatic chronic periradicular lesions rather than acute periradicular periodontitis or acute periradicular abscesses in the category of primary endodontic infections (, ). It has been confirmed that E. faecalis is significantly associated with treatment failures. Whereas this species was detected in 18% of the cases of primary endodontic infections, its prevalence in root-filled teeth was much higher: 67% of the cases ().

Different endodontic irrigation solutions and disinfection techniques have been introduced to decrease the root canal bacterial count. The main effect of irrigation is to both physically and chemically remove organic and inorganic debris, infected materials and soft and hard tissue residues from the root canals. In this way, these materials are inhibited from accumulating, clogging and becoming inaccessible in the apical section of the root canals. Endodontic irrigation solutions usually have antibacterial properties. It has been proven that a combined use of irrigation materials with a different antimicrobial spectrum can even increase the antibacterial activity provided by a single solution through a synergic and/or additive effect for the elimination of hundreds of types of microorganisms forming on the root canal microflora ().

There is a limited number of studies in the literature assessing and comparing the combined use of frequently used endodontic irrigation solutions in terms of their antibacterial characteristics. The positive or negative effects of the antibacterial interactions of current and potential irrigation solutions will especially guide the treatment of persistent endodontic infections. Therefore, understanding the interactions between irrigation solutions will be very helpful in implementing the most effective treatment procedure for persisting infections in endodontic cases. This current study aimed to investigate the antibacterial activity of both the single and combined uses of sodium hypochlorite (NaOCl), chlorhexidine (CHX), ethylenediaminetetraacetic acid (EDTA), hydrogen peroxide (H2O2), BioPure MTAD (Densply, Tulsa Dental, Tulsa, OK, USA). SmearClear (SC) (Sybron Endo, Orange, CA, USA) and chlorine dioxide (ClO2) against E. faecalis and to compare the antibacterial effects of this study’s solutions against E. faecalis.

Materials and Methods

The current study was started after receiving approval from the Ethical Board of Selcuk University, Faculty of Dentistry (Document # 203). One hundred eighty single-rooted human lower premolars that had been extracted for orthodontic or periodontal reasons were used. The hard and soft tissue residuals on the teeth were cleaned with curettes and then the teeth were kept at 4° C in a 100% moist environment until the laboratory procedures were performed.

The teeth were de-coronated with a diamond disk (Ortho Technology Inc., Tampa, FL, USA) under water cooling to a standardized root length of 14 ± 0.5 mm. The canal lengths were standardized with 15 K-File hand devices (Mani Inc., Tochigi, Japan) and enlarged with ProTaper universal NiTi rotary files (Dentsply, Tulsa Endodontics, Tulsa, OK, USA) using the crown-down method. The apical parts of the root canals were finished at F3. During the preparations, the root canals were irrigated with 1 ml of 5.25% NaOCl solution (Caglayan Kimya San., Konya, Turkey).

In order to remove the smear layer of root canal walls, the roots were exposed to an ultrasonic bath (USG 4000 Ultraschall, Dentaurum, Ispringen, Germany) in 17% EDTA (AppliChem GmbH, Darmstadt, Germany), 5.25% NaOCl and distilled water for 10 min in each solution, in that order (). The samples were then embedded perpendicular to the long axes in a silicone impression material (Zetaplus, Zhermack SpA, Badia Polesine (RO), Italy) and placed in metal plates filled with distilled water and capped, 10 in each group, and sterilized at 121° C for 20 min in an autoclave (Hirayama, Saitama, Japan). The metal plates were then opened inside a Biosafety Level 2 (BSL 2) lamina air-flow cabin and each sample was coated with two layers of nail polish (Loreal Jet-Set Diamond, Paris, France) in order to prevent bacterial leak during the experimental procedures. In order to contaminate the sterilized root canals with E. faecalis for experimental purposes, fresh cultures of E. faecalis (ATCC 29212) microorganisms were obtained after 24 h of incubation inside Brain Heart Infusion Broth (BHI) (bioMerieux® sa 69280, Marcy I’Etoile, France). The optical density (OD) of the E. faecalis suspension inside BHI was adjusted according to McFarland No: 0.5 standard to approximately 1.5 x 108 colony/ml. Except for the negative control group, the E. faecalis suspension was planted to the root canals with the help of a sterile 1 ml tuberculin syringe. Then, the samples in the metal plates were incubated at 37° C for 24 h. After incubation, the metal plates were opened in a BSL 2 air-flow cabin and irrigation was performed with the experimental solutions.

A flow chart of the study design is shown in Figure 1. The basic irrigation solutions and their combinations used in the study are presented in Table 1. All solutions except for EDTA were ready to use. The 17% EDTA solution was prepared in the laboratory according to the instructions of Sen et al. ().

Figure 1

Flow Chart of the Study Design

Table 1

Basic irrigation Solutions and Manufacturers

The Basic Irrigation Solutions	Manufacturer
5.25% Sodium hypochlorite (NaOCl)	Caglayan Kimya San., Konya/Turkey
2% Chlorhexidine gluconate (CHX)	Klorhex, Drogsan ilaç san., Ankara/Turkey
13.8% Chlorine dioxide (ClO2)	Bioclenz, Frontier Pharmaceutical, Melville, NY, USA
BioPure MTAD (MTAD)	Dentsply, Tulsa Dental, Tulsa, OK, USA
SmearClear (SC)	Sybron Endo, Orange, CA, USA
3% Hydrogen peroxide (H2O2)	Kimpa ilaç lab. Ve tic. Ltd. sti.; Istanbul, Turkey
17% Ethylene Diamine Tetra Acetic Acid (EDTA)	Prepared in the laboratory

Each root canal was irrigated with a single or combined irrigation solution for 5 min using 30-gauge endodontic irrigation needles (KerrHawe SA, Bioggio, Switzerland), according to the irrigation regimens indicated in Figure 1. Sterile F3 paper cones were placed into the root canals for 1 min to allow for complete absorption. The paper cones were then put into sterile tubes containing 1 ml BHI Broth and placed into a vortex device (MS 1 Minishaker IKA®, Darmstadt, Germany) for 5 min. A 200 µl sample was taken from the shaken medium and transferred to a well in a 96-well sterile ELISA plate (Costar 3599, Corning, NY, USA). Each sample went through this procedure twice for a total of two wells per sample to get averages for the measurements. The plates were then placed in an ELISA reader (BioTek ELx800, Absorbance Microplate Reader, Winooski, VT, USA) to complete the first optic density (OD) test (hour 0) at a wavelength of 450 nm, and the data were recorded. The data were taken every 6 h in the ELISA reader and repeated twice. The plates were placed in the incubator and kept at 37° C in a 100% moist environment during the experiment. Data were obtained for each sample at hours 0, 6, 12, 18, 24, 30, 36, 42 and 48. The averages of the data collected at each measurement were calculated separately for each group, and an average OD value was determined for each period. Average OD data obtained for each sample at each time period were used, and a time-dependent OD change graph was created for each experimental group (Figure 2,4–10)

Figure 2

Bacterial growth curves resulting from time-dependent OD values

Figure 4

CHX and Combinations

Figure 5

NaOCl and Combinations

Figure 6

ClO2 and Combinations

Figure 7

MTAD and Combinations

Figure 8

SC and Combinations

Figure 9

H2O2 and Combinations

Figure 10

EDTA and Combinations

For statistical analysis, the Kruskal–Wallis and Mann–Whitney U tests were used to find any significant differences among the study groups. Significance level was accepted as p>0.05.

Results

The post-incubation, time-dependent OD values (at 450 nm) of the samples tested for antibacterial activity following single and combined uses of the root canal solutions are shown in Figure 2.

Overall statistical results showed that significant similarity was found between the OD values of samples taken from root canals irrigated with CHX, NaOCl, MTAD, SC and ClO2 and the negative control group (p>0.05). On the other hand, the positive control group showed significant similarities with OD values of the samples taken from the root canals irrigated with H2O2 and EDTA (p>0.05).

CHX and CHX combinations

No statistically significant difference was found between the negative control group and CHX and its combinations at all times, including the NaOCl + CHX group, which showed an increase in OD values (Figure 2, 3, 4) (p>0.05).

Figure 3

Mean OD values of irrigation regimens used in the experiment

NaOCl and NaOCl combinations

No statistically significant difference was found between the NaOCl and NaOCl + MTAD groups and the negative control group at all times (p>0.05). The values of samples irrigated with H2O 2+ NaOCl and EDTA + NaOCl at hours 24, 36 and 48 were found to be similar with the positive control group (p>0.05) (Figure 5). The increase in the OD value of the SC + NaOCl group after hour 6 was found to be significantly different from the negative control group after hour 36 (p<0.05) (Figure 5).

ClO2 and ClO2 combinations

ClO2 and all its combinations were found to be significantly similar with the negative control group at all times (p>0.05) (Figure 6).

MTAD and MTAD combinations

No difference was found between OD values of MTAD and all its combinations and the negative control group (p>0.05). (Figure 7).

SC and SC combinations

SC, SC + CHX, SC + MTAD and SC + H2O2 were found to be statistically similar with the negative control group at all times (p>0.05) (Figure 8). A continuous increase was found in the OD values of the groups irrigated with SC + ClO2 and SC + NaOCl at all time periods (Figure 8). While this increase was not found to be statistically significant in the group irrigated with SC + ClO2 (p>0.05), in the group irrigated with SC + NaOCl, it was found to be significantly different for the measurements at hours 30, 36 and 48 when compared with the negative control group (p<0.05).

H2O2 and H2O2 combinations

There were no statistically significant differences between the OD values of the groups irrigated with SC + H2O2, H2O2 + ClO2, H2O2 + CHX and H2O2 + MTAD and those of the negative control group (Figure 9). The group irrigated with H2O2 was found to be statistically significantly similar with the positive control group (p>0.05). The increase in the first 24-hour period in the groups irrigated with H2O2 + EDTA and H2O2 + NaOCl was not statistically significant when compared with the positive control group (p>0.05) (Figure 9). However, the increase continuing at hours 24 and 48 in the groups irrigated with H2O2 + EDTA and H2O2 + NaOCl was significantly different when compared with the positive control group (p<0.05) (Figure 9).

EDTA and EDTA combinations

The time-dependent increase in OD values of the groups irrigated with EDTA and EDTA + H2O2 was found to be significantly similar with the positive control group (p>0.05) (Figure 10). The increase observed for the first 6 hours in the EDTA + H2O2 group was found to be significantly similar with the negative control group (p>0.05), while that increase was found to be significantly similar with the positive control group at hours 6 and 24 (p>0.05). In the remaining periods, the increase in the OD values was found to be significantly different from those of the positive control group (p<0.05). The OD values of the group irrigated with EDTA + ClO2 were found to be similar with the negative control group at all times (p>0.05) (Figure 10). When the time-dependent OD value of EDTA + NaOCl was compared with the negative control group, it was found to be significantly different after hour 18 (p<0.05) (Figure 10). However, no statistically significant difference was observed between the groups irrigated with EDTA + CHX and EDTA + MTAD and the negative control group (p>0.05) (Figure 10).

Discussion

In this study, the bacterial growth in samples from infected root canals irrigated with antibacterial irrigation solutions was compared with normal bacterial growth (positive control group), and bacterial growth from when the sterilized roots were incubated (negative control group). During the incubation period, it was clearly determined in which period the reproduction occurred, slowed down and regressed.

The antibacterial activity of irrigation solutions is known to increase with the increase in volume and application time of the irrigation (, ). In this study, the standard irrigation application time was determined to be a total of 5 min of irrigation with a 5 ml solution for all canals. In combined uses, the total volume used was 2.5 ml + 2.5 ml for each solution. For MTAD, the manufacturer recommended an application regimen of 5 ml for each canal. Thus, MTAD also used the same volume and time interval as the other solutions, making all solutions in the experiment comparable. All of the root canals were also irrigated at a post-experiment stage with 5ml saline to achieve maximum dilution of the residual solution. In this way, the impact of the transferred solution on the medium was minimized and the residual antibacterial effect was reduced.

In this study, the order of the solution application recommended by researchers was used (, ). It is acknowledged that a residual antimicrobial effect might still be present unless the activity of the solution is neutralized by means of an inactivator (). However, to be able to standardize the experiments undertaken in this study, neutralization of the test solutions was not carried out. One of the other reasons for this is that some of the test solutions, such as MTAD and SC, are proprietary products and there are no chemical inactivators known to exist for them. Therefore, to achieve uniformity in the experiment methodology and to make it an easier procedure to compare the relatively high number of solutions, none of the test specimens were inactivated.

It has been reported by many researchers that CHX shows antibacterial activity against E. faecalis (-). In accordance with other studies, no significant increase in the OD values of CHX and its combinations (except NaOCl + CHX) were observed in this recent study. However, NaOCl + CHX showed less antibacterial activity with increased OD values. This may be due to the orange-colored residue made of parachlorophenol (PCU) or chloropfenilguanidil-1,6-diguanidil-hexaze (PCGH), which can be obtained when CHX and NaOCl are combined (, ). These residues may have had negative effects on the interaction between the root filling and the canal wall dentin by blocking dentin tubules (, ). In order to prevent these solution’s interaction, it is recommended to irrigate the root canals with saline, sterile distilled water or alcohol before irrigation with CHX, and NaOCl left in the canal can be aspirated with a needle, dried with paper cones or ultrasonic activation with EDTA (-). However, in the present study, the recommended processes to prevent the interaction of these two solutions mentioned above were not used because of their variations in the experimental procedure. Another important issue noted with the NaOCl + CHX group is that the antibacterial activity did not decrease in the first 12 hours but did decrease after hour 12 (Figure 4). This may be because of the residue that accumulated on the root canal while taking the sample, and/or the possibility of PCU and PCGH being toxic to E. faecalis and the decrease observed in these effects at the end of hour 12.

Since no increase was found in the EDTA + CHX group’s OD values over time (p>0.05), this combination has the potential to show effective antibacterial activity against E. faecalis. Liu et al. () reported that the combined use of CHX and EDTA had antibacterial activity and it was also. better than MTAD and EDTA + NaOCl. The present study also supports these results. Gonzalez-Lopez et al. () observed a pink-colored residue when CHX and EDTA were mixed. In their study, Rasimick et al. () showed a white-colored salt when these two materials were mixed. Later, Prado et al. () examined the interaction of CHX and EDTA and observed a milky residue and concluded that this was a result of an acid-base reaction of the combination. The results of the present study confirmed that the white and/or salt residue formed by these two materials does not negatively affect the antibacterial activity of CHX in infected root canals.

No studies have been conducted so far on whether ClO2 + CHX shows antibacterial activity against E. faecalis. According to the results of the present study, ClO2 + CHX is effective in the elimination of E. faecalis. These two agents do not form any reactions nor do they negatively influence each other (, ) and they maintain their antibacterial activity at all times when used in combinations (Figure 4,6).

Although it has been reported in a great number of studies that MTAD has antibacterial activity against E. faecalis (, ), no studies have been found comparing the antibacterial characteristics of CHX + MTAD to MTAD. The results of this study showed that the CHX + MTAD and MTAD groups have similar antibacterial activities against E. faecalis (p>0.05) (Figure 7). The CHX + MTAD combination did not cause any negative change in the antibacterial characteristics of either solution. This important characteristic can be evaluated with further studies not only on E. faecalis but also on the elimination of other bacteria that are also responsible for endodontic infections.

In this study, an increase was found in the OD value of the H2O2 + NaOCl group after 12 hours, and this increase was found to be different from the negative control group’s (p<0.05) (Figure 5,9). The combined use of these two solutions has been recommended to ease organic and inorganic debris from the root canal, and it has disinfecting and whitening properties through its foaming effect (). The antibacterial activity of an irrigant increases as its volume increases (). Since, NaOCl’s volume in combined use is less than when it is used alone, a decrease in antibacterial activity is expected. The antibacterial activity of H2O2 was also found to be insufficient in the present study. Therefore, it was speculated that the combined use of these two solutions did not positively contribute to the solutions’ antibacterial characteristics. Another reason for the decreased antibacterial activity may be due to decreased hydroxyl radicals, which actually creates antibacterial activity with the reaction of the two solutions ().

In the EDTA + NaOCl and SC + NaOCl groups, a time-dependent increase was found in measurements after hour 6 (Figure 5). When compared to the negative control group, the antibacterial activity in the EDTA + NaOCl group was found to be less than the activity in the SC + NaOCl group.The reason for the decrease in antibacterial activity may be related to EDTA’s negative effect on the tissue-dissolving capacity of NaOCl and decrease in the amount of active chlorine in the combination ().

It has previously been reported that NaOCl + MTAD is successful in E. faecalis elimination (). Tay et al. () reported a brown liquid developed when NaOCl and MTAD were combined and that this combination decreased the dentine substantivity of MTAD (). For these reasons, it has been reported that the canals need to be irrigated in intervals with saline. In the present study, no decrease was found in the combination’s antibacterial activity. Thus, the results of the current study support Shabahang and Torabinejad’s study (), which states that the antibacterial effect of the combination of 1.3% NaOCl and MTAD is an effective solution in eradicating E. faecalis.

ClO2 is a strong oxidizing agent, and it effectively kills pathogenic microorganisms (). ClO2 actually has smear layer removing characteristics and the capacity to dissolve organic tissue (-). In this study, it was found that when used alone and in combination with other solutions, ClO2 showed similar antibacterial characteristics with NaOCl and its combinations against E. faecalis. However, ClO2 and the SC + ClO2, EDTA + ClO2, H2O2 + ClO2 and ClO2 + CHX combinations showed relatively higher antibacterial characteristics compared to the NaOCl combinations (Figure 6). These results are parallel to Eddy et al.’s findings (), which stated 10% and 13.8% Chlorine dioxide and 5.25% NaOCl were both effective in eliminating E. faecalis from the dentinal disks within 30 min.

According to the results of the present study, a fair amount of increase was found in the OD values of the EDTA + ClO2 and SC + ClO2 groups; however, this increase was found to be statistically similar to the negative control group (p>0.05) (Figure 6). This increase in the OD levels of the EDTA + ClO2 group may be due to the decrease in antibacterial activity as a result of the ClO2-oxidizing effect against EDTA (). In addition, this increase in the OD values of the samples treated with SC+ClO2 was found to be lower than that of EDTA + ClO2 (Figure 6). This may be due to the antibacterial characteristic of the surface-active agent (cetrimide) in SC (). Since there are no studies researching the antibacterial activity of SC + ClO2 combinations, further studies are needed to explain the reasons for this increase.

There was a continuous increase in the OD values of the SC + ClO2 and SC + NaOCl groups in the OD measurements after hour 6 (Figure 8). While the SC + ClO2 group’s increase was found to be significantly similar to the negative control group’s (p>0.05) at all hours (Figure 8), the measurements at hours 30, 36 and 48 in the SC + NaOCl group were found to be significantly higher than those of the negative control group’s (p<0.05) (Figure 8). Thus, SC + ClO2 showed better antibacterial activity than SC + NaOCl. This may be lower inhibitory effect of SC on ClO2 than the inhibitory effect of NaOCl.

A time-dependent increase was found in the EDTA + H2O2 group. Although the increase in the OD values of EDTA + H2O2 within the first 18 hours was significantly similar to the positive control group (p>0.05), it was significantly different from the positive control group after hour 30 (p<0.05) (Figure 9). The reason for the increase in this group may be because the medium created with the combination of these solutions was a suitable medium for E. faecalis to grow. However, further studies are needed to confirm these results.

Conclusions

Within the limitations of this study, the following conclusions can be drawn:

EDTA is not a suitable solution for providing antibacterial activity in the irrigation process of root canals. Although it did not have any negative effects in combinations with CHX and MTAD, combined uses of EDTA and H2O2 solutions may be harmful for disinfecting the root canals since they can potentially provide a medium for bacteria reproduction; Chelation agents, such as EDTA and SC, can be used for the removal of the smear layer from the root canal walls. However, their application together with NaOCl reduces the antibacterial effect of these agents. For these reasons, additional irrigation with another irrigation solution and/or solution combination may be suggested; ClO2 is chemically similar to NaOCl and when used in combination with H2O2, CHX, EDTA or SC, its antibacterial activities are less affected than with combinations of NaOCl. Therefore, ClO2, with its positive properties, may be considered as an alternative to NaOCl and suitable for routine clinical use; Overall it can be concluded that the SC + CHX combinations can be recommended as the most effective irrigation regimen against E. faecalis in persistent endodontic infections.