Comparative evaluation of effect of different irrigation solutions against Enterococcus faecalis: A polymerase chain reaction-based study.

UNLABELLED: Enterococcus faecalis is the most isolated or detected species from oral infections including marginal periodontitis, infected root canals, periradicular abscesses and also detected in cases of failed endodontic therapy. To prevent endodontic treatment failure irrigation is mandatory for the effective removal of smear layer, pulp tissue, and microorganisms. Cultivation and other traditional identification methods have been demonstrated to have several limitations when it comes to microbiological identification. Polymerase chain reaction was selected because it has an added advantage over traditional microbiological methods.
MATERIALS AND METHODS: Twenty single rooted premolars were taken were taken stored in 0.1% thymol solution at 4°C decoronated to obtain 12 mm length, teeth were autoclaved at 121°C, canals were instrumented up to 35k file (International Organization for Standardization). The samples were randomly divided into three groups Group I - 5.25% sodium hypochlorite (NaOCL) and 17% ethylenediaminetetraacetic acid (EDTA), Group - II 5.25% NaOCL and 2% chlorhexidine (CHX), Group III - 5.25% NaOCL and 17% EDTA and 2% CHX.
RESULTS: The results showed that Group III which is 5% NaOCl followed by 17% EDTA and followed by 2% CHX showed maximum antimicrobial activity in all the three different time intervals.

Of the major diseases occurring in the human body, the most unique disease occurring in the human body is the infection because the infection establishes itself in an area where micro-organisms have previously not been present. In 1965, Kakehashi et al. convincingly established the role of bacterial infection in pulpitis and apical periodontitis. In 1970, Sundqvist determined that most bacteria in endodontic infections are strict anaerobes.

Enterococcus faecalis is the most commonly isolated species from oral infections including marginal periodontitis, infected root canals, periradicular abscesses, and also detected in cases of failed endodontic therapy. E. faecalis has the potential to get adapted for the survival in a variety of adverse environments. Moreover, hence it can survive in root canal infections where the availability of nutrients.

Shaping and cleaning is the most important step in root canal treatment. This eliminates the microbial infection from the root canal space and from the dentinal tubules. This can be achieved with the help of irrigants for the removal of organic tissue, smear layer, as well as the microorganisms. Irrigation dynamics also play an important role, and the effectiveness of irrigation depends on many factors like the working mechanism of the irrigant and the ability to contact with the microorganisms and the tissue debris present in the root canal complex.

There are many innovative approaches in irrigation protocol to enhance the action of irrigants. The combined use of many irrigants in sequential order may help in eliminating the organisms effectively. However, caution should be exercised when using the combined irrigation protocol because of the chemical interaction between the irrigation solutions that result in precipitation in the root canal space. Hence, the application of final irrigating solution should remain active at the time of application, as well as over a long period of time, for preventing bacterial recolonization or eliminating the bacteria that persist after root canal treatment. The most common final irrigation protocol includes various irrigants specifically, a chelating agent followed by an antimicrobial irrigant to remove both the inorganic and organic components of smear layer from the surface of instrumented root canals. The alternate use of sodium hypochlorite (NaOCl), a deproteinizing agent, and ethylenediaminetetraacetic acid (EDTA), a calcium chelating agent has been recommended for its efficient removal.[12] These irrigants must be brought into direct contact with the entire canal wall for effective action.[3]

Most of the studies, done on the microbiota of root canals in teeth with failed endodontic treatment have used culture techniques for bacterial identification. Cultivation, which is the traditional identification method has limitations when it comes to microbiological identification. In contrast, polymerase chain reaction (PCR)-based detection methods enable rapid identification of both uncultivable and cultivable microbial species with high specificity and sensitivity. Hence, in the current study, PCR was selected to evaluate the effectiveness of different irrigants against E. faecalis.

Materials and Methods

Twenty single rooted premolars were taken were taken stored in 0.1% thymol solution at 4°C decoronated to obtain 12 mm length, teeth were autoclaved at 121°C, canals were instrumented up to 35k file (International Organization for Standardization). The samples were randomly divided into three groups

Group I - 5.25% NaOCl and 17% EDTA

Group II - 5.25% NaOCl and 2% chlorhexidine (CHX)

Group III - 5.25% NaOCl and 17% EDTA and 2% CHX.

After irrigation, root canals were dried, and dentinal shavings were taken using H-files. The regrowth of E. faecalis was checked at 1-day, 7 days, and 14 days. The samples were transferred to an eppendorf tube containing 5 ml of brain heart infusion broth. The broth was transferred to blood agar plates to check the regrowth of bacteria.

Polymerase Chain Reaction Analysis

Microorganism and inoculum preparation

A suspension of 50 μl of E. faecalis (ATCC 29212) strain was incubated in 5 ml of Trypticase Soy Agar broth culture medium (Difco, Sparks, MD, USA) at 37°C in an incubator for 4 h. The concentration of the inoculation was then adjusted to a degree of turbidity (0.5) according to the McFarland scale (Bio Merieux, Marcy l’Etoile, France), which corresponds to a bacterial load of 3 × 108 cells/ml and an optical density of 600 nm [Table 1].

Table 1

The Real-time PCR assay was carried out in a thermal cycler (7900HT Real-time PCR system)

Real-time polymerase chain reaction using Enterococcus faecalis specific 16S ribosomal deoxyribonucleic acid primer

The final volume of 20 μl was prepared from the reaction mix and loaded in a 96-well plate, which was then covered with an optical adhesive sheet [Graph 1 and Tables 2 and 3].

Graph 1

Polymerase chain reaction cycling analysis

Table 2

Primer sequence

Table 3

Steps in PCR cycling

Results

Statistical analysis was done using software SPSS version 21.0 and one-way analysis of variance and Fisher extract test at a significance level of 0.05%. The results showed that Group III, which is 5% NaOCl followed by 17% EDTA and followed by 2% PCR analysis. CHX showed maximum antimicrobial activity in all the three different time intervals [Table 4].

Table 4

Evaluation of total CFU after three different intervals

Discussion

The basis of endodontic treatment is to the control of endodontic microbiota. Many irrigation solutions have been used in the treatment of infected root canal.[45] In the current study, E. faecalis had been chosen as a testing organism because PCR, though making up a small proportion of flora in untreated canals, plays an important role in persistent periradicular lesions after root canal treatment. It is commonly associated with root canal failure cases, and it is able to survive in the root canal as a single organism or as a major component of the flora. E. faecalis, a Gram-positive, facultative anaerobe, and is most commonly found in the root canals of failing endodontically treated cases.[6] It can survive as a monoinfection in root canals.[7] E. faecalis has displayed resistance to chemo-mechanical preparation[8] and intracanal medication.[9] The persistence of E. faecalis may stem, from its capability to form biofilms in root canals.[10] If bacteria are located only on the root canal surface, the intracanal medicaments can reach and may present efficacy against E. faecalis. E. faecalis showed resistant to the antibacterial action of irrigants when they are lodged in deep layers that are within the dentinal tubules.

In the current study, PCR was used as measuring tool since it provides many advantages than the traditional culture methods. PCR is currently a more precise method for detection of E. faecalis.[1112] This method provides to be faster, more sensitive, and more accurate than culturing methods and has enabled researchers to detect bacteria that were difficult.[12] When compared to detection of E. faecalis by culturing (24–70%), E. faecalis has been found at consistently higher percentages (67–77%) when a PCR detection method is used.[6] An important distinction needs to be made between what is evaluated by culture and PCR assays.[13] The viable bacterial cells were counted by culturing as colony-forming units. Molecular methods measure nucleotide sequences, and the PCR method allow amplification of very minute quantities of deoxyribonucleic acid to detectable levels.

The selection of endodontic irrigants that synergistically have the ideal properties was a major point in the present study. CHX, in a 2% gel or liquid concentration, is effective at eliminating E. faecalis from the dentinal tubules.[141516] A 2 min rinse of 2% CHX liquid can be used to remove E. faecalis from the superficial layers of dentinal tubules up to 100 _m.[14] Concentrations of 1–2% CHX when combined with calcium hydroxide had the efficacy at killing E. faecalis.[151718] When CHX was combined with calcium hydroxide, the combined mixture had a greater ability to kill E. faecalis than calcium hydroxide mixed with water.[18] Two percentage CHX gel, when combined with calcium hydroxide, achieves a pH of 12.8 and can completely eradicate E. faecalis within dentinal tubules.[15] It is important that CHX alone has been shown to provide as good or even better, antimicrobial action against E. faecalis than calcium hydroxide/CHX combinations.[1516] Until further studies have been conducted, an intracanal dressing of 2% CHX placed for 7 days may be the best way to eradicate E. faecalis from dentinal tubules and the root canal space[1516]. In other in vitro studies, CHX -impregnated, and iodoform-containing gutta-percha points have shown little inhibitory action against E. faecalis.[1920] Unlike NaOCl, CHX lacks a tissue dissolving property. Therefore, NaOCl is still considered to be the primary irrigation solution used in endodontics.

This study, Group III treated with 5.25% NaOCL and 17% EDTA and 2% CHX showed the least regrowth, followed by Group II 17% EDTA and 2% CHX after 14 days and rest of the groups showed increased regrowth after 14 days.

The final irrigation with CHX provides good antimicrobial activity and imparts substantivity. This Group III irrigation combination and the order of irrigation solution used both provide higher antimicrobial activity when compared to Group I and II. The NaOCl at 5.25% dissolves organic tissue and kill bacteria. And EDTA was used as the next irrigant, which act as chelating agent removes the smear layer. Since EDTA effectively abolishes the tissue dissolving effect of NaOCl this was not used as the final irrigant. Alternating use of NaOCl and EDTA during instrumentation is a common practice. Since EDTA abolishes the tissue dissolving effect of NaOCl and should not be used until at the end of the treatment as the final rinse.[2122] CHX was used as final irrigant that increases the antimicrobial spectral activity. This result is in accordance with Zamany et al. in 2003 reported a further reduction in the proportion of positive cultures in the CHX group. Their results showed a better disinfection of the root canals using CHX compared to saline as a final rinse.[23]

The main use of an irrigant is to reduce the bacterial load during root canal treatment. The application of a final irrigating solution that remains active not only at the time of application but over a fairly long period of time is paramount.

The sequential use of NaOCl and a chelating agent or acid that dissolves inorganic tissue is required. Hence, the protocol for smear layer removal is NaOCl followed by EDTA or citric acid. Even though EDTA or citric acid removes the smear layer, they cannot do it completely without the preceding use of NaOCl. Water, saline CHX or iodine compounds have no dissolving effect on the smear layer.[24]

The Group II, which is 5.25% NaOCl, and CHX provide better antimicrobial activity than Group I.

Moreover, Group I showed the least antimicrobial activity. Moreover, when using the irrigants in combination, the chemical interaction between the irrigants should also be considered.

Bui et al.[25] evaluated the effect of irrigating root canals with a combination of NaOCl and CHX on root dentin and dentinal tubules by using scanning electron microscope and indicated that there were no significant differences in the amount of debris remaining between the negative control group and the experimental groups, although there were significantly fewer patent tubules when compared with the negative control group and concluded that the NaOCl/CHX precipitate tends to occlude the dentinal tubules and suggested that, and caution should be exercised when irrigating with both NaOCl and CHX. However, CHX lacks a tissue dissolving property. Therefore, NaOCl is still considered to be the primary irrigation solution used in endodontics.

Grawehr, et al.[26] studied the interactions of EDTA with NaOCl and concluded that EDTA retained its calcium-complexing ability when mixed with NaOCl. However, NaOCl loses its tissue dissolving capacity when used with EDTA. Hence, EDTA and NaOCl should be used separately. In few situations the copious amount of NaOCl should be administered to wash out remnants of EDTA.

Sodium hypochlorite is the most important irrigating solution during root canal treatment and should be used throughout the instrumentation. In higher concentrations, it kills bacteria and lower concentrations it dissolves organic tissue. Final irrigation with EDTA should be done to complete the removal of the smear layer and to continue the antimicrobial effect. Using CHX as final irrigation after EDTA provides good antimicrobial activity because of its substantivity. Alternating the use of NaOCl and EDTA during instrumentation is not advocated as EDTA effectively abolished the tissue dissolving effect of NaOCl and may increase erosion of dentin. For the same reason, a final NaOCL rinse after smear layer removal is not recommended.

Conclusion

Within the limitations of this study combination of irrigants and its sequential use have a valuable role in root canal treatment in eliminating the microorganisms. PCR can be used as a valuable tool for microbial identification than the traditional culture methods, which aid in more precise diagnosis.

Financial support and sponsorship

Nil.

Conflicts of Interest

There are no conflicts of interest.