DISCUSSION
Microbial elimination is important in the success of endodontic treatment. E.faecalis is the major bacterial species isolated from root canals in endodontic failure. It has been reported thatE.faecalis is mostly monoculture in 30-89% of teeth with post-endodontic failures (George et al., 2010). Also, it can cause life-threatening infections such as infective endocarditis (Madsen et al., 2017). E.faecalis is a Gram-positive, facultatively anaerobic, non-spore-forming, non-motile, cocciform bacterium (Kayaoğlu et al.). It is a microorganism resistant to harsh conditions. The virulence factors of E.faecalis associated with endodontic infection are lipoteichoic acid, sex pheromones, surface adhesins, extracellular superoxide, aggregation substances, lytic enzymes (gelatinase and hyaluronidase), and the cytolysin toxin, furthermore, new factors continue to be explored (Kayaoglu & Ørstavik, 2004).E.faecalis is related to endodontic infections, whileS.mutans play the main role in the etiology of dental caries (Blancas et al., 2021). S.mutans can cause oral infections as well as induce systemic infections such as cardiovascular disorders (Lucchese, 2017). The importance of microbiological control in root canals increases when the relationship between focal oral infections and human systemic diseases is regarded. Considering all these conditions,E.faecalis and S.mutans species, which are the main factors in the pathology of oral and endodontic diseases, were used in this study.
According to the results of the current study, when used alone inS.mutans, the highest antimicrobial activity was observed in CHX and HOCl (20 mm inhibition zone diameter), while approximately the same activity was observed in NaOCl (6 mm) and PHMB (5 mm). In combined use, the highest efficiency was determined in CHX+HOCl (20mm). InE.faecalis , the highest antimicrobial activity was observed in CHX (5 mm) when used alone, while the efficacy of HOCl and PHMB was higher than NaOCl. In combination, the highest efficacy was observed in CHX+HOCl (12 mm) and CHX+PHMB (10 mm). It was observed that the antibacterial activity of HOCl and PHMB, which are alternative irrigation solutions, were higher than NaOCl. Especially inE.faecalis , when chlorhexidine was used in combination with alternative solutions, it was determined that the antibacterial activity increased by 2 times.
In a study evaluating the antibacterial activity of root canal irrigation solutions based on NaOCl and electrolyzed oxidizing (EO) water (HOCl), it was concluded that EO waters containing HOCl had a bactericidal effect like that of conventional NaOCl againstE.faecalis and S.mutans. It has been stated that low-concentration HOCl is equally antibacterial as 1.5% and 5.25% NaOCl and considering its low toxicity, it has the potential to replace NaOCl as an alternative irrigation solution for vital pulp therapy (Hsieh et al., 2020).
HOCl, naturally produced by the human immune system to fight infection, has antimicrobial activity (Ateş; Hsieh et al., 2020). Like HOCl produced by human immunity by the myeloperoxidase-H2O2-Cl system of phagocytic cells (Pullar et al., 2000), HOCl artificially produced for disinfection also can fight invading pathogens and infections (Ateş; Hsieh et al., 2020; Lapenna & Cuccurullo, 1996). HOCl has been reported to have a toxic effect on bacteria by causing complete disruption of bacterial ATP production (Barrette et al., 1989). Compared with NaOCl in the current study, it showed greater antibacterial activity againstE.faecalis and S.mutans . There are similar results in the use of HOCl as an irrigation solution for cleaning root canals and removing the smear layer in endodontic treatment (Garcia et al., 2010; Hsieh et al., 2020).
PHMB is a powerful antimicrobial solution effective against Gram-positive and Gram-negative bacteria (Messick et al., 1999), yeasts (Larkin et al., 1992), and viruses (Medvedec Mikić et al., 2018). In one study, it was reported that 0.2% PHMB and 2.5% NaOCl solutions both successfully eliminated E.faecalis from mature dentin biofilm, but 0.2% CHX was not effective enough (Medvedec Mikić et al., 2018). Another study showed that 0.2% PHMB exerted significantly greater persistence on human dentin than 2% CHX (Chandki et al., 2020). PHMB is recommended as a suitable alternative to CHX as it reduces oral biofilm and has no reported side effects (Santos et al., 2021). In the current study, it was observed that 0.1% PHMB has antibacterial activity that can be an alternative to NaOCl. There is limited data in the literature on the use of PMHB as an irrigation solution in the endodontic literature, so more studies are needed on it.
This study showed that 5.25% NaOCl was not efficient enough to eliminate E.faecalis like the observations of other studies (Liu et al., 2010; Peciuliene et al., 2001; Portenier et al., 2003). It has been suggested that the resistance of E.faecalis to NaOCl may be due to its binding affinity to collagen fibers and hydroxyapatite (Chivatxaranukul et al., 2008; Kayaoglu et al., 2008). Although similar results were seen in different studies, there was no host factor such as dentin in the current study.
In this study, the most effective antimicrobial solution was 2% CHX in eliminating E.faecalis and S.mutans, and these findings are consistent with the literature (Önçağ et al., 2003; Vianna et al., 2006). Few studies have tested E.faecalis against disinfectants without including host factors (Kayaoğlu et al., 2008). In a study,E.faecalis suspensions were treated with 2% CHX for 1 hour, and negative culture was observed (Fouad & Barry, 2005). In another study, it was found to be eliminated in 1 minute with 2% CHX gel (Gomes et al., 2006). It has been reported that 0.5% CHX has a good antibacterial effect after 1 and 24 hours of application (Kayaoğlu et al., 2008).
A greater antibacterial effect can be achieved in the root canal microflora with the combination of solutions compared to the single solution, because of the synergistic and/or additive effects of irrigants with different antimicrobial activities (Ozkan et al., 2020; Sundqvist, 1992). In the current study, it was observed that the antibacterial activity of CHX in combination with HOCl and PHMB was 2 times higher than the use of CHX alone on E.faecalis . Since there is no study in the literature on the combined use of relatively new alternative irrigation solutions, the findings cannot be compared with other studies. In addition, NaOCl + CHX showed less antibacterial activity compared to the use of CHX with alternative solutions, like in another study (Ozkan et al., 2020). It has been stated that the reason for this may be the orange-colored precipitate formed by parachlorophenol (PCU) or chlorophenylguanidyl-1,6-diguanidyl-hexane (PCGH) when CHX and NaOCl are used together (Basrani et al., 2007; Nowicki & Sem, 2011). To prevent the interaction of different solutions, it is recommended to irrigate the root canals with saline, sterile distilled water, or alcohol between solutions, and aspirate the remaining irrigant in the canal with a needle and dry it with paper cones or perform ultrasonic activation with EDTA (Bui et al., 2008; Keles et al., 2020; Prado et al., 2013). However, in this study, the above-mentioned methods were not applied to prevent the interaction of the solutions due to the difference in the experimental design.
In the present study, the absence of host factors such as blood, serum, dentin, and collagen in the experimental design prevents the results from being reflected in the in-vivo environment and constitutes the limitation of the study. In this technique, the absence of dentinal tubules that protect bacteria can be advantageous for disinfectants. However, this study evaluated the protection mechanisms of bacteria against different irrigation solutions in a standard in-vitroenvironment without host factors. There are many studies in the literature with different techniques and materials. Further studies are needed on endodontic alternative irrigation solutions, such as cleaning, antimicrobial, biocompatibility, effects on tooth structure, etc.
CONCLUSION
Within the limitations of this study, HOCl and PHMB irrigation solutions were found to have sufficient antimicrobial activity to be an alternative to conventional solutions for S.mutans andE.faecalis. It was determined that the combination of CHX and alternative irrigants increased the antibacterial activity. In particular, the combined use of HOCl with CHX may provide microbiological advantages in clinical use.
ACKNOWLEDGMENTS
Funding: None
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