<div>DISCUSSION</div><div>Microbial elimination is important in the success of endodontic
treatment. <i>E.faecalis</i> is the major bacterial species isolated
from root canals in endodontic failure. It has been reported that<i>E.faecalis</i> 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). <i>E.faecalis</i> 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 <i>E.faecalis</i> 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 &amp; Ørstavik, 2004).<i>E.faecalis</i> is related to endodontic infections, while<i>S.mutans</i> play the main role in the etiology of dental caries
(Blancas et al., 2021). <i>S.mutans</i> 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,<i>E.faecalis</i> and <i>S.mutans</i> species, which are the main
factors in the pathology of oral and endodontic diseases, were used in
this study.</div><div>According to the results of the current study, when used alone in<i>S.mutans,</i> 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). In<i>E.faecalis</i> , 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 in<i>E.faecalis</i> , when chlorhexidine was used in combination with
alternative solutions, it was determined that the antibacterial activity
increased by 2 times.</div><div>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 against<i>E.faecalis</i> and <i>S.mutans.</i> 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).</div><div>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 &amp; 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 against<i>E.faecalis</i> and <i>S.mutans</i> . 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).</div><div>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 <i>E.faecalis</i> 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.</div><div>This study showed that 5.25% NaOCl was not efficient enough to
eliminate <i>E.faecalis</i> 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 <i>E.faecalis</i> 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.</div><div>In this study, the most effective antimicrobial solution was 2% CHX in
eliminating <i>E.faecalis</i> and <i>S.mutans,</i> and these findings
are consistent with the literature (Önçağ et al., 2003; Vianna et al.,
2006). Few studies have tested <i>E.faecalis</i> against disinfectants
without including host factors (Kayaoğlu et al., 2008). In a study,<i>E.faecalis</i> suspensions were treated with 2% CHX for 1 hour, and
negative culture was observed (Fouad &amp; 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).</div><div>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 <i>E.faecalis</i> . 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 &amp; 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.</div><div>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 <i>in-vivo</i> 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 <i>in-vitro</i>environment 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.</div><div>CONCLUSION</div><div>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 <i>S.mutans</i> and<i>E.faecalis.</i> 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.</div><div>ACKNOWLEDGMENTS</div><div>Funding: None</div><div>REFERENCES</div><div><b>Arican, M., Çuhadar, F., &amp; Zamirbekova, N. (2020).</b>Investigation of the Clinical Effectiveness of
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