## 4 Discussion
[10] reported that the Reciproc Blue file system exhibited lower antibacterial action when compared to the ProTaper Next file system. These results can be attributed to the fact that the ProTaper file system removes the dentin surface more aggressively therefore leading to lower bacterial counts in samples after instrumentation [8]. In contrast with these results, other previous studies have indicated no significant differences in the efficacy of bacterial removal between WaveOne and ProTaper Gold rotary files [6,24]. In the current study, 2 Shape instrumentation led to significantly greater bacterial reduction within root canals compared with Recipro Blue in the TEC design group. Moreover, significant differences were revealed in _E. faecalis_ counts between TruNatomy and ProTaper Gold file systems in the TEC design group. There is no previous research in the literature on the levels of bacterial reduction in root canals after mechanical instrumentation applied to teeth using the 2 Shape and TruNatomy file systems. The results of this study highlighted significantly lower bacterial reductions in the CEC group than in the TAC and TEC groups following instrumentation with the ProTaper Gold and 2 Shape file systems. In contrast with our results, [PERSON] and [PERSON] [1] determined the impact of root canal instrumentation on the amount of _E. faecalis_ reduction within the root canal system by utilizing various file systems in teeth with the TEC and CEC access cavity designs, finding no significant differences between the cavities. Similarly, in their study, [PERSON] et al. [17] examined the efficacy of canal instrumentation in microbial reduction in mandibular molar teeth, revealing no significant differences in microbial reduction. The authors also obtained similar results for unprepared areas between three access cavities. These results are in accordance with the findings obtained by [PERSON] et al. [31], which indicated similar results for unprepared areas. According to the results of our study, mechanical instrumentation might be affected by the access cavity design. In particular, there may have been more unprepared areas in the root canals utilizing a CEC design due to the absence of straight-line access. This study has specific limitations. One of the limitations was that only sterile saline was used as an irrigation solution. This allowed examination of the mechanical action of endodontic instruments; however, it did not reflect the real-world clinical conditions of endodontic treatment. In addition, this was an in vitro study that could not exactly mimic in vivo conditions. Further studies should be performed utilizing different irrigation solutions in vitro and in vivo to evaluate the effectiveness of the instrument systems used in this study. Conceptualization, G.A., A.K. and F.B. ; Methodology, G.A., F.B. and B.B. ; Software, G.A. and B.B. ; Validation. G.A., A.K. and F.B. ; Formal Analysis, G.A. and B.B. ; Investigation, G.A. ; Resources, B.B. ; Data Curation, G.A. and A.K. ; Writing--Original Draft Preparation, G.A., F.B. and B.B. ; Writing--Review and Editing, G.A., F.B. and A.K. ; Visualization, G.A. ; Supervision, G.A. and F.B. ; Project Administration, G.A. and A.K. ; Funding Acquisition, G.A. All authors have read and agreed to the published version of the manuscript. This research received no external funding. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Near East University (YDU/2020/85-1207 and 26 November 2020). The authors declare no conflict of interest.