## 2 Materials and Methods  
Fifty-four freshly extracted teeth were collected. All of them were extracted due to periodontal disease or orthodontic purposes. All patients gave their informed consent for inclusion before commencing the study. The study was conducted in accordance with the revised Helsinki declaration and the local Ethical Committee. All collected teeth had a single root and a single canal. The coronal portions of the teeth were cut with a diamond-coated cylindrical bur to obtain standard 12 mm segments. Each root canal was mechanically shaped with a ProTaper(r) system (Dentsply, Maillefer, Ballagues, Switzerland) till reaching the F1 file (\(\O\) = 0.20 mm). The canals were irrigated with sterile saline and were dried with sterile paper points. The apex of each tooth was sealed with a flowable light-cured resin (Tetric Flow(r), Ivoclar Vivadent, Italy). Each tooth was put into a 1.5 mL Eppendorf tube in an upright position. Then they were sterilized in an autoclave at 121 \({}^{\circ}\)C for 15 min. _E. faecalis_ strain (CCM2541) was grown in brain heart infusion (BHI) (Oxoid Ltd., Hampshire, UK) broth and incubated for 24 h at 37 \({}^{\circ}\)C in a humid atmosphere. A dilution (1:50) with a sterile 0.9% NaCl was prepared and incubated for 3 h at 37 \({}^{\circ}\)C. After that, the optical density (OD\({}_{600}\)) was measured to obtain a suspension with OD\({}_{600}\) equal to 0.1 (corresponding to about \(5\pm 1.3\times 10^{8}\) colony-forming units/mL (CFUs)/mL) with which teeth were inoculated [6]. A total of 5 \(\upmu\)L of this suspension, containing about \(2.5\pm 0.7\times 10^{6}\) CFUs, was carefully injected in each canal using a lab pipette to avoid any contamination of the outer surface of the tooth. Each tooth was placed in an Eppendorf tube and incubated for 72 h in a humid atmosphere at 37 \({}^{\circ}\)C. During the experimental procedures, four teeth were damaged and eliminated from the experiment due to the presence of cracks in 3 teeth after the cutting of the coronal portion and due to the occurrence of overcutting in one tooth (<12 mm). The remaining 50 teeth were randomly distributed into six groups (Table 1). The first group (9 teeth) was considered a negative control group, since the teeth were not subjected to any treatment. Group A (8 teeth) was considered as a positive control group and only chemo-mechanical endodontic treatment was performed. Groups B (8 teeth) and C (8 teeth) were used to evaluate the PDT efficacy; therefore, the teeth were subjected to mechanical instrumentation (with irrigation of 0.9% NaCl) and PDT application. Groups D (8 teeth) and E (9 teeth) were employed to analyze the efficacy of the combination of PDT and chemo-mechanical treatment. The chemo-mechanical debridement treatment (groups A, D, and E) was as follows: Irrigation with 5% of NaOCl; then mechanical preparation till reaching a file F2 of the Pro Taper(r) system (\(\O\) = 0.25 mm). Irrigation with 5% of NaOCl for 1 min, washing with 5% sodium thiosulfate in order to neutralize the NaOCl, and washing with sterile saline. The mechanical instrumentation (groups B and C) was as follows: Irrigation with 0.9% NaCl; then mechanical preparation till reaching a file F2 of the ProTaper(r) system (\(\O\) = 0.25 mm), and finally, irrigation with sterile saline.