Antibacterial effectiveness of multi-strain probiotics supernatants intracanal medication on Enterococcus faecalis biofilm in a tooth model

The manuscript of this laboratory study has been written according to Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021 guidelines [23] (Fig. 1). Ethical approval was obtained from the Research Ethics Committee, Faculty of Dentistry, Alexandria University (IRB 00010556 – IORG 0008839). Informed consent was obtained from all subjects and/or their legal guardian(s) who voluntarily agreed to dontae their extracted teeth for the current study.

Sample size calculations were performed using MedCalc Statistical Software (MedCalc Software bvba, Ostend, Belgium; http://​www.​medcalc.​org; 2018). The sample size was planned on a significance level of 5% α and a power of 80%. It was calculated to be 5 per group increased to 7 to compensate for any potential laboratory errors [24, 25]. The total sample size required to compare the antibacterial effectiveness of different intra-canal medications at different follow-up intervals = number of subgroups × number of specimens per subgroup = 8 × 7 = 56 [26]. One extra sample was added to each main group for biofilm validation by Scanning electron microscope (SEM). Therefore, a total of 60 freshly extracted, for periodontal involvement, and caries-free human permanent single-rooted teeth with a single canal were used in the current study. Extracted teeth were collected from the outpatient clinic of the Oral and Maxillofacial Surgery Department, Faculty of Dentistry, Alexandria University.

Poloxamer gel 30% (w/w) was prepared using the ‘cold technique’ under a magnetic stirrer (SCILOGEX, Rocky Hill, USA.) by adding poloxamer 407 powder (Sigma Aldrich Chemical Co., Gillingham, UK.) to sterile distilled water (4–8°C) then kept in the refrigerator for 24 h. Three strains of Lactobacilli Probiotics (Lactobacillus plantarum ATCC 14917, Lactobacillus rhamnosus ATCC 7469, and Lactobacillus acidophilus ATCC 4356) were obtained from Microbiological Resources Centre (MIRCEN). They were grown individually on deMan Rogsa Sharpe (MRS) broth (HiMedia Laboratories Pvt. Ltd, India) aerobically for 48 hours at 37°C [27]. The pure isolate of Lactobacilli species suspension was propagated in a 100 ml flask containing MRS broth and incubated for 72 hours at 37°C. The cell-free supernatant (CFS) was obtained by centrifuging the culture at 10,000 rpm for 10 minutes, then lyophilized using a vacuum freeze dryer (Model FDF 0350, Korea). In a previous study [27], the minimum concentration of MSP to initiate an inhibitory response against E. faecalis was 50 mg/ml as verified by agar well diffusion assay. Higher concentrations of MSP of 100, 200, and 300 mg/ml showed bigger zones of inhibition. In the current study, 300 mg of the obtained lyophilized CFS for each supernatant was mixed with 1ml poloxamer gel under a magnetic stirrer in an ice bath to get the MSP gel at a targeted concentration of 300 mg/ml.

An ultrasonic scaler (Woodpecker,China.) was used to remove all calculus deposits if found, followed by ultrasonic cleaning in distilled water for 10 min and then immersed in 5.25% sodium hypochlorite for 30 min to remove soft tissue attached to the root surface. Teeth were decoronated, to a standard 14 mm root length, using a diamond disc mounted on a low-speed handpiece under water coolant. The working length (WL) was established by subtracting one millimeter from the full length of the decoronated teeth. A k-file no. 15 (MANI,Japan.) was inserted gently into the root canal at the estimated working length for radiograph taking to confirm the WL. The remaining roots were mechanically cleaned and shaped using the ProTaper Next rotary system (Dentsply Sirona Maillefer, Ballaigues, Switzerland.) up to size X3 (0.30/7%) and irrigated with 3ml of 2.5% sodium hypochlorite. The smear layer was removed by flushing the root canal with 3 ml 17% EDTA solution ( Dharma, Miami, Florida, USA) for 5 min followed by 3ml of 2.5% sodium hypochlorite. Final irrigation was employed using 5% sodium thiosulfate (PIOCHEM,Egypt) to inactivate sodium hypochlorite. The outer surfaces of the roots were covered with a double layer of nail varnish then the apical foramen of the teeth was sealed with a light-cured resin composite (Nexcomp, Dentsply Maillefer, Ballaigues, Switzerland).

Each root was individually placed in a cryovial containing 500 µl of brain heart infusion (BHI) broth (HiMedia Laboratories Pvt. Ltd, India) and autoclaved at 121° C for 30 min. Roots were then separately mounted vertically on silicon impression material (zeta plus condensation silicone, Zhermack, Badia Polesine, Rovigo, Italy) and underwent a second round of autoclave cycle to ensure the sterility of the setup. Subcultures on blood agar from randomly selected samples were obtained to verify sterilization after incubation for 48 h at 37° C.

E. faecalis (ATCC29212) was grown on a blood agar-sealed petri dish at 37° C for 24 h. The colonies were suspended in 5 ml of BHI broth and adjusted by spectrophotometer to a concentration of 1 McFarland. Each root canal was inoculated with E. faecalis suspension using a sterile insulin syringe and incubated at 37 °C for 21 days under aseptic aerobic conditions. The inoculation was repeated every 3 days to ensure the viability of the bacteria [28, 29].

Four randomly selected specimens were sectioned longitudinally using a diamond disc, fixed by immersion in 4F1G solution (40% formalin and 25% glutaraldehyde in phosphate buffer) for 3 h at 4 °C then immersed in 2% osmium tetroxide OsO₄ for 2 h at 4 °C, dehydrated at 4 °C through a graded series of ethanol, dried at room temperature, mounted using carbon paste on a copper stub, and sputter-coated with gold Surface roughness. Secondary electron images at 5,000 X and 10,000 X were obtained using SEM (JEOL JSM-IT200,Tokyo, Japan.) to examine and validate the formed E. faecalis biofilm on each root canal.

The remaining 56 roots were dried with sterile paper points and then randomly assigned, for treatment by ICM, into four groups of 14. Groups were treated, respectively, by one of three ICM using insulin syringes: (i) Probiotics supernatants: MSP 300 mg/ml in poloxamer gel, (ii) Ca(OH)₂: calcium hydroxide paste (35% Ultra Cal XS Ca(OH)₂), (iii) Poloxamer: poloxamer vehicle gel, while untreated E. faecalis-loaded roots served as a control group. The coronal surface of the roots was sealed with a sterile Teflon pellet topped with temporary filling material (Ultratemp, Ultradent) and incubated at 37℃. Half of the specimens in each treated group were incubated for 1 day, while the other half was incubated for 7 successive days [25, 30].

At the end of each incubation period, the temporary filling material was removed and each root canal from all tested and control groups was irrigated with 6 ml of 17% EDTA followed by 6 ml of distilled water [31]. Ca(OH)₂ groups were additionally washed with 1 ml of 0.5% citric acid solution to neutralize Ca(OH)₂ then final irrigation with 3 ml sterile saline was done [22]. In an anaerobic workstation for bacterial culture analysis, each root was then mounted on a custom-made holder device to hold each root. At the base of the holder, a cryovial containing BHI was placed 1 cm from the root apex. A representative sample was collected by one same operator from each root canal by gentle manual scraping of dentinal walls, 1 mm shorter than WL, by Hedstrom file (H-file) #30 (MANI, Japan.) for 30 s to obtain dentin chips [30]. Dentin chips were collected by inserting three paper points of equivalent size 1 mm short of the WL and kept for 60 s. The contaminated paper points and H-files were placed into a cryovial containing 1ml BHI and were vortexed for 60 s and then 10-fold serially diluted. Sterile spreaders were used to collect 10µL from the resulting serial dilution to be immediately plated in 4 sealed blood agar petri dish, for each tested group, and incubated at 37° C for up to 24 h [22]. The antibacterial activity was then assessed quantitatively through the calculation of the Colony-forming units per milliliter (CFUs/ml) for each dish using a colony counter (Fisher Scientific, Waltham, MA, USA.) following the methods of Barbosa-Ribeiro et al. [32] and Martinho and Gomes [33]. Bacterial load reduction percentages between 24 h and 7 days were calculated following this mathematical formula [34]:

A further qualitative antibacterial assessment was done on the treated groups under the SEM. Two randomly treated roots from each group were longitudinally sectioned. Their apical third were dissicated, gold/palladium-coated and investigated under SEM to assess the presence of E. faecalis biofilm at a microscopic level (Fig. 2).