Twenty freshly extracted single rooted fully developed intact human teeth were stored in 0.05% sodium hypochlorite solution. Informed consent was obtained from the extracted teeth donors.

This study was approved by the Tel Aviv university ethics committee.

The crowns of the selected teeth were removed in order to obtain multiple root specimens of 13 mm length, and the apical 3 mm of the root end was resected without a bevel using Zakaria high speed bur (Maillefer, Ballaigues, Switzerland). The root canal lumen was then enlarged to a minimum of 0.5 mm using low speed burs (Gates Glidden Drills, Dentsply Maillefer, Tulsa, OK, USA). The roots were embedded in self-cure acrylic repair material (UNIFAST Trad, GC America). To prepare the dentin slabs, the roots were cut perpendicular to the long axis of the root under water cooling with a diamond saw rotating at 500 rpm (Isomet, Buehler Ltd., Lake Bluff, IL, USA). Two dentin slabs of 1 mm thickness each were obtained from each root (Kuci et al., 2014), see Supplementary Figure S1. The specimens were then placed in small dishes and sterilized overnight using ethylene oxide gas (Brosco et al., 2010). For each of the indicated treatment at least nine independent dentin slabs were evaluated under the same conditions.

All of the experiments were performed in a clinical isolate of Enterococcus faecalis 29212 (Minogue et al., 2014). To confirm reproducible results we evaluated biofilm formation of a single virulent strain on-top of artificial and biological surfaces.

The strains were routinely manipulated in LB broth (Difco), or in TSB broth (Difco), enriched with 0.5% glucose (Sigma) (Lopez and Kolter, 2010).

Cells were grown in LB from a single colony isolated over LB plates to a mid-logarithmic phase of growth (6 h at 37°C with shaking). To grow biofilms, 1.5 μl of starter culture was inoculated into the TSB glucose media in 96-well polystyrene plates and further incubated for 24 h. The growth media were either applied or not with one of the following substances: (i) A Sodium hypochlorite was added to the final concentration of 0.6% from a stock solution of sodium hypochlorite solution, reagent grade, 10–15% (sigma), (ii) D-Leucine (Sigma–Aldrich) was added to the final concentration of 2 mM from a stock solution of 76 mM D-Leucine in DDW, (iii) flavomycin (AK Scientific) was added to the final concentration of 2 μg/ml from a stock solution of 2 mg/ml (iv) EDTA (Sigma–Aldrich) was added to the final concentration of 0.5 mM from a stock solution of 50 mM (0.5 mM), and (v) 2-2′ bipyridyl (Sigma–Aldrich) was added to a final concentration of 10 μg/ml from a stock solution of 10 mg/ml in ethanol. In the case of the 96-well polystyrene plate, the crystal violet (Sigma–Aldrich) assay was performed as described by Friedman and Kolter (2004).

Cells were grown from a single colony isolated from LB plates to a stationary phase of growth (12 h at 37°C with shaking). The culture was then diluted 1:25 in 5 ml liquid TSB glucose medium (Thermo Scientific). Cells were grown with agitation at 37°C for 10 h in a growth chamber, and the optical density at 600 nm (OD₆₀₀) was measured every 2 h. Cells were either grown in presence or absence of cell-wall inhibiting molecules, as indicated in the corresponding figure legend.

Cells were grown in LB from a single colony isolated over LB plates to a mid-logarithmic phase of growth (6 h at 37°C with shaking). To grow biofilms, 1.5 μl of starter culture was inoculated into 6 ml TSB glucose media dispensed into a Petri dish containing the dentin disks. Cells were grown on-top of dentin disks as described above. Following 24 h of growth, the media was removed from the dentin disks, and the associated bacteria were incubated in different substances and treated further, as specified in the legends for each figure. Following incubation, the suspension solution was collected and further evaluated for the live cell counts as well as the biofilm fraction. The biofilm fraction was obtained by three washes of the dentin disks with phosphate buffer to the final volume of the suspension solution. To determine the number of live cells, cells were serially diluted in phosphate-buffered saline (PBS; Biological Industries, Israel), plated on LB plates, and colony forming units (CFU) were counted after incubation at 37°C overnight as done by us previously (Bucher et al., 2015).

To determine culture density and live cell counts of cells grown on the disks, cells were harvested from a dentin biofilm dispersal assay (described above): Cells were grown on-top of dentin disks as described above. Following 24 h of growth, the media was removed from the dentin disks, and the associated bacteria were incubated in different substances and treated further, as specified in the legends for each figure. Samples of biofilms grown for 24 h and treated as indicated were stained using LIVE/DEAD BacLight Bacterial Viability kit L-7012 for microscopy and quantitative assays (Molecular Probes, Eugene, OR, USA) containing separate vials of the two component dyes (SYTO 9 and propidium iodide in 1:1 mixture) in solution was used for staining of the biofilm following the manufacturer’s instructions. The excitation/emission maxima for these dyes is approximately 480–500 nm for the SYTO 9 stain and 490–635 nm for propidium iodide (Aziz et al., 2010). Fluorescence from the stained cell was viewed under a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems CMS GmbH Germany). Single channel and simultaneous dual-channel imaging was used to display green and red fluorescence (Zapata et al., 2008). Confocal laser scanning microscope images of the biofilms were acquired by the LAS AF software (version 2.6.0.7266; Leica Microsystems CMS GmbH) at a resolution of 512 × 512 pixels. The mounted specimens were observed using a X4 lens. Confocal LIVE/DEAD images were analyzed and quantitated using the above mentioned software (LAS AF; Zapata et al., 2008; Shen et al., 2010; Kuci et al., 2014). The specimens were coded for blind evaluation.

All studies were performed in duplicates or triplicates at least three separate and independent times. Data are expressed as average values ± standard deviations of the means. Parametric testing was performed after confirming that raw data were normally distributed. Data were analyzed by student’s t-test, used to determine if the set of treated versus the untreated control are different from each other (A paired t-test comparing two sets of measurements) differ and P values of less than 0.1 were considered significant.

Systematic evaluation of small molecule biofilm inhibitors was performed on planktonic growth (Figure 1) and biofilm formation, according to the Microtiter Dish Biofilm Formation Assay (Friedman and Kolter, 2004). Three categories of biofilm inhibitors were tested: (i) Small molecules that target the cell envelope: Flavomycin, an antibiotic that inhibits transglycosylation directly by binding the transglycosylation domain of PBP enzymes (Dengler et al., 2011); and D-Leucine, a non-canonical D-amino acid, which competes with D-Alanine for the fifth position in the B. subtilis pentapeptide (Bucher et al., 2015), and interferes with transpeptidation (Lam et al., 2009; Cava et al., 2011; Lupoli et al., 2011) and transglycosylation (Lam et al., 2009). (ii) Cation chelators: EDTA, a chelating agent that sequesters a variety of polyvalent cations such as calcium; and 2,2′-bipyridyl, an organic bidentate chelating ligand, forming complexes with many transition metals, with a strong affinity to iron. (iii) Sodium hypochlorite (bleach) – commonly used concentrations between 0.5 and 6% for irrigation in root canal treatments (Bystrom and Sundqvist, 1985; Gomes et al., 2001; Haapasalo et al., 2014).

For planktonic growth, sodium hypochlorite turned out to be most toxic, eliminating E. faecalis growth altogether (Figure 1), EDTA, flavomycin and 2-2′ bipyridyl inhibited (reversibly) bacterial growth at indicated concentrations, and D-Leucine at concentrations of up to 2 mg/ml had little or no effect on planktonic growth.

In contrast, the results from the assay on biofilm formation in polystyrene wells were as following: the mildly toxic 2-2′ bipyridyl and flavomycin had a modest inhibitory effect on biofilm formation, while EDTA (mildly toxic), D-Leucine (non-toxic), and sodium hypochlorite (highly toxic) inhibited biofilm formation comparably and significantly. These results indicate that inhibition of biofilm formation may not be directly correlated to inhibition of planktonic growth.

In order to establish a more ecological dentin model, root dentin slabs of 1 mm thickness each were cut as described in the section “Materials and Methods” (Supplementary Figure S1). The sterile dentin disk was then inoculated with E. faecalis and further incubated in biofilm media. As shown, E. faecalis cells formed a thick biofilm on the dentin slab within 24 h. Once a biofilm was established, we used the several biofilm inhibitors that proved to effectively inhibit biofilm formation on-top of polystyrene plates (Figure 2) and evaluated their effect on dispersing root-dentin associated biofilms. For this purpose, we first scored the remaining biofilm using the Live/Dead BacLight Viability Kit (Figure 3). The outcome of the application of biofilm inhibitors to an established dentin-associated biofilm differed dramatically between treatments. Surprisingly, the sodium hypochlorite treatment, found to be most toxic to planktonic growth, had little effect on removal of the biofilm biomass, and only modestly impacted the overall viability of the biofilm cells. The concentration of flavomycin which halted planktonic growth and biofilm formation (Figures 1 and 2) had no impact on established biofilms (Supplementary Figure S2), and chelation of cations by EDTA had little or no effect on the overall biofilm’s biomass. However, EDTA treatment significantly increased the proportion of dead cells (Figures 3 and 4). Specific chelation of iron by 2-2′ bipyridyl greatly increased cell death within dentin-associated biofilms (Figures 3 and 4). The most efficient treatment was D-Leucine, as it significantly dispersed the biofilm’s biomass (Figure 3) and modestly increased the fraction of dead cells compared with the control. Importantly, the D-Leucine treatment was found to be effective in sub-toxic concentration compatible with endodontic therapy.

To further evaluate the viability of the E. faecalis following different treatments, we assessed the replicative cell counts from the treated biofilms and the growth media. Strikingly, though the conservation of the biofilm biomass following sodium hypochlorite treatment was evident between different experiments (Figure 3), very few culturable cells could be eluted from the dentin disks and the inoculation media (Figure 5). In contrast, replicative cell counts from other treatments correlated better with the confocal examination. This result could be an indication that the sodium hypochlorite treatment is promoting a viable but not culturable state (VBNC) in root-associated biofilms.