# Therapeutic Potential of Gramicidin S in the Treatment of Root Canal Infections
## 2 Results
### Biofilm-Killing Effect of GS  
For these experiments, it was necessary to determine the biofilm forming capacity of all _E. faecalis_ isolates to choose the strongest biofilm formers and the most suitable medium. A standard crystal violet staining assay was applied to evaluate the mass of the biofilms that were pre-grown for 24 h in 96-well microtiter plates (Figure 1A).  
The strongest biofilm growth was found to occur in the Todd Hewitt (TH) broth, which contains glucose. The Mueller Hinton (MH) broth and minimal medium were less suitable for biofilm formation. The biofilm-forming capacity of the control strain DSM 2570 was comparable with that of the tetracycline-resistant WW4 (cheese isolate) and the tetracycline-susceptible WW6 (root canal isolate) (Table 1). The tetracycline-resistant clinical isolate _E. faecalis_ TRE2 was the best biofilm former. Scanning electron microscopy (SEM) showed that the cells in this biofilm completely covered the surface of the hydroxyapatite disc (HAD) and were attached to the HAD nanoparticles (Figure 1B1).  
Figure 2: Reduction of the number of _E. faecalis_ bacteria during exposure to GS: (**A**) control strain DSM 2570; (**B**) cheese isolate WW4; (**C**) root canal isolate WW6; and (**D–F**) clinical tetracycline-resistant isolates TRE1, TRE2, and TRE4, respectively. The bacterial number remained unchanged in the controls (dotted lines), but decreased drastically during exposure to 5 \(\times\) MIC of GS (solid lines). The dashed lines show the faster bactericidal effect of 10 \(\times\) MIC for DSM 2570 and WW6 strains; 10 CFU/mL was taken as the detection limit although no colonies were grown from undiluted sample. The standard deviations were calculated from at least two independent experiments.  
After exposure to 400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of GS for 18 h, the cell morphology of _E. faecalis_ TRE2 was drastically altered (Figure 1D1) in comparison to the control or to the treatment with 400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of demeclocycline (Figure 1C1). The GS-treated cells decreased in size from 1 to 0.6-0.8 \(\mathrm{\SIUnitSymbolMicro m}\), the cell surface got wrinkled, and the cells detached from the HAD particles (Figure 1D1). Treatment of the pre-grown biofilms with 400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of GS led to a disappearance of the matrix layer and to a partial elimination of biofilm cells, so that the nanoparticles of the HAD were clearly exposed underneath the cells. No re-growth of the robust TRE2 biofilm in the fresh TH broth was observed, even when only 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL GS has been applied (Figure 1D2). This concentration represents the minimal biofilm bactericidal concentration (MBBC) of GS against _E. faecalis_ TRE2, because subsequent plating of this non re-grown culture on nutrient agar did not reveal any viable cells. The control biofilm (Figure 1B2) as well as the biofilm after demeclocycline treatment (Figure 1C2) were clearly found to re-grow in the fresh TH nutrient broth after cultivation at 37 \(\mathrm{\SIUnitSymbolCelsius}\) for 24 h. The second strongest biofilm former, the tetracycline-susceptible root canal isolate _E. faecalis_ WW6 (Figure 1A), did not exhibit re-growth after treatment with 400 \(\mathrm{\SIUnitSymbolMicro g}\)/mL demeclocycline, as expected due to its high susceptibility (see Table 1). We note, however, that the MBBC of GS against this strain was even lower (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) (data not shown).