Figure 4. Interactions of KSL with oral biofilms. (A) Effect of KSL on intact vs. disrupted 45-hour biofilms formed on saliva-coated HA disks by salivary bacteria, analyzed by the in vitro plaque assay. A Mann-Whitney test was used for comparison of log reductions in CFU between the experimental groups (KSL-treated intact or disrupted biofilms) and the control groups (dH₂O-treated intact or disrupted biofilms). The single asterisk represents a statistically significant difference between KSL- and dH O-treated intact biofilms (p < 0.05). Likewise, double asterisks represent a statistically significant difference between KSL- and dH₂O-treated disrupted biofilms (p < 0.01). While KSL caused slight reductions in CFU of treated, intact biofilms, chlorhexidine (CHX) caused more reduction in viability of intact biofilms. (B) Effect of benzalkonium chloride in promoting the bactericidal activity of KSL against 66-hour-old intact oral biofilms formed on saliva-coated HA disks in the in vitro plaque system. We used a Kruskal-Wallis test to compare log reductions in CFU among various treatment groups, including the control group (dH₂O-treated). The single asterisk represents a statistically significant difference between the combined treatment of KSL and benzalkonium chloride (Bzl) and dH₂O (p < 0.001)-, KSL (p < 0.01)-, or Bzl (p < 0.01)-treated intact biofilms. Double asterisks represent a statistically significant difference between CHX- and dH₂O (p < 0.001)- or Bzl (p < 0.05)-treated intact biofilms. While KSL or Bzl alone, as compared with the dH₂O-treated group, caused no significant reductions in the viability of intact biofilms, the combined use of KSL and Bzl had a significant effect on the viability (over one log reduction of viable counts) of these 66-hour-old oral biofilms. No significant difference in viability counts was observed between CHX-treated vs. the combined use of KSL and Bzl. For (A) and (B), the data represent the determinations of 1 of 3 separate experiments, each performed in quadruplicate. Bars represent standard deviations. (C) Confocal images of control and treated biofilms grown on saliva-coated HA surfaces. We used the Live/Dead BacLight^TM Viability kit (Molecular Probes, Eugene, OR, USA) to assess the viability of biofilm cells exposed to different treatments. A BacLight assay solution was prepared as described by the manufacturer, and the specimens were stained in the dark at room temperature for 15 min. After being washed 3x with water, samples were observed with an Axioplan light microscope fitted with an Ar-Kr laser (Zeiss LSM 510 Meta, Thornwood, NY, USA) and water immersion (long working distance) objectives. An excitation wavelength of 488 nm was used, and the fluorescence light emitted was collected by 2 separate emission filters, 500-530 nm (SYTO 9, live), and 650–710 nm (propidium iodide, dead). As compared with the control (1a,1b), which showed mostly green-staining biofilm cells (indicating live), CHX (2a,2b) or combined use of KSL and Bzl (5a,5b) significantly reduced the viability of biofilm cells, indicated by the presence of mostly red-staining biofilm cells (indicating dead). KSL (3a,3b) or Bzl (4a,4b) alone, at indicated concentrations, had less impact on the viability of biofilm cells. Panels 1a-5a represent horizontal (xy) sections through biofilms, whereas panels 1b-5b are sagittal (xz) images of biofilms (indicated by the line on the horizontal xy sections) treated with different agents. Bars represent 50 µm.