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Control of Oral Biofilm Formation by an Antimicrobial Decapeptide
K.-P. Leung1,*,
T.D. Crowe1,
J.J. Abercrombie1,
C.M. Molina1,
C.J. Bradshaw1,
C.L. Jensen1,
Q. Luo2 and
G.A. Thompson2
1 Microbiology Branch and
2 Biomaterials Branch, US Army Dental and Trauma Research Detachment, Walter Reed Army Institute of Research, 310B, B Street, Building 1H, Great Lakes, IL 60088, USA
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Figure 1. Schematic diagram of the dual-flow cell model. (A) The flow system. Arrowheads indicate the direction of the flow. The system is connected by 14-gauge Masterflex tubing (Cole-Palmer, Vernon Hills, IL, USA). For pulsed treatment of biofilms with KSL, a syringe pump (KD Scientific, Holliston, MA, USA) with 2 injectable syringes containing respective treatment and control solutions was directly connected to each of the flow chambers through a three-way valve. (B) The dual-flow cell. The flow cell consists of 2 parallel flow chambers, each of which contains 3 recesses for holding Ge disks. The inner diameter and depth of each recess are 10.25 mm and 2.0 mm, respectively. Holes with a diameter of 2.0 mm for flow inlets and outlets are drilled in each end of the flow chamber. The flow chambers are contained on one side by the polycarbonate bottom plate, and on the other side by an aluminum cover plate containing 2 parallel 60 mm x 24 mm no. 2 cover glasses. (C) Cross-section of the flow chamber, showing the dimensions of the flow channel (0.4 mm deep, 13 mm wide, and 25 mm long).
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Figure 2. Effect of KSL on oral biofilm development in a dual-flow cell, as revealed by DIC microscopy. (A) The continuous perfusion of a biofilm flow cell with KSL-containing (50 µg/mL) medium prevents biofilm formation. Images of untreated biofilm cells (a-c, negative control), showing the development of biofilms from salivary bacteria adhered to saliva-conditioned Ge surfaces in the flow chamber perfused with KSL-free medium. Images of KSL (50 µg/mL)-treated biofilm cells (d-f). Side-by-side images of treated vs. untreated cells were obtained at intervals of 2 hrs (a,d), 5 hrs (b,e), and 8 hrs (c,f) following inoculation of the parallel chambers of the dual-flow cell. (B) Perfusion of the chamber with a lower concentration of KSL-containing medium (10 µg/mL) was less effective in preventing biofilm formation. Untreated (a,b) and treated (c,d). Images were obtained at intervals of 2 hrs (a,c) and 8 hrs (b,d) following inoculation. Results represent 1 of the 3 experiments. Magnification, 200X. Bars represent 50 µm.
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Figure 3. DIC images of oral biofilm cells on Ge surfaces pulse-treated with KSL-free (a-c) and KSL-containing (50 µg/mL) medium (d-f). Pulsed treatment (30 min at 0.2 mL/min at two-hour intervals) initiated 4 hrs (A) or 6 hrs (B) after inoculation. Growth of biofilms was greatly inhibited in the flow chamber pulse-treated with KSL 4 hrs, but not 6 hrs, after inoculation. Images of treated vs. untreated biofilm cells were obtained at intervals of 2 hrs (a,d), 6 hrs (b,e), and 10 hrs (c,f) after inoculation of salivary bacteria into the parallel chambers of the dual-flow cell. The data represent the results of 1 of the 3 separate experiments. Magnification, 200X. Bars represent 50 µm.
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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 BacLightTM 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.
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Journal of Dental Research, Vol. 84, No. 12,
1172-1177 (2005)
DOI: 10.1177/154405910508401215
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