Inhibitory effects of lactoferrin on growth and biofilm formation of Porphyromonas gingivalis and Prevotella intermedia

Abstract

Lactoferrin (LF) is an iron-binding antimicrobial protein present in saliva and gingival crevicular fluids, and it is possibly associated with host defense against oral pathogens, including periodontopathic bacteria. In the present study, we evaluated the in vitro effects of LF-related agents on the growth and biofilm formation of two periodontopathic bacteria, Porphyromonas gingivalis and Prevotella intermedia, which reside as biofilms in the subgingival plaque. The planktonic growth of P. gingivalis and P. intermedia was suppressed for up to 5 h by incubation with >or=130 microg/ml of human LF (hLF), iron-free and iron-saturated bovine LF (apo-bLF and holo-bLF, respectively), and >or=6 microg/ml of bLF-derived antimicrobial peptide lactoferricin B (LFcin B); but those effects were weak after 8 h. The biofilm formation of P. gingivalis and P. intermedia over 24 h was effectively inhibited by lower concentrations (>or=8 microg/ml) of various iron-bound forms (the apo, native, and holo forms) of bLF and hLF but not LFcin B. A preformed biofilm of P. gingivalis and P. intermedia was also reduced by incubation with various iron-bound bLFs, hLF, and LFcin B for 5 h. In an examination of the effectiveness of native bLF when it was used in combination with four antibiotics, it was found that treatment with ciprofloxacin, clarithromycin, and minocycline in combination with native bLF for 24 h reduced the amount of a preformed biofilm of P. gingivalis compared with the level of reduction achieved with each agent alone. These results demonstrate the antibiofilm activity of LF with lower iron dependency against P. gingivalis and P. intermedia and the potential usefulness of LF for the prevention and treatment of periodontal diseases and as adjunct therapy for periodontal diseases.

FIG. 1.

Inhibition of planktonic growth of P. gingivalis by LF-related agents. About 10⁴ cells/ml of P. gingivalis JCM 8525 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs (n = 3) of one representative result from two similar results. *, P < 0.05 compared with the result for the control (no test agent) at 5 or 8 h; **, P < 0.001 compared with the result for the control (no test agent) at 5 or 8 h.

FIG. 2.

Inhibition of planktonic growth of P. intermedia by LF-related agents. About 10⁴ cells/ml of P. intermedia ATCC 25611 were incubated with the indicated concentrations of hLF (A), apo-bLF (B), holo-bLF (C), or LFcin B (D) in TSB for 8 h. Bacterial growth was determined by ATP-based luminescence quantification and is expressed as RLU. Values are means ± SDs (n = 3) of one representative result from two similar results. *, P < 0.05 compared with the result for the control (no test agent) at 5 or 8 h; **, P < 0.001 compared with the result for the control (no test agent) at 5 or 8 h.

FIG. 3.

Inhibition of biofilm formation of P. gingivalis and P. intermedia by LF-related agents. About 10⁷ cells/ml were incubated in TSB with hemin, vitamin K₁, and yeast extract in polyvinylchloride plates for 24 h. Biofilm formation was determined by crystal violet staining and is expressed as the OD₅₅₀. P. gingivalis JCM 8525, ATCC 33277, and ATCC 53978 and P. intermedia ATCC 25611 and ATCC 49046 were treated with the indicated concentrations of native bLF (A). P. gingivalis ATCC 33277 (B) and P. intermedia ATCC 25611 (C) were treated with the indicated concentrations of hLF, apo-bLF, native bLF, holo-bLF, or LFcin B. Values are means ± SDs (n = 3) of one representative result from two similar results. *, P < 0.05 compared with the result for the control (no test agent); **, P < 0.001 compared with the result for the control (no test agent).

FIG. 4.

Phase-contrast micrographs of a P. gingivalis biofilm. About 10⁷ cells/ml of P. gingivalis ATCC 33277 were incubated with 0 mg/ml (A), 0.031 mg/ml (B), or 0.5 mg/ml (C) of native bLF in TSB with hemin, vitamin K₁, and yeast extract in polystyrene plates for 24 h. After crystal violet staining, the biofilm that formed on the plate was observed microscopically. P. gingivalis developed a substantial biofilm (A), but biofilm formation was significantly inhibited in the presence of native bLF (B and C). Bars, 50 μm.

FIG. 5.

Reduction of preformed biofilm of P. gingivalis and P. intermedia by LF-related agents. About 10⁷ cells/ml of P. gingivalis ATCC 33277 (A) or P. intermedia ATCC 25611 (B) were incubated in TSB with hemin, vitamin K₁, and yeast extract in polyvinylchloride plates for 24 h. The preformed biofilm was then incubated with the indicated concentrations of hLF, apo-bLF, native bLF, holo-bLF, or LFcin B in saline for 5 h. The amount of remaining biofilm was measured by crystal violet staining and is expressed as the OD₅₅₀. Values are means ± SDs (n = 3) of one representative result from two similar results. *, P < 0.05 compared with the result for the control (no test agent); **, P < 0.001 compared with the result for the control (no test agent).

FIG. 6.

Effects of combination of LF and antibiotics on a preformed P. gingivalis biofilm. About 10⁷ cells/ml of P. gingivalis ATCC 33277 were incubated in TSB with hemin, vitamin K₁, and yeast extract in polyvinylchloride plates for 24 h. After the removal of the planktonic bacteria, the preformed biofilm was incubated with the indicated concentrations (μg/ml) of ABPC, CPFX, CAM, or MINO and 0.5 mg/ml of native bLF (A) or 0.5 mg/ml of apo-bLF (B) in saline for 24 h. (A) The amount of remaining biofilm was measured by crystal violet staining and is expressed as the mean percentage ± SD of the amount of the control biofilm. The experiment was performed in triplicate, and the results of two different experiments were combined and are presented here. (B) The viability of the bacteria in the biofilm was measured by ATP-based luminescence quantification in triplicate and is expressed as the mean RLU ± SDs. *, P < 0.05 compared with the result obtained with no bLF; #, P < 0.05 compared with the results obtained with no antibiotic.