Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia

Hiroyuki Wakabayashi; Koji Yamauchi; Tetsuo Kobayashi; Tomoko Yaeshima; Keiji Iwatsuki; Hiromasa Yoshie

doi:10.1128/AAC.01688-08

. 2009 May 18;53(8):3308–3316. doi: 10.1128/AAC.01688-08

Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia^▿

Hiroyuki Wakabayashi ^1,^*, Koji Yamauchi ¹, Tetsuo Kobayashi ^2,3, Tomoko Yaeshima ¹, Keiji Iwatsuki ¹, Hiromasa Yoshie ²

PMCID: PMC2715627 PMID: 19451301

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 ≥130 μg/ml of human LF (hLF), iron-free and iron-saturated bovine LF (apo-bLF and holo-bLF, respectively), and ≥6 μg/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 (≥8 μg/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.

Lactoferrin (LF) is an 80-kDa iron-binding glycoprotein of the transferrin family. LF is a component of exocrine secretions, such as saliva, tears, and milk; and it is present in neutrophil granules (25). LF is thought to play a role in innate defense and exhibits a diverse range of biological activities, including antimicrobial, antiviral, antioxidant, and immunomodulatory activities. In addition, it modulates cell growth and binds to several bioactive compounds, such as lipopolysaccharide (9, 13). LF inhibits the inflammatory response caused by lipopolysaccharide by binding to that molecule (9). LF inhibits bacterial growth by sequestering the iron essential for bacterial growth (31). In addition, potent antimicrobial peptides, lactoferricin H (LFcin H) and LFcin B, are derived from the N-terminal region of human LF (hLF) and bovine LF (bLF) by pepsin digestion, respectively (11). LFcin kills various microorganisms by membrane disruption, and the LFcin region seems to be the bactericidal domain of the LF molecule (42).

LF is one of the antimicrobial factors in saliva (39), and under inflammatory conditions, it is secreted from neutrophils to gingival crevicular fluid (GCF) (16). Therefore, LF has been studied for its activity against oral pathogens, including Streptococcus mutans (7), Candida albicans (38, 41), and periodontopathic bacteria (2, 21). Porphyromonas gingivalis and Prevetella intermedia are gram-negative black-pigmenting anaerobes that are etiologic agents of chronic adult periodontitis (26) and that reside as biofilms in the subgingival plaque (14, 27). hLF was shown to exhibit growth-inhibitory activity against P. gingivalis but not P. intermedia (2, 35), and LFcin B was found to have weak activity against P. gingivalis (35). hLF and bLF also inhibited the adhesion of P. intermedia to epithelial cells (4). Additionally, the hLF level in GCF correlates with the clinical severity of periodontal diseases, as well as the number of neutrophils (1). A decrease in hLF levels was observed in GCF and saliva after surgical periodontal treatment in patients with chronic periodontitis (19). Thus, LF in GCF may play a role in host defense against periodontopathic bacteria. Recently, in a small-scale clinical trial, we observed that bLF administration to patients with chronic periodontitis decreased the number of P. gingivalis and P. intermedia cells in the subgingival plaque (23). The aim of the current study was to assess the in vitro effect of LF-related agents, including various iron-bound forms of bLF and LFcin B, on the growth, biofilm formation, and preformed biofilms of P. gingivalis and P. intermedia.

MATERIALS AND METHODS

LF-related agents.

hLF was purified from the milk of healthy volunteers by a previously reported method (34). bLF was produced by Morinaga Milk Industry (Tokyo, Japan) and was used as native bLF. Iron-free apo-bLF was prepared by the depletion of iron through the incubation of native bLF in a 0.1 M citric acid solution and dialysis against distilled water. Iron-saturated holo-bLF was prepared from native bLF by the method reported previously (3). The binding of iron to the metal-chelating site of bLF was confirmed by measuring the maximum absorbance at 465 nm (3). The metal contents of these preparations were measured by atomic absorption spectrometry. The iron saturation rates were 3.5% for apo-bLF, 8.1% for native bLF, and 83.6% for holo-bLF. LFcin B was purified from bLF pepsin hydrolysate, as described previously (40).

Antibiotics.

Ampicillin (ABPC), ciprofloxacin (CPFX), clarithromycin (CAM), and minocycline hydrochloride (MINO) were obtained from Sigma-Aldrich (Tokyo, Japan). The antibiotics were dissolved in dimethyl sulfoxide and added to the test medium.

Bacteria.

P. gingivalis ATCC 33277^T and ATCC 53978 and P. intermedia ATCC 25611^T and ATCC 49046 were obtained from the American Type Culture Collection (Manassas, VA). P. gingivalis JCM 8525 was obtained from the Japan Collection of Microorganisms (Wako, Japan). All strains were grown and maintained on Trypticase soy agar (TSA; BD, Franklin Lakes, NJ) supplemented with 5% sheep blood, hemin (5 μg/ml), vitamin K₁ (0.5 μg/ml), and yeast extract (10 mg/ml) under an anaerobic atmosphere (10% CO₂, 5% H₂, 85% N₂) in a forced-air incubator (model 2000; Coy Laboratory Products, Grass Lake, MI). The bacteria were incubated at 37°C in Trypticase soy broth (TSB; BD) supplemented with hemin (5 μg/ml), vitamin K₁ (5 μg/ml), and yeast extract (1 mg/ml) overnight to prepare the inocula.

Susceptibility tests with planktonic bacteria.

To determine the growth-inhibitory activity, about 10⁴ bacteria/ml were incubated in 2 ml of TSB in the presence of fourfold serial dilutions of the test agents for 8 h. Bacterial growth was determined at defined intervals by ATP-based luminescence quantification. One hundred microliters of the bacterial culture was incubated with 100 μl of BacTiter-Glo microbial cell viability assay reagent (Promega, Madison, WI) in each well of a 96-well luminescence white plate (Greiner Bio-One, Tokyo, Japan) for 5 min. The luminescence was recorded by a Veritas microplate luminometer (Promega). Bacterial growth was calculated by subtracting the number of relative luminescence units (RLUs) at 0 h from that at the defined time. To determine bactericidal activity, about 10⁷ bacteria/ml were incubated in physiological saline (0.85% NaCl) with or without the test agents for 4 h. At defined intervals, each culture was collected, serially diluted, and incubated on a TSA plate for 3 to 5 days; and the numbers of CFU were counted.

Susceptibility tests with biofilms.

The amount of bacterial biofilm was measured by a microtiter plate biofilm assay by use of a previously reported protocol (28). In brief, about 10⁷ bacteria/ml were incubated in 100 μl of TSB supplemented with hemin (5 μg/ml), vitamin K₁ (5 μg/ml), and yeast extract (1 mg/ml) in the presence of fourfold serial dilutions of test agents in the wells of round-bottom polyvinylchloride 96-well microtiter plates (BD) for 24 h. After removal of the planktonic bacteria by washing the plates with water, 125 μl of 0.1% crystal violet solution was placed in each well and the plates were incubated for 10 min. After the plates were washed with water and air dried, 150 μl of 95% ethanol was placed in each well and the plates were incubated for 10 min. The optical density at 550 nm (OD₅₅₀) of 125 μl of the solution was measured with a microplate reader (MTP-32; Corona Electric, Ibaraki, Japan) and was used to define the amount of biofilm. For microscopic observation, the biofilm was developed in the wells of flat-bottom polystyrene 96-well microtiter plates (BD) and stained with crystal violet, and phase-contrast micrographs were taken. To determine the effects of the test agents on the preformed biofilm, the bacteria were incubated anaerobically in polyvinylchloride 96-well microtiter plates for 24 h, as described above for incubation without test agents. After the planktonic bacteria were removed, the biofilm was incubated anaerobically with 100 μl of physiological saline containing the test agents for 5 or 24 h. The amount of biofilm was then measured by crystal violet staining. The viability of the bacteria in the biofilm was measured by ATP-based luminescence quantification, as indicated above, except that each well was incubated with 100 μl of the BacTiter-Glo reagent for 15 min, after the removal of the planktonic bacteria, and 80 μl of the resultant solution was transferred to the luminescence white plate for measurement.

Statistical analysis.

Data are expressed as means ± standard deviations (SDs). Statistical analysis was done between two groups by two-tailed Student's t test. P values of <0.05 were considered to indicate a significant difference.

RESULTS

Effects of LF on planktonic P. gingivalis and P. intermedia.

Approximately 10⁴ cells/ml of P. gingivalis JCM 8525 (Fig. 1) and P. intermedia ATCC 25611 (Fig. 2) were incubated with different concentrations of LF-related agents in TSB not supplemented with an iron source for 8 h, and the growth was monitored by a viability assay by the use of ATP-based luminescence. All agents tested, including hLF, apo-bLF, and holo-bLF at 0.13 to 8 mg/ml and LFcin B at 0.006 to 0.4 mg/ml, suppressed the growth of P. gingivalis for up to 5 h (Fig. 1). The RLU for the control bacteria (which were incubated with no test agent) declined at 8 h, which may reflect growth arrest in a medium free of an iron source. Inhibition of the RLU (viability) by the test agents was weak or was not seen after 8 h, especially by lower concentrations of the test agents (0.13 to 2 mg/ml of LF and 0.006 mg/ml of LFcin B). The growth of P. intermedia was suppressed by hLF through 8 h of incubation (Fig. 2A). apo-bLF and holo-bLF suppressed the growth of P. intermedia until 5 h, but the effect was weak or tended to be opposite of the earlier effect at 8 h (Fig. 2B and C). The inhibitory effect of LFcin B against P. intermedia was seen more clearly at 8 h than at 5 h (Fig. 2D). Because LFcin B exhibits broad bactericidal activity (10) and because even hLF shows killing activity against some bacteria, including S. mutans, in saline (7), we tested the effects of bLF and LFcin B on the numbers of CFU of P. gingivalis and P. intermedia in saline. Approximately 10⁷ cells/ml of P. gingivalis JCM 8525 and P. intermedia ATCC 25611 were incubated with native bLF (8 mg/ml) or LFcin B (0.4 mg/ml) in physiological saline for 4 h (Table 1). The numbers of CFU of P. gingivalis and P. intermedia spontaneously decreased in saline. However, LFcin B further reduced the numbers of CFU of P. gingivalis and P. intermedia after 2 or 4 h of incubation compared with numbers of CFU in the control cultures, although bLF did not show such an inhibitory effect.

TABLE 1.

Effects of native bLF and LFcin B on number of bacteria^a

Bacterium	Treatment	CFU/ml at the following times (h):
Bacterium	Treatment	0	0.5	2	4
P. gingivalis	Control	3.1 × 10⁷	1.5 × 10⁷	5.8 × 10⁴	1.0 × 10³
	Native bLF	NA	2.0 × 10⁷	5.8 × 10⁶	<5.0 × 10²
	LFcin B	NA	8.0 × 10⁶	<5.0 × 10²	<5.0 × 10²
P. intermedia	Control	2.1 × 10⁷	8.6 × 10⁶	5.1 × 10⁵	3.4 × 10⁴
	Native bLF	NA	4.8 × 10⁶	3.1 × 10⁵	2.2 × 10⁴
	LFcin B	NA	4.3 × 10⁶	9.5 × 10³	<5.0 × 10²

Open in a new tab

P. gingivalis JCM 8525 or P. intermedia ATCC 25611 was incubated with 8 mg/ml of native bLF or 0.4 mg/ml of LFcin B in physiological saline for 4 h. The numbers of CFU of the bacteria were determined by counting the colonies in diluted samples grown on a TSA plate. The detection limit was 5 × 10² CFU/ml. The values indicate one representative result from two similar results. NA, not assayed.

Inhibition of biofilm formation by LF.

We investigated whether LF-related agents affected the biofilm formation of P. gingivalis and P. intermedia. The level of biofilm formation of P. gingivalis JCM 8525, ATCC 33277, and ATCC 53978 and P. intermedia ATCC 25611 and ATCC 49046 in the presence of several concentrations of native bLF in polyvinylchloride round wells was estimated by measuring the OD₅₅₀ after staining of the bacteria with crystal violet (Fig. 3A). The amount of biofilm that was formed varied among the bacterial strains. However, native bLF inhibited biofilm formation by all strains tested. Since P. gingivalis ATCC 33277 and P. intermedia ATCC 25611 formed large amounts of biofilm, these strains were used for the subsequent experiments. hLF, apo-bLF, native bLF, and holo-bLF inhibited the biofilm formation of P. gingivalis ATCC 33277 at a lower concentration (0.008 mg/ml, a concentration lower than that required to inhibit the planktonic growth) in a dose-dependent manner (Fig. 3B). LFcin B at lower concentrations (0.006 and 0.025 mg/ml) promoted biofilm formation, but at 0.4 mg/ml, the peptide markedly inhibited biofilm formation. For P. intermedia ATCC 25611, apo-bLF, native bLF, and holo-bLF at more than 0.031 mg/ml or hLF at more than 0.13 mg/ml inhibited biofilm formation in a dose-dependent manner (Fig. 3C). The inhibition of biofilm formation by LFcin B was apparent at 0.4 mg/ml. For microscopic observation, the biofilm of P. gingivalis ATCC 33277 was formed on polystyrene flat-bottom wells and stained with crystal violet. Culture of P. gingivalis for 24 h generated a substantial biofilm on the surface of each well (Fig. 4A). Addition of native bLF at 0.031 mg/ml (Fig. 4B) or 0.5 mg/ml (Fig. 4C) to the culture of P. gingivalis drastically inhibited biofilm formation, with only single cells or clumps attached to the plates being observed. Microscopically, it seems likely that the LF inhibition of biofilm formation by P. gingivalis is more drastic when the organism is cultured on a polystyrene plate than on a polyvinylchloride plate (data not shown).

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.

Reduction of preformed biofilms by LF.

Next, we examined whether LF-related agents could reduce preformed biofilms of P. gingivalis and P. intermedia. Biofilms of P. gingivalis ATCC 33277 and P. intermedia ATCC 25611 formed by 24 h of culture were treated with saline containing hLF, apo-bLF, native bLF, holo-bLF, and LFcin B for 5 h. The remaining biofilm was then stained with crystal violet. hLF and apo-bLF reduced the amount of the P. gingivalis biofilm in a dose-dependent manner starting at 0.008 mg/ml (Fig. 5A). The effects of native bLF and holo-bLF were weaker than the effect of apo-bLF. LFcin B was more effective at a lower concentration (at 0.0063 mg/ml but not at 0.0016 mg/ml) than at higher concentrations. For P. intermedia, treatment with all types of LFs resulted in dose-dependent reductions in the level of biofilm formation (Fig. 5B). LFcin B at concentrations ranging from 0.0063 to 0.4 mg/ml reduced the level of P. intermedia biofilm formation by similar amounts.

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).

Effects of combinations of antibiotics and LF on preformed biofilms.

The effects of combinations of LF with antibiotics were examined by a biofilm reduction test. A biofilm of P. gingivalis ATCC 33277 formed through 24 h of culture was treated for 24 h with saline containing 0.5 mg/ml of native bLF and antibiotics at several concentrations, including ABPC, CPFX, CAM, and MINO (Fig. 6A). The amount of remaining biofilm was then determined by crystal violet staining. CPFX, CAM, or MINO in combination with native bLF reduced the amount of the preformed biofilm, but ABPC in combination with native bLF did not (Fig. 6A). The combination of native bLF and 1 or 10 μg/ml of CPFX significantly reduced the amount of the preformed biofilm compared with the level of reduction achieved with each agent alone. Furthermore, the combination of native bLF and 1 μg/ml of CAM or 10 μg/ml of MINO significantly reduced the amount of preformed biofilm compared with the level of reduction achieved with each agent alone. The effects of combinations of antibiotics and apo-bLF on the viability of the bacteria in the preformed biofilm were examined (Fig. 6B). Although, overall, the results obtained with the combinations seemed to be relatively modest, the combination of 0.5 mg/ml of apo-bLF and 10 μg/ml of CPFX significantly reduced the viability of the bacteria in the biofilm compared with that achieved with each agent alone.

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.

DISCUSSION

The iron-withholding ability of LF is the central mechanism of its antimicrobial activity (13, 31). Our results indicated that bLF has the ability to suppress the growth of planktonic P. gingivalis and P. intermedia independently of the iron-bound form of bLF used. Furthermore, we found that various iron-bound forms of bLF inhibit the biofilm formation of these bacteria even at lower concentrations and that LF alone or in combination with antibiotics reduces the amounts of preformed biofilms of these bacteria.

Previous studies showed that hLF has growth-inhibitory activity against P. gingivalis but not P. intermedia or Prevotella nigrescens in a conventional turbidity assay (2, 35). The activity of LF against P. gingivalis was not seen in a medium supplemented with hemin (35), and by a turbidity assay, the growth of P. gingivalis was not detected in a medium not supplemented with hemin (2). For the more sensitive detection of the susceptibility of planktonic P. gingivalis and P. intermedia cells to LF in a medium not supplemented with hemin, we measured the ATP-based luminescence of the bacteria during a short-term incubation starting with a small number of cells (10⁴/ml). The results indicated that P. intermedia, as well as P. gingivalis, is susceptible to the growth-inhibitory activity of LF during a 5-h incubation. However, the effect of LF appears to be weak or absent after a longer incubation (8 h). It is reported that LF is degraded by the proteases produced by P. gingivalis and P. intermedia (5, 15). The decline in the antibacterial activity of LF after longer incubations with P. gingivalis and P. intermedia may be due to the degradation of this protein. Our results revealed that both iron-free apo-bLF and iron-saturated holo-bLF suppress the short-term growth of P. gingivalis and P. intermedia, suggesting that the growth-inhibitory activity against these bacteria is independent of an iron-withholding mechanism. It was suggested that hLF exhibits a bacteriostatic action on P. gingivalis by removal of a hemoglobin receptor from the bacterial cell surface in a medium containing hemoglobin as the sole iron source but not in a medium containing hemin as the sole iron source (35). Although we used a medium not supplemented with heme-related compounds in our assay, the bacteria may have used a trace amount of hemin from the inoculum culture for their growth. It is conceivable that LF may have an additional antibacterial mechanism by which the growth of P. gingivalis and P. intermedia is suppressed, apart from the iron-withholding mechanism and the removal of the hemoglobin receptor.

LFcin B is a potent antimicrobial peptide that shows activity against various microorganisms at concentrations of 0.3 to 45 μg/ml (10, 43), and it is recognized as the bactericidal domain of bLF (42). However, the growth-inhibitory activity of LFcin B against P. gingivalis and P. intermedia did not appear to be very strong. Although the number of P. gingivalis and P. intermedia cells in physiological saline spontaneously decreased, LFcin B at a relatively high concentration (0.4 mg/ml) further reduced the numbers of these bacteria. These results obtained with LFcin B seem to be in accord with those obtained in previous studies with other antimicrobial peptides, in which their activities against P. gingivalis and P. intermedia were weak or were not seen at all (8, 29, 32).

P. gingivalis and P. intermedia colonize and grow as biofilms within subgingival plaque (14, 27). Recently, our preliminary clinical trial indicated that the bLF orally administered to patients with chronic periodontitis was transferred to the subgingival pocket and led to decreases in the numbers of total bacteria, P. gingivalis, and P. intermedia in the subgingival plaque (23). Therefore, we explored the effects of LF-related agents on the biofilms of P. gingivalis and P. intermedia in vitro. hLF, apo-bLF, native bLF, and holo-bLF inhibited the biofilm formation of P. gingivalis in a dose-dependent manner; and these agents were effective even at a low concentration of 0.008 mg/ml. Likewise, hLF at 0.13 mg/ml and apo-bLF, native bLF, and holo-bLF at 0.031 mg/ml inhibited P. intermedia biofilm formation. There are reports indicating that LF prevents the biofilm formation of Pseudomonas aeruginosa, Burkholderia cepacia, enteroaggregative Escherichia coli, and an oral pathogen, S. mutans (12, 18, 30, 33, 36). It has been documented that hLF inhibited the biofilm formation of P. aeruginosa by an iron-chelating mechanism (36). In contrast, apo-bLF increased the biofilm formation of S. mutans, while holo-bLF decreased the biofilm formation of S. mutans (18). bLF both with and without iron inhibited the biofilm formation of enteroaggregative E. coli (30). The inhibition of P. gingivalis and P. intermedia biofilm formation by LF was independent of the iron status of the protein, and the mechanism of action of LF against P. aeruginosa and S. mutans biofilm formation seems to be different from that against P. gingivalis and P. intermedia biofilm formation but similar to that against enteroaggregative E. coli biofilm formation. LFcin B inhibited the biofilm formation of P. gingivalis and P. intermedia only at a concentration of 0.4 mg/ml, suggesting that the LFcin region is not solely responsible for the biofilm-inhibitory activity of LF. It was reported that the cleavage of hLF by cathepsin results in the loss of its inhibitory activity on P. aeruginosa biofilm formation (33). It is assumed that an intact LF structure may be important for its inhibitory effect on biofilm formation.

We obtained results showing that various iron-bound forms of bLF, hLF, and LFcin B also reduce the amounts of preformed biofilms of P. gingivalis and P. intermedia. In this experiment, LFcin B was effective even at a low concentration (0.0063 mg/ml). It was reported that hLF and bLF bind to P. gingivalis and P. intermedia cells (20). bLF also interferes with the binding of P. intermedia to subepithelial matrix proteins, including fibronectin, collagen types I and IV, and laminin, as well as fibroblasts and epithelial cells (4, 6). Therefore, LF may have interacted with the cell surface of P. gingivalis and P. intermedia and interfered with or disconnected the binding of biofilm-forming cells to the plastic wall. It is thought that the biofilm-inhibiting and -reducing effects of LF at least partly contributed to the reductions in the amounts of P. gingivalis and P. intermedia in the subgingival plaque of patients with chronic periodontitis, which was observed in a small-scale clinical study (23). The physiological concentration of hLF in stimulated whole saliva of patients with chronic periodontitis was 9.8 μg/ml in our previous clinical study (data not shown) and 9.0 μg/ml in another study (19). The hLF concentration in the GCF of patients with chronic periodontitis was calculated to be 51 μg/ml in our previous clinical study (data not shown) and 80 μg/ml in another study (19), on the basis of the assumption that 2 μl of GCF was collected. These concentrations of hLF in saliva and GCF may have a role in the inhibition of biofilm formation by P. gingivalis and P. intermedia. However, the oral administration of LF would boost the antibiofilm environment in the oral cavity because a higher LF concentration is more effective at inhibiting biofilm formation.

Furthermore, specific concentrations of CPFX, CAM, or MINO combined with bLF significantly reduced the amounts of preformed P. gingivalis biofilms in vitro. Although a combination of CPFX and bLF also suppressed the viability of the bacteria in the biofilm, the overall effectiveness of combinations of antibiotics and bLF on the viability of the bacteria in the biofilm was somewhat modest. These results imply that the use of combinations of antibiotics and LF may be effective at reducing the biofilm materials rather than suppressing bacterial activity. In relation to these results, hLF has been reported to enhance the B. cepacia biofilm formation-inhibitory activities of antibiotics such as CPFX (12). Although tetracycline antibiotics, including MINO, have been employed as local drug delivery systems for the treatment of periodontal diseases (22), the low levels of activity of antibiotics, including tetracyclines, against P. gingivalis and P. intermedia biofilm formation provide a reason for the difficulty of developing antibiotic therapy for periodontal diseases (17, 24, 37). LF may aid with the chemotherapy of periodontitis with antibiotics such as MINO, CPFX, and CAM. Thus, larger-scale clinical tests are warranted for assessment of the preventive and therapeutics effects of LF alone or LF with antibiotics on periodontal diseases.

Footnotes

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Published ahead of print on 18 May 2009.

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10.Bellamy, W., M. Takase, H. Wakabayashi, K. Kawase, and M. Tomita. 1992. Antibacterial spectrum of lactoferricin B, a potent bacterial peptide derived from the N-terminal region of bovine lactoferrin. J. Appl. Bacteriol. 73:472-479. [DOI] [PubMed] [Google Scholar]
11.Bellamy, W., M. Takase, K. Yamauchi, H. Wakabayashi, K. Kawase, and M. Tomita. 1992. Identification of the bactericidal domain of lactoferrin. Biochim. Biophys. Acta 1121:130-136. [DOI] [PubMed] [Google Scholar]
12.Caraher, E. M., K. Gumulapurapu, C. C. Taggart, P. Murphy, S. McClean, and M. Callaghan. 2007. The effect of recombinant human lactoferrin on growth and the antibiotic susceptibility of the cystic fibrosis pathogen Burkholderia cepacia complex when cultured planktonically or as biofilms. J. Antimicrob. Chemother. 60:546-554. [DOI] [PubMed] [Google Scholar]
13.Chierici, R. 2001. Antimicrobial actions of lactoferrin. Adv. Nutr. Res. 10:247-269. [DOI] [PubMed] [Google Scholar]
14.Davey, M. E., and J. W. Costerton. 2006. Molecular genetics analyses of biofilm formation in oral isolates. Periodontology 2000 42:13-26. [DOI] [PubMed] [Google Scholar]
15.de Lillo, A., R. Teanpaisan, J. F. Fierro, and C. W. I. Douglas. 1996. Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens. FEMS Immunol. Med. Microbiol. 14:135-143. [DOI] [PubMed] [Google Scholar]
16.Eberhard, J., Z. Drosos, M. Tiemann, S. Jepsen, and J. M. Schröder. 2006. Immunolocalization of lactoferrin in healthy and inflamed gingival tissues. J. Periodontol. 77:472-478. [DOI] [PubMed] [Google Scholar]
17.Eick, S., T. Seltmann, and W. Pfister. 2004. Efficacy of antibiotics to strains of periodontopathogenic bacteria within a single species biofilm—an in vitro study. J. Clin. Periodontol. 31:376-383. [DOI] [PubMed] [Google Scholar]
18.Francesca, B., M. Ajello, P. Bosso, C. Morea, P. Andrea, A. Giovanni, and V. Piera. 2004. Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans. Biometals 17:271-278. [DOI] [PubMed] [Google Scholar]
19.Jentsch, H., Y. Sievert, and R. Göcke. 2004. Lactoferrin and other markers from gingival crevicular fluids and saliva before and after periodontal treatment. J. Clin. Periodontol. 31:511-514. [DOI] [PubMed] [Google Scholar]
20.Kalfas, S., M. Andersson, S. Edwardsson, A. Forsgren, and A. S. Naidu. 1991. Human lactoferrin binding to Porphyromonas gingivalis, Prevotella intermedia and Prevotella melaninogenica. Oral Microbiol. Immunol. 6:350-355. [DOI] [PubMed] [Google Scholar]
21.Kalmar, J. R., and R. R. Arnold. 1988. Killing of Actinobacillus actinomycetemcomitans by human lactoferrin. Infect. Immun. 56:2552-2557. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Killoy, W. J. 1998. Chemical treatment of periodontitis: local delivery of antimicrobials. Int. Dent. J. 48:305-315. [DOI] [PubMed] [Google Scholar]
23.Kondo, I., T. Kobayashi, H. Wakabayashi, K. Yamauchi, K. Iwatsuki, and H. Yoshie. 2008. Effects of oral administration of bovine lactoferrin on periodontitis patients. Jpn. J. Conserv. Dent. 51:281-291. (In Japanese.) http://wwwsoc.nii.ac.jp/jscd/member/pdf/vol51_no3/291.pdf. [Google Scholar]
24.Larsen, T. 2002. Susceptibility of Porphyromonas gingivalis in biofilms to amoxicillin, doxycycline and metronidazole. Oral Microbiol. Immunol. 17:267-271. [DOI] [PubMed] [Google Scholar]
25.Levay, P. F., and M. Viljoen. 1995. Lactoferrin: a general review. Haematologica 80:252-267. [PubMed] [Google Scholar]
26.Loesche, W. J. 1999. The antimicrobial treatment of periodontal disease: change the treatment paradigm. Crit. Rev. Oral Biol. Med. 10:245-275. [DOI] [PubMed] [Google Scholar]
27.Marsh, P. D. 1992. Microbiological aspects of the chemical control of plaque and gingivitis. J. Dent. Res. 71:1431-1438. [DOI] [PubMed] [Google Scholar]
28.Merritt, J. H., D. E. Kadouri, and G. A. O'Toole. 2005. Growing and analyzing static biofilms, p. 1B.1.1-1B.1.17. In Current protocols in microbiology. John Wiley & Sons, Inc., Hoboken, NJ. [DOI] [PMC free article] [PubMed]
29.Novak, K. F., W. J. Diamond, S. Kirakodu, R. Peyyala, K. W. Anderson, R. C. Montelaro, and T. A. Mietzner. 2007. Efficacy of the de novo-derived antimicrobial peptide WLBU2 against oral bacteria. Antimicrob. Agents Chemother. 51:1837-1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Ochoa, T. J., E. L. Brown, C. E. Guion, J. Z. Chen, R. J. McMahon, and T. G. Cleary. 2006. Effect of lactoferrin on enteroaggregative E. coli (EAEC). Biochem. Cell Biol. 84:369-376. [DOI] [PubMed] [Google Scholar]
31.Oram, J. D., and B. Reiter. 1968. Inhibition of bacteria by lactoferrin and other iron-chelating agents. Biochim. Biophys. Acta 170:351-365. [DOI] [PubMed] [Google Scholar]
32.Ouhara, K., H. Komatsuzawa, S. Yamada, H. Shiba, T. Fujiwara, M. Ohara, K. Sayama, K. Hashimoto, H. Kurihara, and M. Sugai. 2005. Susceptibilities of periodontopathogenic and cariogenic bacteria to antimicrobial peptides, β-defensins and LL37, produced by human epithelial cells. J. Antimicrob. Chemother. 55:888-896. [DOI] [PubMed] [Google Scholar]
33.Rogan, M. P., C. C. Taggart, C. M. Greene, P. G. Murphy, S. J. O'Neill, and N. G. McElvaney. 2004. Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fibrosis. J. Infect. Dis. 190:1245-1253. [DOI] [PubMed] [Google Scholar]
34.Sawatzki, G., and B. Kubanek. 1983. Isolation and ELISA of mouse and human lactoferrin, p. 441-443. In I. Urushizaki, P. Aisen, I. Listowsky, and J. W. Drysdale (ed.), Structure and function of iron storage and transport protein. Elsevier Science Publishers, Amsterdam, The Netherlands.
35.Shi, Y., W. Kong, and K. Nakayama. 2000. Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis. J. Biol. Chem. 275:30002-30008. [DOI] [PubMed] [Google Scholar]
36.Singh, P. K., M. R. Parsek, E. P. Greenberg, and M. J. Welsh. 2002. A component of innate immunity prevents bacterial biofilm development. Nature 417:552-555. [DOI] [PubMed] [Google Scholar]
37.Takahashi, N., K. Ishihara, R. Kimizuka, K. Okuda, and T. Kato. 2006. The effects of tetracycline, minocycline, doxycycline and ofloxacin on Prevotella intermedia biofilm. Oral Microbiol. Immunol. 21:366-371. [DOI] [PubMed] [Google Scholar]
38.Takakura, N., H. Wakabayashi, H. Ishibashi, S. Teraguchi, Y. Tamura, H. Yamaguchi, and S. Abe. 2003. Oral lactoferrin treatment of experimental oral candidiasis in mice. Antimicrob. Agents Chemother. 47:2619-2623. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Tenovuo, J. 2002. Clinical applications of antimicrobial host proteins lactoperoxidase, lysozyme and lactoferrin in xerostomia: efficacy and safety. Oral Dis. 8:23-29. [DOI] [PubMed] [Google Scholar]
40.Tomita, M., H. Wakabayashi, K. Yamauchi, S. Teraguchi, and H. Hayasawa. 2002. Bovine lactoferrin and lactoferricin derived from milk: production and applications. Biochem. Cell Biol. 80:109-112. [DOI] [PubMed] [Google Scholar]
41.Wakabayashi, H., S. Abe, S. Teraguchi, H. Hayasawa, and H. Yamaguchi. 1998. Inhibition of hyphal growth of azole-resistant strains of Candida albicans by triazole antifungal agents in the presence of lactoferrin-related compounds. Antimicrob. Agents Chemother. 42:1587-1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Wakabayashi, H., M. Takase, and M. Tomita. 2003. Lactoferricin derived from milk protein lactoferrin. Curr. Pharm. Des. 9:1277-1287. [DOI] [PubMed] [Google Scholar]
43.Yamauchi, K., M. Tomita, T. J. Giehl, and R. T. Ellison III. 1993. Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment. Infect. Immun. 61:719-728. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r1] 1.Adonogianaki, E., N. A. Moughal, and D. F. Kinane. 1993. Lactoferrin in the gingival crevice as a marker of polymorphonuclear leucocytes in periodontal diseases. J. Clin. Periodontol. 20:26-31. [DOI] [PubMed] [Google Scholar]

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[r8] 8.Bachrach, G., H. Altman, P. E. Kolenbrander, N. I. Chalmers, M. Gabai-Gutner, A. Mor, M. Friedman, and D. Steinberg. 2008. Resistance of Porphyromonas gingivalis ATCC 33277 to direct killing by antimicrobial peptides is protease independent. Antimicrob. Agents Chemother. 52:638-642. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r9] 9.Baveye, S., E. Elass, J. Mazurier, G. Spik, and D. Legrand. 1999. Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process. Clin. Chem. Lab. Med. 37:281-286. [DOI] [PubMed] [Google Scholar]

[r10] 10.Bellamy, W., M. Takase, H. Wakabayashi, K. Kawase, and M. Tomita. 1992. Antibacterial spectrum of lactoferricin B, a potent bacterial peptide derived from the N-terminal region of bovine lactoferrin. J. Appl. Bacteriol. 73:472-479. [DOI] [PubMed] [Google Scholar]

[r11] 11.Bellamy, W., M. Takase, K. Yamauchi, H. Wakabayashi, K. Kawase, and M. Tomita. 1992. Identification of the bactericidal domain of lactoferrin. Biochim. Biophys. Acta 1121:130-136. [DOI] [PubMed] [Google Scholar]

[r12] 12.Caraher, E. M., K. Gumulapurapu, C. C. Taggart, P. Murphy, S. McClean, and M. Callaghan. 2007. The effect of recombinant human lactoferrin on growth and the antibiotic susceptibility of the cystic fibrosis pathogen Burkholderia cepacia complex when cultured planktonically or as biofilms. J. Antimicrob. Chemother. 60:546-554. [DOI] [PubMed] [Google Scholar]

[r13] 13.Chierici, R. 2001. Antimicrobial actions of lactoferrin. Adv. Nutr. Res. 10:247-269. [DOI] [PubMed] [Google Scholar]

[r14] 14.Davey, M. E., and J. W. Costerton. 2006. Molecular genetics analyses of biofilm formation in oral isolates. Periodontology 2000 42:13-26. [DOI] [PubMed] [Google Scholar]

[r15] 15.de Lillo, A., R. Teanpaisan, J. F. Fierro, and C. W. I. Douglas. 1996. Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens. FEMS Immunol. Med. Microbiol. 14:135-143. [DOI] [PubMed] [Google Scholar]

[r16] 16.Eberhard, J., Z. Drosos, M. Tiemann, S. Jepsen, and J. M. Schröder. 2006. Immunolocalization of lactoferrin in healthy and inflamed gingival tissues. J. Periodontol. 77:472-478. [DOI] [PubMed] [Google Scholar]

[r17] 17.Eick, S., T. Seltmann, and W. Pfister. 2004. Efficacy of antibiotics to strains of periodontopathogenic bacteria within a single species biofilm—an in vitro study. J. Clin. Periodontol. 31:376-383. [DOI] [PubMed] [Google Scholar]

[r18] 18.Francesca, B., M. Ajello, P. Bosso, C. Morea, P. Andrea, A. Giovanni, and V. Piera. 2004. Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans. Biometals 17:271-278. [DOI] [PubMed] [Google Scholar]

[r19] 19.Jentsch, H., Y. Sievert, and R. Göcke. 2004. Lactoferrin and other markers from gingival crevicular fluids and saliva before and after periodontal treatment. J. Clin. Periodontol. 31:511-514. [DOI] [PubMed] [Google Scholar]

[r20] 20.Kalfas, S., M. Andersson, S. Edwardsson, A. Forsgren, and A. S. Naidu. 1991. Human lactoferrin binding to Porphyromonas gingivalis, Prevotella intermedia and Prevotella melaninogenica. Oral Microbiol. Immunol. 6:350-355. [DOI] [PubMed] [Google Scholar]

[r21] 21.Kalmar, J. R., and R. R. Arnold. 1988. Killing of Actinobacillus actinomycetemcomitans by human lactoferrin. Infect. Immun. 56:2552-2557. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r22] 22.Killoy, W. J. 1998. Chemical treatment of periodontitis: local delivery of antimicrobials. Int. Dent. J. 48:305-315. [DOI] [PubMed] [Google Scholar]

[r23] 23.Kondo, I., T. Kobayashi, H. Wakabayashi, K. Yamauchi, K. Iwatsuki, and H. Yoshie. 2008. Effects of oral administration of bovine lactoferrin on periodontitis patients. Jpn. J. Conserv. Dent. 51:281-291. (In Japanese.) http://wwwsoc.nii.ac.jp/jscd/member/pdf/vol51_no3/291.pdf. [Google Scholar]

[r24] 24.Larsen, T. 2002. Susceptibility of Porphyromonas gingivalis in biofilms to amoxicillin, doxycycline and metronidazole. Oral Microbiol. Immunol. 17:267-271. [DOI] [PubMed] [Google Scholar]

[r25] 25.Levay, P. F., and M. Viljoen. 1995. Lactoferrin: a general review. Haematologica 80:252-267. [PubMed] [Google Scholar]

[r26] 26.Loesche, W. J. 1999. The antimicrobial treatment of periodontal disease: change the treatment paradigm. Crit. Rev. Oral Biol. Med. 10:245-275. [DOI] [PubMed] [Google Scholar]

[r27] 27.Marsh, P. D. 1992. Microbiological aspects of the chemical control of plaque and gingivitis. J. Dent. Res. 71:1431-1438. [DOI] [PubMed] [Google Scholar]

[r28] 28.Merritt, J. H., D. E. Kadouri, and G. A. O'Toole. 2005. Growing and analyzing static biofilms, p. 1B.1.1-1B.1.17. In Current protocols in microbiology. John Wiley & Sons, Inc., Hoboken, NJ. [DOI] [PMC free article] [PubMed]

[r29] 29.Novak, K. F., W. J. Diamond, S. Kirakodu, R. Peyyala, K. W. Anderson, R. C. Montelaro, and T. A. Mietzner. 2007. Efficacy of the de novo-derived antimicrobial peptide WLBU2 against oral bacteria. Antimicrob. Agents Chemother. 51:1837-1839. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r30] 30.Ochoa, T. J., E. L. Brown, C. E. Guion, J. Z. Chen, R. J. McMahon, and T. G. Cleary. 2006. Effect of lactoferrin on enteroaggregative E. coli (EAEC). Biochem. Cell Biol. 84:369-376. [DOI] [PubMed] [Google Scholar]

[r31] 31.Oram, J. D., and B. Reiter. 1968. Inhibition of bacteria by lactoferrin and other iron-chelating agents. Biochim. Biophys. Acta 170:351-365. [DOI] [PubMed] [Google Scholar]

[r32] 32.Ouhara, K., H. Komatsuzawa, S. Yamada, H. Shiba, T. Fujiwara, M. Ohara, K. Sayama, K. Hashimoto, H. Kurihara, and M. Sugai. 2005. Susceptibilities of periodontopathogenic and cariogenic bacteria to antimicrobial peptides, β-defensins and LL37, produced by human epithelial cells. J. Antimicrob. Chemother. 55:888-896. [DOI] [PubMed] [Google Scholar]

[r33] 33.Rogan, M. P., C. C. Taggart, C. M. Greene, P. G. Murphy, S. J. O'Neill, and N. G. McElvaney. 2004. Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fibrosis. J. Infect. Dis. 190:1245-1253. [DOI] [PubMed] [Google Scholar]

[r34] 34.Sawatzki, G., and B. Kubanek. 1983. Isolation and ELISA of mouse and human lactoferrin, p. 441-443. In I. Urushizaki, P. Aisen, I. Listowsky, and J. W. Drysdale (ed.), Structure and function of iron storage and transport protein. Elsevier Science Publishers, Amsterdam, The Netherlands.

[r35] 35.Shi, Y., W. Kong, and K. Nakayama. 2000. Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis. J. Biol. Chem. 275:30002-30008. [DOI] [PubMed] [Google Scholar]

[r36] 36.Singh, P. K., M. R. Parsek, E. P. Greenberg, and M. J. Welsh. 2002. A component of innate immunity prevents bacterial biofilm development. Nature 417:552-555. [DOI] [PubMed] [Google Scholar]

[r37] 37.Takahashi, N., K. Ishihara, R. Kimizuka, K. Okuda, and T. Kato. 2006. The effects of tetracycline, minocycline, doxycycline and ofloxacin on Prevotella intermedia biofilm. Oral Microbiol. Immunol. 21:366-371. [DOI] [PubMed] [Google Scholar]

[r38] 38.Takakura, N., H. Wakabayashi, H. Ishibashi, S. Teraguchi, Y. Tamura, H. Yamaguchi, and S. Abe. 2003. Oral lactoferrin treatment of experimental oral candidiasis in mice. Antimicrob. Agents Chemother. 47:2619-2623. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r39] 39.Tenovuo, J. 2002. Clinical applications of antimicrobial host proteins lactoperoxidase, lysozyme and lactoferrin in xerostomia: efficacy and safety. Oral Dis. 8:23-29. [DOI] [PubMed] [Google Scholar]

[r40] 40.Tomita, M., H. Wakabayashi, K. Yamauchi, S. Teraguchi, and H. Hayasawa. 2002. Bovine lactoferrin and lactoferricin derived from milk: production and applications. Biochem. Cell Biol. 80:109-112. [DOI] [PubMed] [Google Scholar]

[r41] 41.Wakabayashi, H., S. Abe, S. Teraguchi, H. Hayasawa, and H. Yamaguchi. 1998. Inhibition of hyphal growth of azole-resistant strains of Candida albicans by triazole antifungal agents in the presence of lactoferrin-related compounds. Antimicrob. Agents Chemother. 42:1587-1591. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r42] 42.Wakabayashi, H., M. Takase, and M. Tomita. 2003. Lactoferricin derived from milk protein lactoferrin. Curr. Pharm. Des. 9:1277-1287. [DOI] [PubMed] [Google Scholar]

[r43] 43.Yamauchi, K., M. Tomita, T. J. Giehl, and R. T. Ellison III. 1993. Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment. Infect. Immun. 61:719-728. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia^▿

Hiroyuki Wakabayashi

Koji Yamauchi

Tetsuo Kobayashi

Tomoko Yaeshima

Keiji Iwatsuki

Hiromasa Yoshie

Abstract