| Sign In to gain access to subscriptions and/or personal tools. |
Control of Oral Biofilm Formation by an Antimicrobial Decapeptide
1 Microbiology Branch and Correspondence: * Corresponding author, kai.leung{at}us.army.mil
Oral biofilms are mixed-species microbial communities, and their uncontrolled outgrowth can express as oral diseases. Antimicrobial peptides represent alternative classes of antimicrobials that exhibit selectivity for prokaryotes. We wanted to test the effect of a synthetic decapeptide antimicrobial, KSL, on the development of oral biofilms formed by isolated human salivary bacteria. We used differential interference contrast microscopy, coupled with a dual-flow cell system, to determine the effect of KSL on oral biofilm development. We used reductions of viable counts and confocal microscopy to assess the bactericidal activity of KSL on mature oral biofilms. KSL effectively blocked biofilm development. A significant effect on the viability of mature biofilms was observed when KSL was used in the presence of a surface-active agent, or after biofilms were mechanically disrupted. This study shows that KSL may be a useful adjunct for conventional oral hygiene to prevent plaque-mediated dental diseases.
Key Words: oral biofilms • salivary bacteria • antimicrobial peptide • dual-flow cell • surface-active agent
Human oral biofilms are complex three-dimensional structures consisting of diverse and multispecies microbial communities formed on colonizable surfaces (Kolenbrander and London, 1993; Marsh and Bradshaw, 1995; Foster et al., 2004; Kolenbrander and Palmer, 2004). Aside from the substrata’s physical and chemical surface properties, which have a significant impact on bacterial accumulation (Quirynen et al., 2000), the formation of oral biofilms involves a series of events. This includes the initial formation of a conditioning saliva-derived film (the acquired salivary pellicle) on colonizable surfaces, the attachment of primary colonizers to host-derived receptor molecules present in the acquired pellicle, the subsequent interactions of secondary colonizers with the attached early colonizers, followed by the proliferation of the adhered bacteria (colonization), and the development of mature microbial communities (Kolenbrander and London, 1993; Marsh and Bradshaw, 1995; Quirynen et al., 2000). Uncontrolled growth of certain resident microbes in these communities may contribute to the development of oral diseases (Loesche, 1999).
Antimicrobial peptides show a broad range of antibacterial activity and could play a role in protection against infections (Raj and Dentino, 2002; Zasloff, 2002; Koczulla and Bals, 2003; Finlay and Hancock, 2004). For example, epithelial-derived antimicrobial peptides like human β-defensin-3 could protect the host from infection caused by periodontal pathogens (Bissell et al., 2004). We previously demonstrated (Concannon et al., 2003) that an In this study, we adopted the use of saliva-coated hydroxyapatite (HA) and germanium (Ge) (Herles et al., 1994) disks as growing surfaces for oral biofilms formed by salivary bacteria. We used a dual-flow cell system, consisting of 2 individual flow chambers, to test the effect of our antimicrobial decapeptide, as compared with controls, in influencing biofilm formation, and against developed biofilms.
Synthesis of the Antimicrobial Decapaptide, KSL KSL (KKVVFKVKFK-NH2) was synthesized by standard solid-phase procedures, with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, on an automatic peptide synthesizer (Model 90, Advanced ChemTech, Louisville, KY, USA), and its purity was determined as previously described (Concannon et al., 2003).
Buffers and Media
Collection of Saliva and Isolation of Salivary Bacteria
Dual-flow Cell System
To form biofilms, we conditioned Ge disks (Mindrum, Rancho Cucamonga, CA, USA) in the dual-flow cell for 1 hr with sterile 50% human whole saliva. Isolated salivary bacteria, adjusted to approximately 1.0 x 107 cells/mL in 50% saliva (total, 1.5 mL), were injected into the flow chambers. After 2 hrs of initial adherence of bacteria onto disk surfaces, the flow of culture medium (20% Todd-Hewitt broth) was started at a rate of 0.2 mL/min (Foster et al., 2004). The flow rate used generated a shear rate of approximately 9.65 s–1 on the substrate surface, compatible with the fluid flow reported in the oral cavity (Bakker et al., 2003). To evaluate the effects of KSL in controlling the development and maturation of oral biofilms, we continuously perfused surface-adhered cells (after initial colonization) with KSL-free or KSL-containing media (KSL at 10 or 50 µg/mL). Alternatively, biofilms at different stages of maturation (i.e., 4 or 6 hrs after inoculation) were pulse-treated, by means of an injection pump, at 0.2 mL/min with 50 µg/mL KSL in 20% Todd-Hewitt broth or with control medium for 30 min at two-hour intervals. Direct comparison of the effects of antimicrobials on the growth of biofilms between treated and untreated cells was made in real time by DIC microscopy.
Bactericidal Activities of KSL against Oral Biofilms To determine the inhibitory activity of KSL on developed oral biofilms, we transferred disks containing 45-hour-old biofilms to wells containing aqueous KSL (200 µg/mL; 1 mL/well). Following 30 min of exposure at 37°C, disks were rinsed 3 times with 1 mL of saline and transferred to sterile 15-mL polypropylene tubes containing 1 mL PBS. Biofilm cells adherent to surfaces (after treatment) were recovered by sonication for 2 min at 5 Watts with a Microson ultrasonic cell disrupter equipped with a cup horn (Misonix Inc., Farmingdale, NY, USA). Settings, including the time interval used in the sonication, were pre-determined empirically to yield maximal recovery of adherent biofilm cells. We assessed the effect of KSL on disrupted biofilms by recovering biofilm cells from HA disks (45-hour-old biofilms) through sonication as described above. The detached biofilm cells (in sterile dH2O) were mixed with an equal volume of aqueous KSL to obtain a final peptide concentration of 200 µg/mL, and the reaction mixtures were incubated at 37°C for 30 min. We terminated the interactions of KSL and suspended biofilm cells by washing them in PBS. To determine the effect of the surface-active agent (benzalkonium chloride; Sigma, St. Louis, MO, USA) in promoting the killing of intact biofilms by KSL, we treated 66-hour-old oral biofilms with KSL (200 µg/mL), benzalkonium chloride (0.001%), or a combination of the two agents, followed by viable counts determinations and confocal laser scanning microscopy of treated samples. The benzalkonium chloride dosages were pre-determined empirically for the selection of concentrations of the agent exhibiting minimal bactericidal activity. We determined viable counts of biofilm cells derived from treated disks or disrupted biofilms by spiral plating serially diluted samples onto blood agar plates. Distilled water or 0.12% aqueous chlorhexidine digluconate (Sigma) was used as the negative or positive control, respectively. The 45-hour-old biofilms were exposed to chlorhexidine for 1 min, and to water for 30 min at 37°C.
Interactions of KSL with Oral Biofilms Formed in a Dual-flow Cell To determine whether KSL has anti-biofilm activity, we examined the effects of various concentrations of KSL on oral biofilm development. Using DIC microscopy, we observed the adherence of salivary bacteria to the saliva-conditioned Ge surface in the flow chambers 2 hrs after the inoculation of the flow cell (Figs. 2Aa, 2Ad, 2Ba, 2Bc  ). After the attachment of bacteria to the surface, the flow chambers were perfused continuously with culture medium with or without KSL. In the flow chamber perfused with medium lacking KSL, microcolonies were formed 5 hrs after the inoculation (Figs. 2Ab) and continued to develop into film-like structures after 8 hrs (Fig. 2Ac). In contrast, KSL at 50 µg/mL disrupted biofilm development. Bacteria remained attached, but failed to form microcolonies and film-like structure (Figs. 2Ad–2Af). Further, KSL at 10 µg/mL was partially effective in inhibiting biofilm formation. Microcolonies formed 8 hrs after inoculation (Figs. 2Bc–2Bd), whereas the untreated adhered salivary bacteria formed a film-like structure (Figs. 2Ba, 2Bb).
While continuous perfusion of medium containing KSL to the flow chambers prevented the attached salivary bacteria from differentiating into biofilms on conditioned Ge surfaces, we were also interested in determining whether KSL could disrupt the development process by pulsed treatment of biofilm cells at different time points after inoculation. As shown in Fig. 3A (a-c), pulsed treatment (30 min at 0.2 mL/min for every two-hour interval) of biofilm cells 4 hrs after inoculation with KSL-free medium did not prevent attached salivary bacteria from developing into biofilms. In contrast, pulsed treatment of biofilm cells 4 hrs after inoculation with KSL-containing medium (50 µg/mL) inhibited biofilm formation (Figs. 3Ad–3Af). However, as compared with the controls (Figs. 3Ba–3Bc), pulsed treatment of biofilms 6 hrs after inoculation with KSL-containing medium failed to inhibit or alter the development of biofilm structures (Figs. 3Bd–3Bf).
Interactions of KSL with Intact and Disrupted Oral Biofilms Our flow-cell experiments showed that mature oral biofilms were less susceptible to KSL. In contrast, exposure of adherent salivary bacteria or biofilm cells to KSL at earlier stages of development inhibited their further development into mature growing biofilms. In this context, we were interested in determining, using the in vitro plaque assay, whether the organized structure of developed oral biofilms contributed to the resistance of mature oral biofilms to KSL. There was a small reduction of viable counts (p < 0.05) when intact 45-hour-old oral biofilms formed on saliva-conditioned HA disks were exposed to KSL (Fig. 4A ). A larger reduction of viable counts was observed with intact biofilms treated with 0.12% chlorhexidine. When these biofilms were mechanically disrupted by sonication before KSL treatment, there was a much greater (1.8 log) reduction of viability of KSL-treated cells as compared with dH2O-treated control cells. There was likewise a significant reduction of viability in disrupted biofilms as compared with intact biofilms treated with the same concentration of KSL.
Interactions of KSL with Intact Oral Biofilms in the Presence of a Surface-active Agent Since the organized structure of biofilms might influence biofilm susceptibility to antimicrobials, we were interested in determining, using the in vitro plaque assay, the effect of a surface-active agent, benzalkonium chloride, in promoting the killing of biofilm cells by KSL. As compared with water treatment, KSL, in the presence of benzalkonium chloride (0.001%), significantly reduced the viability (over one log reduction) of 66-hour-old oral biofilms to an extent similar to that caused by chlorhexidine (Fig. 4B  ). KSL (200 µg/mL) or benzalkonium chloride (0.001%) alone had less effect on the viability of these biofilms. These results were confirmed by live/dead staining of treated samples as revealed by confocal microscopy (Fig. 4C).
The use of a dual-flow cell containing removable colonizable surfaces, together with isolated salivary bacteria, provides an alternative method for the examination of the effects of antimicrobials on oral biofilm formation. The use of human salivary bacteria as the plaque seeds is particularly relevant, since these bacteria are derived from biofilms formed on hard and soft tissues in the oral cavity (Helmerhorst et al., 1999). The system allows for the nondestructive, direct comparison of biofilm development between the treated and negative control groups. In this test system, KSL markedly prevented biofilm development as compared with the control. We reasoned that the observed inhibition was probably due to the antimicrobial activity of KSL. We have shown that KSL exerts its antimicrobial activity by destabilizing target bacterial membranes (Concannon et al., 2003). In contrast, exposing established oral biofilms (45-hour-old biofilms) to KSL did not disrupt their structure or cause any large reductions of viability of biofilm cells. The results indicate that, once developed, biofilms were more resistant to KSL. Interestingly, similar properties were observed with lactoferrin, a native antimicrobial component that is abundantly present in surface secretions. Continuous perfusion of lactoferrin at sub-inhibitory concentrations prevented biofilm development by Pseudomonas aeruginosa. However, lactoferrin, like KSL, failed to alter the structure of mature biofilms (Singh et al., 2002). Several factors influence biofilm susceptibility to antimicrobials (Gilbert et al., 1997; Campanac et al., 2002; Stewart et al., 2004). We hypothesized that the reduced susceptibility of developed oral biofilms to KSL could be due to retarded diffusion or exclusion of our antimicrobial, imposed by the three-dimensional biofilm structures and/or the presence of exopolymeric substances. To test this, we disrupted the oral biofilms grown on saliva-coated HA surfaces formed by salivary bacteria, and determined the susceptibility of these disrupted biofilm cells to KSL as compared with intact biofilms. We reasoned that the disruption of the biofilm structure would improve the accessibility of the targeted biofilm cells to our antimicrobial agent. Indeed, the disruption procedure greatly enhanced the susceptibility of biofilm cells from disrupted as compared with intact oral biofilms, suggesting that the organized structure of biofilms might play a role in influencing the susceptibility of intact biofilms to antimicrobials. However, we are uncertain whether the reduced susceptibility observed with intact biofilms is also attributable to the exopolymers that might be associated with the biofilm cells. Further, sub-bactericidal concentrations of benzalkonium chloride, a known cationic surface-active agent (Baker et al., 1978), significantly promoted biofilm susceptibility to KSL. Though we are not clear about the underlying mechanisms, one possible explanation is that the presence of sub-inhibitory concentrations of benzalkonium chloride might facilitate the accessibility of biofilm cells residing in intact oral biofilms to KSL by influencing biofilm structures. Alternatively, the cationic agent benzalkonium chloride could provide a synergistic effect on the bactericidal activity of KSL in killing salivary bacteria. In conclusion, the findings that KSL prevented the development of oral biofilms raise the possibility that KSL could be a valuable adjunct to toothpastes for preventing plaque-mediated dental diseases. This is particularly relevant since KSL was effective in killing disrupted oral biofilm cells. This disruption could be generated by mechanical brushing and/or flossing during oral hygiene procedures.
This work was supported by the US Army Medical Research and Materiel Command. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Army, the Department of Defense, or the US Government.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication January 4, 2005. Revision received July 1, 2005. Accepted for publication August 30, 2005.
Journal of Dental Research, Vol. 84, No. 12,
1172-1177 (2005) This article has been cited by other articles:
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
-helical antimicrobial decapeptide, KSL, possesses a broad range of microbicidal activity, and effectively kills selected strains of oral pathogens, including mutans streptococci. However, its ability to control oral biofilm formation and destruct intact oral biofilms has not been determined. 
