Journal of Antimicrobial Chemotherapy (2006) 58, 198–201 doi:10.1093/jac/dkl181 Advance Access publication 10 May 2006
In vitro assessment of antimicrobial peptides as potential agents against several oral bacteria H. Altman1,2, D. Steinberg1†, Y. Porat3, A. Mor3, D. Fridman1, M. Friedman2 and G. Bachrach1*† 1
Institute of Dental Sciences, Hebrew University-Hadassah School of Dental Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel; 2Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel; 3Laboratory of Antimicrobial Peptides Investigation (LAPI), Department of Biotechnology & Food Engineering, Technion–Israel Institute of Technology, Haifa, Israel Received 16 December 2005; returned 2 March 2006; revised 2 April 2006; accepted 16 April 2006
Background: Antimicrobial peptides are components of the innate immunity that play an important role in systemic and oral health. Objectives: The antibacterial activity of the amphibian-derived K4-S4(1-15)a antimicrobial peptide was tested against oral pathogens associated with caries and periodontitis and compared with the activities of the human-derived antimicrobial peptides LL-37 and dhvar4a. Methods: Growth inhibition of planktonic bacteria was tested using standard microdilution assays. Live/ Dead staining followed by confocal scanning laser microscopy (CSLM) was used to determine the bactericidal effect of K4-S4(1-15)a on Streptococcus mutans attached to a glass surface or grown as biofilm. Results: The cariogenic species S. mutans, Streptococcus sobrinus, Lactobacillus paracasei and Actinomyces viscosus were resistant to LL-37 found in the oral cavity. Porphyromonas gingivalis was the species most resistant to the three tested peptides. K4-S4(1-15)a demonstrated the highest activity against the tested planktonic bacteria. In addition, K4-S4(1-15)a was bactericidal to surface-attached S. mutans as well as to S. mutans biofilms grown in vitro. However, surface attachment increased S. mutans resistance to the antimicrobial peptide. Conclusions: Our results support growing evidence suggesting the use of antimicrobial peptides for prevention and treatment of oral disease. Keywords: oral infection, biofilm, LL-37, dermaseptin, histatin-5
Introduction Microbial resistance to antibiotics raises the need to develop novel compounds and approaches for microbial control. Antimicrobial peptides offer a promising opportunity to combat microbial pathogens. Acquisition of resistance to antimicrobial peptides in susceptible strains is slower and less common compared with that developed against other antimicrobial agents. A net positive charge and the ability to adopt an amphipathic structure are properties thought to enhance the affinity of peptides for negatively charged phospholipids at the outer surfaces of bacterial membranes. The peptides interfere with the membrane’s integrity and may further affect cytoplasmic targets. In
vertebrates, antimicrobial peptides are synthesized and secreted from phagocytic cells and epithelia and contribute to the innate immunity. The oral cavity, which is colonized by numerous microorganisms, contains a wide selection of antibacterial peptides that play an important role in maintaining its complex ecological homeostasis.1 Tooth decay and periodontal disease are associated with the presence of dental plaque, a microbial biofilm comprising multiple species anchored to oral surfaces and protected by a selfproduced polymeric matrix. Bacterial traits including susceptibility to antimicrobial agents are altered in biofilm compared with planktonic environment.2 Controlling dental plaque bacteria is important for prevention and treatment of oral diseases.
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[email protected] †These authors contributed equally to the study. .............................................................................................................................................................................................................................................................................................................................................................................................................................
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Effect of three antimicrobial peptides on selected oral pathogens Three antimicrobial peptides were tested in the present study. K4-S4(1-15)a is an analogue of dermaseptin S4, a member of the dermaseptin family, originally isolated from tree frog skin.3 The antibacterial activity of K4-S4(1-15)a was compared with those of two human-derived antibacterial peptides common in the oral cavity. LL-37 is produced and secreted from epithelia and granules of neutrophils. Deficiency of LL-37 in neutrophils and saliva has been correlated with occurrence of periodontal disease.4 Dhvar4 is a potent derivative of histatin-5, a member of a family of antifungal, histidine-rich proteins, secreted from salivary glands.5 Dhvar4a, a C-terminally amidated form of dhvar4, was used in our experiments.
Materials and methods Peptide synthesis K4-S4(1-15)a (LWKTLLKKVLKAAA-NH2), dhvar4a (KRLFKKLLFSLRKY-NH2) and LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) were prepared by automated solid phase method applying Fmoc active ester chemistry and subsequently purified by reversed phase HPLC.3 K4-S4(1-15)a and LL-37 showed a purity level >95% and dhvar4a (Biosight, Israel) >80% as determined by analytical HPLC.
Species and strains are listed in Table 1. Actinomyces viscosus, Lactobacillus paracasei, Streptococcus mutans and Streptococcus sobrinus were cultured in brain–heart infusion broth (BHI) (Difco, MD, USA), at 37 C in an atmosphere enriched with 5% CO2. Porphyromonas gingivalis and Fusobacterium nucleatum were grown in Wilkin’s broth (Oxoid, UK) and BHI supplemented with 0.05% glutamate, respectively, in jars containing an anaerobic atmosphere generation system (Oxoid). Actinobacillus actinomycetemcomitans was cultured in 0.5% yeast extract, 1.5% Bacto Tryptone, 0.75% D-glucose, 0.25% NaCl, 0.075% L-cysteine, 0.05% Table 1. Susceptibilities of selected bacteria to the antibacterial peptides K4-S4(1-15)a, dhvar4a and LL-37 MIC (mg/L) Organism
K4-S4(1-15)a
S. mutans DJ1 S. mutans ATCC 27351 S. mutans UA159 S. sobrinus 6715 L. paracasei DJ1 A. viscosus ATCC 43146 A. actinomycetemcomitans Y4 A. actinomycetemcomitans ATCC 29523 F. nucleatum ATCC 10953 P. gingivalis ATCC 33277 P. gingivalis W50 E. coli ATCC 25922
20 5 20 5 10 5–10 (5) 25–50 (50) 25–50 (50)
50–100 (100)a 20–40 (25) 50–100 (100) 20–40 (30) 100 30–100 (50) 200 200
NIb NI NI NI NI NI NI NI
5 NI NI 5
50–100 (50) NI NI 100
100 NI NI 50–100 (75)
dhvar4a
Calculated median values are shown in parentheses. No inhibition at concentrations £200 mg/L.
b
MIC against planktonic bacteria This was determined using a microdilution assay: overnight cultures of bacteria were diluted 1:10 (1:5000 for E. coli, 1:5 for A. actinomycetemcomitans) in culture medium, sonicated for 1 min in a sonication bath (Transsonic T 640, ELMA, Germany) and distributed in aliquots of 190 mL into 96-well plates. Ten microlitres of serial dilutions of the peptides were added. Plates were incubated at the appropriate growth conditions. Bacterial growth was determined by optical density (OD) measurements (650 nm) using a Thermo Max microplate spectrophotometer (Molecular Devices, CA, USA). MIC was determined as the lowest peptide concentration that prevented increase in OD. Chlorhexidine digluconate 5 mg/L (CHX) (Sigma, MO, USA) served as a positive control in all experiments. Each peptide concentration was tested in triplicate in three independent experiments.
Inhibition of biofilm formation
Bacterial strains and growth conditions
a
sodium thioglycolate and 4% NaHCO3 at 37 C in 5% CO2. Escherichia coli was grown in BHI under aerobic conditions. S. mutans DJ1 and L. paracasei DJ1 were isolated from saliva using the CRT kit (Ivoclar Vivadent, Schaan, Liechtenstein) and confirmed by 16S rDNA PCR amplification and sequencing (GenBank accession numbers DQ462439 and DQ462440, respectively), performed as described previously.6
S. mutans ATCC 27351 biofilms were generated by placing 10 mL aliquots of an overnight culture on each of the chambered cover glasses (Nunc, Denmark) and allowing attachment of bacteria for 30 min at room temperature. BHI (200 mL) supplemented with 4% sucrose and increasing concentrations (0, 5, 50, 500 mg/L) of K4-S4(1-15)a or CHX was added to each well, and chambers were incubated for 15–17 h at 37 C in 5% CO2. In order to test bactericidal activity on mature biofilms, the overnight biofilm cultures were washed and incubated for an additional 1 h in 50 mL fresh BHI, untreated or supplemented with K4-S4(1-15)a or CHX. Biofilm formation and viability were assessed using confocal laser scanning microscopy (CSLM).7 Slides were washed, incubated for 15 min in a solution containing propidium iodide and SYTO9 (live/dead BacLight viability kit, Molecular Probes, OR, USA) and washed again. Fluorescence emission was detected using a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany).
LL-37
Results and discussion K4-S4(1-15)a and dhvar4a inhibited growth of the oral streptococci, lactobacillus and A. viscosus at MIC values ranging from 5–20 and 20–100 mg/L, respectively. LL-37 did not inhibit growth of these species. This finding is in agreement with a recent study that found no correlation between dental caries and levels of salivary LL-37, in contrast to other antibacterial peptides.8 Among the selected Gram-negative species, F. nucleatum and the enteric E. coli were the most susceptible to the tested antibacterial peptides (Table 1). P. gingivalis was resistant to the peptides at all tested concentrations. Similarly, A. actinomycetemcomitans demonstrated low susceptibility to LL-37 and dhvar4a. K4-S4(1-15)a, however, inhibited A. actinomycetemcomitans growth, but at higher concentrations than those required for inhibiting the other susceptible bacteria. Out of the three peptides, K4-S4(1-15)a demonstrated the highest potency towards the
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Altman et al. tested species. Previous studies observed an MIC of 10 mg/L and 50–100 mg/L for LL-37 and a shorter analogue, against A. actinomycetemcomitans.9,10 MIC discrepancies may be attributable to differences in the experimental conditions such as exposure times, media and salt concentrations used in the antibacterial assays. Dental caries and periodontal disease are associated with bacterial biofilm. Bacteria in biofilm display lower susceptibility to antimicrobial agents compared with planktonic bacteria.2 As seen in Figure 1, K4-S4(1-15)a killed surface-attached S. mutans and inhibited biofilm formation. Killing of the surface-attached bacteria occurred at a higher concentration (50 mg/L) than that required to inhibit planktonic growth (5 mg/L). It appears that attachment of S. mutans to a surface reduces its susceptibility to K4-S4(1-15)a. Chlorhexidine digluconate inhibited biofilm formation at 5 mg/L. The ability of K4-S4(1-15)a to affect viability of S. mutans in a mature biofilm was also tested (Figure 1). Following 1 h of exposure to 500 mg/L K4-S4(1-15)a, a bactericidal effect was
evident throughout the 100 mm biofilm. At a concentration 10fold lower, partial killing effect could be observed, corresponding with the effect of chlorhexidine digluconate (Figure 1). Information regarding the effects of antibacterial peptides on biofilm compared with planktonic bacteria is limited. Dhvar4 reduced viability of a complex oral biofilm to a lower degree compared with homogeneous planktonic cultures,5 corresponding with our observations regarding the S. mutans biofilm. Mechanisms for microbial resistance to polycationic peptides include mutations affecting the membrane structure and charge distribution, modifications in the lipopolysaccharide structure of Gram-negative bacteria and active pumping of peptides out of the cell. P. gingivalis, which is implicated in periodontal diseases, is a highly proteolytic organism that has been found to degrade a variety of antimicrobial peptides.1 This may account for its resistance to the tested antibacterial peptides. In conclusion, it seems feasible to employ antibacterial peptides in future therapy of oral infection, as one approach in
Figure 1. CSLM cross-section images of S. mutans biofilms. Dead cells were stained red, while live cells were stained green using the BacLight LIVE/DEAD viability stain. Numbers in the bottom-left corner of each panel represent % viable cells – SD (n = 4), as measured by Image ProPlus software. (A–G) Mature biofilms, untreated (A) or exposed for 1 h to 5 (B), 50 (C), 500 (D) mg/L of K4-S4(1-15)a or to 5 (E), 50 (F), 500 (G) mg/L CHX. (H–K) Surface-attached S. mutans, untreated (H) or exposed overnight to 5 (I) or 50 (J) mg/L K4-S4(1-15)a or to 5 mg/L CHX (K).
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Effect of three antimicrobial peptides on selected oral pathogens dealing with the increase in microbial resistance to antibiotics. The oral cavity, being readily accessible for local application, may be particularly suitable for peptide therapy.
4. Putsep K, Carlsson G, Boman HG et al. Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. Lancet 2002; 360: 1144–9.
Acknowledgements
5. Helmerhorst EJ, Hodgson R, van’t Hof W et al. The effects of histatin-derived basic antimicrobial peptides on oral biofilms. J Dent Res 1999; 78: 1245–50.
This paper is a part of AH’s PhD dissertation and supported by the GSK/IADR innovation in oral care award.
Transparency declarations
6. Paster BJ, Boches SK, Galvin JL et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001; 183: 3770–83. 7. Zaura-Arite E, van Marle J, ten Cate JM. Conofocal microscopy study of undisturbed and chlorhexidine-treated dental biofilm. J Dent Res 2001; 80: 1436–40.
None to declare.
8. Tao R, Jurevic RJ, Coulton KK et al. Salivary antimicrobial peptide expression and dental caries experience in children. Antimicrob Agents Chemother 2005; 49: 3883–8.
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9. Tanaka D, Miyasaki KT, Lehrer RI. Sensitivity of Actinobacillus actinomycetemcomitans and Capnocytophaga spp. to the bactericidal action of LL-37: a cathelicidin found in human leukocytes and epithelium. Oral Microbiol Immunol 2000; 15: 226–31. 10. Ouhara K, Komatsuzawa H, Yamada S et al. Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells. J Antimicrob Chemother 2005; 55: 888–96.
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