A novel hydroxyapatite-binding antimicrobial peptide against oral biofilms

  • Yan Yang
  • Lingyun Xia
  • Markus Haapasalo
  • Wei Wei
  • Duo Zhang
  • Jingzhi Ma
  • Ya Shen
Original Article



Novel synthetic antimicrobial peptides which consist of a new immunomodulatory peptide 1018 and two different modifications with hydroxyapatite-binding affinity were developed. We compared the effect(s) of these peptides against oral plaque biofilms and measured their effectiveness in killing biofilm microbes and in reducing biofilm volume.

Materials and methods

The high affinity hydroxyapatite (HA)-binding peptide 1018 (SHABP), the mild affinity HA-binding peptide 1018 (MHABP), and peptide 1018 without additional amino acid sequence (peptide 1018) were synthesized. Oral multispecies biofilms were grown anaerobically for 3 days. The biofilms were exposed to three peptides at two different concentrations (0.65 and 3.25 μmol/L) for 24, 48, and 72 h. The biofilms were also treated for 3 or 9 min with the peptides (3.25 μmol/L). The percentage of killed biofilm bacteria and biofilm volume were determined by using LIVE/DEAD viability staining and confocal laser scanning microscopy.


SHABP was superior to MHABP and peptide 1018 in its killing efficacy of the pre-formed biofilms, especially at concentration of 3.25 μmol/L (p < 0.05). SHABP performed also better than MHABP and peptide 1018 in reducing the overall biofilm volume at both concentrations (p < 0.05). During the 3 days of long-term exposure, MHABP and peptide 1080 killed more bacteria in the top half of the biofilms, compared to bottom half. SHABP killed more bacteria in the bottom half (39%) of the biofilms than in the top half (29%) at day 1 (p < 0.05), whereas more bacteria were killed in the upper layers on days 2 and 3. SHABP killed a much higher percentage of plaque biofilm bacteria when used on 3-day-old biofilms for one or three times for 3 min than MHABP or peptide 1018 at high concentration (p < 0.05).


The modified peptide 1018 with high HA-binding affinity had higher antimicrobial activity against biofilm microbes and reduced biofilm volume more than the other peptides tested.

Clinical relevance

Modified peptide 1018 with high hydroxyapatite-binding affinity is a promising agent for use in oral antibiofilm strategies in the future.


Antimicrobial Binding Biofilm Hydroxyapatite Peptide 1018 



This work was partly supported by National Natural Science Foundation of China (NSFC, No. 81641035, 81700961, 81873714, and 81401524) and by Canada Foundation for Innovation (CFI: 32623).


This work was partly supported by National Natural Science Foundation of China (NSFC, No. 81641035, 81700961, 81873714, and 81401524) and by Canada Foundation for Innovation (CFI: 32623).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Ethics permission was obtained from the University of British Columbia Office of Research Services, Clinical Research Ethics Board (certificate number H15-02793).

Informed consent

Informed consent was obtained from the volunteer providing oral plaque in this study.


  1. 1.
    Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL, Boches SK, Dewhirst FE, Griffen AL (2002) Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol 40:1001–1009. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Socransky SS, Smith C, Haffajee AD (2002) Subgingival microbial profiles in refractor periodontal disease. J Clin Periodontol 29:260–268. CrossRefPubMedGoogle Scholar
  3. 3.
    Brito LC, Teles FR, Teles RP, FrancËa EC, Ribeiro-Sobrinho AP, Haffajee AD, Socransky SS (2007) Use of multiple-displacement amplification and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. J Clin Microbiol 45:3039–3049. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Haapasalo M, Shen Y, Ricucci D (2008) Reasons for persistent and emerging post-treatment endodontic disease. Endod Top 18:31–50. CrossRefGoogle Scholar
  5. 5.
    Shen Y, Zhao J, de la Fuente-Núñez C et al (2016) Experimental and theoretical investigation of multispecies oral biofilm resistance to chlorhexidine treatment. Sci Rep 6:27537. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Davey ME, O’Toole GA (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64:847–867. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K (2004) Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230:13–18. CrossRefPubMedGoogle Scholar
  8. 8.
    Lewis K (2007) Persister cells, dormancy and infectious disease. Nat Rev Microbiol 5:48–56. CrossRefPubMedGoogle Scholar
  9. 9.
    Shen Y, Stojicic S, Haapasalo M (2011) Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development. J Endod 37:657–661. CrossRefPubMedGoogle Scholar
  10. 10.
    Stojicic S, Shen Y, Haapasalo M (2013) Effect of the source of biofilm bacteria, level of biofilm maturation, and type of disinfecting agent on the susceptibility of biofilm bacteria to antibacterial agents. J Endod 39:473–477. CrossRefPubMedGoogle Scholar
  11. 11.
    Yeung AT, Gellatly SL, Hancock RE (2011) Multifunctional cationic host defence peptides and their clinical applications. Cell Mol Life Sci 68:2161–2176. CrossRefPubMedGoogle Scholar
  12. 12.
    Fjell CD, Hiss JA, Hancock REW, Schneider G (2012) Designing antimicrobial peptides: form follows function. Nat Rev Drug Discov 11:37–51. CrossRefGoogle Scholar
  13. 13.
    Hancock REW, Haney EF, Gill EE (2016) The immunology of host defence peptides: beyond antimicrobial activity. Nat Rev Immunol 16:321–334. CrossRefPubMedGoogle Scholar
  14. 14.
    de la Fuente-Nu ñez C, Reffuveille F, Haney EF, Straus SK, REW H (2014) Broad-spectrum anti-biofilm peptide that targets a cellular stress responses. PLoS Pathog e1004152:10. CrossRefGoogle Scholar
  15. 15.
    Wang Z, de la Fuente-Nu ñez C, Shen Y, Haapasalo M, Hancock RE (2015) Treatment of oral multispecies biofilms by an anti-biofilm peptide. PLoS One 10:e0132512. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zhang T, Wang Z, Hancock RE, de la Fuente-Núñez C, Haapasalo M (2016) Treatment of oral biofilms by a D-enantiomeric peptide. PLoS One 11:e0166997. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Sieprawska-Lupa M, Mydel P, Krawczyk K, Wo jcik K, Puklo M, Lupa B et al (2004) Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases. Antimicrob Agents Chemother 48:4673–4679. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Molhoek EM, van Dijk A, Veldhuizen EJ, Haagsman HP, Bikker FJ (2011) Improved proteolytic stability of chicken cathelicidin-2 derived peptides by D-amino acid substitutions and cyclization. Peptides 32:875–880. CrossRefPubMedGoogle Scholar
  19. 19.
    Kim H, Jang JH, Kim SC, Cho JH (2014) De novo generation of short antimicrobial peptides with enhanced stability and cell specificity. J Antimicrob Chemother 69:121–132. CrossRefPubMedGoogle Scholar
  20. 20.
    Dawes C (1987) Physiological factors affecting salivary flow rate, oral sugar clearance, and the sensation of dry mouth in man. J Dent Res 66:648–653. CrossRefPubMedGoogle Scholar
  21. 21.
    Fincham AG, Moradian-Oldak J, Simmer JP (1999) The structural biology of the developing dental enamel matrix. J Struct Biol 126:270–299. CrossRefPubMedGoogle Scholar
  22. 22.
    Gungormus M, Fong H, Kim IW, Evans JS, Tamerler C, Sarikaya M (2008) Regulation of in vitro calcium phosphate mineralization by combinatorially selected hydroxyapatite-binding peptides. Biomacromolecules 9:966–973. CrossRefPubMedGoogle Scholar
  23. 23.
    Ribeiro SM, de la Fuente-Núñez C, Baquir B, Faria-Junior C, Franco OL, Hancock RE (2015) Antibiofilm peptides increase the susceptibility of carbapenemase-producing Klebsiella pneumoniae clinical isolates to β-lactam antibiotics. Antimicrob Agents Chemother 59:3906–3912. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Stomatology, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
  2. 2.Faculty of Dentistry, Division of Endodontics, Department of Oral Biological and Medical SciencesUniversity of British ColumbiaVancouverCanada
  3. 3.Department of Stomatology, Taihe hospitalHubei University of MedicineShiyanChina

Personalised recommendations