Antimicrobial peptide GH12 as root canal irrigant inhibits biofilm and virulence of Enterococcus faecalis.

AIM The objectives of this laboratory-based study were to investigate the effects of GH12 on E. faecalis biofilm and virulence. METHODOLOGY Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of GH12 against E. faecalis were first determined. Time-kill assay was further conducted. The effects of GH12 on the expression of virulence and stress genes in E. faecalis were evaluated by RT-qPCR. Crystal violet stain was used to investigate the effects of GH12 on E. faecalis biofilm formation and 1-day-old biofilm. Finally, an ex vivo tooth model contaminated with E. faecalis was used to evaluate the antimicrobial activity of GH12 as an irrigant by CFU counting, SEM and CLSM. One-way ANOVA and Tukey's multiple comparisons test were used to compare the differences among groups (α = 0.05). RESULTS The MICs and MBCs of GH12 against E. faecalis were 8.0 ± 0.0 mg/L and 16.0 ± 0.0 mg/L, respectively and GH12 at 32.0 mg/L reduced the bacterial numbers by more than 99.9% within 1 min. Various virulence genes (efaA, esp, gelE) and stress genes (dnaK, groEL, ctsR, clpPBCEX) in E. faecalis were significantly downregulated by GH12 at sub-MIC levels (P < 0.05). Additionally, both E. faecalis biofilm formation and the biomass of 1-day-old E. faecalis biofilms were significantly reduced by GH12 (P < 0.05). Elimination of E. faecalis in biofilms from root canal walls was achieved through irrigation with 64.0 mg/L GH12 for 30 min. CLSM analysis revealed that GH12 at 64.0 mg/L was most effective in eliminating bacteria within dentinal tubules (P < 0.05). CONCLUSION In a laboratory setting, and when used as an irrigant, GH12 suppressed E. faecalis, downregulated specific virulence and stress-associated genes, eliminated intracanal E. faecalis protected by biofilms and killed bacteria in dentinal tubules. These results emphasise the need for preclinical and clinical studies to explore the potential of GH12 as an antimicrobial agent in root canal treatment.

[1]  T. Lu,et al.  Peptide Design Principles for Antimicrobial Applications. , 2019, Journal of molecular biology.

[2]  L. Lo Muzio,et al.  Inspection of the Microbiota in Endodontic Lesions , 2019, Dentistry journal.

[3]  J. A. Vale,et al.  The clinical toxicology of sodium hypochlorite , 2019, Clinical toxicology.

[4]  M. Burrow,et al.  Effect on the mechanical properties of human and bovine dentine of intracanal medicaments and irrigants , 2018, Australian dental journal.

[5]  M. Jafelicci,et al.  Effect of the combination of several irrigants on dentine surface properties, adsorption of chlorhexidine and adhesion of microorganisms to dentine , 2018, International endodontic journal.

[6]  Linglin Zhang,et al.  Antimicrobial peptide GH12 targets Streptococcus mutans to arrest caries development in rats , 2018, Journal of Oral Microbiology.

[7]  K. Ganbarov,et al.  Effect of acidic and alkali shocks on expression of efaA gene in Enterococcus faecalis, isolated from root canal infection. , 2018, Cellular and molecular biology.

[8]  Xuedong Zhou,et al.  Effects of Antimicrobial Peptide GH12 on the Cariogenic Properties and Composition of a Cariogenic Multispecies Biofilm , 2018, Applied and Environmental Microbiology.

[9]  J. Schrenzel,et al.  Root Microbiota in Primary and Secondary Apical Periodontitis , 2018, Front. Microbiol..

[10]  Raquel Zanin Midena,et al.  Antibacterial properties of silver nanoparticles as a root canal irrigant against Enterococcus faecalis biofilm and infected dentinal tubules , 2018, International endodontic journal.

[11]  M. Mathew,et al.  Total Eradication of Bacterial Infection in Root Canal Treatment: An Electrochemical Approach. , 2018, ACS biomaterials science & engineering.

[12]  M. Pourhajibagher,et al.  Ex vivo assessment of synergic effect of chlorhexidine for enhancing antimicrobial photodynamic therapy efficiency on expression patterns of biofilm-associated genes of Enterococcus faecalis. , 2018, Photodiagnosis and photodynamic therapy.

[13]  M. Del Fabbro,et al.  Antimicrobial effectiveness of combinations of oxidant and chelating agents in infected dentine: an ex vivo confocal laser scanning microscopy study , 2018, International endodontic journal.

[14]  J. Maillard,et al.  Enterococcus faecalis Demonstrates Pathogenicity through Increased Attachment in an Ex Vivo Polymicrobial Pulpal Infection , 2018, Infection and Immunity.

[15]  W. Fan,et al.  Effects of prolonged exposure to moderate static magnetic field and its synergistic effects with alkaline pH on Enterococcus faecalis. , 2018, Microbial pathogenesis.

[16]  R. Nixon,et al.  Immediate hypersensitivity to chlorhexidine , 2018, The Australasian journal of dermatology.

[17]  Xuedong Zhou,et al.  Antimicrobial peptide GH12 suppresses cariogenic virulence factors of Streptococcus mutans , 2018, Journal of oral microbiology.

[18]  Thaís da Silva Moraes,et al.  Mikania glomerata Sprengel extract and its major compound ent-kaurenoic acid display activity against bacteria present in endodontic infections. , 2017, Anaerobe.

[19]  K. Kum,et al.  The synthetic human beta-defensin-3 C15 peptide exhibits antimicrobial activity against Streptococcus mutans, both alone and in combination with dental disinfectants , 2017, Journal of Microbiology.

[20]  H. Sharghi,et al.  Antibiofilm Efficacy of Positively Charged Imidazolium-Based Silver Nanoparticles in Enterococcus faecalis Using Quantitative Real-Time PCR , 2017 .

[21]  O. Franco,et al.  Antimicrobial and immunomodulatory activity of host defense peptides, clavanins and LL-37, in vitro: An endodontic perspective , 2017, Peptides.

[22]  C. Keskin,et al.  Pyrosequencing Analysis of Cryogenically Ground Samples from Primary and Secondary/Persistent Endodontic Infections , 2017, Journal of endodontics.

[23]  Xuedong Zhou,et al.  De novo synthetic short antimicrobial peptides against cariogenic bacteria. , 2017, Archives of oral biology.

[24]  M. Pourhajibagher,et al.  The evaluation of cultivable microbiota profile in patients with secondary endodontic infection before and after photo-activated disinfection. , 2017, Photodiagnosis and photodynamic therapy.

[25]  F. Tay,et al.  Potential applications of antimicrobial peptides and their mimics in combating caries and pulpal infections. , 2017, Acta biomaterialia.

[26]  M. Pourhajibagher,et al.  Sub-lethal doses of photodynamic therapy affect biofilm formation ability and metabolic activity of Enterococcus faecalis. , 2016, Photodiagnosis and photodynamic therapy.

[27]  Zhongchun Tong,et al.  The Starvation Resistance and Biofilm Formation of Enterococcus faecalis in Coexistence with Candida albicans, Streptococcus gordonii, Actinomyces viscosus, or Lactobacillus acidophilus. , 2016, Journal of endodontics.

[28]  H. Fong,et al.  Disinfection Efficacy of Current Regenerative Endodontic Protocols in Simulated Necrotic Immature Permanent Teeth. , 2016, Journal of endodontics.

[29]  A. de-Jesus-Soares,et al.  Antimicrobial Susceptibility and Characterization of Virulence Genes of Enterococcus faecalis Isolates from Teeth with Failure of the Endodontic Treatment. , 2016, Journal of endodontics.

[30]  S. Han,et al.  Lipoteichoic Acid of Enterococcus faecalis Inhibits the Differentiation of Macrophages into Osteoclasts. , 2016, Journal of endodontics.

[31]  L. Gonçalves,et al.  The Effect of Sodium Hypochlorite and Chlorhexidine as Irrigant Solutions for Root Canal Disinfection: A Systematic Review of Clinical Trials. , 2016, Journal of endodontics.

[32]  Xuedong Zhou,et al.  Activity of Synthetic Antimicrobial Peptide GH12 against Oral Streptococci , 2016, Caries Research.

[33]  M. H. Hungaro Duarte,et al.  Antimicrobial activity of Chlorhexidine, Peracetic acid and Sodium hypochlorite/etidronate irrigant solutions against Enterococcus faecalis biofilms. , 2015, International endodontic journal.

[34]  I. G. de Moraes,et al.  A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy , 2015, Journal of applied oral science : revista FOB.

[35]  P. Samant,et al.  To comparatively evaluate the antimicrobial efficacy of chlorhexidine, nisin and linezolid as an intracanal medicament on Enterococcus faecalis: An in vitro study. , 2015, Indian journal of dental research : official publication of Indian Society for Dental Research.

[36]  I. N. Rôças,et al.  Total and Specific Bacterial Levels in the Apical Root Canal System of Teeth with Post-treatment Apical Periodontitis. , 2015, Journal of endodontics.

[37]  W. Jiang,et al.  Assessment of dentinal tubule invasion capacity of Enterococcus faecalis under stress conditions ex vivo. , 2015, International endodontic journal.

[38]  V. Shetty,et al.  Biofilm in endodontics: A review , 2015, Journal of International Society of Preventive & Community Dentistry.

[39]  O. Franco,et al.  Antimicrobial peptide-based treatment for endodontic infections--biotechnological innovation in endodontics. , 2015, Biotechnology advances.

[40]  W. Fan,et al.  Effects of adsorbed and templated nanosilver in mesoporous calcium-silicate nanoparticles on inhibition of bacteria colonization of dentin , 2014, International journal of nanomedicine.

[41]  E. Watanabe,et al.  Antibiofilm activity of irrigating solutions associated with cetrimide. Confocal laser scanning microscopy. , 2014, International endodontic journal.

[42]  D. Deng,et al.  Effects of Intracanal Irrigant MTAD Combined with Nisin at Sub-Minimum Inhibitory Concentration Levels on Enterococcus faecalis Growth and the Expression of Pathogenic Genes , 2014, PloS one.

[43]  M. Jafelicci,et al.  Wettability of chlorhexidine treated non-carious and caries-affected dentine. , 2014, Australian dental journal.

[44]  Y. Shen,et al.  Irrigation in endodontics , 2014, BDJ.

[45]  A. Al-Ahmad,et al.  Antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth. , 2014, Journal of endodontics.

[46]  B. Üreyen Kaya,et al.  Efficacy of endodontic applications of ozone and low‐temperature atmospheric pressure plasma on root canals infected with Enterococcus faecalis , 2014, Letters in applied microbiology.

[47]  M. Versluis,et al.  Radiographic healing after a root canal treatment performed in single-rooted teeth with and without ultrasonic activation of the irrigant: a randomized controlled trial. , 2013, Journal of endodontics.

[48]  C. Löst,et al.  Ultrasonic monitoring of the effect of sodium hypochlorite on the elasticity of dentine. , 2013, International endodontic journal.

[49]  Z. Schauperl,et al.  Antimicrobial efficacy of a high-power diode laser, photo-activated disinfection, conventional and sonic activated irrigation during root canal treatment. , 2013, International endodontic journal.

[50]  M. González-Rodríguez,et al.  Antimicrobial activity of essential oils and chloroform alone and combinated with cetrimide against Enterococcus faecalis biofilm. , 2013, European journal of microbiology & immunology.

[51]  B. Yingyongnarongkul,et al.  Antibacterial substances from Albizia myriophylla wood against cariogenic Streptococcus mutans , 2013, Archives of Pharmacal Research.

[52]  C. Huang,et al.  Effect of intracanal dentine wettability on human dental pulp cell attachment. , 2012, International endodontic journal.

[53]  Yi Zhou,et al.  Effects of compounds found in Nidus Vespae on the growth and cariogenic virulence factors of Streptococcus mutans. , 2012, Microbiological research.

[54]  Ya Shen,et al.  A new noninvasive model to study the effectiveness of dentin disinfection by using confocal laser scanning microscopy. , 2011, Journal of endodontics.

[55]  C. Bramante,et al.  Biofilm dissolution and cleaning ability of different irrigant solutions on intraorally infected dentin. , 2011, Journal of endodontics.

[56]  Xuedong Zhou,et al.  Effect of the antimicrobial decapeptide KSL on the growth of oral pathogens and Streptococcus mutans biofilm. , 2011, International journal of antimicrobial agents.

[57]  H. Flemming,et al.  The biofilm matrix , 2010, Nature Reviews Microbiology.

[58]  R. Vogel,et al.  Sub-lethal stress effects on virulence gene expression in Enterococcus faecalis. , 2010, Food microbiology.

[59]  L. E. Chávez de Paz,et al.  The effects of antimicrobials on endodontic biofilm bacteria. , 2010, Journal of endodontics.

[60]  W. Stark,et al.  Effect of sodium hypochlorite on human root dentine--mechanical, chemical and structural evaluation. , 2007, International endodontic journal.

[61]  H. Ingmer,et al.  Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram‐positive bacteria , 2007, Molecular microbiology.

[62]  L. Zurek,et al.  Ecology of Antibiotic Resistance Genes: Characterization of Enterococci from Houseflies Collected in Food Settings , 2006, Applied and Environmental Microbiology.

[63]  S. Schwartz,et al.  nterococcus faecalis: Its Role in Root Canal Treatment ailure and Current Concepts in Retreatment , 2006 .

[64]  D. Ørstavik,et al.  The susceptibility of starved, stationary phase, and growing cells of Enterococcus faecalis to endodontic medicaments. , 2005, Journal of endodontics.

[65]  D. Ørstavik,et al.  Virulence factors of Enterococcus faecalis: relationship to endodontic disease. , 2004, Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists.

[66]  D. Snydman,et al.  Association between the presence of enterococcal virulence factors gelatinase, hemolysin, and enterococcal surface protein and mortality among patients with bacteremia due to Enterococcus faecalis. , 2002, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[67]  M. Gilmore,et al.  Differential Expression of Virulence-Related Genes in Enterococcus faecalis in Response to Biological Cues in Serum and Urine , 2002, Infection and Immunity.

[68]  K Gulabivala,et al.  Effect of sodium hypochlorite on mechanical properties of dentine and tooth surface strain. , 2001, International endodontic journal.

[69]  L. Watanabe,et al.  Acid-etching and Hydration Influence on Dentin Roughness and Wettability , 1999, Journal of dental research.