Real-time quantitative reverse transcription-PCR analysis of expression stability of Aggregatibacter actinomycetemcomitans fimbria-associated gene in response to photodynamic therapy

Photodiagnosis Photodyn Ther. 2017 Jun:18:78-82. doi: 10.1016/j.pdpdt.2017.02.009. Epub 2017 Feb 16.

Abstract

Background: Aggregatibacter actinomycetemcomitans is an etiological agent of both chronic and aggressive periodontitis. Dissemination of A. actinomycetemcomitans from the oral cavity and initiation of systemic infections has led to new approaches for treatment being needed. In this study, a series of experiments presented investigated the effect of methylene blue (MB)-mediated antimicrobial photodynamic therapy (aPDT) on cell viability and expression of fimbria-associated gene (rcpA) in A. actinomycetemcomitans.

Materials and methods: To determine the dose-depended effects of aPDT, A. actinomycetemcomitans ATCC 33384 strain photosensitized with MB was irradiated with diode laser following bacterial viability measurements. Cell-surviving assay and expression ratio of rcpA were assessed by colony forming unit and real-time quantitative reverse transcription-PCR (qRT-PCR) assays, respectively.

Results: In the current study, MB-mediated aPDT using 100μg/mL showed significant reduction in A. actinomycetemcomitans growth when compared to the control (P<0.05). Sub-lethal dose of aPDT against A. actinomycetemcomitans was 25μg/mL MB at fluency of 93.75J/cm2. Sub-lethal dose of aPDT could lead to about four-fold suppression of expression of rcpA.

Conclusion: High doses of MB-mediated aPDT could potentially exhibit antimicrobial activity, and the expression of rcpA as an important virulence factor of this strain is reduced in cells surviving aPDT with MB. So, aPDT can be a valuable tool for the treatment of A. actinomycetemcomitans infections.

Keywords: Aggregatibacter actinomycetemcomitans; Biofilm; Diode laser; Methylene blue; Photodynamic therapy; Virulence factor; qRT-PCR; rcpA.

MeSH terms

  • Aggregatibacter actinomycetemcomitans / genetics
  • Aggregatibacter actinomycetemcomitans / metabolism*
  • Aggregatibacter actinomycetemcomitans / radiation effects*
  • Bacterial Load
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Outer Membrane Proteins / metabolism*
  • Biomarkers / metabolism
  • Cell Survival / drug effects
  • Cell Survival / radiation effects
  • Dose-Response Relationship, Drug
  • Dose-Response Relationship, Radiation
  • Fimbriae Proteins / genetics
  • Fimbriae Proteins / metabolism
  • Fimbriae, Bacterial / genetics
  • Fimbriae, Bacterial / metabolism
  • Gene Expression Regulation, Bacterial / drug effects*
  • Gene Expression Regulation, Bacterial / radiation effects*
  • Photochemotherapy / methods*
  • Photosensitizing Agents / administration & dosage
  • Radiation Dosage
  • Real-Time Polymerase Chain Reaction / methods
  • Treatment Outcome

Substances

  • Bacterial Outer Membrane Proteins
  • Biomarkers
  • Photosensitizing Agents
  • RcpA protein, Actinobacillus actinomycetemcomitans
  • Fimbriae Proteins