title: A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm.
abstract: BACKGROUND
  Antimicrobial photodynamic therapy (APDT) is a process that generates reactive oxygen species (ROS) in presence of photosensitizer, visible light and oxygen which destroys the bacterial cells. We investigated the photoinactivation efficiency of phenothiazinium dyes and the effect of ROS generation on Gram positive and Gram negative bacterial cell as well as on biofilm.
  
  
  METHODS
  Enterococcus faecalis and Klebsiella pneumonia were incubated with all the three phenothiazinium dyes and exposed to 630nm of light. After PDT, colony forming unit (CFU) were performed to estimate the cell survival fraction. Intracellular reactive oxygen species (ROS) was detected by DCFH-DA. Crystal violet (CV) assay and extracellular polysaccharides (EPS) reduction assay were performed to analyze antibiofilm effect. Confocal laser electron microscope (CLSM) scanning electron microscope (SEM) was performed to assess the disruption of biofilm.
  
  
  RESULTS
  8log10reduction in bacterial count was observed in Enterococcus faecalis while 3log10in Klebsiella pneumoniae. CV and EPS reduction assay revealed that photodynamic inhibition was more pronounced in Enterococcus faecalis. In addition to this CLSM and SEM study showed an increase in cell permeability of propidium iodide and leakage of cellular constituents in treated preformed biofilm which reflects the antibiofilm action of photodynamic therapy.
  
  
  CONCLUSIONS
  We conclude that Gram-positive bacteria (Enterococcus faecalis) are more susceptible to APDT due to increased level of ROS generation inside the cell, higher photosensitizer binding efficiency and DNA degradation. Phenothiazinium dyes are proved to be highly efficient against both planktonic and biofilm state of cells.
authors.affiliation: Medical Microbiology and Molecular Biology Lab., Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP, 202 002, India.
authors.forename: Lama
authors.initials: L
authors.lastname: Misba
authors.affiliation: Medical Microbiology and Molecular Biology Lab., Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP, 202 002, India.
authors.forename: Sahar
authors.initials: S
authors.lastname: Zaidi
authors.affiliation: Medical Microbiology and Molecular Biology Lab., Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, UP, 202 002, India. Electronic address: asad.k@rediffmail.com.
authors.forename: Asad U
authors.initials: AU
authors.lastname: Khan
mesh_terms: D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development* / Q000528:radiation effects; D004396:Coloring Agents / Q000008:administration & dosage; D004203:Disinfection / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000254:growth & development / Q000528:radiation effects; D007709:Klebsiella / Q000187:drug effects* / Q000254:growth & development / Q000528:radiation effects; D010640:Phenothiazines / Q000008:administration & dosage*; D010778:Photochemotherapy / Q000379:methods*; D017319:Photosensitizing Agents / Q000008:administration & dosage; D016896:Treatment Outcome
chemical_list: D004396:Coloring Agents; D010640:Phenothiazines; D017319:Photosensitizing Agents
keywords: Biofilm; DCFH-DA; DNA fragmentation; Gram negative; Gram positive; Photodynamic therapy