title: Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model.
abstract: OBJECTIVE
  Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment.
  
  
  METHODS
  PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 μm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance.
  
  
  RESULTS
  When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10 . P. gingivalis showed a vitality reduction in the biofilm of 3 log10 , while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction.
  
  
  CONCLUSIONS
  These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.
authors.affiliation: Research Laboratory, Faculty of Dentistry, Complutense University of Madrid, Spain.
authors.forename: M C
authors.initials: MC
authors.lastname: Sánchez
authors.forename: M J
authors.initials: MJ
authors.lastname: Marín
authors.forename: E
authors.initials: E
authors.lastname: Figuero
authors.forename: A
authors.initials: A
authors.lastname: Llama-Palacios
authors.forename: R
authors.initials: R
authors.lastname: León
authors.forename: V
authors.initials: V
authors.lastname: Blanc
authors.forename: D
authors.initials: D
authors.lastname: Herrera
authors.forename: M
authors.initials: M
authors.lastname: Sanz
mesh_terms: D019840:2-Propanol / Q000494:pharmacology; D000190:Actinomyces / Q000187:drug effects / Q000302:isolation & purification; D016976:Aggregatibacter actinomycetemcomitans / Q000187:drug effects / Q000302:isolation & purification*; D000890:Anti-Infective Agents / Q000494:pharmacology; D001386:Azides; D058491:Bacterial Load / Q000187:drug effects; D018441:Biofilms / Q000145:classification* / Q000187:drug effects; D004396:Coloring Agents; D004269:DNA, Bacterial / Q000032:analysis; D016967:Fusobacterium nucleatum / Q000187:drug effects / Q000302:isolation & purification*; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects; D016966:Porphyromonas gingivalis / Q000187:drug effects / Q000302:isolation & purification*; D011419:Propidium / Q000031:analogs & derivatives*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012463:Saliva / Q000737:chemistry; D019533:Streptococcus oralis / Q000187:drug effects / Q000302:isolation & purification; D013997:Time Factors; D014678:Veillonella / Q000187:drug effects / Q000302:isolation & purification
chemical_list: D000890:Anti-Infective Agents; D001386:Azides; D004396:Coloring Agents; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium; D019840:2-Propanol
keywords: Aggregatibacter actinomycetemcomitans; Fusobacterium nucleatum; Porphyromonas gingivalis; antimicrobial agents; oral biofilm; propidium monoazide; quantitative real-time PCR