title: Monitoring the prevalence of viable and dead cariogenic bacteria in oral specimens and in vitro biofilms by qPCR combined with propidium monoazide.
abstract: BACKGROUND
  Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important for understanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR).
  
  
  RESULTS
  We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. In addition, we applied this assay to analyze viable cell numbers in oral specimens. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H₂O₂). In planktonic cells, the number of viable cells decreased significantly with increasing H₂O₂ concentration, whereas only a small decrease was observed in biofilm cell numbers.
  
  
  CONCLUSIONS
  PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens and the oral status.
authors.affiliation: Division of Community Oral Health Development, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku Kitakyushu 803-8580, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Yasunaga
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yoshida
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Morikawa
authors.forename: Kenshi
authors.initials: K
authors.lastname: Maki
authors.forename: Suguru
authors.initials: S
authors.lastname: Nakamura
authors.forename: Inho
authors.initials: I
authors.lastname: Soh
authors.forename: Shuji
authors.initials: S
authors.lastname: Awano
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Ansai
mesh_terms: D001386:Azides / Q000378:metabolism; D058491:Bacterial Load / Q000379:methods; D018441:Biofilms; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D050296:Microbial Viability; D009055:Mouth / Q000382:microbiology*; D011419:Propidium / Q000031:analogs & derivatives / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D013295:Streptococcus mutans / Q000302:isolation & purification / Q000502:physiology*; D016986:Streptococcus sobrinus / Q000302:isolation & purification / Q000502:physiology*
chemical_list: D001386:Azides; D017931:DNA Primers; C533957:propidium monoazide; D011419:Propidium