title: Microcalorimetric determination of the effects of amoxicillin, metronidazole, and their combination on in vitro biofilm.
abstract: BACKGROUND
  The mechanism of action of adjuvant antibiotic therapy in the treatment of peri-implantitis is not well understood. The aim of this study is to investigate antibiotic susceptibility of an in vitro biofilm by isothermal microcalorimetry (IMC).
  
  
  METHODS
  Titanium disks containing a 72-hour three-species biofilm (Streptococcus sanguinis DSM20068, Fusobacterium nucleatum ATCC10953, and Porphyromonas gingivalis DSM20709) were placed in a series of IMC ampoules with nutrient agar supplemented with increasing concentrations of amoxicillin, metronidazole, or their combination and incubated anaerobically for 10 days. Lag time and maximum growth rate were determined from continuous heat-flow recordings of metabolic activity. To validate the IMC biofilm results, adherent S. sanguinis and P. gingivalis were incubated anaerobically in medium supplemented with antibiotics at 37°C for 24 hours, and their vitality was determined by live/dead staining, conventional culturing, and IMC.
  
  
  RESULTS
  In all biofilm samples incubated with antibiotics, a prolonged lag phase was observed compared with controls (P <0.05). Maximum growth rate was significantly lower for samples treated with either amoxicillin or metronidazole compared with controls (P <0.05). Combining the antibiotics did not improve this effect. Concentrations exceeding 10 times the minimum inhibitory concentration completely inhibited the growth of adherent S. sanguinis and P. gingivalis, whereas lower concentrations resulted in only a delay in the lag phase. A poor correlation was observed between live/dead staining and conventional culturing.
  
  
  CONCLUSIONS
  IMC gives new evidence about antibiotic effects on oral biofilms and is more informative than conventional culture and live/dead assays. The combination of antibiotics was found to be more efficient than metronidazole alone; however, only minor differences in growth inhibition were detected compared with amoxicillin alone.
authors.affiliation: Clinic of Preventive Dentistry and Oral Microbiology, School of Dental Medicine, University of Basel, Basel, Switzerland.
authors.forename: Monika
authors.initials: M
authors.lastname: Astasov-Frauenhoffer
authors.forename: Olivier
authors.initials: O
authors.lastname: Braissant
authors.forename: Irmgard
authors.initials: I
authors.lastname: Hauser-Gerspach
authors.forename: Roland
authors.initials: R
authors.lastname: Weiger
authors.forename: Clemens
authors.initials: C
authors.lastname: Walter
authors.forename: Nicola U
authors.initials: NU
authors.lastname: Zitzmann
authors.forename: Tuomas
authors.initials: T
authors.lastname: Waltimo
mesh_terms: D000658:Amoxicillin / Q000008:administration & dosage / Q000494:pharmacology*; D000693:Anaerobiosis; D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000494:pharmacology*; D001422:Bacterial Adhesion; D058491:Bacterial Load / Q000187:drug effects; D001431:Bacteriological Techniques; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D002151:Calorimetry / Q000379:methods; D003470:Culture Media; D004338:Drug Combinations; D024881:Drug Resistance, Bacterial; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D016967:Fusobacterium nucleatum / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008795:Metronidazole / Q000008:administration & dosage / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D016966:Porphyromonas gingivalis / Q000187:drug effects; D013298:Streptococcus sanguis / Q000187:drug effects; D013696:Temperature; D013997:Time Factors
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D004338:Drug Combinations; D005456:Fluorescent Dyes; D008795:Metronidazole; D000658:Amoxicillin; D016650:Fluorescein-5-isothiocyanate