Selective killing of Aggregatibacter actinomycetemcomitans by ciprofloxacin during development of a dual species biofilm with Streptococcus sanguinis

Abstract

Objectives: Periodontal disease is associated with a pathogen-induced transition to a chronic destructive inflammatory response. Since commensals may either passively or actively contribute to immune homeostasis, therapies aimed at selectively reducing the competitive advantage of pathogens may be effective supplements to traditional methods. We developed an in vitro system to grow biofilms composed of the pathogen (Aggregatibacter actinomycetemcomitans) and the commensal (Streptococcus sanguinis). We used the biofilm model to determine the feasibility of selectively killing the pathogen using the fluoroquinolone, ciprofloxacin.Design: Biofilms were exposed to relevant ciprofloxacin doses during the first 24 h of development, with subsequent removal of the ciprofloxacin for a 24 h period. Biofilm growth was assessed by confocal laser scanning microscopy, crystal violet staining and DNA abundance.Results: Exposure to 0.01 mg/L or 0.5 mg/L ciprofloxacin significantly reduced the microcolony size and cell surface density of A. actinomycetemcomitans in the dual species biofilm over a 24 h period whilst allowing uninhibited S. sanguinis biofilm formation. A. actinomycetemcomitans biofilm development was insignificant over a subsequent 24 h period after removal of the ciprofloxacin indicating that A. actinomycetemcomitans cells were killed.Conclusions: A. actinomycetemcomitans residing in a dual species biofilm with the commensal, S. sanguinis can be selectively killed, or at least rendered metabolically inactive, by treatment with ciprofloxacin. The dual species biofilm model will be a useful tool for designing in vivo studies to determine the efficacy of selective killing agents as an adjunct treatment of localized aggressive forms of periodontal disease.