A quantitative method to study co-adhesion of microorganisms in a parallel plate flow chamber: basic principles of the analysis

Intermicrobial aggregation is described as one of the factors contributing to dental plaque formation. Intermicrobial aggregation is usually measured by mixing potential partners suspended in a liquid phase (‘coaggregation’). However, even if aggregation in the liquid phase occurs, adhesion of microorganisms to partners already adhering to a substratum surface may also occur (‘co-adhesion’). Coaggregation assays have been performed in order to measure coaggregation and to model co-adhesion, although it is not yet clear which the two prevails in vivo. Apart from being semi-quantitative (scores run from 0 to 4) it is questionable whether coaggregation assays really mimic co-adhesion. This study was designed to develop a method to quantitative assess co-adhesion of microbial pairs in order to gain a better understanding of the mechanisms governing co-adhesion. Co-adhesion of coaggregating and non-coaggregating partners (S. oralis, S. sanguis and A. naeslundii) to glass has been studied in a parallel plate flow chamber using real time image analysis. The spatial arrangements of adhering bacteria were analyzed by radial pair distribution functions, revealing the relative density of adhering bacteria around adhering bacteria from the same (g₂₂(r)) or a partner strain (g₂₁(r)). Pair distribution functions g₂₁(r) of coaggregating pairs clearly reveal a preference of coaggregating streptococci (S. oralis J22 and S. sanguis PK2951) to adhere around the actimomyces (A. naeslundii PK213 or T14V-J1), which were used to coat the bare glass substratum. Besides, the distribution function g₂₁(r) showed differences in co-adhesion patterns for strains with the same coaggregation score. From the results presented in this paper it can be concluded that with a parallel plate flow chamber, co-adhesion can be quantified on a continuous scale under well controlled conditions, more closely resembling those occurring in vivo.