Highâ throughput quantitative method for assessing coaggregation among oral bacterial species

Abstract

This paper describes a highâ throughput method that relies upon a microplate reader to score coaggregation 60 min postmixing, and use of a highâ speed realâ time imaging technology to describe the rate of coaggregation over time. The results of visual, microplate, and FlowCamâ ¢ aggregation scores for oral bacteria Streptococcus gordonii, Streptococcus oralis, and Actinomyces oris, whose ability to coaggregate are well characterized, are compared. Following mixing of all possible pairs, the top fraction of the supernatant was added to a microplate to quantify cellâ density. Pairs were also passed through a flow cell within a FlowCamâ ¢ to quantify the rate of coaggregation of each pair. Results from both the microplate and FlowCamâ ¢ approaches correlated with corresponding visual coaggregation scores and microscopic observations. The microplateâ based assay enables highâ throughput screening, whereas the FlowCamâ ¢â based assay validates and quantifies the extent that autoaggregation and coaggregation occur. Together these assays open the door for future inâ depth studies of autoaggregation and coaggregation among large panels of test strains.Significance and Impact of the StudyCoaggregation between bacterial species is integral to multiâ species biofilm development. Difficulties in rapidly and reproducibly identifying and quantifying coaggregation have limited mechanistic studies. This paper demonstrates two complementary quantitative methods to screen for coaggregation. The first approach uses a microplateâ based highâ throughput approach and the other uses a FlowCamâ ¢ device. The microplateâ based approach enables rapid detection of coaggregation between candidate coaggregating pairs of strains simultaneously while controlling for variation between replicates. The FlowCamâ ¢ approach allows for inâ depth analysis of the rates of coaggregation and size of aggregates formed.Significance and Impact of the Study: Coaggregation between bacterial species is integral to multiâ species biofilm development. Difficulties in rapidly and reproducibly identifying and quantifying coaggregation have limited mechanistic studies. This paper demonstrates two complementary quantitative methods to screen for coaggregation. The first approach uses a microplateâ based highâ throughput approach and the other uses a FlowCamâ ¢ device. The microplateâ based approach enables rapid detection of coaggregation between candidate coaggregating pairs of strains simultaneously while controlling for variation between replicates. The FlowCamâ ¢ approach allows for inâ depth analysis of the rates of coaggregation and size of aggregates formed.