title: High-throughput quantitative method for assessing coaggregation among oral bacterial species.
abstract: This paper describes a high-throughput method that relies upon a microplate reader to score coaggregation 60 min postmixing, and use of a high-speed real-time imaging technology to describe the rate of coaggregation over time. The results of visual, microplate, and FlowCam(™) 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 cell-density. Pairs were also passed through a flow cell within a FlowCam(™) to quantify the rate of coaggregation of each pair. Results from both the microplate and FlowCam(™) approaches correlated with corresponding visual coaggregation scores and microscopic observations. The microplate-based assay enables high-throughput screening, whereas the FlowCam(™) -based assay validates and quantifies the extent that autoaggregation and coaggregation occur. Together these assays open the door for future in-depth studies of autoaggregation and coaggregation among large panels of test strains.
  
  
  CONCLUSIONS
  Coaggregation between bacterial species is integral to multi-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 microplate-based high-throughput approach and the other uses a FlowCam(™) device. The microplate-based approach enables rapid detection of coaggregation between candidate coaggregating pairs of strains simultaneously while controlling for variation between replicates. The FlowCam(™) approach allows for in-depth analysis of the rates of coaggregation and size of aggregates formed.
authors.affiliation: Epidemiology Department, School of Public Health, University of Michigan, Ann Arbor, MI, USA.
authors.forename: E
authors.initials: E
authors.lastname: Levin-Sparenberg
authors.affiliation: Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Shin
authors.affiliation: Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA.
authors.forename: E M
authors.initials: EM
authors.lastname: Hastings
authors.affiliation: Epidemiology Department, School of Public Health, University of Michigan, Ann Arbor, MI, USA.
authors.forename: M
authors.initials: M
authors.lastname: Freeland
authors.affiliation: Epidemiology Department, School of Public Health, University of Michigan, Ann Arbor, MI, USA.
authors.forename: H
authors.initials: H
authors.lastname: Segaloff
authors.affiliation: Epidemiology Department, School of Public Health, University of Michigan, Ann Arbor, MI, USA.
authors.forename: A H
authors.initials: AH
authors.lastname: Rickard
authors.affiliation: Epidemiology Department, School of Public Health, University of Michigan, Ann Arbor, MI, USA. bfoxman@umich.edu.
authors.forename: B
authors.initials: B
authors.lastname: Foxman
mesh_terms: D000190:Actinomyces / Q000254:growth & development / Q000502:physiology*; D001422:Bacterial Adhesion / Q000502:physiology*; D018441:Biofilms / Q000254:growth & development*; D057166:High-Throughput Screening Assays / Q000379:methods; D018613:Microscopy, Confocal; D009055:Mouth / Q000382:microbiology*; D013291:Streptococcus / Q000254:growth & development / Q000502:physiology*
keywords: Actinomycetes; Biofilms; Biotechnology; Coaggregation; Streptococci