A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials.
OBJECTIVES
Few model systems are amenable to developing multi-species biofilms in parallel under environmentally germane conditions. This is a problem when evaluating the potential real-world effectiveness of antimicrobials in the laboratory. One such antimicrobial is cetylpyridinium chloride (CPC), which is used in numerous over-the-counter oral healthcare products. The aim of this work was to develop a high-throughput microfluidic system that is combined with a confocal laser scanning microscope (CLSM) to quantitatively evaluate the effectiveness of CPC against oral multi-species biofilms grown in human saliva.


METHODS
Twenty-four-channel BioFlux microfluidic plates were inoculated with pooled human saliva and fed filter-sterilized saliva for 20 h at 37°C. The bacterial diversity of the biofilms was evaluated by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). The antimicrobial/anti-biofilm effect of CPC (0.5%-0.001% w/v) was examined using Live/Dead stain, CLSM and 3D imaging software.


RESULTS
The analysis of biofilms by bTEFAP demonstrated that they contained genera typically found in human dental plaque. These included Aggregatibacter, Fusobacterium, Neisseria, Porphyromonas, Streptococcus and Veillonella. Using Live/Dead stain, clear gradations in killing were observed when the biofilms were treated with CPC between 0.5% and 0.001% w/v. At 0.5% (w/v) CPC, 90% of the total signal was from dead/damaged cells. Below this concentration range, less killing was observed. In the 0.5%-0.05% (w/v) range CPC penetration/killing was greatest and biofilm thickness was significantly reduced.


CONCLUSIONS
This work demonstrates the utility of a high-throughput microfluidic-CLSM system to grow multi-species oral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectiveness of antimicrobials.
['Nance|William C|WC|', 'Dowd|Scot E|SE|', 'Samarian|Derek|D|', 'Chludzinski|Jeffrey|J|', 'Delli|Joseph|J|', 'Battista|John|J|', 'Rickard|Alexander H|AH|']
D000328:Adult D000890:Anti-Infective Agents D018441:Biofilms D002594:Cetylpyridinium D003773:Dental Plaque D006801:Humans D059013:Microbial Consortia D044085:Microfluidics D018613:Microscopy, Confocal D012463:Saliva
microfluidics multi-species biofilm pyrosequencing Live/Dead staining confocal scanning laser microscopy