期刊
JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY
卷 68, 期 11, 页码 2550-2560出版社
OXFORD UNIV PRESS
DOI: 10.1093/jac/dkt211
关键词
multi-species biofilm; confocal scanning laser microscopy; Live/Dead staining; microfluidics; pyrosequencing
资金
- National Institutes of Health [NIH: R21DE018820]
- University of Michigan
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 multispecies oral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectiveness of antimicrobials.
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