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ABSTRACT In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro.IMPORTANCEAlthough most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

Abstract Background Caries and periodontitis are amongst the most prevalent diseases worldwide, leading to pain and loss of oral function for those affected. Prevention relies heavily on mechanical removal of dental plaque biofilms but for populations where this is not achievable, alternative plaque control methods are required. With concerns over undesirable side-effects and potential bacterial resistance due to the use of chlorhexidine gluconate (CHX), new antimicrobial substances for oral use are greatly needed. Here we have investigated the antimicrobial effect of hypochlorous acid (HOCl), stabilized with acetic acid (HAc), on oral biofilms and compared it to that of CHX. Possible adverse effects of stabilized HOCl on hydroxyapatite surfaces were also examined. Methods Single- and mixed-species biofilms of six common oral bacteria (Streptococcus mutans, Streptococcus gordonii, Actinomyces odontolyticus, Veillonella parvula, Parvimonas micra and Porphyromonas gingivalis) within a flow-cell model were exposed to HOCl stabilized with 0.14% or 2% HAc, pH 4.6, as well as HOCl or HAc alone. Biofilm viability was assessed in situ using confocal laser scanning microscopy following LIVE/DEAD® BacLight™ staining. In-situ quartz crystal microbalance with dissipation (QCM-D) was used to study erosion of hydroxyapatite (HA) surfaces by stabilized HOCl. Results Low concentrations of HOCl (5 ppm), stabilized with 0.14% or 2% HAc, significantly reduced viability in multi-species biofilms representing supra- and sub-gingival oral communities, after 5 min, without causing erosion of HA surfaces. No equivalent antimicrobial effect was seen for CHX. Gram-positive and Gram-negative bacteria showed no significant differential suceptibility to stabilized HOCl. Conclusions At low concentrations and with exposure times which could be achieved through oral rinsing, HOCl stabilized with HAc had a robust antimicrobial activity on oral biofilms, without causing erosion of HA surfaces or affecting viability of oral keratinocytes. This substance thus appears to offer potential for prevention and/or treatment of oral biofilm-mediated diseases.