Abstract
The present study aims to establish a standardized model that makes it possible to evaluate the dynamic dissolution of biofilm, killing of biofilm microbes and inhibition of growth of biofilm by disinfecting solutions. Biofilm was grown from dental plaque bacteria on collagen-coated hydroxyapatite (HA) disks for 3 days or 3 weeks under anaerobic conditions. Biofilms were stained with the LIVE/DEAD viability stain and subjected to sterile water, 2% sodium hypochlorite (NaOCl), 6% NaOCl, or 2% chlorhexidine (CHX) for 32 min. Dynamic change in fluorescence on bacterial cells and extracellular polymeric substance (EPS) during the exposure was analyzed using Alexa Fluor 647-labeled dextran conjugate and a live-cell imaging confocal laser scanning microscopy (LC-CLSM). The biofilm structures after treatments were visualized by scanning electron microscopy (SEM). The treated biofilms on HA disks were collected and subjected to colony forming unit (CFU) counting. Another set of sterile HA disks were coated with CHX prior to the monitoring of plaque biofilm growth for 12 h. The LC-CLSM results showed that NaOCl dissolved biofilm effectively, more so at a higher concentration and longer exposure time. Six percent NaOCl was the most effective at dissolving and killing bacteria (e.g., 99% bacterial reduction in 3-day-old biofilm and 95% bacterial reduction in 3-week-old biofilm in 32 min) followed by 2% NaOCl and CHX. Sodium hypochlorite dissolved over 99.9% of the EPS whereas CHX only slightly reduced the EPS biovolume in 32 min. CFU results indicated that the dispersed biofilm bacteria are more resistant than planktonic bacteria to disinfectants. SEM showed the disruption of biofilm after exposures to CHX and NaOCl. The use of 2% CHX and sterile water did not result in biofilm dissolution. However, prior exposure of the HA disks to 2 and 0.2% CHX for 3 min prevented biofilm from growing on the HA disk surfaces for at least 12 h. This new platform has the potential to aid in a better understanding of the antibiofilm properties of oral disinfectants.
Highlights
Bacteria organized in multicellular biofilm communities pose a considerable clinical challenge because they are capable of causing oral diseases (Leung et al, 2005)
Only 5 to 6% of the bacteria were stained with propidium iodide (PI), indicating dead bacteria (Supplementary Table 2)
Forty-one percent of the bacteria in the undissolved biofilm were killed during the first 2 min of the 2% NaOCl treatment, and the percentage of killed bacteria gradually increased to 72% (P < 0.001) by 32 min (Supplementary Table 2 and Figures 1D,N)
Summary
Bacteria organized in multicellular biofilm communities pose a considerable clinical challenge because they are capable of causing oral diseases (Leung et al, 2005). One of the most complex biofilm systems in nature, human dental plaque is responsible for a variety of oral diseases and infections (Wood et al, 2000). The use of antimicrobial solutions plays a key role in biofilm eradication among the antiplaque strategies by detaching and dissolving the biofilms and by facilitating the killing of biofilm microorganisms (Haapasalo et al, 2010; Howlin et al, 2015; Lecic et al, 2016). Different disinfecting solutions have been used in dental clinics, currently there are limited minimally invasive methods to evaluate and quantitatively measure their ability to dissolve biofilm bacteria and EPS (Sule et al, 2009; Corte et al, 2019)
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