Wang Zhejun, Shen Ya, Haapasalo Markus
Division of Endodontics, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, The University of British Columbia, Vancouver, BC, Canada.
Front Microbiol. 2020 May 20;11:964. doi: 10.3389/fmicb.2020.00964. eCollection 2020.
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.
本研究旨在建立一个标准化模型，以评估生物膜的动态溶解、生物膜微生物的杀灭以及消毒溶液对生物膜生长的抑制作用。生物膜由牙菌斑细菌在胶原包被的羟基磷灰石（HA）圆盘上于厌氧条件下培养3天或3周形成。生物膜用LIVE/DEAD活力染色剂染色，然后分别用无菌水、2%次氯酸钠（NaOCl）、6% NaOCl或2%洗必泰（CHX）处理32分钟。使用Alexa Fluor 647标记的葡聚糖共轭物和活细胞成像共聚焦激光扫描显微镜（LC-CLSM）分析处理过程中细菌细胞和细胞外聚合物（EPS）上荧光的动态变化。处理后的生物膜结构通过扫描电子显微镜（SEM）观察。收集HA圆盘上处理过的生物膜并进行菌落形成单位（CFU）计数。另一组无菌HA圆盘在监测牙菌斑生物膜生长12小时之前用CHX包被。LC-CLSM结果表明，NaOCl能有效溶解生物膜，浓度越高、暴露时间越长效果越好。6% NaOCl在溶解和杀灭细菌方面最有效（例如，在32分钟内，3天龄生物膜中的细菌减少99%，3周龄生物膜中的细菌减少95%），其次是2% NaOCl和CHX。次氯酸钠在32分钟内溶解了超过99.9%的EPS，而CHX在32分钟内仅略微降低了EPS的生物量。CFU结果表明，分散的生物膜细菌比浮游细菌对消毒剂更具抗性。SEM显示，暴露于CHX和NaOCl后生物膜受到破坏。使用2% CHX和无菌水未导致生物膜溶解。然而，HA圆盘预先暴露于2%和0.2% CHX 3分钟可防止生物膜在HA圆盘表面生长至少12小时。这个新平台有可能有助于更好地理解口腔消毒剂的抗生物膜特性。
