Supplementary Material for: The effect of enzymatic treatment with mutanase, beta-glucanase and DNase on a saliva-derived biofilm model

Introduction: The dental biofilm matrix is an important determinant of virulence for caries development and comprises a variety of extracellular polymeric substances that contribute to biofilm stability. Enzymes that break down matrix components may be a promising approach to caries control, and in light of the compositional complexity of the dental biofilm matrix, treatment with multiple enzymes may enhance the reduction of biofilm formation compared to single enzyme therapy. The present study investigated the effect of the three matrix-degrading enzymes mutanase, beta-glucanase and DNase, applied separately or in combinations, on biofilm prevention and removal in a saliva-derived in vitro-grown model.

Methods: Biofilms were treated during growth to assess biofilm prevention or after 24 h of growth to assess biofilm removal by the enzymes. Biofilms were quantified by crystal violet staining and impedance-based real-time cell analysis, and the biofilm structure was visualized by confocal microscopy and staining of extracellular DNA (eDNA) and polysaccharides.

Results: The in vitro model was dominated by Streptococcus spp., as determined by 16S rRNA gene amplicon sequencing. All tested enzymes and combinations had a significant effect on biofilm prevention, with reductions of >90% for mutanase and all combinations including mutanase. Combined application of DNase and beta-glucanase resulted in an additive effect (81.0%±1.3% SD vs. 36.9%±21.9% SD and 48.2%±14.9% SD). For biofilm removal, significant reductions of up to 73.2%±5.5% SD were achieved for combinations including mutanase, whereas treatment with DNase had no effect. Glucans, but not eDNA decreased in abundance upon treatment with all three enzymes.

Conclusion: Multi-enzyme treatment is a promising approach to dental biofilm control that needs to be validated in more diverse biofilms.

引言：牙菌斑生物膜基质是龋病发病过程中重要的毒力决定因素，其由多种胞外聚合物（extracellular polymeric substances）组成，可维持生物膜的结构稳定性。能够降解基质组分的酶类或许是龋病防控的潜在手段；鉴于牙菌斑生物膜基质的组成复杂性，相较于单一酶疗法，联合使用多种酶或许能更有效地抑制生物膜形成。本研究探究了变聚糖酶（mutanase）、β-葡聚糖酶（beta-glucanase）与脱氧核糖核酸酶（DNase）这三种基质降解酶，分别单独使用或联合使用时，在唾液来源的体外培养模型中对生物膜形成的预防及去除效果。
方法：本研究分别在生物膜生长过程中（以评估其对生物膜形成的预防作用），以及生物膜生长24小时后（以评估其对生物膜的去除效果）施加酶处理。通过结晶紫染色法、基于阻抗的实时细胞分析法对生物膜进行定量，并借助共聚焦显微镜及细胞外DNA（extracellular DNA, eDNA）、多糖染色技术对生物膜结构进行可视化观察。
结果：经16S rRNA基因扩增子测序（16S rRNA gene amplicon sequencing）鉴定，该体外模型的优势菌群为链球菌属（Streptococcus spp.）。所有受试酶及酶组合均对生物膜形成具有显著的预防效果，其中变聚糖酶及所有包含变聚糖酶的酶组合可使生物膜形成量降低超过90%。脱氧核糖核酸酶与β-葡聚糖酶联合使用时可产生相加效应，其生物膜形成抑制率为81.0%±1.3%标准差（SD），而单独使用脱氧核糖核酸酶、β-葡聚糖酶时的抑制率分别为36.9%±21.9%标准差（SD）与48.2%±14.9%标准差（SD）。在生物膜去除实验中，所有包含变聚糖酶的酶组合均可实现最高达73.2%±5.5%标准差（SD）的显著去除效果，而单独使用脱氧核糖核酸酶则无明显作用。经三种酶处理后，葡聚糖的丰度显著降低，而细胞外DNA的丰度未发生明显变化。
结论：多酶疗法是一种颇具潜力的牙科生物膜防控手段，未来需在菌群更为多样的生物膜模型中对其进行验证。