16S rRNA sequence of saliva-derived multispecies biofilm

Biofilms were formed ex vivo using cell-free saliva as a medium, and total salivary cells pooled from six healthy subjects as an inoculum. Pre-formed multispecies biofilms grown anaerobically for 24 hr were treated with either nisin A (50 ug/mL) or two concentrations of salivaricin 10 (25 and 50 ug/mL) for 20 min. The biofilms were then washed to remove tested compounds and scraped to make a total cell suspension which was inoculated into a fresh growth medium to allow non-inhibited cells to grow overnight. 16S rRNA gene amplicon sequencing was used to profile the species composition of viable cells that survived the peptides' bactericidal action and were able to regrow again post-treatment compared to viable cells derived from the PBS treatment as a negative control.

本研究以无细胞唾液为培养基，以6名健康受试者混合的总唾液细胞为接种物，体外（ex vivo）构建生物膜（Biofilm）。将经厌氧培养24小时形成的预构建多物种生物膜，分别用乳链菌肽A（nisin A，50 μg/mL）以及两种浓度的唾液菌素10（salivaricin 10，25 μg/mL与50 μg/mL）处理20分钟。随后冲洗生物膜以去除受试化合物，刮取样本制备总细胞悬液，将其接种至新鲜生长培养基中，使未被抑制的细胞过夜培养增殖。采用16S rRNA基因扩增子测序（16S rRNA gene amplicon sequencing），表征经肽类杀菌作用存活且可在处理后重新增殖的活细胞的物种组成，并与作为阴性对照的磷酸盐缓冲液（PBS）处理组来源的活细胞进行比对。