Abstract：
OBJECTIVE: To investigate the bacteriome present in teeth with primary endodontic infection (PEI) and apical periodontitis (AP) and to determine quantitatively and qualitatively the impact of chemomechanical preparation (CMP) using 2.5% sodium hypochlorite NAOCl on the bacteriome found in PEI with AP using the Illumina MiSeq platform.
METHODS: Thirty-six paired samples from 18 patients were successfully sequenced and analysed. Samples were collected at two sampling times: before (s1) and after (s2) CMP using 2.5% NaOCl. The DNA was extracted from s1 and s2 samples and quantified using quantitative PCR (qPCR). All 36 samples were sequenced using the Illumina MiSeq platform. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants (ASVs). Alpha diversity metrics representing abundance (Chao1) and diversity and evenness (Shannon, Simpson) were computed. The paired-sample Wilcoxon\'s test was used to compare alpha diversity metrics and qPCR counts between s1 and s2. The PERMANOVA method (with 999 permutations) was applied to compare community composition between sample types (s1 versus s2) and between patient IDs. ALDEx2 (ANOVA-like differential expression tool for high-throughput sequencing data) to investigate differentially abundant taxa between s1 and s2. A paired-sample Wilcoxon\'s test was used to compare alpha diversity metrics and qPCR counts between s1 and s2.
RESULTS: The qPCR counts were significantly higher in s1 compared to s2 (p = .0007). The Chao1 index indicated no difference in alpha diversity (p < .7019); whereas Shannon (p = .0056) and Simpson (p = .02685) indexes showed higher values in s2. The PERMANOVA test using Adonis2 showed a significant effect of sample time on community composition (R2 = .0630, p = .012). Patient ID also showed a significant effect on community composition (R2 = .6961, p = .001). At the genus level, Dialister, Mogibacterium, Prevotella, and Olsenella were differentially enriched at s1, while Actinomyces, Stenotrophomonas_unclassified, Enterococcus_unclassified, and Actinomyces_unclassified were differentially enriched in s2.
CONCLUSIONS: The bacteriome present in teeth with PEI with AP is complex and diverse. CMP using 2.5% NaOCl showed a high quantitatively and qualitatively disinfectant impact on the bacteriome present in PEI with AP.
METHODS: Thirty-six paired samples from 18 patients were successfully sequenced and analysed. Samples were collected at two sampling times: before (s1) and after (s2) CMP using 2.5% NaOCl. The DNA was extracted from s1 and s2 samples and quantified using quantitative PCR (qPCR). All 36 samples were sequenced using the Illumina MiSeq platform. Raw V3-V4 amplicon sequencing data were processed with the DADA2 pipeline to generate amplicon sequence variants (ASVs). Alpha diversity metrics representing abundance (Chao1) and diversity and evenness (Shannon, Simpson) were computed. The paired-sample Wilcoxon\'s test was used to compare alpha diversity metrics and qPCR counts between s1 and s2. The PERMANOVA method (with 999 permutations) was applied to compare community composition between sample types (s1 versus s2) and between patient IDs. ALDEx2 (ANOVA-like differential expression tool for high-throughput sequencing data) to investigate differentially abundant taxa between s1 and s2. A paired-sample Wilcoxon\'s test was used to compare alpha diversity metrics and qPCR counts between s1 and s2.
RESULTS: The qPCR counts were significantly higher in s1 compared to s2 (p = .0007). The Chao1 index indicated no difference in alpha diversity (p < .7019); whereas Shannon (p = .0056) and Simpson (p = .02685) indexes showed higher values in s2. The PERMANOVA test using Adonis2 showed a significant effect of sample time on community composition (R2 = .0630, p = .012). Patient ID also showed a significant effect on community composition (R2 = .6961, p = .001). At the genus level, Dialister, Mogibacterium, Prevotella, and Olsenella were differentially enriched at s1, while Actinomyces, Stenotrophomonas_unclassified, Enterococcus_unclassified, and Actinomyces_unclassified were differentially enriched in s2.
CONCLUSIONS: The bacteriome present in teeth with PEI with AP is complex and diverse. CMP using 2.5% NaOCl showed a high quantitatively and qualitatively disinfectant impact on the bacteriome present in PEI with AP.
摘要：
目的：研究患有原发性牙髓感染（PEI）和根尖周炎（AP）的牙齿中存在的细菌组，并定量和定性地确定使用2.5％次氯酸钠NAOCl的化学机械制剂（CMP）对使用IlluminaMiSeq平台在PEI和AP中发现的细菌组的影响。
方法：对18例患者的36个配对样本进行了成功测序和分析。在两个采样时间收集样品:在使用2.5%NaOCl的CMP之前(s1)和之后(s2)。从s1和s2样品中提取DNA并使用定量PCR(qPCR)定量。使用IlluminaMiSeq平台对所有36个样品进行测序。用DADA2流水线处理原始V3-V4扩增子测序数据以产生扩增子序列变体(ASV)。代表丰度(Chao1)和多样性和均匀度(Shannon，辛普森）进行了计算。配对样品Wilcoxon检验用于比较s1和s2之间的α多样性指标和qPCR计数。应用PERMANOVA方法(具有999个排列)来比较样品类型(s1对s2)之间和患者ID之间的群落组成。ALDEx2（用于高通量测序数据的ANOVA样差异表达工具）研究s1和s2之间的差异丰富分类单元。使用配对样品Wilcoxon检验来比较s1和s2之间的α多样性指标和qPCR计数。
结果：与s2相比，s1中的qPCR计数明显更高（p=.0007）。Chao1指数表明α多样性没有差异（p<.7019）；而Shannon（p=.0056）和Simpson（p=.02685）指数在s2中显示出较高的值。使用Adonis2的PERMANOVA检验显示样本时间对群落组成有显著影响(R2=.0630,p=.012)。患者ID对社区组成也显示出显着影响（R2=.6961，p=.001）。在属一级，Dialister,小杆菌属,普雷沃氏菌,和Olsenella在s1差异富集，而放线菌，窄食单胞菌_未分类，肠球菌_未分类，和未分类的放线菌在s2中差异富集。
结论：PEI伴AP的牙齿中存在的细菌组复杂多样。使用2.5％NaOCl的CMP对带有AP的PEI中存在的细菌组显示出较高的定量和定性消毒剂影响。
方法：对18例患者的36个配对样本进行了成功测序和分析。在两个采样时间收集样品:在使用2.5%NaOCl的CMP之前(s1)和之后(s2)。从s1和s2样品中提取DNA并使用定量PCR(qPCR)定量。使用IlluminaMiSeq平台对所有36个样品进行测序。用DADA2流水线处理原始V3-V4扩增子测序数据以产生扩增子序列变体(ASV)。代表丰度(Chao1)和多样性和均匀度(Shannon，辛普森）进行了计算。配对样品Wilcoxon检验用于比较s1和s2之间的α多样性指标和qPCR计数。应用PERMANOVA方法(具有999个排列)来比较样品类型(s1对s2)之间和患者ID之间的群落组成。ALDEx2（用于高通量测序数据的ANOVA样差异表达工具）研究s1和s2之间的差异丰富分类单元。使用配对样品Wilcoxon检验来比较s1和s2之间的α多样性指标和qPCR计数。
结果：与s2相比，s1中的qPCR计数明显更高（p=.0007）。Chao1指数表明α多样性没有差异（p<.7019）；而Shannon（p=.0056）和Simpson（p=.02685）指数在s2中显示出较高的值。使用Adonis2的PERMANOVA检验显示样本时间对群落组成有显著影响(R2=.0630,p=.012)。患者ID对社区组成也显示出显着影响（R2=.6961，p=.001）。在属一级，Dialister,小杆菌属,普雷沃氏菌,和Olsenella在s1差异富集，而放线菌，窄食单胞菌_未分类，肠球菌_未分类，和未分类的放线菌在s2中差异富集。
结论：PEI伴AP的牙齿中存在的细菌组复杂多样。使用2.5％NaOCl的CMP对带有AP的PEI中存在的细菌组显示出较高的定量和定性消毒剂影响。
