Molecular Identification of Bacteria by Total Sequence Screening: Determining the Cause of Death in Ancient Human Subjects

Research of ancient pathogens in ancient human skeletons has been mainly carried out on the basis of one essential historical or archaeological observation, permitting specific pathogens to be targeted. Detection of ancient human pathogens without such evidence is more difficult, since the quantity and quality of ancient DNA, as well as the environmental bacteria potentially present in the sample, limit the analyses possible. Using human lung tissue and/or teeth samples from burials in eastern Siberia, dating from the end of 17th to the 19th century, we propose a methodology that includes the: 1) amplification of all 16S rDNA gene sequences present in each sample; 2) identification of all bacterial DNA sequences with a degree of identity ≥95%, according to quality criteria; 3) identification and confirmation of bacterial pathogens by the amplification of the rpoB gene; and 4) establishment of authenticity criteria for ancient DNA. This study demonstrates that from teeth samples originating from ancient human subjects, we can realise: 1) the correct identification of bacterial molecular sequence signatures by quality criteria; 2) the separation of environmental and pathogenic bacterial 16S rDNA sequences; 3) the distribution of bacterial species for each subject and for each burial; and 4) the characterisation of bacteria specific to the permafrost. Moreover, we identified three pathogens in different teeth samples by 16S rDNA sequence amplification: Bordetella sp., Streptococcus pneumoniae and Shigella dysenteriae. We tested for the presence of these pathogens by amplifying the rpoB gene. For the first time, we confirmed sequences from Bordetella pertussis in the lungs of an ancient male Siberian subject, whose grave dated from the end of the 17th century to the early 18th century.针对古代人类骨骼中古病原体的研究，此前主要基于一项关键的历史或考古学观测开展，以此锁定特定病原体进行研究。若无此类观测证据，检测古代人类病原体的难度则更高，这是由于古DNA的数量与质量，以及样本中可能存在的环境细菌，均会限制可行的分析手段。本研究采用西伯利亚东部墓葬出土的人类肺组织及/或牙齿样本（年代为17世纪末至19世纪），提出一套包含以下步骤的研究方法：1）对每份样本中存在的所有16S核糖体DNA（16S rDNA）基因序列进行扩增；2）依据质量标准，对所有相似度≥95%的细菌DNA序列进行鉴定；3）通过扩增rpoB基因，对细菌病原体进行鉴定与确认；4）建立古DNA的真实性判定标准。本研究证明，利用古代人类遗骸的牙齿样本，可实现以下目标：1）依据质量标准准确鉴定细菌分子序列特征；2）区分环境细菌与致病性细菌的16S rDNA序列；3）明确每位受试者及每处墓葬的细菌物种分布情况；4）表征永冻土（permafrost）特异性细菌的特征。此外，本研究通过16S rDNA序列扩增，在不同牙齿样本中鉴定出三种病原体：博德特菌属（Bordetella sp.）、肺炎链球菌（Streptococcus pneumoniae）以及痢疾志贺菌（Shigella dysenteriae）。本研究通过扩增rpoB基因，对上述病原体的存在与否进行了验证。本研究首次在一位古代西伯利亚男性受试者的肺部组织中，检测到百日咳博德特菌（Bordetella pertussis）的序列，该墓葬的年代为17世纪末至18世纪初。