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

Adler, Ben, Thèves, Catherine, Senescau, Alice, Vanin, Stefano, Keyser, Christine, Ricaut, François Xavier, Alekseev, Anatoly N., Dabernat, Henri, Ludes, Bertrand, Fabre, Richard and Crubézy, Eric (2011) Molecular Identification of Bacteria by Total Sequence Screening: Determining the Cause of Death in Ancient Human Subjects. PLoS ONE, 6 (7). e21733. ISSN 1932-6203

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Abstract

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.

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