title: Molecular identification of bacteria by total sequence screening: determining the cause of death in ancient human subjects.
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 17(th) to the 19(th) 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 17(th) century to the early 18(th) century.
authors.affiliation: Laboratoire AMIS, UMR 5288, Université Toulouse IIII/CNRS/Université de Strasbourg, Toulouse, France. ctheves@cict.fr
authors.forename: Catherine
authors.initials: C
authors.lastname: Thèves
authors.forename: Alice
authors.initials: A
authors.lastname: Senescau
authors.forename: Stefano
authors.initials: S
authors.lastname: Vanin
authors.forename: Christine
authors.initials: C
authors.lastname: Keyser
authors.forename: François Xavier
authors.initials: FX
authors.lastname: Ricaut
authors.forename: Anatoly N
authors.initials: AN
authors.lastname: Alekseev
authors.forename: Henri
authors.initials: H
authors.lastname: Dabernat
authors.forename: Bertrand
authors.initials: B
authors.lastname: Ludes
authors.forename: Richard
authors.initials: R
authors.lastname: Fabre
authors.forename: Eric
authors.initials: E
authors.lastname: Crubézy
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000379:methods*; D001483:Base Sequence; D001842:Bone and Bones / Q000382:microbiology; D002423:Cause of Death; D004247:DNA / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004783:Environmental Microbiology; D005580:Fossils; D005615:Freezing; D049670:History, 17th Century; D049671:History, 18th Century; D049672:History, 19th Century; D006801:Humans; D008297:Male; D058889:Molecular Typing / Q000379:methods*; D010802:Phylogeny; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012800:Siberia
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004247:DNA