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.
Highlights
Determination of human bacterial pathogens and their animal vectors is one of the main objectives in paleomicrobiology and in the study of human-environment interactions [1]
4) Authenticity criteria: controlling for contamination and validation of data In this study, we considered the criteria for authenticity [31] as essential, but since they cannot always be followed we used the strong logical approach of Gilbert et al.[32] to validate the ancient DNA results. (i) Extensive precautions were taken to avoid contamination by modern DNA, as well as cross-contamination between ancient samples, in the laboratories dedicated to ancient DNA analysis and during PCR cycling; (ii) For human and bacterial DNA no amplifications were observed in the extraction blanks or PCR blanks, demonstrating no contamination during pre-PCR preparation and analytical steps
In this study we demonstrate that bacterial pathogens can be identified in ancient human subjects without preliminary observations such as historical texts or pathological lesions on bodies
Summary
Determination of human bacterial pathogens and their animal vectors is one of the main objectives in paleomicrobiology and in the study of human-environment interactions [1]. Recent studies have shown that Helicobacter pylori existed in the New World prior to the arrival of Columbus and phylogenetic analysis have indicated that ancient strain clusters are closely related to Asian strains [7,8]. In these cases historic manuscripts, pathological lesions on bones or specific phylogenetic strains have oriented the choice of pathogen to be researched
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