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 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.

Shotgun microbial profiling of fossil remains

“…This is in line with previous results based on low‐depth 16S rDNA sequencing that showed the presence of microbes typical of Antarctic and Arctic permafrost environments in 100‐ to 300‐year‐old human remains collected in Yakutia (Thèves et al . ).…”

The rediscovery of smallpox

“…Reprinted with permission. Palaeogenetic studies conducted over >10 years in our laboratory have clearly demonstrated the persistence of fragmented DNA molecules . It is important to note that such archaeological samples consist of many DNA molecules, originating not only from the subject (human nuclear and mitochondrial DNA ) and the pathogen potentially responsible for the death of the subject (bacterial or viral DNA ), but also environmental bacteria from this complex environment: soil, water, and plants.…”

“…It is important to note that such archaeological samples consist of many DNA molecules, originating not only from the subject (human nuclear and mitochondrial DNA ) and the pathogen potentially responsible for the death of the subject (bacterial or viral DNA ), but also environmental bacteria from this complex environment: soil, water, and plants. Metagenomic or bacterial genome analyses show that the majority of bacteria in this kind of sample are environmental .…”

Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

“…The human microbiome has long been studied for a better understanding of its influence upon human development, physiology, immunity, and nutrition [ 1 ]. In most of these studies, microbial identification methods rely on sample collection followed by DNA isolation and sequencing [ 2 , 3 ]. Despite providing important information on the communities that inhabit the human body, these methods disrupt the spatial structure of the sample, meaning that important information about human/microorganism or microorganism/microorganism interactions might be lost.…”