A genomic and historical synthesis of plague in 18th century Eurasia.

Plague continued to afflict Europe for more than five centuries after the Black Death. Yet, by the 17th century, the dynamics of plague had changed, leading to its slow decline in Western Europe over the subsequent 200 y, a period for which only one genome was previously available. Using a multidisciplinary approach, combining genomic and historical data, we assembled Y. pestis genomes from nine individuals covering four Eurasian sites and placed them into an historical context within the established phylogeny. CHE1 (Chechnya, Russia, 18th century) is now the latest Second Plague Pandemic genome and the first non-European sample in the post-Black Death lineage. Its placement in the phylogeny and our synthesis point toward the existence of an extra-European reservoir feeding plague into Western Europe in multiple waves. By considering socioeconomic, ecological, and climatic factors we highlight the importance of a noneurocentric approach for the discussion on Second Plague Pandemic dynamics in Europe.

Commensal and Pathogenic Members of the Dental Calculus Microbiome of Badia Pozzeveri Individuals from the 11th to 19th Centuries.

The concept of the human oral microbiome was applied to understand health and disease, lifestyles, and dietary habits throughout part of human history. In the present study, we augment the understanding of ancient oral microbiomes by characterizing human dental calculus samples recovered from the ancient Abbey of Badia Pozzeveri (central Italy), with differences in socioeconomic status, time period, burial type, and sex. Samples dating from the Middle Ages (11th century) to the Industrial Revolution era (19th century) were characterized using high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. Consistent with previous studies, individuals from Badia Pozzeveri possessed commensal oral bacteria that resembled modern oral microbiomes. These results suggest that members of the oral microbiome are ubiquitous despite differences in geographical regions, time period, sex, and socioeconomic status. The presence of fecal bacteria could be in agreement with poor hygiene practices, consistent with the time period. Respiratory tract, nosocomial, and other rare pathogens detected in the dental calculus samples are intriguing and could suggest subject-specific comorbidities that could be reflected in the oral microbiome.

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

Taxonomic and predicted metabolic profiles of the human gut microbiome in pre-Columbian mummies.

Characterization of naturally mummified human gut remains could potentially provide insights into the preservation and evolution of commensal and pathogenic microorganisms, and metabolic profiles. We characterized the gut microbiome of two pre-Columbian Andean mummies dating to the 10-15th centuries using 16S rRNA gene high-throughput sequencing and metagenomics, and compared them to a previously characterized gut microbiome of an 11th century AD pre-Columbian Andean mummy. Our previous study showed that the Clostridiales represented the majority of the bacterial communities in the mummified gut remains, but that other microbial communities were also preserved during the process of natural mummification, as shown with the metagenomics analyses. The gut microbiome of the other two mummies were mainly comprised by Clostridiales or Bacillales, as demonstrated with 16S rRNA gene amplicon sequencing, many of which are facultative anaerobes, possibly consistent with the process of natural mummification requiring low oxygen levels. Metagenome analyses showed the presence of other microbial groups that were positively or negatively correlated with specific metabolic profiles. The presence of sequences similar to both Trypanosoma cruzi and Leishmania donovani could suggest that these pathogens were prevalent in pre-Columbian individuals. Taxonomic and functional profiling of mummified human gut remains will aid in the understanding of the microbial ecology of the process of natural mummification.

Natural mummification of the human gut preserves bacteriophage DNA.

The natural mummification process of the human gut represents a unique opportunity to study the resulting microbial community structure and composition. While results are providing insights into the preservation of bacteria, fungi, pathogenic eukaryotes and eukaryotic viruses, no studies have demonstrated that the process of natural mummification also results in the preservation of bacteriophage DNA. We characterized the gut microbiome of three pre-Columbian Andean mummies, namely FI3, FI9 and FI12, and found sequences homologous to viruses. From the sequences attributable to viruses, 50.4% (mummy FI3), 1.0% (mummy FI9) and 84.4% (mummy FI12) were homologous to bacteriophages. Sequences corresponding to the Siphoviridae, Myoviridae, Podoviridae and Microviridae families were identified. Predicted putative bacterial hosts corresponded mainly to the Firmicutes and Proteobacteria, and included Bacillus, Staphylococcus, Clostridium, Escherichia, Vibrio, Klebsiella, Pseudomonas and Yersinia. Predicted functional categories associated with bacteriophages showed a representation of structural, replication, integration and entry and lysis genes. The present study suggests that the natural mummification of the human gut results in the preservation of bacteriophage DNA, representing an opportunity to elucidate the ancient phageome and to hypothesize possible mechanisms of preservation.

Gut Microbiome of an 11th Century A.D. Pre-Columbian Andean Mummy.

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.

Positioning the red deer (Cervus elaphus) hunted by the Tyrolean Iceman into a mitochondrial DNA phylogeny.

In the last years several phylogeographic studies of both extant and extinct red deer populations have been conducted. Three distinct mitochondrial lineages (western, eastern and North-African/Sardinian) have been identified reflecting different glacial refugia and postglacial recolonisation processes. However, little is known about the genetics of the Alpine populations and no mitochondrial DNA sequences from Alpine archaeological specimens are available. Here we provide the first mitochondrial sequences of an Alpine Copper Age Cervus elaphus. DNA was extracted from hair shafts which were part of the remains of the clothes of the glacier mummy known as the Tyrolean Iceman or Ötzi (5,350-5,100 years before present). A 2,297 base pairs long fragment was sequenced using a mixed sequencing procedure based on PCR amplifications and 454 sequencing of pooled amplification products. We analyzed the phylogenetic relationships of the Alpine Copper Age red deer's haplotype with haplotypes of modern and ancient European red deer. The phylogenetic analyses showed that the haplotype of the Alpine Copper Age red deer falls within the western European mitochondrial lineage in contrast with the current populations from the Italian Alps belonging to the eastern lineage. We also discussed the phylogenetic relationships of the Alpine Copper Age red deer with the populations from Mesola Wood (northern Italy) and Sardinia.

Phylogenetic position of a copper age sheep (Ovis aries) mitochondrial DNA.

BACKGROUND: Sheep (Ovis aries) were domesticated in the Fertile Crescent region about 9,000-8,000 years ago. Currently, few mitochondrial (mt) DNA studies are available on archaeological sheep. In particular, no data on archaeological European sheep are available. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the first portion of mtDNA sequence of a Copper Age European sheep. DNA was extracted from hair shafts which were part of the clothes of the so-called Tyrolean Iceman or Ötzi (5,350-5,100 years before present). Mitochondrial DNA (a total of 2,429 base pairs, encompassing a portion of the control region, tRNA(Phe), a portion of the 12S rRNA gene, and the whole cytochrome B gene) was sequenced using a mixed sequencing procedure based on PCR amplification and 454 sequencing of pooled amplification products. We have compared the sequence with the corresponding sequence of 334 extant lineages. CONCLUSIONS/SIGNIFICANCE: A phylogenetic network based on a new cladistic notation for the mitochondrial diversity of domestic sheep shows that the Ötzi's sheep falls within haplogroup B, thus demonstrating that sheep belonging to this haplogroup were already present in the Alps more than 5,000 years ago. On the other hand, the lineage of the Ötzi's sheep is defined by two transitions (16147, and 16440) which, assembled together, define a motif that has not yet been identified in modern sheep populations.

Tracking plant, fungal, and bacterial DNA in honey specimens.

Consuming honey can result in adverse effects owing to poisoning by bacterial (botulism) or plant toxins. We have devised a method to extract polymerase chain reaction (PCR) amplifiable DNA of up to c. 400 bp in length based on dialysis of a 15-mL honey sample for 18 h against deionized water followed by sequential extraction using phenol, phenol/chloroform/isoamyl alcohol, chloroform/isoamyl alcohol, and ether. Sequence analysis of PCR products obtained using "universal" plant, fungal, and bacterial primers targeted to the ribosomal RNA genes has allowed us to identify six different orders of plants (Apiales, Fabales, Asterales, Solanales, Brassicales, and Sapindales), two orders of fungi (Entylomatales and Saccharomycetales), and six orders of bacteria (Sphingomonadales, Burkholderiales, Pseudomonadales, Enterobacteriales, Actinomycetales, and Bifidobacteriales) in a single honey specimen.

Characterization of nucleotide misincorporation patterns in the iceman's mitochondrial DNA.

BACKGROUND: The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation ("miscoding lesions") has been the object of extensive investigations. METHODOLOGY/PRINCIPAL FINDINGS: To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp) mitochondrial (mt) DNA sequences obtained from the 5,350-5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Otzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp) mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85%) of the observed nucleotide misincorporations in ancient sequences. CONCLUSIONS/SIGNIFICANCE: This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine-->thymine/guanine-->adenine) transitions and that type 1 transitions are essentially PCR artifacts.

Complete mitochondrial genome sequence of the Tyrolean Iceman.

The Tyrolean Iceman was a witness to the Neolithic-Copper Age transition in Central Europe 5350-5100 years ago, and his mummified corpse was recovered from an Alpine glacier on the Austro-Italian border in 1991 [1]. Using a mixed sequencing procedure based on PCR amplification and 454 sequencing of pooled amplification products, we have retrieved the first complete mitochondrial-genome sequence of a prehistoric European. We have then compared it with 115 related extant lineages from mitochondrial haplogroup K. We found that the Iceman belonged to a branch of mitochondrial haplogroup K1 that has not yet been identified in modern European populations. This is the oldest complete Homo sapiens mtDNA genome generated to date. The results point to the potential significance of complete-ancient-mtDNA studies in addressing questions concerning the genetic history of human populations that the phylogeography of modern lineages is unable to tackle.

Fine characterization of the Iceman's mtDNA haplogroup.

Starting from specimens of the intestinal contents of the so-called Tyrolean Iceman or Otzi (5,350-5,100 years before present), it was possible by polymerase chain reaction to amplify fragments of the human mitochondrial DNA (mtDNA) control region that correspond to the sequence found in 1994 at the Munich and Oxford laboratories and which had been attributed to the original DNA of the mummy. The particularly favorable condition of the specimens, showing very low contamination levels, made it easier to extend the analyses to the coding region, which had not previously been considered. The mtDNA of the European population is currently divided into nine (H, T, U, V, W, X, I, J, and K) main groups (haplogroups). The K haplogroup, in particular, is composed of two (K1 and K2) subclusters. The results demonstrate that the Iceman's mtDNA belongs to the K1 subcluster, yet it does not fit any of the three known branches (a, b, and c) into which the K1 subcluster is presently divided. In addition, some other sites, reported to be linked to environmental adaptation or pathologies, were investigated.

Studies on the preservation of the intestinal microbiota's DNA in human mummies from cold environments.

Analysis of ancient microorganism DNA represents one of the newest and most promising branches of molecular archaeology. In particular, microbial DNA associated with human remains can provide direct evidence of the occurrence and frequency of infectious diseases in historic times. Human mummies represent very interesting subjects for palaeomicrobiological investigations as they retain soft tissues. Recently reports on the identification of ancient bacterial pathogens in human mummies by DNA analysis are steadily becoming more numerous. However, despite this favourable trend, the analysis of ancient microbial DNA is still a contentious issue. As a model system, we studied the preservation of the intestinal microbiota's DNA in two naturally freeze-dried human mummies found on the Alps. This kind of mummy is an ideal subject for ancient DNA investigations. The first is a male body historically dated 1918 A.D. while the second is the famous Tyrolean Iceman (3.350-3.100 BC).

Otzi's last meals: DNA analysis of the intestinal content of the Neolithic glacier mummy from the Alps.

Samples of the intestinal content were collected from the ileum and colon of the Neolithic glacier mummy popularly known as the Tyrolean Iceman, or Otzi. DNA was extracted from the samples and PCR amplified, using a variety of primer pairs designed to bind to different genes (mammal mitochondrial 12S ribosomal RNA gene, plant/fungal nuclear 18S ribosomal RNA gene, plant chloroplast ribulose bisphosphate carboxylase large subunit gene). This made it possible to distinguish between animal and plant food residues (macroremains) and pollen (microremains). According to the DNA reconstruction, the man's last meal was composed of red deer (Cervus elaphus) meat, and, possibly, cereals; this meal had been preceded by another one based on ibex (Capra ibex), different species of dicots, and cereals. The DNA spectrum corresponding to pollen residues in the colon, on the other hand, fits with the hypothesis that the last journey of the Neolithic hunter/warrior was made through a subalpine coniferous forest to the site at over 3,200 m above sea level, where his mummified body was to be discovered 5,000 years later.

DNA decay rate in papyri and human remains from Egyptian archaeological sites.

The writing sheets made with strips from the stem (caulis) of papyri (Cyperus papyrus) are one of the most ingenious products of ancient technology. We extracted DNA from samples of modern papyri varying in age from 0-100 years BP and from ancient specimens from Egypt, with an age-span from 1,300-3,200 years BP. The copy number of the plant chloroplast DNA in the sheets was determined using a competitive PCR system designed on the basis of a short (90 bp) tract of the chloroplast's ribulose bisphosphate carboxylase large subunit (rbcL) gene sequence. The results allowed us to establish that the DNA half-life in papyri is about 19-24 years. This means that the last DNA fragments will vanish within no more than 532-672 years from the sheets being manufactured. In a parallel investigation, we checked the archaeological specimens for the presence of residual DNA and determined the extent of racemization of aspartic (Asp) acid in both modern and ancient specimens, as a previous report (Poinar et al. [1996], Science 272:864-866) showed that racemization of aspartic acid and DNA decay are linked. The results confirmed the complete loss of authentic DNA, even in the less ancient (8th century AD) papyri. On the other hand, when the regression for Asp racemization rates in papyri was compared with that for human and animal remains from Egyptian archaeological sites, it proved, quite surprisingly, that the regressions are virtually identical. Our study provides an indirect argument against the reliability of claims about the recovery of authentic DNA from Egyptian mummies and bone remains.