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The Mycobacterium tuberculosis complex (MTC) has affected South American populations since ca. 200 years BCE. In Argentina, possible cases date from ca. 1000-1400 Common Era (CE). This paper describes the oldest (905-1030 CE) confirmed case of tuberculosis (TB) in a young adult male from Lomitas de Saujil (Tinogasta, Catamarca, Argentina). Osteolytic lesions on the bodies of the lower spine were macroscopically and radiographically identified. Bilateral new bone formation was seen on the visceral vertebral third of several ribs and in long bones, compatible with hypertrophic osteoarthropathy. Representative rib and hand bones gave profiles for MTC-specific C 27 -C 32 mycocerosic acid lipid biomarkers; these were strongest in one heavily-lesioned lower rib, which also had MTC-diagnostic C 76 -C 89 mycolic acids and positive amplification of MTC-typical IS6110 aDNA fragments. During the first millennium CE, the intense social interaction, the spatial circumscription of villages among the pre-Hispanic societies in the mesothermal valleys of Catamarca and the fluid contacts with the Eastern lowlands, valleys and puna, were factors likely to favor disease transmission. It is proposed that TB arrived from northern Chile and dispersed towards the northeast into the Yocavil valley, where several cases of TB infection were macroscopically identified for a later chronology., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Andean paleopathological research has significantly enhanced knowledge about the geographical distribution and evolution of tuberculosis (TB) in pre-Columbian South America. In this paper, we review the history and progress of research on ancient tuberculosis (TB) in the Andean region, focusing on the strengths and limitations of current approaches for the molecular detection of ancient pathogens, with special attention to TB. As a case study, we describe a molecular screening approach for the detection of ancient Mycobacterium tuberculosis in individuals from Late Intermediate Period (1000-1400 CE) contexts at the site of Huari, Peru. We evaluate 34 commingled human vertebrae and combine morphological assessments of pathology with high throughput sequencing and a non-selective approach to ancient pathogen DNA screening. Our method enabled the simultaneous detection of ancient M. tuberculosis DNA and an evaluation of the environmental microbial composition of each sample. Our results show that despite the dominance of environmental DNA, molecular signatures of M. tuberculosis were identified in eight vertebrae, six of which had no observable skeletal pathology classically associated tuberculosis infection. This screening approach will assist in the identification of candidate samples for downstream genomic analyses. The method permits higher resolution disease identification in cases where pathology may be absent, or where the archaeological context may necessitate a broad differential diagnosis based on morphology alone., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: We assessed whether Petrus Donders (died 1887), a Dutch priest who for 27 years cared for people with leprosy in the leprosarium Batavia, Suriname, had evidence of Mycobacterium (M.) leprae infection. A positive finding of M. leprae ancient (a)DNA would contribute to the origin of leprosy in Suriname., Materials: Skeletal remains of Father Petrus Donders; two additional skeletons excavated from the Batavia cemetery were used as controls., Methods: Archival research, paleopathological evaluation and aDNA-based testing of skeletal remains., Results: Neither archives nor inspection of Donders skeletal remains revealed evidence of leprosy, and aDNA-based testing for M. leprae was negative. We detected M. leprae aDNA by RLEP PCR in one control skeleton, which also displayed pathological lesions compatible with leprosy. The M. leprae aDNA was genotyped by Sanger sequencing as SNP type 4; the skeleton displayed mitochondrial haplogroup L3., Conclusion: We found no evidence that Donders contracted leprosy despite years of intense leprosy contact, but we successfully isolated an archaeological M. leprae aDNA sample from a control skeleton from South America., Significance: We successfully genotyped recovered aDNA to a M. leprae strain that likely originated in West Africa. The detected human mitochondrial haplogroup L3 is also associated with this geographical region. This suggests that slave trade contributed to leprosy in Suriname., Limitations: A limited number of skeletons was examined., Suggestions for Further Research: Broader review of skeletal collections is advised to expand on diversity of the M. leprae aDNA database., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent developments in High-Throughput DNA sequencing (HTS) technologies and ancient DNA (aDNA) research have opened access to the characterization of the microbial communities within past populations. Most studies have, however, relied on the analysis of dental calculus as one particular material type particularly prone to the molecular preservation of ancient microbial biofilms and potential of entire teeth for microbial characterization, both of healthy communities and pathogens in ancient individuals, remains overlooked. In this study, we used shotgun sequencing to characterize the bacterial composition from historical subjects showing macroscopic evidence of oral pathologies. We first carried out a macroscopic analysis aimed at identifying carious or periodontal diseases in subjects belonging to a French rural population of the 18th century AD. We next examined radiographically six subjects showing specific, characteristic dental pathologies and applied HTS shotgun sequencing to characterize the microbial communities present in and on the dental material. The presence of Streptococcus mutans and also Rothia dentocariosa, Actinomyces viscosus, Porphyromonas gingivalis, Tannerella forsythia, Pseudoramibacter alactolyticus, Olsenella uli and Parvimonas micra was confirmed through the presence of typical signatures of post-mortem DNA damage at an average depth-of-coverage ranging from 0.5 to 7X, with a minimum of 35% (from 35 to 93%) of the positions in the genome covered at least once. Each sampled tooth showed a specific bacterial signature associated with carious or periodontal pathologies. This work demonstrates that from a healthy independent tooth, without visible macroscopic pathology, we can identify a signature of specific pathogens and deduce the oral health status of an individual.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom-a skeleton from the Great Chesterford cemetery with a calibrated age of 415-545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.

Background: Paleomicrobiology is a special branch of micropaleontology concerned with the study of bacterial fossils. We have used the term 'oral paleomicrobiology', as in this review we have focused on the ancient oral microflora. Recently, dental calculus and dental pulp have been identified as rich sources of ancient microbial DNA. Study of this ancient genetic material opens a new door to the ancient world. This review gives an overview of history of ancient DNA research, various techniques of analyzing ancient DNA in dental calculus and dental pulp, and the implications of the oral paleomicrobiology., Materials and Methods: A comprehensive literature search was performed in the following databases-pubmed, medline and google scholar for studies published before 10 April, 2015. The following keywords were used- 'ancient DNA', 'ancient oral flora, 'oral paleomicrobiology' and 'oral microbiome', '16S rRNA sequencing'. To obtain additional data, a manual search was performed using the reference lists of selected articles., Result: As a result of literature search, 27 articles were found in pubmed, 12 in google scholar and one in medline. Eight more articles were selected from the reference list of selected articles., Conclusion: The combination of microbiology and paleontology has brought a revolution in the study of human evolution and microbial communities. The naturally well-preserved samples of microbial DNA from dental pulp and microbial colonies trapped in dental calculus are a potential source of microbial genetic material, which will prove invaluable in resolving mysteries of the past. This may be a beginning of a new era of oral paleomicrobiology, which will contribute in our studies about prevention of disease by establishing symbiosis between human beings and their microbiome.

Leprosy used to be a widespread, dreaded disease in Europe during the middle ages, and it still remains an important health problem in some parts of the world today. Herein, we present data on the earliest 'Austrian' (an adult female from the early medieval period) proven to have suffered from leprosy. Manifestations of the disease were first identified during a systematic screening of pathological changes in skeletons recovered from an archaeological site in Pottenbrunn (Lower Austria). In the present study, DNA extracts from selected cranial and postcranial bone samples were investigated using polymerase chain reaction primers specific to the Mycobacterium leprae (M. leprae) repetitive element (RLEP). M. leprae traces were detected in extracts from nasal and palatine bones. Sequence analysis of informative polymorphic sites supports previous reports indicating that European M. leprae strains fall into single nucleotide polymorphism group 3. In summary, these findings put Austria on the map of confirmed leprosy cases in ancient Europe.

Bone samples investigated in this study derive from the pathologic-anatomical collection of the Natural History Museum of Vienna. In order to explore the survival of treponemes and treponemal ancient DNA in museal dry bone specimens, we analyzed three individuals known to have been infected with Treponema pallidum pallidum. No reproducible evidence of surviving pathogen's ancient DNA (aDNA) was obtained, despite the highly sensitive extraction and amplification techniques (TPP15 and arp). Additionally, decalcification fluid of bone sections was smear stained with May-Gruenwald-Giemsa. The slides were examined using direct light microscope and dark field illumination. Remnants of spirochetal structures were detectable in every smear. Our results demonstrate that aDNA is unlikely to survive, but spirochetal remains are stainable and thus detectable.

The introduction of agriculture and animal husbandry at the end of the Mesolithic era, despite enabling a significant demographic growth through an increase in food storage and availability, caused new infectious noxae to enter the pathocoenosis. However in the Palaeolithic era, hunter-gatherers were already in contact with infectious diseases of animal origin, albeit episodically. Modern biomedical technologies allow us to estimate, with better approximation, how long mankind has been in contact with Mycobacterium tuberculosis. Archaeological finds, including human and animal remains (especially the aurochs), are particularly studied by palaeopathologists, as mycobacteria frequently cause bone involvement and this characteristic is of particular interest for palaeopathological (even macroscopic) studies; the interest is to detect the ancient DNA of MT, which is the cause of bone tuberculosis in skeletal remains as well as in mummies. According to our present knowledge, palaeopathological findings, confirmed by molecular techniques, suggest that tuberculosis in human skeletons goes back at most to 9000 years ago, while, in a veterinary environment, the most ancient DNA of MTBC to be detected in an American bison dates back about 17,000 years. The possibility of discovering archaeological finds making even more ancient human remains available leaves opens up the possibility of dating back to previous eras the transmission of MTBC infection to mankind. Phylogenetic works examining the available materials (DNAa) suggest that Mycobacterium tuberculosis, the cause of tuberculosis infection in humans and cattle (Aurochs), would have had a co-evolutionary process. On the basis of recent phylogenetic studies, the MTBC genome would have had a wide span of time to reach a suitable adjustment, co-evolving in geographical environments both at high and low host density. It is likely that the strains that did not show this strong "flexibility" underwent extinction, in favour of more versatile, adaptable strains, that are able to infect susceptible hosts "always" and in any environmental condition.

The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Secretion of a metabolite enabling Streptococci to undergo genetic transformation was discovered. The metabolite combined with an optimization process were applied to increase the transformation yield about 20-fold. It was observed that large amounts of DNA exert a bactericidal effect, indicating the ability of at least 70% of cells to uptake the polymer. While studying the molecular mechanism of transformation of Bacillus subtilis it was shown that the uptaken DNA forms complexes with bacterial proteins, which hinders determination of its structure. A method was found to dissociate these complexes which enabled to determine the single-stranded structure of the uptaken DNA. Donor DNA fragments incorporated into the host DNA were of about 10 Da. Non-transforming DNA can be uptaken similarly but does not undergo incorporation into the host DNA. The selectivity of Bacillus subtilis receptors was determined towards DNA of phages containing modified bases: uracil, putrescinyl-thymine and its acetylated derivative, 5'-hydroxymethylcytosine and its glycosylated derivative and also towards double-stranded RNA of f2 phage. All these modifications were tolerated by the cellular receptors, with the exception of glycosylation and the 2'-OH group in RNA.

In 1998, a cultivation-independent survey of the microbial community in Obsidian Pool, Yellowstone National Park, detected 12 new phyla within the Domain Bacteria. These were dubbed 'candidate divisions' OP1 to OP12. Since that time the OP10 candidate division has been commonly detected in various environments, usually as part of the rare biosphere, but occasionally as a predominant community component. Based on 16S rRNA gene phylogeny, OP10 comprises at least 12 class-level subdivisions. However, despite this broad ecological and evolutionary diversity, all OP10 bacteria have eluded cultivation until recently. In 2011, two reference species of OP10 were taxonomically validated, removing the phylum from its 'candidate' status. Construction of a highly resolved phylogeny based on 29 universally conserved genes verifies its standing as a unique bacterial phylum. In the following paper we summarize what is known and what is suspected about the newest described bacterial phylum, the Armatimonadetes., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Paleogenetic analysis for tuberculosis (TB) was conducted on bone and sediment samples dating from the 17th to 19th centuries from the archeological site of Nossa Senhora do Carmo Church in Rio de Janeiro, Brazil. Forty samples were analyzed, corresponding to 32 individuals from 28 burials, 22 of primary type and 6 of secondary type. The samples were collected following strict paleogenetic investigation guidelines and submitted to ancient DNA (aDNA) extraction. In order to detect TB infection, aDNA hybridizations with the molecular targets of Mycobacterium tuberculosis complex (MTC) IS6110 and IS1081 were applied. Additionally, the ancestry of individuals was assessed by human mitochondrial DNA (mtDNA) analysis of hypervariable segment I (HVS-I) sequence polymorphisms. The results of aDNA hybridizations demonstrated varying levels of MTC intensity in 17/32 individuals (53.1%), using the IS6110 target. The IS1081 MTC target showed lower sensitivity, confirming TB positivity in 10/32 (31.2%) individuals. The mtDNA analysis allowed the recovery of HVS-I sequences in 23/32 individuals (71.8%). The majority of these individuals (21/23, 91.3%) were of European ancestry, especially in primary burials. Haplogroups U, J, V, T, K, N, H and R, were identified with haplogroup U being the most frequent at 6/23 (26.1%). African and Amerindian mtDNA haplogroups were observed in two individuals in secondary burials. In spite of the ecclesiastic and aristocratic bias of the population of the study, human ancestry analysis revealed the prominent contribution of Europeans in the introduction or spread of TB in the New World., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The distribution, antiquity and epidemiology of tuberculosis (TB) have previously been studied in osteoarchaeological material in the eastern part of Hungary, mainly on the Great Plain. The purpose of this study is to map the occurrence of skeletal TB in different centuries in the western part of Hungary, Transdanubia, and to present new cases we have found. Palaeopathological analysis was carried out using macroscopic observation supported by radiographic and molecular methods. A large human osteoarchaeological sample (n=5684) from Transdanubian archaeological sites ranging from the 2nd to the 18th centuries served as a source of material. Spinal TB was observed in seven individuals (in three specimens with Pott's disease two of which also had cold abscess) and hip TB was assumed in one case. The results of DNA for Mycobacterium tuberculosis were positive in seven of the eight cases identified by paleopathology, and negative in the assumed case of hip TB. However, the molecular results are consistent with highly fragmented DNA, which limited further analysis. Based on the present study and previously published cases, osteotuberculosis was found in Transdanubia mainly during the 9th-13th centuries. However, there are no signs of TB in many other 9th-13th century sites, even in those that lie geographically close to those where osteotuberculous cases were found. This may be due to a true absence of TB caused by the different living conditions, way of life, or origin of these populations. An alternative explanation is that TB was present in some individuals with no typical paleopathology, but that death occurred before skeletal morphological features could develop., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The chemical characterization of the substance responsible for the phenomenon of "transformation" of pneumococci was presented in the now famous 1944 paper by Avery, MacLeod, and McCarty. Reception of this work was mixed. Although interpreting their results as evidence that deoxyribonucleic acid (DNA) is the molecule responsible for genetic changes was, at the time, controversial, this paper has been retrospectively celebrated as providing such evidence. The mixed and changing assessment of the evidence presented in the paper was due to the work's interpretive flexibility--the evidence was interpreted in various ways, and such interpretations were justified given the neophytic state of molecular biology and methodological limitations of Avery's transformation studies. I argue that the changing context in which the evidence presented by Avery's group was interpreted partly explains the vicissitudes of the assessments of the evidence. Two less compelling explanations of the reception are a myth-making account and an appeal to the wartime historical context of its publication.

Here we tell a 20-year long story. It began with an easily overlooked DNA degradation (Dnd) phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes. This unusual DNA modification, called phosphorothioation, is the first physiological modification identified on the DNA backbone, in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner. Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community. Here, we summarize the progress in chemical, genetic, enzymatic, bioinformatical and analytical aspects of this novel postreplicative DNA modification. We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.

Tuberculosis has plagued humankind since prehistoric times, as is evident from characteristic lesions on human skeletons dating back to the Neolithic period. The disease in man is due predominantly to infection with either Mycobacterium tuberculosis or Mycobacterium bovis, both members of the M. tuberculosis (MTB) complex. A number of studies have shown that when conditions permit, surviving mycobacterial DNA may be amplified from bone by PCR. Such ancient DNA (aDNA) analyses are subject to stringent tests of authenticity and, when feasible, are invariably limited by DNA fragmentation. Using PCRs based on single-nucleotide polymorphic loci and regions of difference (RDs) in the MTB complex, a study was made of five Iron Age individuals with spinal lesions recovered from the cemetery of Aymyrlyg, South Siberia. A sensitive screening PCR for MTB complex mycobacteria was positive in four out of the five cases. Genotyping evidence indicated that all four cases were due to infection with M. bovis rather than M. tuberculosis and the data were consistent with the proposed phylogenetic model of the MTB complex. This is believed to be the first report of M. bovis causing Pott's disease in archaeological human remains. The study shows that genotyping of ancestral strains of MTB complex mycobacteria from contexts of known date provides information which allows the phylogeny of the model to be tested. Moreover, it shows that loss of DNA from RD4, which defines classic M. bovis, had already occurred from the genome over 2000 years before the present.

In contrast to other serotypes of Salmonella enterica, S. Typhi is exclusively adapted to human hosts. Recently, S. Typhi was identified in ancient skeletal material, thereby incriminating typhoid fever for the Plague of Athens. Since, according to Thucydides' report, animals were also affected by the disease, a working hypothesis is constituted that the causative agent of the Plague might be the anticipated original strain of S. Typhi, purportedly capable of infecting animals as well as humans. Possible future sequencing of the discovered ancient strain of S. Typhi may help towards identifying its genomic differences responsible for its modern specification to humans.

The precise nature of the pathogen having caused early plague pandemics is uncertain. Although Yersinia pestis is a likely candidate for all three plague pandemics, the very rare direct evidence that can be deduced from ancient DNA (aDNA) analysis is controversial. Moreover, which of the three biovars, Antiqua, Medievalis or Orientalis, was associated with these pandemics is still debated. There is a need for phylogenetic analysis performed on Y. pestis strains isolated from countries from which plague probably arose and is still endemic. In addition there exist technical difficulties inherent to aDNA investigations and a lack of appropriate genetic targets. The recently described CRISPRs (clustered regularly interspaced short palindromic repeats) may represent such a target. CRISPR loci consist of a succession of highly conserved regions separated by specific "spacers" usually of viral origin. To be of use, data describing the mechanisms of evolution and diversity of CRISPRs in Y. pestis, its closest neighbors, and other species which might contaminate ancient DNA, are necessary. The investigation of closely related Y. pestis isolates has revealed recent mutation events in which elements constituting CRISPRs were acquired or lost, providing essential insight on their evolution. Rules deduced represent the basis for subsequent interpretation. In the present study, the CRISPR loci from representative Y. pestis and Yersinia pseudotuberculosis strains were investigated by PCR amplification and sequence analysis. The investigation of this wider panel of strains, including other subspecies or ecotypes within Y. pestis and also Y. pseudotuberculosis strains provides a database of the existing CRISPR spacers and helps predict the expected CRISPR structure of the Y. pestis ancestor. This knowledge will open the way to the development of a spoligotyping assay, in which spacers can be amplified even from highly degraded DNA samples. The data obtained show that CRISPR analysis can provide a very powerful typing tool, adapted to the systematic, large-scale genotyping of Y. pestis isolates, and the creation of international typing databases. In addition, CRISPRs do constitute a very promising new tool and genetic target to investigate ancient DNA. The corresponding genetic targets are small (<70bp), present in multiple copies (usually more than 10), highly conserved and specific. In addition, the assay can be run in any laboratory. Interpretation of the data is not dependent on accurate sequence data.

"Common regions" (CRs), such as Orf513, are being increasingly linked to mega-antibiotic-resistant regions. While their overall nucleotide sequences show little identity to other mobile elements, amino acid alignments indicate that they possess the key motifs of IS91-like elements, which have been linked to the mobility ent plasmids in pathogenic Escherichia coli. Further inspection reveals that they possess an IS91-like origin of replication and termination sites (terIS), and therefore CRs probably transpose via a rolling-circle replication mechanism. Accordingly, in this review we have renamed CRs as ISCRs to give a more accurate reflection of their functional properties. The genetic context surrounding ISCRs indicates that they can procure 5' sequences via misreading of the cognate terIS, i.e., "unchecked transposition." Clinically, the most worrying aspect of ISCRs is that they are increasingly being linked with more potent examples of resistance, i.e., metallo-beta-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia. Furthermore, if ISCR elements do move via "unchecked RC transposition," as has been speculated for ISCR1, then this mechanism provides antibiotic resistance genes with a highly mobile genetic vehicle that could greatly exceed the effects of previously reported mobile genetic mechanisms. It has been hypothesized that bacteria will surprise us by extending their "genetic construction kit" to procure and evince additional DNA and, therefore, antibiotic resistance genes. It appears that ISCR elements have now firmly established themselves within that regimen.

Background: Until now, in the absence of direct microbiological evidence, the cause of the Plague of Athens has remained a matter of debate among scientists who have relied exclusively on Thucydides' narrations to introduce several possible diagnoses. A mass burial pit, unearthed in the Kerameikos ancient cemetery of Athens and dated back to the time of the plague outbreak (around 430 BC), has provided the required skeletal material for the investigation of ancient microbial DNA., Objective: To determine the probable cause of the Plague of Athens., Method: Dental pulp was our material of choice, since it has been proved to be an ideal DNA source of ancient septicemic microorganisms through its good vascularization, durability and natural sterility., Results: Six DNA amplifications targeted at genomic parts of the agents of plague (Yersinia pestis), typhus (Rickettsia prowazekii), anthrax (Bacillus anthracis), tuberculosis (Mycobacterium tuberculosis), cowpox (cowpox virus) and cat-scratch disease (Bartonella henselae) failed to yield any product in 'suicide' reactions of DNA samples isolated from three ancient teeth. On the seventh such attempt, DNA sequences of Salmonella enterica serovar Typhi were identified providing clear evidence for the presence of that microorganism in the dental pulp of teeth recovered from the Kerameikos mass grave., Conclusion: The results of this study clearly implicate typhoid fever as a probable cause of the Plague of Athens.

The history of Homo sapiens dispersal around the world and inherent interpopulation contacts and conflicts has given rise to several transitions in his relationships with the natural world, with the final result of changes in the patterns of infectious disease (McMichael [2001] Ecosystem Health 7:107-115). Of particular interest, in this context, is the contact between Amerindians and Europeans that started at the end of the 15th century, and the resulting exchange of microbes. We successfully recovered ancient DNA from a pre-Columbian mummy from Cuzco (Peru), radiocarbon-dated to 980-1170 AD, for which consistent mtDNA amplifications and sequences were obtained. The analysis of mtDNA revealed that the mummy's haplogroup was characteristic of Native American populations. We also investigated a sample of feces directly isolated from the intestines of the mummy, using a polymerase chain reaction system designed to detect the broadest spectrum of bacterial DNAs. The analysis of results, following a criterion of "paleoecological consistency" (Rollo and Marota [1998] Philos. Trans. R. Soc. Lond. [Biol.] 354: 111-119), demonstrated that some vestiges of the original microbial flora of the feces were preserved. In particular, we were able to identify the DNA of Haemophylus parainfluenzae, thus suggesting that this recently recognized pathogen was present in precontact Native Americans., (Copyright 2006 Wiley-Liss, Inc.)

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).

DNA treated with alkylating agents is incised at sites of damage by cell extracts. A key component of this DNA repair function was shown by Verly and co-workers to be an endonuclease acting at secondary lesions, apurinic sites, rather than directly at alkylated nucleotide residues.

The discovery of a DNA glycosylase that specifically removes uracil from DNA, opened the door for uncovering a large class of such enzymes that are fundamental to the process of base excision repair of DNA.

During the past 10 years palaeomicrobiology, a new scientific discipline, has developed. The study of ancient pathogens by direct detection of their DNA has answered several historical questions and shown changes to pathogens over time. However, ancient DNA (aDNA) continues to be controversial and great care is needed to provide valid data. Here we review the most successful application of the technology, which is the study of tuberculosis. This has provided direct support for the current theory of Mycobacterium tuberculosis evolution, and suggests areas of investigation for the interaction of M tuberculosis with its host.

The concept of chromosomes with a ring structure was born during the early studies of bacterial sexuality, and the discovery of fertility factors- episomes or plasmids-provided much later the key tools for gene cloning and biotechnology. But the plasmid-mediated transfer of antibiotic and other resistances, as well as pathogenicity, has served bacteria well in their own adaptive evolution.

We describe the molecular identification of human tuberculosis (TB) from vertebral bone tissue samples from three different populations of ancient Egypt. The specimens were obtained from the predynastic to early dynastic necropolis of Abydos (7 individuals, c. 3500-2650 B.C.), from a Middle Kingdom to Second Intermediate Period tomb of the necropolis of Thebes-West (37. c. 2100-1550 B.C.) and from five further Theban tombs used in the New Kingdom and the Late Period (39, c. 1450-500 B.C.). A total of 18 cases tested positive for the presence of ancient DNA (aDNA) of the M. tuberculosis complex. Out of the 9 cases with typical macromorphological signs of tuberculous spondylitis, 6 were positive for mycobacterial aDNA (66.7%). Of 24 cases with non-specific pathological alterations, 5 provided a positive result (20.8%). In 50 cases of normally appearing vertebral bones 7 tested positive (14.0%). There were only minor differences in the frequencies between the three populations. These data strongly support the notion that tuberculosis was present and prevalent in ancient Egypt since very early periods of this civilization. The unexpectedly high rate of mycobacterial aDNA in normal bone samples is presumably due to a pre- to perimortal systemic spread of the bacteria and indicates a generalized infection by M. tuberculosis.

In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.

There are several specific PCR-based methods to detect Mycobacterium leprae DNA, but the amplicons are quite large. For example, primers that target the 36-kDa antigen gene and are in common diagnostic use yield a 530-bp product. This may be a disadvantage when examining samples in which the DNA is likely to be damaged and fragmented. Therefore, two sets of M. leprae-specific nested primers were designed, based on existing primer pairs which have been shown to be specific for M. leprae. Primers that targeted the 18-kDa antigen gene gave an outer product of 136 bp and inner product of 110 bp. The primers based on the RLEP repetitive sequence yielded a 129-bp outer product and 99-bp nested product. With dilutions of a standard M. leprae killed whole-cell preparation as the source of DNA, both single-stage and nested PCR were performed after optimisation of the experimental conditions. Compared with the 36-kDa antigen gene primers, the 18-kDa antigen gene outer primers were 100-fold more sensitive and the RLEP outer primers were 1000-fold more sensitive. As an illustration of two possible applications of these new primers, positive results were obtained from three skin slit samples from treated lepromatous leprosy patients and three archaeological samples from human remains showing typical leprosy palaeopathology. It was concluded that these new primers are a useful means of detecting M. leprae DNA which is damaged or present at a very low level.

This paleomicrobiologic study was conducted on osseous tissue specimens from ancient Hungarian skeletal samples from the 7-8th and the 17th centuries AD with typical macromorphologic evidence of osseous tuberculosis (n = 3), morphologic alterations probably due to tuberculosis (n = 6), or with nontypical osseous changes of vertebral bodies suggestive of inflammatory reaction (n = 5). From these bone samples, DNA was extracted and amplified by polymerase chain reaction (PCR) by using various primer pairs recognizing DNA segments of different mycobacterial species. To confirm specificity of the analysis, the amplification products of several samples were subjected to restriction enzyme digestion and/or direct sequencing. Of the analyzed 14 cases, 8 were unambiguously positive for mycobacterial DNA of the Mycobacterium tuberculosis complex, as shown by the amplification of the IS6110 sequence. In 13 cases we found a PCR product with primers specific for the 65-kDa antigen gene, including 2 cases without genomic DNA. We conclude that the application of other mycobacterial DNA primers may reveal contamination of bones with atypical saprophytic mycobacteria. A positive result for typical mycobacteria was seen in 2 of 3 cases with typical morphologic signs of tuberculosis and amplifiable DNA, in 3 of 6 probable cases, but also in 3 of 6 cases with nontypical bone changes. This indicates that minor osseous reactions of the surface of vertebral bodies may be due-at least in several cases-to infections with bacteria of the M. tuberculosis complex. In these cases the disease may have proceeded rapidly, and the morphologic osseous changes may represent "early" stages of tuberculous infection of the vertebrae., (Copyright 2000 Wiley-Liss, Inc.)

The analysis of the DNA of ancient micro-organisms in archaeological and palaeontological human remains can contribute to the understanding of issues as different as the spreading of a new disease, a mummification process or the effect of diets on historical human populations. The quest for this type of DNA, however, can represent a particularly demanding task. This is mainly due to the abundance and diffusion of bacteria, fungi, yeasts, algae and protozoans in the most diverse environments of the present-day biosphere and the resulting difficulty in distinguishing between ancient and modern DNA. Nevertheless, at least under some special circumstances, by using rigorous protocols, which include an archaeometric survey of the specimens and evaluation of the palaeoecological consistency of the results of DNA sequence analysis, glimpses of the composition of the original microbial flora (e.g. colonic flora) can be caught in ancient human remains. Potentials and pitfalls of this research field are illustrated by the results of research works performed on prehistoric, pre-Columbian and Renaissance human mummies.