Paleoserological detection of Coronavirus antigens in dental calculus of human remains dating from the beginning of the 19th century, French Ardennes.

OBJECTIVE: Vanishing viral RNA restricts our ability to detect ancient pathogens, so, we used paleo serological approaches to trace the dynamics of the Coronavirus in ancient populations. MATERIALS AND METHODS: We investigated 10 ancient dental calculus samples collected from a cemetery dated to the beginning of the 19th century and excavated in Charleville-Mézières. After paleoserum samples were extracted from dental calculus, paleoserology using mini-line-blot incorporating one alpha-Coronavirus (Coronavirus 229 E) and two beta-Coronavirus (Coronavirus OC 43, SARS-CoV-2) antigens and controls was completed by an automated Western blotting assay. RESULTS: Once appropriate controls had validated the data, mini-line-blot detected antibodies against the two beta-Coronavirus antigens in individuals US1300 and US1339, automated Western blotting confirming one beta-Coronavirus antigen for individual US1300 and an additional individual US1326. DISCUSSION: Combing mini-line blot and automated Western blot assays made it possible to detect immunoreactive immunoglobulin tracing circulation of Coronavirus in France at the very beginning of the 19th century.

Introducing clinical nanorachaeaology: Isolation by co-culture of Nanopusillus massiliensis sp. nov.

BACKGROUND: Nanoarchaeota, obligate symbiont of some environmental archaea with reduced genomes, have been described in marine thermal vent environments, yet never detected in hosts, including humans. METHODS: Here, using laboratory tools geared towards the detection of nanoarchaea including PCR-sequencing, WGS, microscopy and culture. RESULTS: We discovered a novel nanoarchaea, Nanopusillus massiliensis, detected in dental plate samples by specific PCR-based assays. Combining fluorescent in situ hybridization (FISH) with scanning electron microscopy disclosed close contacts between N. massiliensis and the archaea Methanobrevibacter oralis in these samples. Culturing one sample yielded co-isolation of M. oralis and N. massiliensis with a 606,935-bp genome, with 23.6% GC encoded 16 tRNA, 3 rRNA and 942 coding DNA sequences, of which 400 were assigned to clusters of orthologous groups. CONCLUSION: The discovery of N. massiliensis, made publicly available in collection, extended our knowledge of human microbiota diversity, opening a new field of research in clinical microbiology here referred to as clinical nanoarchaeology.

Microbial biodiversity of natural toothbrushes in Mali.

Different oral hygiene practices are used to overcome endemic diseases such as dental caries and oral infections. In Mali (Africa), natural plant-based toothbrushes are used for eliminating bacterial biofilm. The repertoire of microorganisms associated with natural toothbrushes is unknown. The aim of our study is to study microbial flora in particular the methanogenic archaea associated with natural toothbrushes recently recognized as responsible for periodontitis and peri-implantitis. We investigated the methanogens and bacteria associated with 15 different natural plant toothbrushes collected in Bamako local market (Mali). Microbiological investigations consisted in culturing the bacteria on agar plates and searching archaea using molecular techniques. No archaea were demonstrated by molecular biology but 50 bacterial species, including 33 aero-anaerobic and 17 aerobic species, were isolated from natural toothbrushes. We isolated Pseudomonas sp., Staphylococcus sp. and Klebsiella pneumoniae, which are acknowledged as opportunistic human pathogens. This study has highlighted the likely impact of the use of natural toothbrushes in the spread of potentially pathogenic bacteria in the human oral cavity.

Yersinia pestis: the Natural History of Plague.

The Gram-negative bacterium Yersinia pestis is responsible for deadly plague, a zoonotic disease established in stable foci in the Americas, Africa, and Eurasia. Its persistence in the environment relies on the subtle balance between Y. pestis-contaminated soils, burrowing and nonburrowing mammals exhibiting variable degrees of plague susceptibility, and their associated fleas. Transmission from one host to another relies mainly on infected flea bites, inducing typical painful, enlarged lymph nodes referred to as buboes, followed by septicemic dissemination of the pathogen. In contrast, droplet inhalation after close contact with infected mammals induces primary pneumonic plague. Finally, the rarely reported consumption of contaminated raw meat causes pharyngeal and gastrointestinal plague. Point-of-care diagnosis, early antibiotic treatment, and confinement measures contribute to outbreak control despite residual mortality. Mandatory primary prevention relies on the active surveillance of established plague foci and ectoparasite control. Plague is acknowledged to have infected human populations for at least 5,000 years in Eurasia. Y. pestis genomes recovered from affected archaeological sites have suggested clonal evolution from a common ancestor shared with the closely related enteric pathogen Yersinia pseudotuberculosis and have indicated that ymt gene acquisition during the Bronze Age conferred Y. pestis with ectoparasite transmissibility while maintaining its enteric transmissibility. Three historic pandemics, starting in 541 AD and continuing until today, have been described. At present, the third pandemic has become largely quiescent, with hundreds of human cases being reported mainly in a few impoverished African countries, where zoonotic plague is mostly transmitted to people by rodent-associated flea bites.

A 2,000-year-old specimen with intraerythrocytic Bartonella quintana.

Photogrammetry and cascading microscopy investigations of dental pulp specimens collected from 2,000-year-old individuals buried in a Roman necropolis in Besançon, France, revealed unprecedented preserved tissular and cellular morphology. Photogrammetry yielded 3-D images of the smallest archaeological human remains ever recovered. Optical microscopy examinations after standard haematoxylin-phloxine-saffron staining and anti-glycophorin A immunohistochemistry exposed dental pulp cells, in addition erythrocytes were visualised by electron microscopy, which indicated the ancient dental pulp trapped a blood drop. Fluorescence in situ hybridisation applied on red blood cells revealed the louse-borne pathogen Bartonella quintana, a finding confirmed by polymerase chain reaction assays. Through paleohistology and paleocytology, we demonstrate that the ancient dental pulp preserved intact blood cells at the time of the individual's death, offering an unprecedented opportunity to engage in direct and indirect tests to diagnose pathogens in ancient buried individuals.

Parabacteroides pacaensis sp. nov. and Parabacteroides provencensis sp. nov., two new species identified from human gut microbiota.

Strains Marseille-P4001 and Marseille-P3668 are new species from the order Bacteroidales isolated from healthy French volunteers. They are anaerobic Gram-negative rod-shaped bacteria. They exhibited 92.68% and 96.68% 16S rRNA sequence identities with Parabacteroides gordonii strain MS-1 and Parabacteroides chinchillae JCM 17104, respectively, the phylogenetically closest species. Their respective draft genomes measured 5.23 Mb and 3.73 Mb with 39.2 mol% and 40.8 mol% of G + C content. Using a taxonogenomics method, we propose here a brief description of Parabacteroides pacaensis sp. nov., strain Marseille-P4001T and Parabacteroides provencensis sp. nov., strain Marseille-P3668T as new bacterial species.

Corynebacterium dentalis sp. nov., a new bacterium isolated from dental plaque of a woman with periodontitis.

Strain Marseille-P4122T is a new species from the order Corynebacteriales that was isolated from the dental plaque of a woman with periodontitis. It is a facultative anaerobic Gram-positive rod-shaped bacterium. Strain Marseille-P4122T exhibited a 98.19% sequence identity with Corynebacterium suicordis strain P81/02, the phylogenetically closely related species with standing in nomenclature. The draft genome size of strain Marseille-P4122T is 2.49 Mb with 60.1% G + C content. We propose that strain Marseille-P4122T (=CSURP4122) is the type strain of the new species Corynebacterium dentalis sp. nov.

Bacteria and archaea paleomicrobiology of the dental calculus: a review.

Dental calculus, a material observed in the majority of adults worldwide, emerged as a source for correlating paleomicrobiology with human health and diet. This mini review of 48 articles on the paleomicrobiology of dental calculus over 7550 years discloses a secular core microbiota comprising nine bacterial phyla - Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, TM7, Synergistetes, Chloroflexi, Fusobacteria, Spirochetes - and one archaeal phylum Euryarchaeota; and some accessory microbiota that appear and disappear according to time frame. The diet residues and oral microbes, including bacteria, archaea, viruses and fungi, consisting of harmless organisms and pathogens associated with local and systemic infections have been found trapped in ancient dental calculus by morphological approaches, immunolabeling techniques, isotope analyses, fluorescent in situ hybridization, DNA-based approaches, and protein-based approaches. These observations led to correlation of paleomicrobiology, particularly Streptococcus mutans and archaea, with past human health and diet.

Genetic variants of dental plaque Methanobrevibacter oralis.

Methanobrevibacter oralis is the major methanogenic archaea found in the oral cavity. It has been implicated in periodontitis, including the severe form. It is unknown whether certain M. oralis genetic variants are associated with severe periodontitis. Here, we developed multispacer sequence typing (MST) as a sequencing-based genotyping method for the assessment of M. oralis. The sequencing of four intergenic spacers from a collection of 17 dental plaque M. oralis isolates obtained from seven individuals revealed 482 genetic polymorphisms, including 401 single nucleotide polymorphisms (83.2 %), 55 deletions (11.4 %) and 26 insertions (5.4 %). Concatenation of the four spacers yielded nine genotypes, which were clustered into six groups with an index of discrimination of 0.919. One periodontitis patient may have harboured up to three genetic variants of M. oralis, revealing the previously unknown diversity of this archaea. MST will allow for the study of the dynamics of M. oralis populations, including inter-individual transmission and any correlations with the severity of periodontitis.

Molecular detection of Bartonella quintana DNA in the dental pulp of a homeless patient.

Dental pulp has been proposed as a suitable tissue sample for the identification of pathogenic organisms. Using PCR with two specific gene targets, Bartonella quintana DNA was detected in the dental pulp extracted from the tooth of a homeless patient. The patient had been bacteremic 6 months previously but was not when the tooth was sampled.

Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death.

Medieval Black Death is believed to have killed up to one-third of the Western European population during the 14th century. It was identified as plague at this time, but recently the causative organism was debated because no definitive evidence has been obtained to confirm the role of Yersinia pestis as the agent of plague. We obtained the teeth of a child and two adults from a 14th century grave in France, disrupted them to obtain the pulp, and applied the new "suicide PCR" protocol in which the primers are used only once. There were no positive controls: Neither Yersinia nor Yersinia DNA were introduced in the laboratory. A negative result is followed by a new test using other primers; a positive result is followed by sequencing. The second and third primer pair used, coding for a part of the pla gene, generated amplicons whose sequence confirmed that it was Y. pestis in 1 tooth from the child and 19/19 teeth from the adults. Negative controls were negative. Attempts to detect the putative alternative etiologic agents Bacillus anthracis and Rickettsia prowazekii failed. Suicide PCR avoids any risk of contamination as it uses a single-shot primer-its specificity is absolute. We believe that we can end the controversy: Medieval Black Death was plague.

Detection of Coxiella burnetii DNA in dental pulp during experimental bacteremia.

Colonization of dental pulp by blood-borne bacteria in the absence of previous inflammation has been hypothetized but has never been convincingly demonstrated. In order to provide convincing support for this hypothesis we attempted to detect Coxiella burnetii DNA in the dental pulp of bacteremic, intraperitoneally inoculated guinea-pigs by PCR amplification and direct sequencing of two molecular targets. Coxiella burnetii DNA was recovered from 20-50% of the animals depending on the molecular target, from 15-20 days after experimental challenge. These results demonstrated, for the first time, that dental pulp is contaminated by blood-borne bacteria and can be detected by molecular tools.

Detection of 400-year-old Yersinia pestis DNA in human dental pulp: an approach to the diagnosis of ancient septicemia.

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague ("plague teeth") and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human beta-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase beta-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.