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Background: Dental pulp, encapsulating a blood drop, could be used to diagnose pathogen bacteraemia in archaeological materials using DNA-based techniques. We questioned the viability of such ancient pathogens preserved in ancient dental pulp. Methods: After meticulous decontamination of 32 teeth collected from 31 World War I soldiers exhumed in Spincourt, France, dental pulps were extracted and cultured under strict anaerobiosis. Colonies were identified by mass spectrometry and whole genome sequencing. Fluorescent in situ hybridisation (FISH) was used for the direct microscopic detection of pathogens of interest in the dental pulp. All the experimental procedures included negative controls, notably sediments in contact with individual SQ517 to ensure that results did not arise from contamination. Findings: Clostridium tertium was detected by FISH in two dental pulp specimens taken from a 1914 soldier. After a two-day incubation period, both dental pulp samples grew colonies identified by mass spectrometry and genome sequencing as C. tertium; whereas negative controls remained free of C. tertium in all the observations, and no C. tertium was founded in sediments. Skeletal remains of this soldier exhibited two notches in the left tibia evocative of a cold steel wound, and a probably fatal unhealed bullet impact in the hip bone. Interpretation: Data indicated the presence of C. tertium in the dental pulp at the time of the death of one World War I soldier, in 1914. This observation diagnosed C. tertium bacteraemia, with war wounds as the probable portal of entry for C. tertium. Our C. tertium strains ante-dated by three years, the princeps description of this deadly opportunistic pathogen.

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. [ABSTRACT FROM AUTHOR]

Methanobrevibacter oralis (M. oralis) has predominated human oral microbiota methanogenic archaea as far back as the Palaeolithic era in Neanderthal populations and gained dominance from the 18th century onwards. M. oralis was initially isolated from dental plaque samples collected from two apparently healthy individuals allowing its first characterization. The culture of M. oralis is fastidious and has been the subject of several studies to improve its laboratory growth. Various PCR methods are used to identify M. oralis, targeting either the 16S rRNA gene or the mcrA gene. However, only one RTQ-PCR system, based on a chaperonin gene, offers specificity, and allows for microbial load quantification. Next-generation sequencing contributed five draft genomes, each approximately 2.08 Mb (±0.052 Mb) with a 27.82 (±0.104) average GC%, and two ancient metagenomic assembled genomes. M. oralis was then detected in various oral cavity sites in healthy individuals and those diagnosed with oral pathologies, notably periodontal diseases, and endodontic infections. Transmission pathways, possibly involving maternal milk and breastfeeding, remain to be clarified. M. oralis was further detected in brain abscesses and respiratory tract samples, bringing its clinical significance into question. This review summarizes the current knowledge about M. oralis, emphasizing its prevalence, associations with dysbiosis and pathologies in oral and extra-oral situations, and symbiotic relationships, with the aim of paving the way for further investigations.

Dental pulp has been proposed as a suitable tissue sample for the identification of pathogenic organisms. Using PCR with two specific gene targets,Bartonella quintanaDNA 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. [ABSTRACT FROM AUTHOR]

Abstract Background Theoretical models suggest that DNA degradation would sharply limit the PCR-based detection of both eukaryotic and prokaryotic DNA within ancient specimens. However, the relative extent of decay of eukaryote and prokaryote DNA over time is a matter of debate. In this study, the murine macrophage cell line J774, alone or infected with Mycobacterium smegmatis bacteria, were killed after exposure to 90°C dry heat for intervals ranging from 1 to 48 h in order to compare eukaryotic cells, extracellular bacteria and intracellular bacteria. The sizes of the resulting mycobacterial rpoB and murine rpb2 homologous gene fragments were then determined by real-time PCR and fluorescent probing. Findings The cycle threshold (Ct) values of PCR-amplified DNA fragments from J774 cells and the M. smegmatis negative controls (without heat exposure) varied from 26–33 for the J774 rpb2 gene fragments and from 24–29 for M. smegmatis rpoB fragments. After 90°C dry heat incubation for up to 48 h, the Ct values of test samples increased relative to those of the controls for each amplicon size. For each dry heat exposure time, the Ct values of the 146-149-bp fragments were lower than those of 746-747-bp fragments. During the 4- to 24-h dry heat incubation, the non-infected J774 cell DNA was degraded into 597-bp rpb2 fragments. After 48 h, however, only 450-bp rpb2 fragments of both non-infected and infected J774 cells could be amplified. In contrast, the 746-bp rpoB fragments of M. smegmatis DNA could be amplified after the 48-h dry heat exposure in all experiments. Infected and non-infected J774 cell DNA was degraded more rapidly than M. smegmatis DNA after dry heat exposure (ANOVA test, p Conclusion In this study, mycobacterial DNA was more resistant to dry-heat stress than eukaryotic DNA. Therefore, the detection of large, experimental, ancient mycobacterial DNA fragments is a suitable approach for paleomicrobiological studies.

To further assess the spectrum of nanoarchaea in human microbiota, we prospectively searched for nanoarchaea in 110 leftover stool specimens, using the complementary approaches of PCR-sequencing screening, fluorescent in situ hybridization, scanning electron microscopy and metagenomics. These investigations yielded a nanoarchaea, Candidatus Nanopusillus phoceensis sp. nov., detected in stool samples by specific PCR-based assays. Microscopic observations indicated its close contact with the archaea Methanobrevibacter smithii . Genomic sequencing revealed 607,775-bp contig with 24.5% G + C content encoding 30 tRNAs, 3 rRNA genes, and 1,403 coding DNA sequences, of which 719 were assigned to clusters of orthologous groups. Ca . Nanopusillus phoceensis is only the second nanoarchaea to be detected in humans, expanding our knowledge of the repertoire of nanoarchaea associated with the human microbiota and encouraging further research to explore the repertoire of this emerging group of nanomicrobes in clinical samples., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense ( Ca . M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca . M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca . M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca . M. massiliense and possible transfer between hosts.IMPORTANCEMethanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca . M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated., Competing Interests: V.P., M.D., G.A., G.G., and E.T. are co-inventors of a patented culture medium referred to as GG culture medium in this report (patent FR 23 01404).

Among oral microbiota methanogens, Methanobrevibacter massiliense ( M. massiliense ) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii . M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens ( P. piscolens ) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens , as non-motile, 1-2-µm-long and 0.6-0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies.

Introduction: The lack of well-preserved material upon which to base the paleo-microbiological detection of Plasmodium parasites has prevented extensive documentation of past outbreaks of malaria in Europe. By trapping intact erythrocytes at the time of death, dental pulp has been shown to be a suitable tissue for documenting ancient intraerythrocytic pathogens such as Plasmodium parasites., Methods: Total DNA and proteins extracted from 23 dental pulp specimens collected from individuals exhumed from the 9th to 13th century archaeological site in Mariana, Corsica, were analyzed using open-mind paleo-auto-immunohistochemistry and direct metagenomics, Plasmodium -targeting immunochromatography assays. All experiments incorporated appropriate negative controls., Results: Paleo-auto-immunohistochemistry revealed the presence of parasites Plasmodium spp. in the dental pulp of nine teeth. A further immunochromatography assay identified the presence of at least one Plasmodium antigen in nine individuals. The nine teeth, for which the PfHRP-2 antigen specific of P. falciparum was detected, were also positive using paleo-autoimmunohistochemistry and metagenomics., Conclusion: Dental pulp erythrocytes proved to be suitable for the direct paleomicrobiology documentation of malaria in nine individuals buried in medieval Corsica, in agreement with historical data. This provides additional information on the millennial dynamics of Plasmodium spp. in the Mediterranean basin., Competing Interests: MD was a co-owner of AnthropoPOC®, patented name cited in this review. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Boualam, Corbara, Aboudharam, Istria, Signoli, Costedoat, Drancourt and Pradines.)

Nanoarchaea measuring less than 500 nm and encasing an average 600-kb compact genome have been studied for twenty years, after an estimated 4193-million-year evolution. Comprising only four co-cultured representatives, these symbiotic organisms initially detected in deep-sea hydrothermal vents and geothermal springs, have been further distributed in various environmental ecosystems worldwide. Recent isolation by co-culture of Nanopusillus massiliensis from the unique ecosystem of the human oral cavity, prompted us to review the evolutionary diversity of nanaorchaea resulting in a rapidly evolving taxonomiy. Regardless of their ecological niche, all nanoarchaea share limited metabolic capacities correlating with an obligate ectosymbiotic or parasitic lifestyle; focusing on the dynamics of nanoarchaea-bacteria nanoarchaea-archaea interactions at the morphological and metabolic levels; highlighting proteins involved in nanoarchaea attachment to the hosts, as well metabolic exchanges between both organisms; and highlighting clinical nanoarchaeology, an emerging field of research in the frame of the recent discovery of Candidate Phyla radiation (CPR) in human microbiota. Future studies in clinical nanobiology will expand knowledge of the nanaorchaea repertoire associated with human microbiota and diseases, to improve our understanding of the diversity of these nanoorganims and their intreactions with microbiota and host tissues., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Paleomicrobiological data have clarified that Plasmodium spp. was circulating in the past in southern European populations, which are now devoid of malaria. The aim of this study was to evaluate the efficacy of immunodetection and, more particularly, rapid diagnostic tests (RDT), in order to further assess Plasmodium infections in ancient northern European populations., Methods: A commercially available RDT, PALUTOP ®  + 4 OPTIMA, which is routinely used to detect malaria, was used to detect Plasmodium antigens from proteins recovered from ancient specimens extracted from 39 dental pulp samples. These samples were collected from 39 individuals who were buried in the sixth century, near the site of the current Palace of Versailles in France. Positive and negative controls were also used. Antigens detected were quantified using chemiluminescence imaging system analysis., Results: Plasmodium antigens were detected in 14/39 (35.9%) individuals, including Plasmodium vivax antigens in 11 individuals and Plasmodium falciparum antigens co-detected in two individuals, while Pan-Plasmodium antigens were detected in three individuals. Controls all yielded expected results., Conclusions: The data reported here showed that RDTs are a suitable tool for detecting Plasmodium spp. antigens in ancient dental pulp samples, and demonstrated the existence of malaria in Versailles, France, in the sixth century. Plasmodium vivax, which is regarded as being responsible for an attenuated form of malaria and less deadly forms, was the most prevalent species. This illustrates, for the first time in ancient populations, co-infection with P. falciparum, bringing into question the climate-driven ecosystems prevailing at that time in the Versailles area., (© 2023. The Author(s).)

Methanobrevibacter smithii (M. smithii), the most prevalent and abundant gut methanogen, detoxifies hydrogen into methane and is, therefore, of paramount importance for the equilibrium of the gut microbiota. The isolation by culture of M. smithii has routinely relied upon hydrogen‑carbon dioxide-enriched, oxygen-deprived atmospheres. In this study, we developed a medium referred to as "GG", which allowed for M. smithii growth and isolation by culture in an oxygen-deprived atmosphere, with no supply of either hydrogen or carbon dioxide, making it easier to detect M. smithii by culture in clinical microbiology laboratories., Competing Interests: Declaration of Competing Interest VP, ET, GA, MD and GG are co-inventor of patent regarding this work entitled: “Milieu et procédé de culture des archaea methanogens”., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent years have been marked by a paradigm shift in the study of the human microbiota, with a re-emergence of culture-dependent approaches. Numerous studies have been devoted to the human microbiota, while studies on the oral microbiota still remain limited. Indeed, various techniques described in the literature may enable an exhaustive study of the microbial composition of a complex ecosystem. In this article, we report different methodologies and culture media described in the literature that can be applied to study the oral microbiota by culture. We report on specific methodologies for targeted culture and specific culture techniques and selection methodologies for cultivating members of the three kingdoms of life commonly found in the human oral cavity, namely, eukaryota, bacteria and archaea. This bibliographic review aims to bring together the various techniques described in the literature, enabling a comprehensive study of the oral microbiota in order to demonstrate its involvement in oral health and diseases.

Background: Immunohistochemistry (IHC) using monoclonal and polyclonal antibodies is a useful diagnostic method for detecting pathogen antigens in fixed tissues, complementing the direct diagnosis of infectious diseases by PCR and culture on fresh tissues. It was first implemented in a seminal publication by Albert Coons in 1941., Main Body: Of 14,198 publications retrieved from the PubMed, Google, Google Scholar and Science Direct databases up to December 2021, 230 were selected for a review of IHC techniques, protocols and results. The methodological evolutions of IHC and its application to the diagnosis of infectious diseases, more specifically lice-borne diseases, sexually transmitted diseases and skin infections, were critically examined. A total of 59 different pathogens have been detected once in 22 different tissues and organs; and yet non-cultured, fastidious and intracellular pathogens accounted for the vast majority of pathogens detected by IHC. Auto-IHC, incorporating patient serum as the primary antibody, applied to diseased heart valves surgically collected from blood culture-negative endocarditis patients, detected unidentified Gram-positive cocci and microorganisms which were subsequently identified as Coxiella burnetii, Bartonella quintana, Bartonella henselae and Tropheryma whipplei. The application of IHC to ancient tissues dated between the ends of the Ptolemaic period to over 70 years ago, have also contributed to paleomicrobiology diagnoses., Conclusion: IHC plays an important role in diagnostic of infectious diseases in tissue samples. Paleo-auto-IHC derived from auto-IHC, is under development for detecting non-identified pathogens from ancient specimens., (© 2022. The Author(s).)

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., Competing Interests: All the authors declare that there are no conflicts of interest., (© 2021 The Authors. Published by Elsevier B.V.)

Bartonella quintana is a facultative intracellular bacterium responsible for relapsing fever, an example of non-sterilizing immunity. The cellular sanctuary of B. quintana in-between febrile relapses remains unknown but repeated detection of B. quintana in dental pulp specimens suggested long-term half-life dental pulp stem cells (DPSCs) as candidates. As the capacity of DPSCs to internalize microscopic particles was unknown, we confirmed that DPSCs internalized B. quintana bacteria: Gimenez staining and fluorescence microscopy localized B. quintana bacteria inside DPSCs and this internalization did not affect the cellular multiplication of DPSCs during a one-month follow-up despite the increase in the bacterial load. B. quintana-infected DPSCs did not produce Tumor Necrosis Factor-α whereas an important production of Monocytes Chemoattractant Protein-1 was observed. These unprecedented observations suggest the possibility that DPSCs are shelters for the long-term persistence of B. quintana in the host, warranting further experimental and clinical investigations., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objectives: Depicting past epidemics currently relies on DNA-based detection of pathogens, an approach limited to pathogens with well-preserved DNA sequences. We used paleoserology as a complementary approach detecting specific antibodies under a mini line-blot format including positive and negative control antigens., Methods: Mini line blot assay incorporated skim milk as negative control, Staphylococcus aureus as positive control, and antigens prepared from lice-borne pathogens Rickettsia prowazekii, Borrelia recurrentis, Bartonella quintana, and Yersinia pestis. Paleoserums were extracted from rehydrated dental pulp recovered from buried individuals. Mini line blots observed with the naked eye, were quantified using a scanner and appropriate software. Paleoserology was applied to the indirect detection of lice-borne pathogens in seven skeletons exhumed from a 16th-17th century suspected military burial site (Auxi-le-Château); and 14 civils exhumed from a 5th-13th century burial site (Saint-Mont). Direct detection of pathogens was performed using quantitative real-time PCR., Results: In Auxi-le-Château, paleoserology yielded 7/7 interpretable paleoserums including 7/7 positives for B. recurrentis including one also positive for B. quintana. In Saint-Mont, paleoserology yielded 8/14 interpretable paleoserums and none reacted against any of the four pathogens. Antibodies against R. prowazekii and Y. pestis were not detected. The seroprevalence was significantly higher in the military burial site of Auxi-le-Château than in the civil burial site of Saint-Mont. Real-time PCR detection of B. quintana yielded 5/21 positive (3 at Saint-Mont and 2 at Auxi-le-Château) whereas B. recurrentis was not detected., Conclusions: Paleoserology unmasked an outbreak of relapsing B. recurrentis fever in one 16th - 17th century military garrison, missed by real-time PCR. Paleoserology offers a new tool for investigating past epidemics, in complement to DNA sequence-based approaches., (© 2020 Wiley Periodicals LLC.)

Methanogens, the sole microbes producing methane, are archaea commonly found in human anaerobic microbiota. Methanogens are emerging as opportunistic pathogens associated with dysbiosis and are also detected and cultured in anaerobic abscesses. Their presence in the respiratory tract is yet unknown. As a preliminary answer, prospective investigation of 908 respiratory tract samples using polyphasic approach combining PCR-sequencing, real-time PCR, fluorescent in situ hybridization (FISH), and methanogens culture was carried out. Methanobrevibacter smithii and Methanobrevibacter oralis DNA sequences, were detected in 21/527 (3.9%) sputum samples, 2/188 (1.06%) bronchoalveolar lavages, and none of 193 tracheo-bronchial aspirations. Further, fluorescence in situ hybridization detected methanogens in three sputum investigated specimens with stick morphology suggesting M. oralis and in another one bronchoalveolar lavage sample investigated, diplococal morphology suggesting M. smithii . These observations extend the known territory of methanogens to the respiratory tract and lay the foundations for further interpretation of their detection as pathogens in any future cases of isolation from bronchoalveolar lavages and the lungs.

During the two World Wars, Bartonella quintana was responsible for trench fever and is now recognised as an agent of re-emerging infection. Many reports have indicated widespread B. quintana exposure since the 1990s. In order to evaluate its prevalence in ancient populations, we used real-time PCR to detect B. quintana DNA in 400 teeth collected from 145 individuals dating from the 1st to 19th centuries in nine archaeological sites, with the presence of negative controls. Fisher's exact test was used to compare the prevalence of B. quintana in civil and military populations. B. quintana DNA was confirmed in a total of 28/145 (19.3%) individuals, comprising 78 citizens and 67 soldiers, 20.1% and 17.9% of which were positive for B. quintana bacteraemia, respectively. This study analysed previous studies on these ancient samples and showed that the presence of B. quintana infection followed the course of time in human history; a total of 14/15 sites from five European countries had a positive prevalence. The positive rate in soldiers was higher than those of civilians, with 20% and 18.8%, respectively, in the 18th and 19th centuries, but the difference in frequency was not significant. These results confirmed the role of dental pulp in diagnosing B. quintana bacteraemia in ancient populations and showed the incidence of B. quintana in both civilians and soldiers., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Dental pulp with special structure has become a good reference sample in paleomicrobiology-related blood-borne diseases, many pathogens were detected by different methods based on the diagnosis of nucleic acids and proteins., Objectives: This review aims to propose the preparation process from ancient teeth collection to organic molecule extraction of dental pulp and summary, analyze the methods that have been applied to detect septicemic pathogens through ancient dental pulps during the past 20 years following the first detection of an ancient microbe., Methods: The papers used in this review with two main objectives were obtained from PubMed and Google scholar with combining keywords: "ancient," "dental pulp," "teeth," "anatomy," "structure," "collection," "preservation," "selection," "photography," "radiography," "contamination," "decontamination," "DNA," "protein," "extraction," "bone," "paleomicrobiology," "bacteria," "virus," "pathogen," "molecular biology," "proteomics," "PCR," "MALDI-TOF," "LC/MS," "ELISA," "immunology," "immunochromatography," "genome," "microbiome," "metagenomics.", Results: The analysis of ancient dental pulp should have a careful preparation process with many different steps to give highly accurate results, each step complies with the rules in archaeology and paleomicrobiology. After the collection of organic molecules from dental pulp, they were investigated for pathogen identification based on the analysis of DNA and protein. Actually, DNA approach takes a principal role in diagnosis while the protein approach is more and more used. A total of seven techniques was used and ten bacteria (Yersinia pestis, Bartonella quintana, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi C, Mycobacterium leprae, Mycobacterium tuberculosis, Rickettsia prowazeki, Staphylococcus aureus, Borrelia recurrentis, Bartonella henselae) and one virus (Anelloviridae) were identified. Y. pestis had the most published in quantity and all methods were investigated for this pathogen, S. aureus and B. recurrentis were identified by three different methods and others only by one. The combining methods interestingly increase the positive rate with ELISA, PCR and iPCR in Yersinia pestis diagnosis. Twenty-seven ancient genomes of Y. pestis and one ancient genome of B. recurrentis were reconstructed. Comparing to the ancient bone, ancient teeth showed more advantage in septicemic diagnosis. Beside pathogen identification, ancient pulp help to distinguish species., Conclusions: Dental pulp with specific tissue is a suitable sample for detection of the blood infection in the past through DNA and protein identification with the correct preparation process, furthermore, it helps to more understand the pathogens of historic diseases and epidemics., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Methanogen cultures require hydrogen produced by fermentative bacteria such as Bacteroides thetaiotaomicron (biological method). We developed an alternative method for hydrogen production using iron filings and acetic acid with the aim of cultivating methanogens more efficiently and more quickly (chemical method). We developed this new method with a reference strain of Methanobrevibacter oralis, compared the method to the biological reference method with a reference strain of Methanobrevibacter smithii and finally applied the method to 50 saliva samples. Methanogen colonies counted using ImageJ software were identified using epifluorescence optical microscopy, real-time PCR and PCR sequencing. For cultures containing pure strains of M. oralis and M. smithii, colonies appeared three days postinoculation with the chemical method versus nine days with the biological method. The average number of M. smithii colonies was significantly higher with the chemical method than with the biological method. There was no difference in the delay of observation of the first colonies in the saliva samples between the two methods. However, the average number of colonies was significantly higher with the biological method than with the chemical method at six days and nine days postinoculation (Student's test, p = 0.005 and p = 0.04, respectively). The chemical method made it possible to isolate four strains of M. oralis and three strains of M. smithii from the 50 saliva samples. Establishing the chemical method will ease the routine isolation and culture of methanogens., (Copyright © 2019. Published by Elsevier Ltd.)

Strain Marseille-P4122 T 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-P4122 T 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-P4122 T is 2.49 Mb with 60.1% G + C content. We propose that strain Marseille-P4122 T (=CSURP4122) is the type strain of the new species Corynebacterium dentalis sp. nov., Competing Interests: None to declare., (© 2019 The Author(s).)

Background: The oral cavity of humans is inhabited by several hundreds of bacterial species and other microorganisms such as fungi and archaeal methanogens. Regarding methanogens, data have been obtained from oral cavity samples collected in Europe, America and Asia. There is no study published on the presence of methanogens in the oral cavity in persons living in Africa. The objective of our study was to bring new knowledge on the distribution of oral methanogens in persons living in Mali, Africa., Methods: A total of 31 patients were included in the study during a 15-day collection period in September. Bacterial investigations consisted in culturing the bacteria in 5% sheep blood-enriched Columbia agar and PolyViteX agar plates. For archaeal research, we used various methods including culture, molecular biology and fluorescent in situ hybridization (FISH)., Results: Eight of 31 (26%) oral samples collected in eight patients consulting for stomatology diseases tested positive in polymerase chain-reaction (PCR)-based assays for methanogens including five cases of Methanobrevibacter oralis and one case each of Methanobrevibacter smithii, Methanobrevibacter massiliense and co-infection Methanobrevibacter oralis and Methanobrevibacter massiliense., Conclusions: In this pilot study, we are reporting here the first characterization of methanogens in the oral cavity in eight patients in Mali. These methanogen species have already been documented in oral specimens collected from individuals in Europe, Asia, North America and Brazil.

The microbial communities of the oral fluid are in direct contact with tobacco smoke, which may thus affect these communities. Few culture-based studies have analyzed the effects of tobacco smoking on the oral fluid microbiota. Using bacterial culture we investigated whether tobacco smoking altered the microbial diversity of the oral fluid, focusing on aerobic and facultative anaerobic Gram-positive bacteria otherwise comprising of major pathogens. Among 90 oral fluid specimens collected in 19 tobacco-smokers and 71 controls, the diversity did not significantly differ with age and with sex. However, diversity was significantly lower in tobacco-smokers (nine different species) than in non-smokers (18 different species) with all the species cultured in tabocco-smokers being also cultured in non-smokers. We isolated the human pathogen Streptococcus australis for the first time from oral fluid. Tobacco smoking significantly alters the saliva Gram-positive bacterial microbiota, including pathogens with potential implication in the pathogenesis of tobacco-related diseases such as periodontitis and peri-implantitis.

Aim: The aim of the present study was to create a tool to evaluate the risk of peri-implantitis according its severity., Methods: After ethics committee approval, 43 patients provided signed consent and were included prospectively. Forty-five observations were recorded. The following criteria were recorded: number of implant faces showing bleeding and/or suppuration, pocket depth on at least two faces of the implant, bone loss as a function of the length of the implant evaluated on X-rays, number of implant faces with bacterial plaque, the parameters required for determination of excess cement (screwed or sealed prosthesis, burying of sealed prostheses), periodontal status, glycemia, and annual consumption of tobacco. Each of these parameters was plotted on a chart using Microsoft Excel., Results: Seventeen of 45 (37.8%) cases were identified as having high peri-implantitis risk, two of 45 (4.4%) had low risk, and 11 of 45 (24.4%) had moderate risk; 33.3% patients did not have peri-implantitis and were considered at very low risk., Conclusion: The observed results applied to the evaluation model are an effective diagnostic tool in assessing the risk of peri-implantitis. The tool takes into account parameters, which have not been taken into account until now. The information is automatically processed and allows early management of peri-implantitis., (© 2019 John Wiley & Sons Australia, Ltd.)

Trichomonas tenax, an anaerobic protist difficult to cultivate with an unreliable molecular identification, has been suspected of involvement in periodontitis, a multifactorial inflammatory dental disease affecting the soft tissue and bone of periodontium. A cohort of 106 periodontitis patients classified by stages of severity and 85 healthy adult control patients was constituted. An efficient culture protocol, a new identification tool by real-time qPCR of T. tenax and a Multi-Locus Sequence Typing system (MLST) based on T. tenax NIH4 reference strain were created. Fifty-three strains of Trichomonas sp. were obtained from periodontal samples. 37/106 (34.90%) T. tenax from patients with periodontitis and 16/85 (18.80%°) T. tenax from control patients were detected by culture (p = 0.018). Sixty of the 191 samples were tested positive for T. tenax by qPCR, 24/85 (28%) controls and 36/106 (34%) periodontitis patients (p = 0.089). By combining both results, 45/106 (42.5%) patients were positive by culture and/or PCR, as compared to 24/85 (28.2%) controls (p = 0.042). A link was established between the carriage in patients of Trichomonas tenax and the severity of the disease. Genotyping demonstrates the presence of strain diversity with three major different clusters and a relation between disease strains and the periodontitis severity (p<0.05). More frequently detected in periodontal cases, T. tenax is likely to be related to the onset or/and evolution of periodontal diseases., Competing Interests: The authors have declared that no competing interests exist.