The discovery of Candidatus Nanopusillus phoceensis sheds light on the diversity of the microbiota nanoarchaea.

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.

Candidatus Methanosphaera massiliense sp. nov., a methanogenic archaeal species found in a human fecal sample and prevalent in pigs and red kangaroos.

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.

Mycobacterium ulcerans-Bordetella trematum chronic tropical cutaneous ulcer: A four-case series, Côte d'Ivoire.

BACKGROUND: Chronic tropical cutaneous ulcers remain a neglected medical condition in West Africa, particularly Buruli ulcer, which is caused by mycolactone cytotoxin-secreting Mycobacterium ulcerans (M. ulcerans). Medical management of this highly debilitating and necrotising skin infection may be modified by colonisation and co-infection of the ulcer by opportunistic and pathogenic microorganisms, which considerably delays and increases the cost of treatment. METHODOLOGY/PRINCIPAL FINDING: We diagnosed chronic tropical cutaneous ulcers in nine patients in Côte d'Ivoire using M. ulcerans-specific PCRs and culturomics. This revealed M. ulcerans in 7/9 ulcer swabs and 5/9 control swabs as well as an additional 122 bacterial species, 32 of which were specific to ulcers, 61 specifics to the controls, and 29 which were shared, adding 40 bacterial species to those previously reported. Whole genome sequencing of four Bordetella trematum (B. trematum) isolates in four Buruli ulcer swabs and no controls indicated cytolethal distending toxins, as confirmed by cytotoxic assay. CONCLUSIONS/SIGNIFICANCE: In four cases of Buruli ulcer in Côte d'Ivoire, B. trematum was a co-pathogen which was resistant to rifampicin and clarithromycin, unmatching M. ulcerans antibiotic susceptibility profile and counteracting the current treatment of Buruli ulcer in West Africa and Australia. Thus, we report here chronic mixed M. ulcerans-B. trematum chronic tropical ulcer as a specific form of Buruli ulcer in West Africa.

Mycobacterium tuberculosis Resides in Macrophages in Laryngeal Tuberculosis: A Case Report.

Laryngeal tuberculosis is a rare form of extrapulmonary tuberculosis that questions the natural history of this infection. We report one such case in which a pathological examination of a laryngeal biopsy revealed granulomatous inflammation with caseous necrosis. Further investigations combining immunofluorescence detection of macrophages and in situ hybridization of Mycobacterium tuberculosis indicated the presence of Mycobacterium tuberculosis (M. tuberculosis) in laryngeal granulomatous inflammatory lesions. This observation suggests that the natural history of laryngeal tuberculosis does not differ from that of other forms, guiding early diagnosis in patients with laryngeal lesions to ensure appropriate check-ups and treatment.

The millennial dynamics of malaria in the mediterranean basin: documenting Plasmodium spp. on the medieval island of Corsica.

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.

Current knowledge and clinical perspectives for a unique new phylum: Nanaorchaeota.

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.

The Enterococcus secretome inhibits the growth of Mycobacterium tuberculosis complex mycobacteria.

Enterococcus mundtii , a commensal intestinal bacterium, was demonstrated to inhibit the growth of some Mycobacterium tuberculosis complex (MTC) species that cause tuberculosis in humans and mammals. To further explore this preliminary observation, we cross-investigated five E. mundtii strains and seven MTC strains representative of four MTC species using a standardized quantitative agar well diffusion assay. All five E. mundtii strains, calibrated at 10 MacFarland, inhibited the growth of all M. tuberculosis strains with various susceptibility profiles, but no inhibition was observed with lower inoculums. Further, eight E. mundtii freeze-dried cell-free culture supernatants (CFCS) inhibited the growth of M. tuberculosis , Mycobacterium africanum, Mycobacterium bovis and Mycobacterium canettii, the most susceptible MTC species (inhibition diameter 25±1 mm), proportionally to CFCS protein concentrations. The data reported here indicate that the E. mundtii secretome inhibited growth of all MTC species of medical interest, which broadens previously reported data. In the gut, the E. mundtii secretome may modulate the expression of tuberculosis, exhibiting an anti-tuberculosis effect, with some protective roles in human and animal health.

Old World Medieval Treponema pallidum Complex Treponematosis: A Case Report.

BACKGROUND: Introduction of 1 Treponema pallidum complex pathogen in naive European populations following the return of Christopher Columbus' troops from Central America in 1493 is a central dogma in venereology. METHODS: Among skeletal elements from the seventh or eighth century uncovered in Roquevaire, France, individual RS-1003 femur macroscopically suspected of having an infectious disease was investigated by means of paleoautoimmunohistochemistry, direct metagenomics, and paleoserology, along with 1 control femur from an apparently healthy individual (R-1003) and experimental negative controls. RESULTS: RS-1003 femur showed infectious bone; paleoautoimmunohistochemistry of the lesions led to microscopic detection of a T. pallidum complex pathogen. Phylogenetic analyses comprising 71 T. pallidum complex-specific reads covering 2.37% of the T. pallidum subsp. pallidum reference genome sequence revealed an ancestral T. pallidum complex pathogen in the lesion. Paleoserology detecting T. pallidum-specific antigens confirmed positive serological findings in individual RS-1003. Individual R-1003 and the negative controls remained negative. CONCLUSIONS: This case, predating by 8 centuries previous detections of T. pallidum complex treponematosis in Europe, indicated that European populations were not naive to these pathogens before the 1493 introduction of a Central American T. pallidum complex pathogen overwhelming the T. pallidum ones previously circulating in the Old World. These data break a century-old dogma in medical microbiology.

Detection of Mycobacterium angelicum in Human Urinary Tract, French Polynesia.

We definitively characterized Mycobacterium angelicum, an aquatic zoonotic opportunistic pathogen of the M. szulgai complex, using a polyphasic approach that included whole-genome sequencing. The sequence was obtained on the island of Tahiti, French Polynesia, from a urine specimen collected from a patient experiencing a urinary tract infection.

Use of rapid diagnostic tests for the detection of ancient malaria infections in dental pulp from the sixth century in Versailles, France.

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.

Culturing clinical Methanobrevibacter smithii using GG medium in a minimal anaerobe atmosphere.

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 hydrogen­carbon 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.

An ancient coronavirus from individuals in France, circa 16th century.

OBJECTIVES: At the time when the COVID-19 pandemic was responsible for more than six million deaths worldwide, the antiquity of coronaviruses remains undefined. We investigated individuals buried during the 16th century in France for the direct and paleoserological diagnosis of the coronavirus. METHODS: The 2011-2012 excavation of Abbey Saint-Pierre in Baume-Les-Messieurs, France uncovered 12 skeletons of individuals from the 13th to the 18th century. The total proteins extracted from dental pulps were subjected to microbial paleoserology, targeting SARS-CoV-2, human-associated coronavirus (HCoV)-229E, and OC43 antigens and for coronavirus peptide research using metaproteomics, in parallel to negative controls. RESULTS: Three peptide sequences totaling 36 amino acids indicative of a coronavirus were retrieved from the dental pulp remains collected from two individuals buried circa 16th century, in whom paleoserology confirmed a specific immunological response against modern-day SARS-CoV-2 and HCoV-229E. CONCLUSION: We provide serological and proteomic evidence for a betacoronavirus with no modern correspondent, infecting populations in the 16th century, extending the antiquity of coronaviruses by more than three centuries. Historical, archaeozoological, and paleoproteomic data suggested close contacts between these two individuals and domestic swine, cattle, and poultry, suggesting an ancient zoonotic coronavirus. Coronaviruses have been undesirable companions of populations long before the ongoing coronavirus disease 2019 outbreak emerged.

Clinical and Echocardiographic Features of Patients With Infective Endocarditis and Bicuspid Aortic Valve According to Echocardiographic Definition of Valve Morphology.

BACKGROUND: The influence of different bicuspid aortic valve (BAV) morphology in the clinical course of infective endocarditis (IE) has not yet been investigated. This study aimed to describe the clinical and echocardiographic features of IE in patients with BAV (BAVIE) according to valve morphology. METHODS: Patients with definite BAVIE prospectively enrolled in 4 high-volume referral centers from 2000 to 2019 were evaluated and divided into 2 groups according to the echocardiographic definition of fused BAV morphology: right-left coronary (RL type) and right noncoronary or left noncoronary (non-RL type) cusp fusion. All patients were followed up for 1 year. RESULTS: One hundred thirty-eight patients with BAVIE were included (77.7% male; median age, 52 [36.83-61.00] years): 112 patients with RL type (81%) and 26 patients with non-RL type BAV (19%), with no significant differences in age, sex, and comorbidities between groups. Although 43% of the cohort had known BAV, the referral was late after symptom onset, particularly for the RL phenotype; time from symptom onset to hospitalization >30 days (31.3% vs 11.5%; P = .032) and New York Heart Association class ≥ II (64.3% vs 42.3%; P = .039) were more frequent in patients with RL type BAV than in patients with non-RL type BAV. Conversely, patients with non-RL type BAV had a higher incidence of hemorrhagic stroke (19.2% vs 5.4%; P = .034) and high-grade atrioventricular block (11.5% vs 0.9%; P = .021). Streptococcus viridans was more frequently isolated in patients with non-RL type BAV than in patients with RL type BAV (44% vs 24.1%; P = .045). No difference in short- and intermediate-term mortality was observed between groups. CONCLUSIONS: Clinical profile and echocardiographic features in BAVIE patients may differ according to valve morphology, and patients with BAVIE appear to be referred late, even when BAV disease is previously known.

Gold Panning-Related Chronic Cutaneous Ulcers in Guinea, West Africa.

Chronic cutaneous ulcers caused potentially by several pathogens are of increasing concern in endemic tropical countries, including Guinea in West Africa, in rural populations exposed to aquatic environments during recreational, domestic, or agricultural activities. By plotting 1,011 cases of chronic cutaneous ulcers classified under the name Buruli ulcer in 24 of 33 Guinea health districts (72%) between 2018 and 2020 against the gold map and gold-panning map of Guinea, we revealed a significant spatial association between chronic cutaneous ulcer foci and gold-panning foci (P < 0.05), but not with nongold-panning foci (P = 0.12) in Guinea. Gold panning should be listed as an additional economic activity exposing populations to chronic cutaneous ulcers. Further research may aim to clarify whether any geological and biologic factors underlie such an association, besides the possibility that the unprotected skin of gold panners may be exposed to opportunistic, pathogen-contaminated environments in gold-panning areas.

Real-time metagenomics-based diagnosis of community-acquired meningitis: A prospective series, southern France.

BACKGROUND: Point-Of-Care (POC) diagnosis of life-threatening community-acquired meningitis currently relies on multiplexed RT-PCR assays, that lack genotyping and antibiotic susceptibility profiling. We assessed the usefulness of real-time metagenomics (RTM) directly applied to the cerebrospinal fluid (CSF) for the identification, typing and susceptibility profiling of pathogens responsible for community-acquired meningitis. METHODS: A series of 52 CSF samples from patients suspected of having community-acquired meningitis, were investigated at POC by direct RTM in parallel to routine real-time multiplex PCR (RT-PCR) and bacterial culture, for the detection of pathogens. RTM-generated sequences were blasted in real-time against an in-house database incorporating the panel of 12 most prevalent pathogens and against NCBI using EPI2ME online software, for pathogen identification. In-silico antibiogram and genotype prediction were determined using the ResFinder bio-tool and MLST online software. FINDINGS: Over eight months, routine multiplex RT-PCR yielded 49/52 positive CSFs, including 21 Streptococcus pneumoniae, nine Neisseria meningitidis, eight Haemophilus influenzae, three Streptococcus agalactiae, three Herpesvirus-1, two Listeria monocytogenes, and one each of Escherichia coli, Staphylococcus aureus and Varicella-Zoster Virus. Parallel RTM agreed with the results of 47/52 CSFs and revealed two discordant multiplex RT-PCR false positives, one H. influenzae and one S. pneumoniae. Both multiplex RT-PCR and RTM agreed on the negativity of three CSFs. While multiplex RT-PCR routinely took 90 min, RTM took 120 min, although the pipeline analysis detected the pathogen genome after 20 min of sequencing in 33 CSF samples; and after two hours in 14 additional CSFs; yielding > 50% genome coverage in 19 CSFs. RTM identified 14 pathogen genotypes, including a majority of H. influenzae b, N. meningitidis B and S. pneumoniae 11A and 3A. In all 16 susceptible cultured bacteria, the in-silico antibiogram agreed with the in-vitro antibiogram in 10 cases, available within 48 h in routine bacteriology. INTERPRETATION: In addition to pathogen detection, RTM applied to CSF samples offered supplementary information on bacterial profiling and genotyping. These data provide the proof-of-concept that RTM could be implemented in a POC laboratory for one-shot diagnostic and genomic surveillance of pathogens responsible for life-threatening meningitis. FUNDING: This work was supported by the French Government under the Investments in the Future programme managed by the National Agency for Research reference: Méditerranée Infection 10-IAHU-03.

Rapid identification of clinically interesting methanogens using an improved MALDI-TOF-MS assay.

Methanogens, the archaea uniquely detoxifying fermentative hydrogen into methane in the digestive tract, are increasingly being detected in pathology situations, rendering their rapid identification mandatory. We improved the experimental protocol to identify broth-cultured methanogens by matrix-assisted laser desorption time-of-flight MS (MALDI-TOF-MS). A database incorporating 34 reference spectra derived from 16 methanogen reference strains representative of eight species supported further identification of 21 Methanobrevibacter smithii and 14 Methanobrevibacter oralis isolates broth-cultured from human stool and oral fluid, respectively, with scores >2. In addition, MALDI-TOF-MS differentiated five Methanobrevibacter smithii genotypes incorporated in the study. The data reported here found MALDI-TOF-MS as a first-line identification method for methanogens recovered from microbiota and clinical samples.

Influence of the healthcare pathway on the outcome of patients with infective endocarditis.

AIMS: To determine the prognosis of patients treated for infective endocarditis (IE) according to their healthcare pathway. To assess how the ESC guidelines are implemented concerning the performance of transoesophageal echocardiography, the use of antibiotic therapy, and the performance of valve surgery; and to compare the epidemiological profile of IE according to the type of centres in which the patients are hospitalized. METHODS AND RESULTS: In a prospective multicentric study including 22 hospitals in the South-East of France, 342 patients were classified into three groups according to their healthcare pathway: 119 patients diagnosed and taken care entirely in a reference centre or hospital with cardiac surgery [Referral Center (RC) group], 111 patients diagnosed and initially taken care in a non-RC (NRC), then referred in a centre including cardiac surgery [transferred to the Referral Center (TRC) group] and 112 patients totally taken care in the NRC (NRC group). One-year mortality was 26% (88 deaths) and was not significantly different between Groups 1 and 2 (20 vs. 21%, P = 0.83). Patients in the NRC group had a higher mortality (37%) compared with patients in the RC and TRC groups (P < 0.001). ESC guidelines were not implemented similarly depending on the healthcare pathway (P = 0.04). Patients in the NRC group were significantly older (P < 0.001) and had more comorbidities (P < 0.001) than patients treated in referral centres. CONCLUSION: Prognosis of patients with IE is influenced by their healthcare pathway. Patients treated exclusively in NRC have a worse prognosis than patients treated in referral or surgical centres.

Direct Metagenomic Diagnosis of Community-Acquired Meningitis: State of the Art.

Current routine diagnosis of community-acquired meningitis (CAM) by multiplex real-time polymerase chain reaction (RT-PCR) is limited in the number of tested pathogens and their full characterisation, requiring additional in vitro investigations to disclose genotype and antimicrobial susceptibility. We reviewed 51 studies published through December 2021 reporting metagenomic next generation sequencing (mNGS) directly applied to the cerebrospinal fluid (CSF). This approach, potentially circumventing the above-mentioned limitations, indicated 1,248 investigated patients, and 617 patients dually investigated by routine diagnosis and mNGS, in whom 116 microbes were detected, including 50 by mNGS only, nine by routine methods only, and 57 by both routine methods and mNGS. Of 217 discordant CSF findings, 103 CSF samples were documented by mNGS only, 87 CSF samples by routine methods only, and 27 CSF samples in which the pathogen identified by mNGS was different than that found using routine methods. Overall, mNGS allowed for diagnosis and genomic surveillance of CAM causative pathogens in real-time, with a cost which is competitive with current routine multiplex RT-PCR. mNGS could be implemented at point-of-care (POC) laboratories as a part of routine investigations to improve the diagnosis and molecular epidemiology of CAM, particularly in the event of failure of routine assays.