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IntroductionCandidate Phyla Radiation (CPR) and more specifically Candidatus Saccharibacteria (TM7) have now been established as ubiquitous members of the human oral microbiota. Additionally, CPR have been reported in the gastrointestinal and urogenital tracts. However, the exploration of new human niches has been limited to date.MethodsIn this study, we performed a prospective and retrospective screening of TM7 in human samples using standard PCR, real-time PCR, scanning electron microscopy (SEM) and shotgun metagenomics.ResultsUsing Real-time PCR and standard PCR, oral samples presented the highest TM7 prevalence followed by fecal samples, breast milk samples, vaginal samples and urine samples. Surprisingly, TM7 were also detected in infectious samples, namely cardiac valves and blood cultures at a low prevalence (under 3%). Moreover, we observed CPR-like structures using SEM in all sample types except cardiac valves. The reconstruction of TM7 genomes in oral and fecal samples from shotgun metagenomics reads further confirmed their high prevalence in some samples.ConclusionThis study confirmed, through their detection in multiple human samples, that TM7 are human commensals that can also be found in clinical settings. Their detection in clinical samples warrants further studies to explore their role in a pathological setting.

While populations at risk for severe SARS-CoV-2 infections have been clearly identified, susceptibility to the infection and its clinical course remain unpredictable. As the nasopharyngeal microbiota may promote the acquisition of several respiratory infections and have an impact on the evolution of their outcome, we studied the nasopharyngeal microbiota of COVID-19 patients in association with baseline disease-related clinical features compared to that of patients tested negative. We retrospectively analyzed 120 nasopharyngeal pseudonymized samples, obtained for diagnosis, divided into groups (infected patients with a favorable outcome, asymptomatic, and deceased patients) and patients tested negative for SARS-CoV-2, by using Illumina-16S ribosomal ribonucleic acid (rRNA) sequencing and specific polymerase chain reaction (PCR) targeting pathogens. We first found a depletion of anaerobes among COVID-19 patients, irrespective of the clinical presentation of the infection (p < 0.029). We detected 9 taxa discriminating patients tested positive for SARS-CoV-2 from those that were negative including Corynebacterium propinquum/pseudodiphtericum (p ≤ 0.05), Moraxella catarrhalis (p ≤ 0.05), Bacillus massiliamazoniensis (p ≤ 0.01), Anaerobacillus alkalidiazotrophicus (p ≤ 0.05), Staphylococcus capitis subsp. capitis (p ≤ 0.001), and Afipia birgiae (p ≤ 0.001) with 16S rRNA sequencing, and Streptococcus pneumoniae (p ≤ 0.01), Klebsiella pneumoniae (p ≤ 0.01), and Enterococcus faecalis (p ≤ 0.05) using real-time PCR. By designing a specific real-time PCR, we also demonstrated that C. propinquum is decreased in asymptomatic individuals compared to other SARS-CoV 2 positive patients. These findings indicate that the nasopharyngeal microbiota as in any respiratory infection plays a role in the clinical course of the disease. Further studies are needed to elucidate the potential role in the clinical course of the disease of M. catarrhalis, Corynebacterium accolens, and more specifically Corynebacterium propinquum/diphteriticum in order to include them as predictors of the severity of COVID-19.

After the end of the first epidemic episode of SARS-CoV-2 infections, as cases began to rise again during the summer of 2020, we at IHU Méditerranée Infection in Marseille, France, intensified the genomic surveillance of SARS-CoV-2, and described the first viral variants. In this study, we compared the incidence curves of SARS-CoV-2-associated deaths in different countries and reported the classification of SARS-CoV-2 variants detected in our institute, as well as the kinetics and sources of the infections. We used mortality collected from a COVID-19 data repository for 221 countries. Viral variants were defined based on ≥5 hallmark mutations along the whole genome shared by ≥30 genomes. SARS-CoV-2 genotype was determined for 24,181 patients using next-generation genome and gene sequencing (in 47 and 11% of cases, respectively) or variant-specific qPCR (in 42% of cases). Sixteen variants were identified by analyzing viral genomes from 9,788 SARS-CoV-2-diagnosed patients. Our data show that since the first SARS-CoV-2 epidemic episode in Marseille, importation through travel from abroad was documented for seven of the new variants. In addition, for the B.1.160 variant of Pangolin classification (a.k.a. Marseille-4), we suspect transmission from farm minks. In conclusion, we observed that the successive epidemic peaks of SARS-CoV-2 infections are not linked to rebounds of viral genotypes that are already present but to newly introduced variants. We thus suggest that border control is the best mean of combating this type of introduction, and that intensive control of mink farms is also necessary to prevent the emergence of new variants generated in this animal reservoir.

Smell and taste disorders are frequent symptoms during acute COVID-19 and may persist long after the resolution of the initial phase. This study aims to estimate the proportion and risk factors for smell and/or taste disorders at the onset of symptoms and their persistence after more than 6 months of follow-up in COVID-19 patients. We analyzed a prospective cohort of COVID-19 patients admitted to our institute in Marseille, France in early 2020. After being discharged from the hospital, patients with smell and/or taste disorders were contacted for a telephone interview. Logistic regression analysis was performed to determine the risk factors for smell and/or taste disorders. A total of 3,737 patients were included, of whom 1,676 reported smell and/or taste disorders at the onset of symptoms. Taste and/or smell disorders were independently associated with being younger and female, a lower likelihood of suffering from diabetes, cardiovascular diseases and cancer, a longer delay between the onset of symptoms and consultation, and non-severe forms of COVID-19 at admission. Of the 605 patients with smell and/or taste disorders who were followed-up, 154 (25.5%) reported the persistence of symptoms for more than 6 months. At the time of follow-up, being female, having a chronic respiratory disease and using angiotensin-converting enzyme inhibitors (ACEis) were factors independently associated with the persistence of smell and/or taste disorders. In conclusion, the long-term persistence of olfactory and gustative disorders is frequent among COVID-19 patients, notably affecting female patients and patients who suffered from chronic respiratory diseases before infection. The role of ACEis needs to be further evaluated in larger numbers of patients.

From January 18th to August 13th, 2021, 13,804 unvaccinated and 1,156 patients who had received at least one COVID-19 vaccine dose were tested qPCR-positive for SARS-CoV-2 in our center. Among vaccinated patients, 949, 205 and 2 had received a single, two or three vaccine doses, respectively. Most patients (80.3%) had received the Pfizer-BioNTech vaccine. The SARS-CoV-2 variants infecting vaccinated patients varied over time, reflecting those circulating in the Marseille area, with a predominance of the Marseille-4/20A.EU2 variant from weeks 3 to 6, of the Alpha/20I variant from weeks 7 to 25, and of the Delta/21A variant from week 26. SARS-CoV-2 infection was significantly more likely to occur in the first 13 days post-vaccine injection in those who received a single dose (48.9%) than two doses (27.4%, p< 10–3). Among 161 patients considered as fully vaccinated, i.e., >14 days after the completion of the vaccinal scheme (one dose for Johnson and Johnson and two doses for Pfizer/BioNTech, Moderna and Sputnik vaccines), 10 (6.2%) required hospitalization and four (2.5%) died. Risks of complications increased with age in a nonlinear pattern, with a first breakpoint at 54, 33, and 53 years for death, transfer to ICU, and hospitalization, respectively. Among patients infected by the Delta/21A or Alpha/20I variants, partial or complete vaccination exhibited a protective effect with a risk divided by 3.1 for mortality in patients ≥ 55 years, by 2.8 for ICU transfer in patients ≥ 34 years, and by 1.8 for hospitalization in patients ≥ 54 years. Compared to partial vaccination, complete vaccination provided an even stronger protective effect, confirming effectiveness to prevent severe forms of COVID-19.

The etiological agent of COVID-19 SARS-CoV-2, is primarily a pulmonary-tropic coronavirus. Infection of alveolar pneumocytes by SARS-CoV-2 requires virus binding to the angiotensin I converting enzyme 2 (ACE2) monocarboxypeptidase. ACE2, present on the surface of many cell types, is known to be a regulator of blood pressure homeostasis through its ability to catalyze the proteolysis of Angiotensin II (Ang II) into Angiotensin-(1-7) [Ang-(1-7)]. We therefore hypothesized that SARS-CoV-2 could trigger variations of ACE2 expression and Ang II plasma concentration in SARS-CoV-2-infected patients. We report here, that circulating blood cells from COVID-19 patients express less ACE2 mRNA than cells from healthy volunteers. At the level of circulating cells, this ACE2 gene dysregulation mainly affects the monocytes, which also show a lower expression of membrane ACE2 protein. Moreover, soluble ACE2 (sACE2) plasma concentrations are lower in prolonged viral shedders than in healthy controls, while the concentration of sACE2 returns to normal levels in short viral shedders. In the plasma of prolonged viral shedders, we also found higher concentrations of Ang II and angiotensin I (Ang I). On the other hand, the plasma levels of Ang-(1-7) remains almost stable in prolonged viral shedders but seems insufficient to prevent the adverse effects of Ang II accumulation. Altogether, these data evidence that the SARS-CoV-2 may affect the expression of blood pressure regulators with possible harmful consequences on COVID-19 outcome.

Although SARS-CoV-2 is considered a lung-tropic virus that infects the respiratory tract through binding to the ACE2 cell-surface molecules present on alveolar lungs epithelial cells, gastrointestinal symptoms have been frequently reported in COVID-19 patients. What can be considered an apparent paradox is that these symptoms (e.g., diarrhea), sometimes precede the development of respiratory tract illness as if the breathing apparatus was not its first target during viral dissemination. Recently, evidence was reported that the gut is an active site of replication for SARS-CoV-2. This replication mainly occurs in mature enterocytes expressing the ACE2 viral receptor and TMPRSS4 protease. In this review we question how SARS-CoV-2 can cause intestinal disturbances, whether there are pneumocyte-tropic, enterocyte-tropic and/or dual tropic strains of SARS-CoV-2. We examine two major models: first, that of a virus directly causing damage locally (e.g., by inducing apoptosis of infected enterocytes); secondly, that of indirect effect of the virus (e.g., by inducing changes in the composition of the gut microbiota followed by the induction of an inflammatory process), and suggest that both situations probably occur simultaneously in COVID-19 patients. We eventually discuss the consequences of the virus replication in brush border of intestine on long-distance damages affecting other tissues/organs, particularly lungs.

Culturomics, a high throughput culture method with rapid identification of the colonies by Matrix Assisted Laser Desorption Ionization/Time Of Flight Mass Spectrometry (MALDI-TOF MS), has demonstrated its contribution to the exploration of the gut microbiota over the past 10 years. However, the cost, work time and workload, considerably limit its use on a large scale or emergency context. Here, by testing two different stool samples, including a stool sample from a patient requiring rapid immunotherapy treatment, we tested a new fast culturomic protocol using two pre-incubation media, blood culture bottle and YCFA modified medium. Both media were supplemented with 2 ml of rumen fluid filtered at 0.2 μm and 2 ml of defibrinated and sterile sheep blood. Unlike the standard culturomics, subculturing of blood culture bottle were performed at reduced incubation time (3 h, 6 h, 9 h, 24 h) and at a longer incubation time (3 days, 7 days, and 10 days) at 37°C. By testing 5,200 colonies per MALDI-TOF MS and obtaining a comparable number of cultured bacterial species (131 to 143) in a stool sample, this new protocol reduced the number of colonies tested by 57%, working time by 78.6% and cost by 72.2%. In addition, we highlighted that the proportion of strict anaerobic species has increased by 24%, known to be the preferential targets for biotherapy, including Faecalibacterium prausnitzii, Akkermansia muciniphila, Christensenella minuta, and Phascolarctobacterium faecium. Finally, this work showed that some bacterial species grew earlier but disappeared with prolonged incubation times.

Human urine was considered sterile for a long time. However, 416 species have been previously cultured, including only 40 anaerobic species. Here, we used culturomics, particularly those targeting anaerobes, to better understand the urinary microbiota. By testing 435 urine samples, we isolated 450 different bacterial species, including 256 never described in urine of which 18 were new species. Among the bacterial species identified, 161 were anaerobes (35%). This study increased the known urine repertoire by 39%. Among the 672 bacterial species isolated now at least once from urine microbiota, 431 (64.1%) were previously isolated from gut microbiota, while only 213 (31.7%) were previously isolated from vagina. These results suggest that many members of the microbiota in the urinary tract are in fact derived from the gut, and a paradigm shift is thus needed in our understanding.

The human gut microbiota plays an important role in human health. Accessing and culturing the gut microbes remains critical in microbiology. The culturomics approach, combined with sequencing strategies, provides new insights in the study of gut microbiota components. However, we are still far from having described all the microbes. Many factors are involved in recovering as much bacteria as possible. One of the most important factors is sample preparation and conservation. The aim of the present work was to evaluate three different types of stool sample conditioning by mainly studying the effect of atmospheric oxygen on bacterial viability and culturability. Quantitative analysis of fecal samples from eight different healthy individuals was carried out using a culture-independent method (flow cytometry) and a culture-dependent method (plating technique). We found that the cultured bacteria reached a 50% yield when the samples were exposed to oxygen for 120 min without any protectant medium, while the percentage of culturability increased to 67% in the presence of antioxidants. More importantly, when samples were exposed to oxygen for less than 2 min, combined with the work under the anaerobic chamber, no discordance was found between the two counting techniques and the culturability increased to 87%. Our study confirmed the importance of sample conditioning to preserve the bacterial viability in samples, especially for oxygen-sensitive intestinal bacteria.

Whipple’s disease (WD) is a rare chronic systemic infection with a wide range of clinical symptoms, routinely diagnosed in biopsies from the small intestine and other tissues by periodic acid–Schiff (PAS) diastase staining and immunohistological analysis with specific antibodies. The aim of our study was to improve the pathological diagnosis of WD. Therefore, we analyzed the potential of fluorescence in situ hybridization (FISH) for diagnosing WD, using a Tropheryma (T.) whipplei-specific probe. 19 formalin-fixed paraffin-embedded (FFPE) duodenal biopsy specimens of 12 patients with treated (6/12) and untreated (6/12) WD were retrospectively examined using PAS diastase staining, immunohistochemistry, and FISH. 20 biopsy specimens with normal intestinal mucosa, Helicobacter pylori, or mycobacterial infection, respectively, served as controls. We successfully detected T. whipplei in tissue biopsies with a sensitivity of 83% in untreated (5/6) and 40% in treated (4/10) cases of WD. In our study, we show that FISH-based diagnosis of individual vital T. whipplei in FFPE specimens is feasible and can be considered as ancillary diagnostic tool for the diagnosis of WD in FFPE material. We show that FISH not only detect active WD but also be helpful as an indicator for the efficiency of antibiotic treatment and for detection of recurrence of disease when the signal of PAS diastase and immunohistochemistry lags behind the recurrence of disease, especially if the clinical course of the patient and antimicrobial treatment is considered.

Severe acute malnutrition is the world-leading cause of children under-five's death. Recent metagenomics studies have established a link between gut microbiota and severe acute malnutrition, describing an immaturity with a striking depletion in oxygen-sensitive prokaryotes. Amoxicillin and therapeutic diet cure most of the children with severe acute malnutrition but an irreversible disruption of the gut microbiota is suspected in the refractory and most severe cases. In these cases, therapeutic diet may be unable to reverse the microbiota alteration leading to persistent impaired development or death. In addition, as enteric sepsis is a major cause of death in this context, identification of missing gut microbes to be tested as probiotics (live bacteria that confer a benefit to the host) to restore rapidly the healthy gut microbiota and prevent the gut pathogenic invasion is of foremost importance. In this study, stool samples of malnourished patients with kwashiorkor and healthy children were collected from Niger and Senegal and analyzed by culturomics and metagenomics. We found a globally decreased diversity, a decrease in the hitherto unknown diversity (new species isolation), a depletion in oxygen-sensitive prokaryotes including Methanobrevibacter smithii and an enrichment in potentially pathogenic Proteobacteria, Fusobacteria and Streptococcus gallolyticus. A complex of 12 species identified only in healthy children using culturomics and metagenomics were identified as probiotics candidates, providing a possible, defined, reproducible, safe, and convenient alternative to fecal transplantation to restore a healthy gut microbiota in malnourished children. Microbiotherapy based on selected strains has the potential to improve the current treatment of severe acute malnutrition and prevent relapse and death by reestablishing a healthy gut microbiota.