Manon Boxberger

On compte deux stratégies principales permettant la culture de microorganismes « difficiles à cultiver », la première repose sur la supplémentation, la personnalisation ou la modification des milieux d'isolement et de culture, et la seconde sur le mimétisme des conditions de croissance en milieu naturel. Fondé sur le principe de cette dernière approche, nous avons développé et utilisé dans cette étude un nouveau milieu sélectif nommé MB2L afin d’isoler et de sélectionner des bactéries cutanées dont la croissance est masquée par les principales espèces cutanées (telles que Staphylococcus). Cette étude à permis de montrer une différence de la composition du microbiote entre les deux groupes d'âge et également d’isoler 20 nouvelles espèces bactériennes jamais décrites auparavant. L'analyse du portage par qPCR de ces nouvelles espèces bactériennes a été reliasse sur de larges populations. Cette analyse a permis de déterminer si ces bactéries étaient commensales ou transitoires. Sur nos 20 nouvelles espèces bactériennes, sept ont été déterminées comme commensales, donc potentiellement d’intérêt particulier pour des investigations plus poussées sur leurs rôles et leur interaction avec la peau, via la coculture utilisant des modèles cutanés 3D. Ces organismes sont le point de départ des investigations futures qui visent à mettre en évidence leur potentiel rôle bénéfique pour la peau et ainsi les utiliser comme probiotiques et postbiotiques. Aussi, nous avons recherché de nouveaux composés bioactifs sécrétés par ces bactéries Ces nouveaux organismes pourraient également être le substrat d’identification de composés prébiotiques.

Extending our knowledge on human skin microbiota is a challenge to better decipher its role in health and disease. Using the culturomics method, we isolated strain Marseille-Q4368 from the healthy forehead of a 59-year-old woman. We describe here the main characteristics of this bacterium using a taxonogenomic approach. This new bacterial species is Gram-positive, non-motile, and non-spore-forming. Its 16S rRNA sequence exhibited a similarity of 99.59% with Leucobacter chromiiresistens, the most closely related species in terms of nomenclature. However, a digital DNA–DNA hybridization analysis between these two species revealed a maximum identity similarity of only 27.5%. We found phenotypical and genomic differences between strain Marseille-Q4368 and its closely related species. These findings underscore the classification of this bacterium as a distinct species. Hence, we propose the name Leucobacter manosquensis sp. nov. strain Marseille-Q4368 (=CSUR Q4368 = DSM 112403) for this newly identified bacterial species.

Over the past two years, several variants of SARS-CoV-2 have emerged and spread all over the world. However, infectivity, clinical severity, re-infection, virulence, transmissibility, vaccine responses and escape, and epidemiological aspects have differed between SARS-CoV-2 variants. Currently, very few treatments are recommended against SARS-CoV-2. Identification of effective drugs among repurposing FDA-approved drugs is a rapid, efficient and low-cost strategy against SARS-CoV-2. One of those drugs is ivermectin. Ivermectin is an antihelminthic agent that previously showed in vitro effects against a SARS-CoV-2 isolate (Australia/VI01/2020 isolate) with an IC₅₀ of around 2 µM. We evaluated the in vitro activity of ivermectin on Vero E6 cells infected with 30 clinically isolated SARS-CoV-2 strains belonging to 14 different variants, and particularly 17 strains belonging to six variants of concern (VOC) (variants related to Wuhan, alpha, beta, gamma, delta and omicron). The in vitro activity of ivermectin was compared to those of chloroquine and remdesivir. Unlike chloroquine (EC₅₀ from 4.3 ± 2.5 to 29.3 ± 5.2 µM) or remdesivir (EC₅₀ from 0.4 ± 0.3 to 25.2 ± 9.4 µM), ivermectin showed a relatively homogeneous in vitro activity against SARS-CoV-2 regardless of the strains or variants (EC₅₀ from 5.1 ± 0.5 to 6.7 ± 0.4 µM), except for one omicron strain (EC₅₀ = 1.3 ± 0.5 µM). Ivermectin (No. EC₅₀ = 219, mean EC₅₀ = 5.7 ± 1.0 µM) was, overall, more potent in vitro than chloroquine (No. EC₅₀ = 214, mean EC₅₀ = 16.1 ± 9.0 µM) (p = 1.3 × 10⁻³⁴) and remdesivir (No. EC₅₀ = 201, mean EC₅₀ = 11.9 ± 10.0 µM) (p = 1.6 × 10⁻¹³). These results should be interpreted with caution regarding the potential use of ivermectin in SARS-CoV-2-infected patients: it is difficult to translate in vitro study results into actual clinical treatment in patients.

Necrotizing enterocolitis is a life-threatening acquired gastrointestinal disorder among preterm neonates and is associated with a high mortality rate and long-term neurodevelopmental morbidity. No etiologic agent has been definitively established; nonetheless, the most implicated bacteria include members of the Clostridium genus. We reported here on a case of Clostridium neonatale bacteremia in a preterm neonate with necrotizing enterocolitis, providing more information regarding the potential role of this bacterium in pathogenesis of necrotizing enterocolitis. We emphasized the sporulating form of C. neonatale that confers resistance to disinfectants usually applied for the hospital environmental cleaning. Further works are needed to establish the causal relationship between the occurrence of NEC and the isolation of C. neonatale, with promising perspectives in terms of diagnostic and therapeutic management.

Marseille-Q4369 is a strain that we isolated from human healthy skin and characterized by taxono-genomic approach. Marseille-Q4369 exhibited 99.80% 16S rRNA sequence similarity with Agrococcus pavilionensisT the phylogenetically closest bacterium with standing in nomenclature. Furthermore, digital DNA–DNA hybridization revealed a maximum identity similarity of only 52.4% and an OrthoANI parameter provided a value of 93.63% between the novel organism and Agrococcus pavilionensisT. Marseille-Q4369 was observed to be a yellowish-pigmented, Gram-positive, coccoïd, facultative aerobic bacterium, and belonging to the Microbacteriaceae family. The major fatty acids detected are 12-methyl-tetradecanoic acid (66%), 14-methyl-hexadecanoic acid (24%) followed by 13-methyl-tetradecanoic acid (5%). The genome size of strain Marseille-Q4369 was 2,737,735-bp long with a 72,27 % G+C content. Taken altogether, these results confirm the status of this strain as a new member of the Agrococcus genus for which the name of Agrococcus massiliensis is proposed (=CSUR-Q4369 = DSM112404).

A Gram-negative bacterium, designated strain Marseille-Q3452^T, was isolated from subgingival dental plaque of a subject suffering from dental plaque biofilm-induced gingivitis on an intact periodontium in Marseille, France. The strain was characterized by 16S rRNA and atpA gene sequence analysis and by conventional phenotypic and chemotaxonomic testing. The average nucleotide identity (ANI) and core genome phylogeny were determined using whole-genome sequences. Although strain Marseille-Q3452^T showed 99.72 % 16S rRNA gene sequence similarity with Campylobacter showae strain ATCC 51146^T, atpA and ANI analyses revealed divergence between the two strains. The two species could also be distinguished phenotypically on the basis of the absence of flagella and nitrate reduction. On the basis of the results from phenotypic, chemotaxonomic, genomic and phylogenetic analyses and data, we concluded that strain Marseille-Q3452^T represents a novel species of the genus Campylobacter , for which the name Campylobacter massiliensis sp. nov. is proposed (=CSUR Q3452=CECT 30263).

A Gram-stain-negative bacterium, designated strain Marseille-Q3039^T, was isolated from subgingival dental plaque of a woman with gingivitis in Marseille, France. Strain Marseille-Q3039^T was found to be an anaerobic, motile and spore-forming crescent-shaped bacterium that grew at 25–41.5 °C (optimum, 37 °C), pH 5.5–8.5 (optimum, pH 7.5) and salinity of 5.0 g l⁻¹ NaCl. The results of 16S rRNA gene sequence analysis revealed that strain Marseille-Q3039^T was closely related to Selenomonas infelix ATCC 43532^T (98.42 % similarity), Selenomonas dianae ATCC 43527^T (97.25 %) and Centipedia periodontii DSM 2778^T (97.19 %). The orthologous average nucleotide identity and digital DNA–DNA hybridization relatedness between strain Q3039^T and its closest phylogenetic neighbours were respectively 84.57 and 28.2 % for S. infelix ATCC 43532^T and 83.93 and 27.2 % for C. periodontii DSM 2778^T. The major fatty acids were identified as C_13 : 0 (27.7 %), C_15 : 0 (24.4 %) and specific C_13 : 0 3-OH (12.3 %). Genome sequencing revealed a genome size of 2 351 779 bp and a G+C content of 57.2 mol%. On the basis of the results from phenotypic, chemotaxonomic, genomic and phylogenetic analyses and data, we concluded that strain Marseille-Q3039^T represents a novel species of the genus Selenomonas , for which the name Selenomonas timonae sp. nov. is proposed (=CSUR Q3039=CECT 30128).

A new severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing coronavirus diseases 2019 (COVID-19), which emerged in Wuhan, China in December 2019, has spread worldwide. Currently, very few treatments are officially recommended against SARS-CoV-2. Identifying effective, low-cost antiviral drugs with limited side effects that are affordable immediately is urgently needed. Methylene blue, a synthesized thiazine dye, may be a potential antiviral drug. Antiviral activity of methylene blue used alone or in combination with several antimalarial drugs or remdesivir was assessed against infected Vero E6 cells infected with two clinically isolated SARS-CoV-2 strains (IHUMI-3 and IHUMI-6). Effects both on viral entry in the cell and on post-entry were also investigated. After 48 h post-infection, the viral replication was estimated by RT-PCR. The median effective concentration (EC₅₀) and 90% effective concentration (EC₉₀) of methylene blue against IHUMI-3 were 0.41 ± 0.34 µM and 1.85 ± 1.41 µM, respectively; 1.06 ± 0.46 µM and 5.68 ± 1.83 µM against IHUMI-6. Methylene blue interacted at both entry and post-entry stages of SARS-CoV-2 infection in Vero E6 cells as retrieved for hydroxychloroquine. The effects of methylene blue were additive with those of quinine, mefloquine and pyronaridine. The combinations of methylene blue with chloroquine, hydroxychloroquine, desethylamodiaquine, piperaquine, lumefantrine, ferroquine, dihydroartemisinin and remdesivir were antagonist. These results support the potential interest of methylene blue to treat COVID-19.

Capnocytophaga species are commensal gliding bacteria that are found in human and animal oral microbiota and are involved in several inflammatory diseases, both in immunocompromised and immunocompetent subjects. This study contributes to increased knowledge of this genus by characterizing a novel species isolated from a dental plaque sample in a male with gingivitis. We investigated morphological and chemotaxonomic characteristics using different growth conditions, temperature, and pH. Cellular fatty acid methyl ester (FAME) analysis was employed with gas chromatography/mass spectrometry (GC/MS). Phylogenetic analysis based on 16S rRNA, orthologous average nucleotide identity (OrthoANI), and digital DNA–DNA hybridization (dDDH) relatedness were performed. The Marseille-Q4570^T strain was found to be a facultative aerobic, Gram-negative, elongated, round-tipped bacterium that grew at 25–56 °C and tolerated a pH of 5.5 to 8.5 and an NaCl content ranging from 5 to 15 g/L. The most abundant fatty acid was the branched structure 13-methyl-tetradecanoic acid (76%), followed by hexadecanoic acid (6%) and 3-hydroxy-15-methyl-hexadecanoic acid (4%). A 16S rDNA-based similarity analysis showed that the Marseille-Q4570^T strain was closely related to Capnocytophaga leadbetteri strain AHN8855^T (97.24% sequence identity). The OrthoANI and dDDH values between these two strains were, respectively, 76.81% and 25.6%. Therefore, we conclude that the Marseille-Q4570^T strain represents a novel species of the genus Capnocytophaga, for which the name Capnocytophaga bilenii sp. nov. is proposed (=CSUR Q4570).

The genus Catonella currently counts a unique species, C. morbi, isolated from periodontal pockets and associated with periodontitis and endodontic infections. This study contributed to the taxonomical and clinical knowledge of this genus by describing a novel species isolated from a saliva sample from a man in clinical gingival health following successful treatment of periodontitis. Morphological and chemotaxonomic characteristics were investigated using different growth conditions, pH, and temperature. Cellular fatty acid methyl ester (FAME) analysis was conducted by gas chromatography/mass spectrometry (GC/MS). Phylogenetic analysis based on 16S rRNA, orthologous average nucleotide identity (OrthoANI), and digital DNA-DNA hybridization (dDDH) relatedness were performed. Strain Marseille-Q4567^T was found to be an anaerobic and non-spore-forming rod-shaped bacterium that grew at 28–41.5 °C (optimum 37 °C), pH 6.5–8.5 (optimum pH 7.5), and 5–10 g/L of NaCl (optimum 5 g/L). The predominant cellular fatty acid was C16:0 (64.2%), followed by unsaturated structures C18:1n9 (12.5%) and C18:2n6 (7.8%). Based on 16S rRNA sequence comparison, the closest phylogenetic neighbor was C. morbi ATCC 51271^T (98.23% similarity). The OrthoANI and dDDH values between strain Q4567^T and C. morbi ATCC 51271^T were respectively 79.43% and 23.8%. Therefore, we concluded that strain Marseille-Q4567^T represents a novel species of the genus Catonella, for which the name Catonella massiliensis sp. nov. is proposed (= CSUR Q4567).