Leucobacter manosquensis sp. nov.-A Novel Bacterial Species Isolated from Healthy Human Skin.

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.

Antiviral Activity of Repurposing Ivermectin against a Panel of 30 Clinical SARS-CoV-2 Strains Belonging to 14 Variants.

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.

Brachybacterium epidermidis Sp. Nov., a Novel Bacterial Species Isolated from the Back of the Right Hand, in a 67-Year-Old Healthy Woman.

Knowledge on human skin microbiota composition has been expanding in recent years. Its role in human health and disease represents an active area of investigation. As part of our culturomics project that consists of exploring the human microbiota by isolating bacteria through innovative culture-dependent methods, we isolated a new bacterial strain from the back of the right hand, in a 67-year-old healthy woman. Here, we characterize the strain Marseille-Q2903 by the taxonogenomic approach. Marseille-Q2903 exhibits a 99.5% 16S rRNA sequence similarity with Brachybacterium muris T but with only 92% of coverage. The closest species based on a 100% coverage of the 16S sequence is Brachybacterium timonense T with an identity similarity of 97.63%. Furthermore, digital DNA-DNA hybridization reveals a maximum identity similarity of only 31.5% and an OrthoANI parameter provided a value of 86.95% between Marseille-Q2903 and Brachybacterium muris T. Marseille-Q2903 is a yellowish-pigmented, Gram-positive, coccoid shaped, and facultative aerobic bacterium, and belonging to the Dermabacteraceae family. The major fatty acids detected are 12-methyl-tetradecanoic acid (69%), 14-methyl-hexadecanoic acid (16%), and 14-methyl-pentadecanoic acid (7%). Marseille-Q2903 genome size is of 3,073,790 bp, with a 70.43% G + C content. Taken altogether, these results confirm the status of this strain as a new member of the Brachybacterium genus for which the name of Brachybacterium epidermidis sp. strain Marseille-Q2903T is proposed (=CSURQ2903T = CECT30363).

Case Report: Clostridium neonatale Bacteremia in a Preterm Neonate With Necrotizing Enterocolitis.

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.

Isolation and characterization of Campylobacter massiliensis sp. nov., a novel Campylobacter species detected in a gingivitis subject.

A Gram-negative bacterium, designated strain Marseille-Q3452T, 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-Q3452T showed 99.72 % 16S rRNA gene sequence similarity with Campylobacter showae strain ATCC 51146T, 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-Q3452T represents a novel species of the genus Campylobacter, for which the name Campylobacter massiliensis sp. nov. is proposed (=CSUR Q3452=CECT 30263).

Isolation and description of Selenomonas timonae sp. nov., a novel Selenomonas species detected in a gingivitis patient.

A Gram-stain-negative bacterium, designated strain Marseille-Q3039T, was isolated from subgingival dental plaque of a woman with gingivitis in Marseille, France. Strain Marseille-Q3039T 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-Q3039T was closely related to Selenomonas infelix ATCC 43532T (98.42 % similarity), Selenomonas dianae ATCC 43527T (97.25 %) and Centipedia periodontii DSM 2778T (97.19 %). The orthologous average nucleotide identity and digital DNA-DNA hybridization relatedness between strain Q3039T and its closest phylogenetic neighbours were respectively 84.57 and 28.2 % for S. infelix ATCC 43532T and 83.93 and 27.2 % for C. periodontii DSM 2778T. The major fatty acids were identified as C13 : 0 (27.7 %), C15 : 0 (24.4 %) and specific C13 : 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-Q3039T represents a novel species of the genus Selenomonas, for which the name Selenomonas timonae sp. nov. is proposed (=CSUR Q3039=CECT 30128).

In Vitro Evaluation of the Antiviral Activity of Methylene Blue Alone or in Combination against SARS-CoV-2.

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.

Isolation and Characterization of Capnocytophaga bilenii sp. nov., a Novel Capnocytophaga Species Detected in a Gingivitis Subject.

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-Q4570T 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-Q4570T strain was closely related to Capnocytophaga leadbetteri strain AHN8855T (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-Q4570T strain represents a novel species of the genus Capnocytophaga, for which the name Capnocytophaga bilenii sp. nov. is proposed (=CSUR Q4570).

Challenges in exploring and manipulating the human skin microbiome.

The skin is the exterior interface of the human body with the environment. Despite its harsh physical landscape, the skin is colonized by diverse commensal microbes. In this review, we discuss recent insights into skin microbial populations, including their composition and role in health and disease and their modulation by intrinsic and extrinsic factors, with a focus on the pathobiological basis of skin aging. We also describe the most recent tools for investigating the skin microbiota composition and microbe-skin relationships and perspectives regarding the challenges of skin microbiome manipulation. Video abstract.

Isolation and Description of Catonella massiliensis sp. nov., a Novel Catonella Species, Isolated from a Stable Periodontitis Subject.

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-Q4567T 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 51271T (98.23% similarity). The OrthoANI and dDDH values between strain Q4567T and C. morbi ATCC 51271T were respectively 79.43% and 23.8%. Therefore, we concluded that strain Marseille-Q4567T represents a novel species of the genus Catonella, for which the name Catonella massiliensis sp. nov. is proposed (= CSUR Q4567).

Isolation and Characterization of Kingella bonacorsii sp. nov., A Novel Kingella Species Detected in a Stable Periodontitis Subject.

Members of the genus Kingella are mostly commensals of the oral cavity, but some of them are involved in invasive infections, especially in young children. This study provides new knowledge on the diversity of this genus by describing a novel species of Kingella isolated from a dental plaque sample from a 51-year-old man with a history of periodontitis. Morphological and chemotaxonomic characteristic were investigated using different growth conditions, pH and temperature. Cellular fatty acid methyl ester (FAME) analysis was performed 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 also performed. Strain Marseille-Q4569T was found to be a facultative aerobic, nonmotile and non-spore-forming rod-shaped bacterium that grows at 28-41.5 °C (optimum 37 °C), pH 5.5-8.5 (optimum pH 7.5) and 5-15 g/L of NaCl. The major fatty acids were Hexadecanoic acid (32.7%), 11-Octadecenoic acid (26.1 %) and 9-Hexadecenoic acid (21.3 %). Despite high 16S rRNA gene sequence similarity (98.72%) between strain Marseille-Q4569T and Kingella oralis strain UB-38T, the degree of OrthoANI was at the limit of the cutoff (95.83%), and the degree of dDDH was lower (63.6%) than thresholds used to delineate prokaryotic species. Therefore, it is proposed that strain Marseille-Q4569T represents a novel species of the genus Kingella, for which the name Kingella bonacorsii sp. nov. is proposed (=CSUR Q4569).

In Vitro Antiviral Activity of Doxycycline against SARS-CoV-2.

In December 2019, a new severe acute respiratory syndrome coronavirus (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19), emerged in Wuhan, China. Despite containment measures, SARS-CoV-2 spread in Asia, Southern Europe, then in America and currently in Africa. Identifying effective antiviral drugs is urgently needed. An efficient approach to drug discovery is to evaluate whether existing approved drugs can be efficient against SARS-CoV-2. Doxycycline, which is a second-generation tetracycline with broad-spectrum antimicrobial, antimalarial and anti-inflammatory activities, showed in vitro activity on Vero E6 cells infected with a clinically isolated SARS-CoV-2 strain (IHUMI-3) with median effective concentration (EC50) of 4.5 ± 2.9 µM, compatible with oral uptake and intravenous administrations. Doxycycline interacted both on SARS-CoV-2 entry and in replication after virus entry. Besides its in vitro antiviral activity against SARS-CoV-2, doxycycline has anti-inflammatory effects by decreasing the expression of various pro-inflammatory cytokines and could prevent co-infections and superinfections due to broad-spectrum antimicrobial activity. Therefore, doxycycline could be a potential partner of COVID-19 therapies. However, these results must be taken with caution regarding the potential use in SARS-CoV-2-infected patients: it is difficult to translate in vitro study results to actual clinical treatment in patients. In vivo evaluation in animal experimental models is required to confirm the antiviral effects of doxycycline on SARS-CoV-2 and more trials of high-risk patients with moderate to severe COVID-19 infections must be initiated.

Methylene blue inhibits replication of SARS-CoV-2 in vitro.

In December 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China. Currently there is no antiviral treatment recommended against SARS-CoV-2. Identifying effective antiviral drugs is urgently required. Methylene blue has already demonstrated in vitro antiviral activity in photodynamic therapy as well as antibacterial, antifungal and antiparasitic activities in non-photodynamic assays. In this study. non-photoactivated methylene blue showed in vitro activity at very low micromolar range with an EC50 (median effective concentration) of 0.30 ± 0.03 µM and an EC90 (90% effective concentration) of 0.75 ± 0.21 µM at a multiplicity of infection (MOI) of 0.25 against SARS-CoV-2 (strain IHUMI-3). The EC50 and EC90 values for methylene blue are lower than those obtained for hydroxychloroquine (1.5 µM and 3.0 µM) and azithromycin (20.1 µM and 41.9 µM). The ratios Cmax/EC50 and Cmax/EC90 in blood for methylene blue were estimated at 10.1 and 4.0, respectively, following oral administration and 33.3 and 13.3 following intravenous administration. Methylene blue EC50 and EC90 values are consistent with concentrations observed in human blood. We propose that methylene blue is a promising drug for treatment of COVID-19. In vivo evaluation in animal experimental models is now required to confirm its antiviral effects on SARS-CoV-2. The potential interest of methylene blue to treat COVID-19 needs to be confirmed by prospective comparative clinical studies.

Draft Genome Sequence of Streptomyces mexicanus Strain Q0842, Isolated from Human Skin.

In 2003, Streptomyces mexicanus was reported as a novel xylanolytic bacterial species isolated from soil; a partial genome sequence was determined. In 2019, a strain from the same species was isolated from a hand skin swab sample from a healthy French woman. Genome sequencing revealed an 8,011,832-bp sequence with a GC content of 72.5%.

Antimalarial drugs inhibit the replication of SARS-CoV-2: An in vitro evaluation.

In December 2019, a new severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China. African countries see slower dynamic of COVID-19 cases and deaths. One of the assumptions that may explain this later emergence in Africa, and more particularly in malaria endemic areas, would be the use of antimalarial drugs. We investigated the in vitro antiviral activity against SARS-CoV-2 of several antimalarial drugs. Chloroquine (EC50 = 2.1 µM and EC90 = 3.8 µM), hydroxychloroquine (EC50 = 1.5 µM and EC90 = 3.0 µM), ferroquine (EC50 = 1.5 µM and EC90 = 2.4 µM), desethylamodiaquine (EC50 = 0.52 µM and EC90 = 1.9 µM), mefloquine (EC50 = 1.8 µM and EC90 = 8.1 µM), pyronaridine (EC50 = 0.72 µM and EC90 = 0.75 µM) and quinine (EC50 = 10.7 µM and EC90 = 38.8 µM) showed in vitro antiviral effective activity with IC50 and IC90 compatible with drug oral uptake at doses commonly administered in malaria treatment. The ratio Clung/EC90 ranged from 5 to 59. Lumefantrine, piperaquine and dihydroartemisinin had IC50 and IC90 too high to be compatible with expected plasma concentrations (ratio Cmax/EC90 < 0.05). Based on our results, we would expect that countries which commonly use artesunate-amodiaquine or artesunate-mefloquine report fewer cases and deaths than those using artemether-lumefantrine or dihydroartemisinin-piperaquine. It could be necessary now to compare the antimalarial use and the dynamics of COVID-19 country by country to confirm this hypothesis.

Antimalarial artemisinin-based combination therapies (ACT) and COVID-19 in Africa: In vitro inhibition of SARS-CoV-2 replication by mefloquine-artesunate.

OBJECTIVES: At the end of November 2019, a novel coronavirus responsible for respiratory tract infections (COVID-19) emerged in China. Despite drastic containment measures, this virus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread in Asia and Europe. The pandemic is ongoing with a particular hotspot in Southern Europe and America; many studies predicted a similar epidemic in Africa, as is currently seen in Europe and the United States of America. However, reported data have not confirmed these predictions. One of the hypotheses that could explain the later emergence and spread of COVID-19 pandemic in African countries is the use of antimalarial drugs to treat malaria, and specifically, artemisinin-based combination therapy (ACT). METHODS: The antiviral activity of fixed concentrations of ACT at concentrations consistent with those observed in human plasma when ACT is administered at oral doses for uncomplicated malaria treatment was evaluatedin vitro against a clinically isolated SARS-CoV-2 strain (IHUMI-3) in Vero E6 cells. RESULTS: Mefloquine-artesunate exerted the highest antiviral activity with % inhibition of 72.1 ± 18.3 % at expected maximum blood concentration (Cmax) for each ACT drug at doses commonly administered in malaria treatment. All the other combinations, artesunate-amodiaquine, artemether-lumefantrine, artesunate-pyronaridine, or dihydroartemisinin-piperaquine, showed antiviral inhibition in the same ranges (27.1 to 34.1 %). CONCLUSIONS: Antimalarial drugs for which concentration data in the lungs are available are concentrated from 10 to 160 fold more in the lungs than in blood. Thesein vitro results reinforce the hypothesis that antimalarial drugs could be effective as an anti-COVID-19 treatment.

In vitro testing of combined hydroxychloroquine and azithromycin on SARS-CoV-2 shows synergistic effect.

Human coronaviruses SARS-CoV-2 appeared at the end of 2019 and led to a pandemic with high morbidity and mortality. As there are currently no effective drugs targeting this virus, drug repurposing represents a short-term strategy to treat millions of infected patients at low costs. Hydroxychloroquine showed an antiviral effect in vitro. In vivo it showed efficacy, especially when combined with azithromycin in a preliminary clinical trial. Here we demonstrate that the combination of hydroxychloroquine and azithromycin has a synergistic effect in vitro on SARS-CoV-2 at concentrations compatible with that obtained in human lung.

Has doxycycline, in combination with anti-malarial drugs, a role to play in intermittent preventive treatment of Plasmodium falciparum malaria infection in pregnant women in Africa?

According to the World Health Organization (WHO), Plasmodium falciparum malaria during pregnancy is responsible for deleterious consequences for the mother and her child. The administration of intermittent preventive treatment (IPTp) with sulfadoxine-pyrimethamine (SP) at each antenatal care visit as early as 13 weeks of gestation until the time of delivery is a strategy that is currently recommended by WHO for the prevention of malaria in pregnancy. However, the emergence and the spread of the resistance to SP in Africa raise the question of the short-term effectiveness of the strategy. Dihydroartemisinin-piperaquine 120 mg/960 mg once a day for 3 consecutive days administered at least three times during the pregnancy might be an option for IPTp. The combination of 200 mg of doxycycline once a day for 3 consecutive days seems to be a good option to retard the emergence and the spread of resistance to artemisinin-based combination therapy (ACT) in Africa and improve the effectiveness of ACT in term of preterm births, neonatal morbidity and mortality. Contrary to preconceived ideas, scientific and medical data suggest that the risk of congenital malformations in the fetus or of tooth staining in infants whose mothers take doxycycline and hepatotoxicity during pregnancy is very low or non-existent. Additionally, the use of doxycycline during the first and second trimesters leads to an increase in gestational age at delivery, a decrease in the number of preterm births and a reduction in neonatal morbidity and mortality due to the beneficial antimicrobial activity of doxycycline against other infections during pregnancy. Furthermore, doxycycline has anti-malarial properties and is already recommended as prophylaxis for travellers and for treatment of falciparum malaria in combination with other anti-malarial drugs.