Étude moléculaire et phylogénétique des bactéries appartenant au genre Nocardia

Les bactéries du genre Nocardia font partie de l'ordre des Actinomycétales. Elles sont largement distribuées dans notre environnement et sont considérées comme des bactéries opportunistes. Chez l'homme, elles sont responsables d'infections graves le plus souvent chez les patients immunodéprimés. Leur identification et traitement restent encore difficiles. Le travail que nous avons entrepris a consisté à développer de nouveaux outils d'identification moléculaire, ainsi qu'une analyse phylogénétique basée sur le séquençage de quatre gènes ménagers. En premier lieu, nous avons réévalué pour le genre Nocardia la méthode de "Restriction Fragments Length Polymorphism" (PCR-RFLP) du gène hsp65 (codant une protéine de choc thermique de 65 kDa) utilisant les enzymes BstEII, HinfI et MspI. Cette technique qui constituait la méthode de référence pour l'identification au niveau spécifique, s'avère incapable de différencier un grand nombre d'espèces récemment décrites. Nous avons cherché à créer, finalement sans succès, un nouvel arbre d'identification avec de nouvelles endonucléases potentiellement plus discriminantes. Nous avons alors choisi une nouvelle approche basée sur une analyse multilocus de 54 souches types appartenant au genre Nocardia. Le séquençage partiel des gènes (ARNr 16S, hsp65, rpoB et sod) nous a permis de construire une banque de données génomique multigénique qui combinée au progiciel dénommé Bio Informatic Bacterial Identification (BIBI) a permis d'obtenir une identification phylogénétique précise des Nocardia et plus généralement des Actinomycètes pathogènes. Cet outil a été validé et utilisé avec différentes collections de souches provenant d'origines diverses (Espagne, France, Koweït et Mexique). Les résultats obtenus ont permis la détection rapide de nouvelles espèces potentiellement pathogènes ainsi que l'identification d'espèces n'ayant jamais été rapportée dans des processus infectieux. Complétés par des études phénotypiques et de sensibilité aux antibiotiques, une nouvelle espèce a été décrite N. mexicana sp. nov. responsable de mycétome. A partir du jeu de séquences disponibles, une analyse phylogénétique globale intégrant les quatre gènes étudiés au sein du genre Nocardia a pu être réalisée et a montré une phylogénie plus robuste et discriminante que celle portant sur le seul gène de l'ARNr 16S . Dans le domaine de la microbiologie clinique, nous avons développé et mis en place une méthode de détection rapide et sensible de Nocardia directement sur des prélèvements cliniques, basée sur une PCR spécifique du genre Nocardia et combinée à une hybridation avec une sonde codant l'ARNr 16S. Parallèlement, une méthode d'amplification spécifique de l'ARNr 16S des bactéries appartenant au genre Streptomyces a été mise au point et validée. L'ensemble de nos résultats représente une avancée importante dans l'identification et l'analyse phylogénétique des bactéries appartenant au genre Nocardia et plus généralement à l'ordre des Actinomycétales.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

La sporotrichose à sporothrix schenckii (diagnostic et traitement : sa répartition mondiale et mexicaine)

LYON1-BU Santé (693882101) / SudocSudocFranceF

Invasive Nocardiosis: Disease Presentation, Diagnosis and Treatment – Old Questions, New Answers?

International audienc

Fulminant Nocardiosis Due to a Multidrug-Resistant Isolate in a 12-Year-Old Immunocompetent Child

International audienceNocardiosis is a rare cause of infection that usually affects immunocompromised adult patients and might not be recognized by pediatricians. We report a fatal case of disseminated nocardiosis in a previously healthy child initially admitted for an abdominal mass with suspicion of a renal malignant tumor. The patient, originating from Mali without any medical history, displayed abdominal pain with progressive altered general status. Laboratory and imaging findings revealed lymphocytic meningitis and disseminated abscesses in the brain and the cerebellum and a large number of cystic lesions of the kidney. Despite being administered wide-spectrum antibiotics and antituberculous and antifungal therapies with an external ventricular drainage for intracranial hypertension, the patient died 6 days after his admission. Nocardia spp was cultured from a renal biopsy and the cerebrospinal fluid. Species identification and antibiotic susceptibility were obtained later, revealing a multidrug-resistant isolate of the Nocardia elegans/aobensis/africana complex. This case reveals the difficulties of diagnosing nocardiosis, in particular in children not known to be immunocompromised, because we face multiple differential diagnoses and the importance of treating nocardiosis appropriately because of intrinsic resistance issues

Antibiotic susceptibility testing and species identification of Nocardia isolates: a retrospective analysis of data from a French expert laboratory, 2010-2015

International audienceObjectives: Nocardia, a Gram-positive bacterium, is responsible for rare and severe infections. Accurate microbiological data are essential to guide antibiotic treatment. Our primary objective was to describe species identification and results of antimicrobial susceptibility testing (AST) for Nocardia isolates analysed over a 6-year period. Secondary objectives were to study temporal trends in species distribution and AST results.Methods: We retrospectively analysed results from Nocardia isolates sent between January 2010 and December 2015 to a French laboratory dedicated to Nocardia (Observatoire Français des Nocardioses). Species identification was obtained by amplification and sequencing of a 600bp fragment of the 16S rRNA gene (for all isolates) and of hsp65 (when required). AST was performed using disk diffusion.Results: We included 793 Nocardia isolates, mostly from the lungs (53.8%). The most frequent species were N. farcinica (20.2%), N. abscessus complex (19.9%) and N. nova complex (19.5%). The proportion of N. farcinica increased significantly over time from 13% in 2010 to 27.6% in 2014. Linezolid, amikacin, trimethoprim-sulfamethoxazole, minocycline, and imipenem were the most frequently identified active antibiotics with, respectively, 0% (0/734), 2.9% (21/730), 5.4% (40/734), 9.4% (69/734) and 19.5% (143/732) of isolates not susceptible. N. farcinica was frequently not susceptible to cefotaxime (118/148, 79.7% of the isolates), but only about 5% of N. cyriacigeorgica and N. abscessus complex isolates were not susceptible to cefotaxime.Conclusions: In this first epidemiological study of Nocardia isolated from human samples in France, N. farcinica was the species most frequently identified and its prevalence increased over time

Comparison of Actinobacteria communities from human‐impacted and pristine karst caves

International audienceActinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization-related visual marks affect this community on cave walls is lacking. We compared Actinobacteria communities among four French limestone caves (Mouflon, Reille, Rouffignac, and Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas-1, Passage, Apse, and Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65-related taxonomic database was constructed for the identification of Actinobacteria to the species level by Illumina-MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% operational taxonomic units cutoff) versus the 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species-level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave

Actinomycetoma Caused by Actinomadura mexicana , A Neglected Entity in the Caribbean

International audienceMycetoma is a chronic infection that is slow to develop and heal. It can be caused by fungi (eumycetoma) or bacteria (actinomycetoma). We describe a case of actinomycetoma caused by Actinomadura mexicana in the Caribbean region

First case of actinomycetoma in France due to a novel nocardia species, nocardia boironii sp nov.

Bacterial mycetoma is a neglected disease mainly observed in tropical area countries and typically associated with rural conditions, making its presence in developed countries of temperate climate areas rare. However, we report the first case of an autochthonous mycetoma case in continental France that originated from a new Nocardia species. A Gram-positive filamentous bacterium (OFN 14.177T) was isolated from a pus sample from the mycetoma of a male French patient 92 years old suffering from chronic lymphocytic leukemia. The isolate was analyzed by a polyphasic taxonomic approach by coupling morphological, biochemical, physiological, and chemotaxonomic aspects to genomic and phylogenetic analyses. Multilocus sequence analysis (MLSA) using four housekeeping genes (16S rRNA gene, secA1, hsp65, and sod) combined with phylogenetic analysis revealed that the strain OFN 14.177T is phylogenetically closer not only to Nocardia altamirensis but also to all other species comprising the Nocardia brasiliensis clade (i.e., N. brasiliensis, N. altamirensis, N. vulneris, N. iowensis, and N. tenerifensis), some of which present cutaneous tropism. The G+C content of isolate OFN 14.177T was 68.2 mol%. DNA-DNA hybridization analyses demonstrated 38.25% relative reassociation with N. altamirensis. The strain OFN 14.177T is different from the closest species at genetic and phenotypical levels, and the data obtained indicate that it should be recognized as a new species, for which the name of Nocardia boironii sp. nov. is proposed. The type strain is OFN 14.177T (= EML 1451 = DSM 101696).IMPORTANCE Bacterial mycetoma is an endemic infection in areas with tropical and subtropical climates. Thus, its presence in temperate climate areas remains rare. We report here the first case of autochthonous actinomycetoma in continental France originating from a Nocardia species other than N. brasiliensis, namely, Nocardia boironii. Considering the history of the patient, the infection source of strain OFN 14.177T may be from frequent contact with the soil over many years because of his gardening activities. The discovery of a French autochthonous Nocardia species responsible for actinomycetoma reveals the importance of considering the possibility of having autochthonous infections of this type in nontropical countries, not only imported cases from tropical countries. However, further studies are needed to elucidate the real incidence of this new species

Clinical Assessment of a Nocardia PCR-Based Assay for Diagnosis of Nocardiosis

International audienceThe diagnosis of nocardiosis, a severe opportunistic infection, is challenging. We assessed the specificity and sensitivity of a 16S rRNA Nocardia PCR-based assay performed on clinical samples. In this multicenter study (January 2014 to April 2015), patients who were admitted to three hospitals and had an underlying condition favoring nocardiosis, clinical and radiological signs consistent with nocardiosis, and a Nocardia PCR assay result for a clinical sample were included. Patients were classified as negative control (NC) (negative Nocardia culture results and proven alternative diagnosis or improvement at 6 months without anti-Nocardia treatment), positive control (PC) (positive Nocardia culture results), or probable nocardiosis (positive Nocardia PCR results, negative Nocardia culture results, and no alternative diagnosis). Sixty-eight patients were included; 47 were classified as NC, 8 as PC, and 13 as probable nocardiosis. PCR results were negative for 35/47 NC patients (74%). For the 12 NC patients with positive PCR results, the PCR assay had been performed with respiratory samples. These NC patients had chronic bronchopulmonary disease more frequently than did the NC patients with negative PCR results (8/12 patients [67%] versus 11/35 patients [31 %]; P = 0.044). PCR results were positive for 7/8 PC patients (88%). There were 13 cases of probable nocardiosis, diagnosed solely using the PCR results; 9 of those patients (69%) had lung involvement (consolidation or nodule). Nocardia PCR testing had a specificity of 74% and a sensitivity of 88% for the diagnosis of nocardiosis. Nocardia PCR testing may be helpful for the diagnosis of nocardiosis in immunocompromised patients but interpretation of PCR results from respiratory samples is difficult, because the PCR assay may also detect colonization

Genome Sequence of the Human- and Animal-Pathogenic Strain Nocardia cyriacigeorgica GUH-2

International audienceThe pathogenic strain Nocardia cyriacigeorgica GUH-2 was isolated from a fatal human nocardiosis case, and its genome was sequenced. The complete genomic sequence of this strain contains 6,194,645 bp, an average G+C content of 68.37%, and no plasmids. We also identified several protein-coding genes to which N. cyriacigeorgica's virulence can potentially be attributed