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

OBJECTIVES: 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 600-bp 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 Nocardia farcinica (20.2%), Nocardia abscessus complex (19.9%) and Nocardia 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. Nocardia farcinica was frequently not susceptible to cefotaxime (118/148, 79.7% of the isolates), but only about 5% of Nocardia 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.

High Intraspecific Genetic Diversity of Nocardia brasiliensis, a Pathogen Responsible for Cutaneous Nocardiosis Found in France: Phylogenetic Relationships by Using sod and hsp65 Genes.

This study aims at genetic characterization and phylogenetic relationships of Nocardia brasiliensis focusing by using housekeeping rrs, hsp65, and sodA genes. N. brasiliensis is the species responsible for 80% of cases of actinomycetoma, one form of cutaneous nocardiosis which occurs mainly in tropical regions reaching immunocompetent patients in which the disease can lead to amputation. We analyze 36 indigenous cases of N. brasiliensis that happened in France. Phylogenetic analysis targeting rrs gene showed no robustness at phylogenetic nodes level. However, the use of a concatenation of hsp65 and sodA genes showed that the tested strains surprisingly ranked in 3 well-defined genotypes. Genotypes 2 and 3 were phylogenetically closer to each other and both diverged from genotype 1 sustained by a high bootstrap of 81%. This last genotype hosts all the cases of pulmonary forms (3), the sole cerebral form, and almost all the cases of immunocompromised patients (3 out of 4). Moreover, excepting one of them, all the strains belonging to this group present a susceptibility to imipenem which is not the case in the other genotypes that rarely count among them strains being susceptible to this drug. The haplotype diversity (Hd) of hsp65 (0.927) and sodA (0.885) genes was higher than that of rrs (0.824). For this gene, we obtained 16 polymorphic sites whereas, for hsp65 and sodA genes, up to 27 and 29 were identified, respectively. This study reveals that these two genes have an important genetic discriminatory power for the evaluation of the intraspecies genetic variability of N. brasiliensis and they may be useful for identification purposes at species level. This study also reveals the possible existence of a new species harbored by genotype 1.

Molecular identification of Nocardia species using the sodA gene: Identificación molecular de especies de Nocardia utilizando el gen sodA.

Currently for bacterial identification and classification the rrs gene encoding 16S rRNA is used as a reference method for the analysis of strains of the genus Nocardia. However, it does not have enough polymorphism to differentiate them at the species level. This fact makes it necessary to search for molecular targets that can provide better identification. The sodA gene (encoding the enzyme superoxide dismutase) has had good results in identifying species of other Actinomycetes. In this study the sodA gene is proposed for the identification and differentiation at the species level of the genus Nocardia. We used 41 type species of various collections; a 386 bp fragment of the sodA gene was amplified and sequenced, and a phylogenetic analysis was performed comparing the genes rrs (1171 bp), hsp65 (401 bp), secA1 (494 bp), gyrB (1195 bp) and rpoB (401 bp). The sequences were aligned using the Clustal X program. Evolutionary trees according to the neighbour-joining method were created with the programs Phylo_win and MEGA 6. The specific variability of the sodA genus of the genus Nocardia was analysed. A high phylogenetic resolution, significant genetic variability, and specificity and reliability were observed for the differentiation of the isolates at the species level. The polymorphism observed in the sodA gene sequence contains variable regions that allow the discrimination of closely related Nocardia species. The clear specificity, despite its small size, proves to be of great advantage for use in taxonomic studies and clinical diagnosis of the genus Nocardia.

Actualmente, para la identificación y clasificación bacteriana se utiliza como método de referencia la secuenciación el gen rrs que codifica al rRNA16S, en el caso del análisis de cepas del género Nocardia, sin embargo, no tiene el suficiente polimorfismo para diferenciarlas a nivel de especie lo que hace necesaria la búsqueda de blancos moleculares que puedan proporcionar una mejor identificación. El gen sodA (que codifica la enzima superóxido dismutasa) ha tenido buenos resultados en la identificación de especies de otros Actinomicetos. En este estudio se propone para la identificación y diferenciación a nivel de especie del género Nocardia. Se utilizaron 41 especies Tipo de diversas colecciones, se amplificó y secuenció un fragmento de 386 pb del gen sodA y se realizó un análisis filogenético comparando los genes rrs (1171 pb) hsp65(401pb) secA1 (494pb), gyrB (1195pb) y rpoB (401pb), las secuencias fueron alineadas utilizando el programa Clustal X, los árboles evolutivos de acuerdo con el método de "Neighbor-Joining"se hicieron con el programa Phylo_win y Mega 6. Se analizó la variabilidad específica del gen sodA del género Nocardia presentando una alta resolución filogenética, una variabilidad genética importante, especificidad y confiabilidad para la diferenciación de los aislados a nivel de especie. El polimorfismo observado en la secuencia del gen sodA contiene regiones variables que posibilitan la discriminación de especies de Nocardia estrechamente relacionadas, y una clara especificidad, a pesar de su pequeño tamaño, demostrando ser de gran ventaja para utilizarse en estudios taxonómicos y en el diagnóstico clínico del género Nocardia.

Phenotypic and genotypic evaluation of 18 Nocardia isolates from human clinical samples in Mexico.

BACKGROUND: Mexico has the largest number of clinical cases of actinomycetoma in North and South America. Species originally identified by less specific methods have been recently reclassified as other known species or as new species. OBJECTIVE: To assess, by 16S rRNA gene sequencing and phenotypic methods, the species distribution of 18 human clinical isolates originally identified as N. brasiliensis, some of them isolated between 1947 and 1959 in Mexico City. METHODS: Clinical isolates came from the Hospital General, "Dr. Manuel Gea Gonzalez", and Instituto Nacional de Diagnóstico y Referencia Epidemiológica (INDRE) in Mexico, D.F. The strains used in this study included 15 clinical strains isolated between 1947 and 1959 that were originally identified as N. brasiliensis and three more strains obtained in 2007 identified as Nocardia spp. The isolates were identified genotypically by sequencing the 16S rRNA gene, and their phenotypic profiles were obtained with the API Coryne(®) system. Antibiotic susceptibility patterns were tested according to the protocol of the Comité de l'antibiogramme de la Société française de microbiologie[4]. RESULTS: According to 16S rRNA gene, sequencing were identified among 18 human clinical isolates as Nocardia farcinica (n=11) and Nocardia brasiliensis (n=7). A high number of the strains were susceptible to the majority of the antibiotics tested. The phenotypic profiles of the strains were quite uniform for N. farcinica and some variability was observed for N. brasiliensis strains. CONCLUSION: N. farcinica was the most prevalent species identified. Modern methodologies should be applied in clinical laboratories to accurately identify etiological agents.

Phaeoacremonium parasiticum subcutaneous infection in a kidney-transplanted patient successfully treated by surgery.

Human infection caused by Phaeoacremonium parasiticum is increasingly being reported. However, only a few case reports show its role as a soil-related contaminant of kidney recipients. We report here a case of a subcutaneous infection by P. parasiticum in a transplanted man. After 4 years, he reported a nonpainful nodule on his forefinger. It was surgically excised. Histological examination revealed dense fibrous connective tissue showing an extensive granulomatous reaction, including the presence of a wooden sliver, and hyaline, branched, and septate hyphae. Six months later, the patient relapsed and underwent large excision. Culture was positive for P. parasiticum. No antifungal drug was administered. Surgical excision was successful.

[Mycotic keratitis and endophthalmitis caused by unusual fungi: Lasiodiplodia theobromae]. / Kératomycose et endophtalmie d'étiologie exceptionnelle: Lasiodiplodia theobromae.

We report a case of endophthalmitis caused by the fungus Lasiodiplodia theobromae. A 68-year-old man was referred to the hospital for right ocular pain since experiencing right ocular branch trauma 2 weeks before. The best-corrected acuity was limited to hand motion. Slit-lamp examination showed a large corneal abscess and an anterior chamber reaction. The patient underwent systemic and local antibiotic therapy, and corneal scraping for microbiological diagnosis. Sabouraud-chloramphenicol-gentamicin agar disclosed filamentous fungus, which was treated with oral itraconazole and topic amphotericin B. Molecular biology revealed Lasiodiplodia theobromae. Despite antimycotic drugs, severe panophthalmia occurred very quickly and led to evisceration. This case report describes Lasiodiplodia theobromae as the cause of keratomycosis and discusses risk factors and clinical features of fungal keratitis in order to improve prognosis by earlier treatment.