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.

First case of cerebral abscess due to a novel Nocardia species in an immunocompromised patient.

We report the first case of cerebral abscess due to a novel species of Nocardia in a heart transplant patient and describe the antimicrobial susceptibility of this isolate. As our patient was intolerant to trimethoprim-sulfamethoxazole, we also discuss alternative therapeutic options in brain abscess due to Nocardia sp.

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.

Cavitary pneumonia caused by nocardia otitidiscaviarum

We report a case of cavitary pneumonia caused by N. otitidiscaviarum in a man with diabetes mellitus and thrombocytopenia treated with systemic corticosteroid. Taxonomic identification involved phenotypic testing and molecular identification that was carried out by DNA sequencing of the 16SrRNA gene.

Cavitary pneumonia caused by Nocardia otitidiscaviarum.

We report a case of cavitary pneumonia caused by N. otitidiscaviarum in a man with diabetes mellitus and thrombocytopenia treated with systemic corticosteroid. Taxonomic identification involved phenotypic testing and molecular identification that was carried out by DNA sequencing of the 16SrRNA gene.

[Nocardia aroensis and lung infection: first case report in Africa ]. / Infection pulmonaire à Nocardia aroensis: première observation en Afrique.

Nocardiosis is a rare disease that is difficult to diagnose. Pulmonary forms are most common in association with a variety of nonspecific symptoms. Up to now isolation of the offending species, i.e., Nocardia aroensis, has been reported only once during the first description in Japan. The purpose of this article is to report the second world case of isolation of the Nocardia aroensis in a 50-year-old immunocompetent African woman.

First Spanish case of nocardiosis caused by Nocardia takedensis.

Nocardia takedensis is a recently described species isolated from soil. The first clinical isolate in Japan has recently been reported. This report describes the first clinical isolate of N. takedensis in Spain from a respiratory specimen.

[Disseminated nocardiosis in a child with acute lymphoblastic leukemia]. / Nocardiose viscérale disséminée chez un enfant atteint de leucémie aiguë lymphoblastique.

Nocardiosis is a rare infectious disease in children. We report here a disseminated nocardiosis in a child with acute lymphoblastic leukemia. The patient presented prolonged febrile neutropenia and nodular pneumopathy. Based on the amplification of a 16S rDNA, a PCR assay detected Nocardia sp. in the patient's bronchoalveolar lavage (BAL) fluid. Culture of BAL samples yielded Nocardia nova colonies after 2 weeks of incubation. Hepatic, splenic, renal and cerebral localisations were detected on extension checkup. trimethoprime-sulfamethoxazole and amikacine were started given the results of PCR assay, with a good response. Improvement of the patient's general condition led to complete chemotherapy under ciprofloxacine and ceftriaxone treatment, without nocardiosis reactivation. Nocardiosis is a rare complication in children with acute lymphoblastic leukemia. trimethoprime-sulfamethoxazole prophylaxis is widely used to prevent Pneumocystis jiroveci infection in children with haematologic malignancies. As Nocardia species are usually sensible, trimethoprime-sulfamethoxazole could play a role in Nocardia prophylaxis in such population. In our patient, compliance with trimethoprime-sulfamethoxazole had been low. Nocardia species are relatively fastidious growth bacteria and are difficult to isolate with classical bacteriological techniques. Molecular methods are now available, with a good sensitivity and fast results allowing to start an appropriate antibiotherapy before culture results, as early treatment is a major prognosis factor in nocardiosis. Nocardia infection should be suspected in case of nodular pneumopathy in immunocompromised children. An extension checkup should be performed to detect secondary localisations.

[Imported mycosis: a review of paracoccidioidomycosis]. / Mycoses d'importation: le point sur la paracoccidioïdomycose.

Population migration is increasing the number of systemic mycosis cases, a condition little or not present in Metropolitan France, thus often weakly documented for medical teams. Paracoccidioidomycosis is a systemic mycosis geographically confined to Latin America. The causative agent is Paracoccidioides brasiliensis, a dimorphic fungus present at ground level. Discovering that the armadillo was a new host allowed documenting the ideal conditions for this fungus development. The mortality rate is currently 1.45 for one million people and the disease affects mainly farmers. The mode of infection is respiratory and all organs and mucous membranes can be affected by lymphatic dissemination. The chronic form with pulmonary, cutaneous and oral lesions is the most important. The diagnosis can be carried out by direct examination of samples revealing the presence of budding yeasts, as well as culture at 25 and 37 degrees C. Antifungals like the sulfamethoxazole-trimethoprime combination, amphotericin B, but especially azole derivatives are use in the therapeutic management of patients. Detection and gp 43 antigen assay is useful for the patient's follow-up.

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.

Brain abscess caused by Nocardia cyriacigeorgica in a patient with human immunodeficiency virus infection.

Nocardia cyriacigeorgica is a recently characterized species within the genus of Nocardia. We report a brain abscess, following a primary pulmonary colonization, due to this species in a human immunodeficiency virus-infected patient. This case confirms that isolation of Nocardia in sputum is associated with a high risk of disseminated infection in immunocompromised patients.

Nocardiosis in a teaching hospital in the Central Anatolia region of Turkey: treatment and outcome.

Predisposing factors, antimicrobial susceptibility patterns, treatment and outcome were analysed for nine consecutive patients with nocardiosis. Predisposing factors were identified in six (67%) of the nine patients. Clinical syndromes of nocardial infection were pulmonary infection (three patients), cerebral infection (five patients) and disseminated infection (one patient). The predominant (60%) species was Nocardia farcinica rather than the Nocardia asteroides complex. Treatment was started empirically, modified according to the antimicrobial susceptibility pattern, and then continued for 6-12 months. Overall mortality was 33%, with death being caused by the Nocardia infection in two cases.

Screening of nonpolyenic antifungal metabolites produced by clinical isolates of actinomycetes.

The purpose of this work was to screen clinical isolates of actinomycetes producing nonpolyenic antifungals. This choice was made to limit the problem of rediscovery of well-known antifungal families, especially polyenic antifungals. One hundred and ten strains were tested, using two diffusion methods and two test media, against three yeast species and three filamentous fungi. Among 54 strains (49%) showing antifungal activity, five strains belonging to the genus Streptomyces were active against all test organisms and appeared promising. These results indicate that clinical and environmental isolates of actinomycetes could be an interesting source of antifungal bioactive substances. The production of nonpolyenic antifungal substances by these five active isolates was investigated using several criteria: antibacterial activity, ergosterol inhibition, and UV-visible spectra of active extracts. One active strain responded to all three selection criteria and produced potentially nonpolyenic antifungal metabolites. This strain was retained for further investigation, in particular, purification, structure elucidation, and mechanism of action of the active product.

Case Reports. Chronic and acute Aspergillus meningitis.

Cerebral aspergillosis usually occurs in severely immunocompromized hosts, is difficult to diagnose, and has a poor prognosis. After 14 months of chronic meningitis, ventriculitis, choroid plexitis, and lumbar arachnoiditis, which was complicated by acute hydrocephalus, Aspergillus, suspected to be from the candidus group, was isolated from the cerebrospinal fluid (CSF) of a previously healthy man. Thereafter Aspergillus antigen was found in stored plasma and CSF samples. He was treated with voriconazole and itraconazole. In a haemodialysis patient affected by an acute meningococcal meningitis, following a 3-day symptom-free interval, symptoms and signs of acute meningitis had reappeared and were unresponsive to a broad antimicrobial coverage. However, they resolved within 5 days after liposomal amphotericin B treatment had been started. From his CSF Aspergillus-DNA was identified and Aspergillus fumigatus isolated by culture. These two different clinical cases show that Aspergillus-DNA and antigen detection tests represent an advance in the diagnosis and liposomal amphotericin B, voriconazole, and itraconazole are an advance in the treatment of Aspergillus meningitis.

[Isolation of three species of Nocardia in a patient receiving long-term corticosteroid therapy]. / Isolement de trois espèces différentes de Nocardia chez un patient recevant une corticothérapie au long cours.

INTRODUCTION: Nocardia are saprophyte bacteria of the environment responsible for systemic infections in immunodepressed patients, due essentially to long-term corticosteroids. OBSERVATION: A patient having received corticosteroids for sarcoidoses for a year was hospitalised because of disseminated granulomatosis (neurological, respiratory, abdominal and cutaneous). Culture of various bacteriological samples isolated three species of Nocardia: N. otitidiscaviarum in uretheral pus and pus from the right gland, N. nova and N. asteroides in respiratory samples (protected distal sampling and broncho-alveolar washing). COMMENTS: Other than the mixed Nocardia infections described habitually, infections with two different species of Nocardia have recently been reported. Our case report is the first to have isolated three concomitant species of Nocardia.