Patrick Boiron

Use of PCR-Restriction Enzyme Pattern Analysis and Sequencing Database for hsp65 Gene-Based Identification of Nocardia Species

ABSTRACT Nocardia identification required laborious and time-consuming phenotypic and chemotaxonomic methods until molecular methods were developed in the mid-1990s. Here we reassessed the capacity of PCR-restriction enzyme pattern analysis (PRA) of the hsp65 gene to differentiate Nocardia species, including 36 new species. Our results confirm that hsp65 PRA must no longer be used for Nocardia species identification, as many species have the same restriction pattern. We then compared sequencing-based strategies using an hsp65 database and a 16S rRNA database and found that the hsp65 region contained sufficient polymorphisms for comprehensive Nocardia species identification.

Phylogeny of the Genus Corynebacterium Deduced from Analyses of Small-Subunit Ribosomal DNA Sequences

We determined almost complete small-subunit ribosomal DNA sequences of 50 reference strains belonging to the genera Corynebacterium, Rhodococcus, and Gordona and compared these sequences with previously published sequences. Three phylogenetic methods (the neighbor-joining, maximum-likelihood, and maximum-parsimony methods), as well as a bootstrap analysis, were used to assess the robustness of each topology which we obtained. The results of comparative phylogenetic analyses confirmed that the genera Corynebacterium, Dietzia, Gordona, Mycobacterium, Nocardia, Tsukamurella, and Turicella form a monophyletic taxon within the phylum containing the high-G+C-content gram-positive bacteria. The genus Corynebacterium appeared to be a monophyletic unit whose members could be divided into four major clusters. The validity of the genus Turicella is doubtful since members of this genus clearly belong to the genus Corynebacterium. The variability of chemotaxonomic characteristics within the genus Corynebacterium suggests that small-subunit ribosomal DNA sequence analysis is probably the most straightforward method for confirming that a bacterium belongs to this genus.

Human Nocardiosis in Northern Italy from 1982 to 1992

We conducted a retrospective survey of nocardiosis in 9 city hospitals in northern Italy from 1982 to 1992. The medical records of 30 patients with documented nocardiosis were reviewed. Microbiological data included morphology, biochemical characteristics, serology and in vitro susceptibility testing. The 29 isolates (1 case was diagnosed on the basis of serological results) were Nocardia asteroides (n = 25) and Nocardia farcinica (n = 4). Predisposing factors including immunosuppression for organ transplant rejection prophylaxis, lung disease (silicotuberculosis and pulmonary fibrosis), solid tumours and hematological malignancies, and AIDS. Three patients had no identified risk factors. 20 cases of pulmonary nocardiosis were observed. Sites of infection in patients without previous pulmonary involvement were: brain abscesses, soft tissues, pericardium, blood, and cerebrospinal fluid. Most strains tested were susceptible to amikacin and imipenem. Resistance to several antimicrobial agents was found, particularly erythromycin, fosfomycin, pefloxacin, sulphonamides and trimethoprim. Antimicrobial chemotherapy included sulphonamides, amikacin, ceftriaxone, imipenem and minocycline. 21 patients survived, although 2 relapsed transiently. Nocardiosis appears to be more common than generally realised by physicians in northern Italy. The local species distribution and disease spectrum are similar to those described elsewhere. Nocardiosis should be part of the differential diagnosis in patients with pulmonary infiltrates or brain abscess, particularly those with predisposing factors.

Direct Detection of Nocardia spp. in Clinical Samples by a Rapid Molecular Method

ABSTRACT We developed a 16S PCR-based assay for the rapid detection of Nocardia spp. directly from human clinical samples. The applicability of the assay was confirmed by using 18 samples from patients with nocardiosis as diagnosed by conventional cultures and 20 clinical samples from patients with confirmed tuberculosis used as negative controls.

Pathogenic and opportunistic microorganisms in caves

Funding from the project RNM-5137, Consejeria de Innovacion, Junta de Andalucia, is acknowledged. This is also a TCP CSD2007-00058 paper.

Nocardia pseudobrasiliensis sp. nov., a New Species of Nocardia Which Groups Bacterial Strains Previously Identified as Nocardia brasiliensis and Associated with Invasive Diseases

We studied five strains of a new Nocardia taxon recently identified among Nocardia brasiliensis strains associated with invasive diseases (R. J. Wallace, Jr., B. A. Brown, Z. Blacklock, R. Ulrich, K. Jost, J. M. Brown, M. M. McNeil, G. Onyi, V. A. Steingrube, and J. Gibson, J. Clin. Microbiol. 33:1528-1533, 1995) to determine their taxonomic status. Several characteristics of these organisms, including the presence of chemotype IV cell walls, nocardomycolic acids, a predominant menaquinone similar to that of Nocardia asteroides ATCC 19247T (T = type strain), and G+C contents ranging from 67 to 68 mol%, are characteristics of the genus Nocardia. Phylogenies based on small-subunit ribosomal DNA sequences clearly confirmed that all five strains belong to the genus Nocardia and occur on a single branch that is clearly distinct from N. brasiliensis. This branch forms a clade with Nocardia vaccinii, Nocardia nova, Nocardia otitidiscaviarum, and Nocardia seriolae. The five new strains exhibited high levels of DNA relatedness with each other, as determined by DNA-DNA hybridization experiments (S1 nuclease procedure), but not with N. brasiliensis strains or with strains of the four phylogenetically related Nocardia species mentioned above. The five new strains differ from N. brasiliensis in the following characteristics: mycolic acid pattern, decomposition of adenine, nitrate reduction, and antimicrobial agent susceptibilities. Therefore, we propose that these strains belong to a new species, Nocardia pseudobrasiliensis. The type strain is strain ATCC 51512, which was isolated from a leg abscess on a patient suffering from ulcerative colitis.

Nocardia mexicana sp. nov., a New Pathogen Isolated from Human Mycetomas

ABSTRACT Three isolates collected from human mycetomas and showing an unusual brownish purple pigmentation on Bennett agar plates were analyzed by a polyphasic taxonomic approach, including morphological, biochemical, physiological, and chemotaxonomic properties coupled with genomic and phylogenetic analysis. It clearly appeared that these microorganisms were distinct from their closest phenotypic and genetic match, the most related species according to 16S rRNA gene sequence analysis being Nocardia pseudobrasiliensis. The data obtained indicated that the three clinical strains should be recognized as a new species for which the name Nocardia mexicana sp. nov. is proposed.

Report of human nocardiosis in Italy between 1993 and 1997

During a 5-year period, from 1993 to 1997, nocardial infection was diagnosed in 26 patients admitted to hospitals in 11 cities in Italy. Pathogens were identified as Nocardia asteroides in 18 cases, as N. farcinica in five cases, as N. nova in two and as N. brasiliensis in one case. All cases were difficult to diagnose, as usually it happens with this disease: physicians have to be alert to suspect nocardial infection so that appropriate therapy can be early given. This is the second retrospective report on Nocardia spp. infection conducted in Italy, suggesting the utility to organise a permanent network for a national survey system for nocardiosis.

Nocardia asteroides in the soil of Kuwait.

A pilot study was undertaken to determine the occurrence and distribution of pathogenic nocardiae in Kuwaiti soil. A total of 102 soil samples collected from two localities were investigated by the paraffin bait technique. Nocardia asteroides was the only species isolated from 42 (41%) soil samples. None of the isolates fulfilled the criteria required for identification of N. farcinica or N. nova. Thirty one (73.8%) isolates showed equivalent growth at 45 °C and 35 °C, 17 (40.4%) isolates utilized acetamide for carbon and nitrogen requirements and 3 (7.1%) isolates showed delayed arylsulphatase activity. Only a solitary isolate was resistant to cefamandole. Soil samples originating from the Kuwait University Campus Shuwaikh, which were rich in humus/organic matter, were more productive for N. asteroides (67%) than the samples which were devoid of it but were mixed with crude oil (39%). Sand samples that lacked organic matter and crude oil samples were least productive of N. asteroides. These preliminary findings do not suggest that massive oil contamination of soil in the Ahmadi oil field area during the Gulf war promoted the natural occurrence of N. asteroides. However, isolation of N. asteroides in as many as 41% of the soil sample is a significant observation warranting further epidemiologic studies including its possible role in the operation desert storm sickness syndrome. This is the first report on the natural occurrence of N. asteroides in Kuwait.

Diversity of Superoxide-Dismutases Among Clinical and Soil Isolates of Streptomyces Species

Abstract. Comparison of the nature, activity, and cellular localization of superoxide-dismutases (SOD) from soil and clinical isolates of Streptomyces species was investigated to identify possible factors that could account for the pathological role of the strains isolated from human lesions. Results showed that all of the studied strains possessed a cytoplasmic Ni-SOD. This particular SOD, found in isolates from patients, could be a new taxonomic criterion to identify Streptomyces species with greater precision. A second minor SOD, assimilated to an Fe/Zn-SOD, was detected in some strains, but no relationship was established between the presence of this enzyme and the clinical origin of the strains.