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.

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.

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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.

The use of restriction endonuclease analysis and Southern hybridization with our new CkF1,2 DNA probe, cold labeled with peroxidase, for the typing of Candida krusei isolates has been investigated. Fifty-five clinical samples isolated from forty-five patients hospitalized in eight centers, one environmental strain, and two reference strains were evaluated. Patterns were analyzed by a computer-assisted method and compared by numerical analysis. Clearer and less ambiguous patterns were obtained by restriction with endonuclease Hinfl. It generated 9 to 14 (average, 11) well-separated fragments in the range of 6.5 to 2.0 kb. Both their numbers and sizes varied greatly among the strains studied. The CkF1,2 probe hybridized with one to seven fragments ofHinfl patterns. A total of 48 distinct types were distinguished among the 58 strains studied. Hinfl and CkF1,2 patterns showed similarities of less than 83 and 75% for unrelated strains and more than 91 and 100% for related strains, respectively. The methods showed 100% typeability, 98% reproducibility, and a discriminatory power of 1. C. krusei isolates from each patient were distinct, whether from one hospital or from different hospitals. Multiple isolates from the same patient were identical, both over time and at different anatomic sites. An endogenous origin is suggested for the colonizing and infecting isolates among the 45 patients. The CkF1,2 probe enhanced discrimination of the strains and provided a definitive comparison for strain identity. Genetic linkages between isolates were assessed at the subspecies level, and 12 clusters were delineated. A typing scheme is proposed for epidemiological studies of C. krusei.

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.The Nocardia genus consists of gram-positive, variably acid fast, strictly aerobic bacteria that form filamentous, branched cells that fragment into pleomorphic, rod-shaped, or coccoid elements (1). It is widely distributed in the environment and causes a variety of suppurative and granulomatous infections of humans and animals, including cutaneous, subcutaneous, lymphocutaneous, pulmonary, cerebral, or disseminated nocardiosis. These bacteria are increasingly recognized as a cause of opportunistic infections (28).The identification of new species was based for a long time on investigation of epidemiology, clinical disease spectrum, and/or drug susceptibility combined to culture features and biochemical or chemotaxonomic characteristics. Such a phenotypic approach was probably responsible for the low number (n ϭ 12) of species described between the first description of the genus by Nocard in 1888 (32) and 1995. In the last decade, the introduction of genomic methods based on the analysis of sequences and polymorphism of the 16S rRNA gene or of the hsp65 gene has been of value for the description of many species. Thus, 17 new species had been validated from 1995 to 2003, leading to a major taxonomic revolution for this genus. It seems that it was just the beginning of the story and that the Nocardia genus will undergo the same dramatic taxonomic changes as Mycobacterium genus, for which almost a hundred species have now been described. This promoted a radical reappraisal of nocardial systematics.According to this new taxonomic background, we decided to reevaluate the taxonomic status of the strains deposited in the culture collection of the French Observatory for Nocardiosis (OFN; Lyon, France). We observed that three of them revealed atypical characteristics. These strains originated from mycetoma samples collected from Mexican patients. The strains were initially identified as Nocardia brasiliensis but showed morphological differences compared to this species. The nocardial systematic position of these three strains was investigated extensively according to an integrated use of phenotypic, chemotaxonomic, and molecular methods. The data obtained indicated that these strains should be recognized as a new species, for which the name Nocardia mexicana sp. nov. is proposed. MATERIALS AND METHODSStrains and culture conditions. The analysis of a large number...

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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.