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.

[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.

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.

Aislamiento de nocardia asteroides de muestras de suelo de cinco estados de Venezuela / Isolation of nocardia asteroides of samples of soil of five state of Venezuela

Los autores presentaron los resultados obtenidos del estudio de 43 muestras recogidas en 25 lugares de cinco estados de Venezuela. Las muestras fueron cultivadas para aislamiento de nocardia sp. usando la técnica del "paraffin bait". Las muestras de suelo fueron recolectadas en el estado Lara (19 muestras), estado Mérida (13 muestras), estado Amazonas (7 muestras), estado Falcón (3 muestras) y estado Apure (1 muestra). De las muestras estudiadas, el 46,51 por ciento se identificaron como nocardia asteroides y el el 16,28 por ciento como nocardias sp. En las muestras de suelo estudiadas del estado Lara se encontró el mayor número de aislados de nocardia asteroides (95 por ciento); esto es un indicador de que este estado podría ser considerado como uno de los reservarios naturales de N. asteroides. Estos datos se compaginan con la alta morbilidad del actinomicetoma, entre los cuales figura como segundo agente etiológico del mismo N. asteroides

Genetic relatedness analysis of nocardia strains by random amplification polymorphic Dna: validation and applications.

Until now, no simple and rapid technique existed for epidemiological study of strains belonging to the Nocardia genus. The application of the arbitrarily primed PCR procedure to generate randomly amplified polymorphic DNA (RAPD) fingerprints for such analysis of Nocardia isolates was investigated. Fifty-one unrelated clinical isolates of N. asteroides were tested. Two conditions of RAPD using two different primers generated RAPD fingerprints that allowed the differentiation of all strains. The patterns were reproducible and discriminating. The results highlight the diversity of N. asteroides species and confirm that RAPD analysis is a highly valuable tool for studying the epidemiology of the Nocardia genus. Several examples describe the advantage of RAPD analysis for establishing the relationship between isolates from a given patient (long-term infections, coinfections) and from different patients (i.e. during an outbreak). In the future, this technique will help us to investigate the source of infection in cases of nosocomial transmission, to understand the outcome of nocardiosis, and to follow the evolution and acquisition of resistance to Nocardia strains.

Rapid identification and fingerprinting of Candida krusei by PCR-based amplification of the species-specific repetitive polymorphic sequence CKRS-1.

A PCR method was developed to identify and fingerprint Candida krusei isolates simply and rapidly. The primer pair Arno1 and Arno2 was designed to amplify the polymorphic species-specific repetitive sequence CKRS-1 (C. krusei repeated sequence 1) that we identified in the nontranscribed intergenic regions (IGRs) of rRNA genes in C. krusei LMCK31. The specificity, sensitivity, reproducibility, and fingerprinting ability of the PCR assay were evaluated. Amplification products were obtained from all 131 C. krusei isolates studied. No other yeast species of medical importance (n = 26), including species similar to C. krusei, species of pathogenic filamentous fungi, or a variety of pathogenic bacteria, yielded a PCR product with these primers. This PCR assay allowed for the identification of C. krusei in less than 6 h. The PCR assay was sensitive enough to detect as little as 10 to 100 fg of C. krusei-purified DNA and proved to be reproducible. Since amplification products varied both in number and in molecular weight according to the strains, PCR patterns allowed strains to be distinguished. To ascertain the epidemiological usefulness of this PCR fingerprinting, the patterns of the 131 isolates were compared. A total of 95 types which corresponded to 95 independent strains were delineated (discriminatory power = 1 with n = 95). Comparison of the results of PCR fingerprinting and those of fingerprinting with the CkF1,2 probe showed that they concurred. In addition, this work yields insights into the mechanisms involved in generating polymorphisms in the IGRs of C. krusei. Since this method is simpler and faster than established identification and genotyping methods of this important pathogenic species, it is a critical improvement for clinical microbiology laboratories relevant not only to diagnosis but also to epidemiology.

Species-specific identification of Candida krusei by hybridization with the CkF1,2 DNA probe.

The species specificity of the Candida krusei DNA fingerprinting probe CkF1,2 has been investigated. A total of 149 pathogenic and nonpathogenic fungal and bacterial DNAs were screened with CkF1,2. The probe was cold labeled with peroxidase, and its specificity was assessed by using Southern blot, dot blot, and colony blot hybridization. Its sensitivity was determined by dot blot hybridization. The CkF1,2 probe proved to be species specific. It hybridized with DNA for the 112 C. krusei strains studied, whereas it failed to hybridize under low-stringency conditions to 37 DNAs from 27 different yeast species, including Candida albicans, Candida glabrata, Candida norvegensis, Candida inconspicua, Candida tropicalis, Candida valida, Candida zeylanoides, and Yarrowia lipolytica, as well as DNAs from the filamentous fungi and bacteria tested. However, CkF1,2 hybridized strongly with DNA of the yeast species Issatchenkia orientalis, the putative ascogenous perfect state of C. krusei. Amounts as small as 60 to 120 ng of C. krusei target DNA were detected by dot blot hybridization with CkF1,2. It permitted the direct screening of colony blots for early identification. The CkF1,2 probe has potential value as a diagnostic reagent for identifying C. krusei.

Nosocomial infection with Candida albicans in a pancreatic transplant recipient investigated by means of restriction enzyme analysis.

Restriction enzyme analysis (REA) of total DNA was used in order to investigate the possible transmission of Candida albicans from the grafted pancreas in a woman with a kidney-pancreas transplant. A strain of Candida albicans was recovered from the pancreas-transplant preservation medium cultured routinely before transplantation. Four infecting isolates and one vulval isolate were recovered from the recipient in the early post-operative stage. In addition, 12 unrelated control strains were studied for comparison. By means of EcoRI and HinfI, restriction patterns of the isolates recovered from the preservation medium and from the patient were found to be identical (100% similitude according to Jaccard's coefficient) apart from that of the strain isolated from the vulva. HinfI gave two characteristic fragments of 5.9 and 4.6 kb. In contrast, the 12 control strains generated 12 different patterns. The percentage of similarity between the patterns of the infecting strains and those of the control and the vulval strains was less than 60%. These findings provide evidence of transmission of the infecting strain via the pancreas transplant and the nosocomial nature of infection.

Typing of Candida krusei clinical isolates by restriction endonuclease analysis and hybridization with CkF1,2 DNA probe.

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 HinfI. 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 of HinfI patterns. A total of 48 distinct types were distinguished among the 58 strains studied. HinfI 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.

[Alcoholic fermentation of inulin by various strains of yeasts]. / Fermentation alcoolique de l'inuline par quelques souches de levures.

Strains of fourteen species of yeasts able to ferment inulin without previous chemical or physical hydrolysis were studied on semi-synthetic medium by evaluation of CO2 production under anaerobic conditions. Among them, Kluyveromyces cicerisporus, Candida macedoniensis and Candida utilis showed the best kinetic characteristics of fermentation. Experiments were carried out to specify the action of different parameters such as temperature, pH and exogenous ethanol concentration. The results obtained on semi-synthetic medium were confirmed on Jerusalem artichoke juice. The optimal temperature for fermentation was 35 degrees C for the three strains, but K. cicerisporus alone conserved its fermentative capacity at 40 degrees C, at low pH (3.5) and with 6% (v/v) exogenous ethanol concentration. This strain appears to be a good yeast for industrial production of ethanol from inulin substrate.