The characteristics of in vitro micafungin (FK463) antifungal activity against six species of dimorphic fungi were investigated in accordance with the NCCLS M27-A microdilution methods. MICs of micafungin, amphotericin B, itraconazole, and fluconazole for Histoplasma capsulatum var. capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Penicillium marneffei, and Sporothrix schenckii were determined both for the yeastlike form and mycelial form. Coccidioides immitis was tested only in its mycelial form. We have clearly demonstrated that the in vitro activity of micafungin depends considerably on the growth form of dimorphic fungi. Micafungin exhibited potent activity against the mycelial forms of H. capsulatum, B. dermatitidis, and C. immitis (MIC range, 0.0078 to 0.0625 g/ml), while it was very weakly active against their yeast-like forms (MIC range, 32 to >64 g/ml). Micafungin was also more active against the mycelial forms than the yeast-like forms of Paracoccidioides brasiliensis, Penicillium marneffei, and S. schenckii. The MICs of amphotericin B were 2 to 5 dilutions lower for the mycelial forms than for the yeast-like forms of B. dermatitidis and Paracoccidioides brasiliensis. There was no apparent difference in the activity of itraconazole between the two forms. The MICs of fluconazole for the yeast-like forms were generally lower than those for the mycelial forms, and considerably so for B. dermatitidis. These results suggest that the growth form employed in antifungal susceptibility testing of dimorphic fungi can considerably influence the interpretation of results. At present, it cannot be judged whether micafungin has clinical usefulness for dimorphic fungus infections, since for most fungi it remains uncertain which growth form correlates better with therapeutic outcome. However, the results of this study warrant further investigations of micafungin as a therapeutic agent for infections caused by dimorphic fungi.

We describe, to our knowledge, the first case of allergic bronchopulmonary mycosis (ABPM) caused by the basidiomycetous fungus Schizophyllum commune in an otherwise healthy woman. Bronchoscopic analysis repeatedly disclosed S. commune hyphae in the bronchi of the lingular lobe; these hyphae were originally misidentified as Aspergillus because the presence of clamp connections was overlooked. A lingular infiltrate with ectatic proximal bronchi, eosinophilia, an elevated serum level of IgE, and antibodies to S. commune supported the diagnosis. It is sometimes difficult to isolate and identify S. commune in clinical specimens, and hence only a limited number of cases of ABPM might have been correctly diagnosed in the past. We suspect, therefore, that some cases of ABPM caused by an allergic reaction to S. commune may be misdiagnosed as allergic bronchopulmonary aspergillosis or eosinophilic pneumonia of unknown origin. The significance of S. commune in allergic bronchopulmonary diseases is discussed.

Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from armadillos from the Amazonian region where the mycosis is uncommon. In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease. Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, Sao Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver, and mesenteric lymph nodes were processed for histology, cultured on Mycosel agar at 37ЊC, and homogenized for inoculation into the testis and peritoneum of hamsters. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37ЊC. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph nodes of hamsters. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three armadillos isolates (Pb-A1, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticularly inoculated hamsters. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos are a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus.

Rifampin is an important chemotherapeutic agent for use against tuberculosis, leprosy, and infections by organisms related to those causing these diseases (3, 7). The antimicrobial activity of rifampin is due to its inhibition of DNA-dependent RNA polymerase, and most rifampin-resistant bacteria have been reported to have an alteration in the ␤-subunit of this enzyme (2). Such a resistance mechanism has been reported for Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium africanum, and Mycobacterium leprae (14, 16). During our studies of fast-growing mycobacterial strains, we found that several strains inactivate rifampin (1, 5). We analyzed the inactivated antibiotics and found them to differ in mass spectrum and chromatographic mobility from those previously reported, i.e., the glycosylated (glucosylated) or phosphorylated compounds produced by pathogenic Nocardia spp. (11,17). These results prompted us to determine the detailed structures of the inactivated compounds. In this paper the isolation, structures, and antimicrobial activities of the inactivated compounds are reported.Rifampin was generously provided by CIBA-GEIGY Pharmaceuticals, Basel, Switzerland. MICs were determined by an agar dilution method with brain heart infusion agar (Difco Laboratories, Detroit, Mich.) medium. The inoculum size of each test organism was adjusted to 10 6 CFU/ml. The plates were spotted with a multipoint inoculator (A 400; Denly Instruments, Ltd., Sussex, England) that delivered 0.005 ml of inoculum, resulting in a spot inoculum of approximately 5 ϫ 10 4 CFU. Inactivation of rifampin was monitored by a bioassay method with Bacillus subtilis PCI 219 as a test organism (17). Inactivated compounds were monitored with a thin-layer chromatography scanner (CS-910; Shimadzu Seisakusho, Kyoto, Japan) (17, 18) at 238 nm.One loopful of the culture from slant cultures of Mycobacterium smegmatis DSM 43756 was inoculated into a 100-ml Erlenmeyer shake flask containing 20 ml of a seed culture medium (2% glycerol-enriched brain heart infusion medium; Difco Laboratories) with 4-mm-diameter glass beads to reduce aggregation of the mycobacterial cells (18). The inoculated flasks were shaken at 250 rpm (5.8-cm stroke) for 4 days at 33ЊC. For large-scale preparation of the inactivation products, the seed culture was used as an inoculum. Ten milliliters of seed culture was inoculated into 500-ml shake flasks containing 100 ml of the same medium. After 24 h of incubation at 33ЊC, rifampin (stock solution in methanol at 100 mg/ml) was added to a final concentration of 50 g/ml, and the mixture was incubated for 3 days. Following separation from mycelia by centrifugation at 6,000 rpm (5,800 ϫ g), the supernatant was adjusted to pH 2.0 with 1 N HCl and extracted three times with an equal volume of ethylacetate. The extracts were combined, dehydrated with Na 2 SO 4 , and concentrated in vacuo to dryness. The dried material was purified by silica gel column chromatography (column size, 3.5 by 15 cm) by using 75 g of Wakogel C-200 (Wako Pu...

Nine multiparous Holstein cows were used in a replicated 3 × 3 Latin square design to determine the effects of substituting corn grain with brown rice (BR) grain in total mixed ration (TMR) silage on milk yield, ruminal fermentation and nitrogen (N) balance. The TMR silages were made from the ensiling of TMR containing (dry matter basis) 50.1% forage in rice silage and corn silage combination, and 49.9% concentrate. The grain portion of the diets contained 31.2% steam-flaked corn, 31.2% steam-flaked BR or an equal mixture of corn and BR. Dietary treatments did not affect dry matter intake, milk yield and milk fat, protein and lactose yields. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The urinary N excretion decreased linearly (P < 0.01) in response to increased levels of BR, with no dietary effect on N intake, N secretion in milk and fecal N excretion. Our results indicate that steam-flaked BR is a suitable replacement for steam-flaked corn in dairy cow diets, and that it can be included in rations to a level of at least 31.2% of dry matter without adverse effects on milk production, when cows were fed rice silage and corn silage-based diets.

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Stationary-phase cells of Cryptococcus neoformans displayed two morphological characteristics: virtually all the cells were unbudded even in the early stationary phase and even when grown in rich media, and average cell size increased from that of exponential-phase cells. DNA contents for small and large stationary-phase cells were determined by quantitative fluorescence microscopy after DNA staining with propidium iodide or DAPI. Small cells contained G1 DNA, whereas large unbudded cells had either a G2 or G1 DNA content, indicating that Cr. neoformans can enter into the stationary phase from either the G1 or G2 period.

Paracoccidioidomycosis is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future.