The first isolation of Nocardia veterana from a human mycetoma

The clinical isolates from biopsy specimen human subcutaneous nodule developed orange-colored and wrinkled colonies on Sabouraud's dextrose agar at 24 C for 2 weeks. The isolates were aerobic and gram-positive. The bacteria were rod-shaped to coccoid and 1 x 5 microm in size. The assimilation tests revealed that the clinical isolate was identical to a reference strain of Nocardia veterana. A nucleotide sequence analysis of the 16S ribosomal DNA from the isolate and a reference strain of N. veterana showed 99.8% similarity. All data are consistent with the conclusion that the isolate in this human case of mycetoma is N. veterana.     

Characteristics of 17Paracoccidioides brasiliensis isolates

We have studied the physiological and morphological features of 17 isolates ofParacoccidioides brasiliensis in order to define their phenotypes. The isolates were cultured at room temperature on potato dextrose agar (PDA, Difco) slants for mycelial growth and in 1% dextrose brain heart infusion agar (BHIA, Difco) at 37°C for the study of yeast forms. Most mycelial and yeast forms grew well between pH 5.6–9.4. In their response to osmotic pressure the isolates were separated in three groups: intolerant, intermediate and tolerant. They also varied in carbohydrate assimilation tests, which indicated important metabolic variation. No clear differences were observed in phenol oxidase tests, KNO₃, starch, casein and arbutin assimilation tests. Only 1 of the isolates, Bt-19, had gelatinase activity. No correlation was observed between the above differences and virulence. Two patterns of growth were observed in the mycelial cultures, glabrous and cottonous, the latter being correlated with increased virulence for ddY mice. Most yeast forms grew as cerebriform colonies, but Pb-HC and Bt-19 colonies had a cobblestone-like surface.     

In vitro anti-Malassezia activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb

AIMS: This study aimed at investigating the anti-Malassezia activity of xanthorrhizol (XTZ) isolated from Curcuma xanthorrhiza Roxb. against Malassezia furfur ATCC 14521 and Malassezia pachydermatis ATCC 14522. METHODS AND RESULTS: The in vitro susceptibility tests for XTZ were carried out in terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), using broth microdilution method with endpoint after 48 h. Time-kill curves were determined at concentrations ranging from 0 to 25 microg ml(-1). The MIC values of XTZ against M. furfur and M. pachydermatis were 1.25 and 0.25 mug ml(-1), respectively. The MFC of XTZ was 5 microg ml(-1) for M. furfur and 2.5 microg ml(-1) for M. pachydermatis. Time-kill curves demonstrated that treatment with 25 microg ml(-1) of XTZ for 5 h was able to kill 100% of M. furfur, while 20 microg ml(-1) of XTZ for 15 min killed M. pachydermatis completely. CONCLUSION: XTZ shows potential as an anti-Malassezia agent for inhibiting the growth of M. furfur ATCC 14521 and M. pachydermatis ATCC 14522 in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: XTZ may be a useful alternative for treating Malassezia-associated diseases.     

First Isolation of Cryptococcus magnus from a Cat

A 6-month-old male Japanese domestic cat with otitis externa due to Aspergillus fumigatus was treated with antifungal agents for 25 days and appeared to be cured. Many yeast colonies however developed from the ear canal samples on Sabouraud's dextrose agar at 27 degrees C for 5 days, instead of A. fumigatus. This yeast colony was cream-colored and slim in texture with smooth and highly glossy surface after 5-day incubation on Sabouraud's dextrose agar at 27 degrees C. The isolate was identified as Cryptococcus magnus by mycological analysis and 28S ribosomal analysis.     

The influence of newly synthesised fenporpimorph derivatives on some pathogen yeasts

The effect of minimum inhibitory concentrations (MICs) of six novel fenpropimorph derivatives on lipid and sterol composition of Candida albicans, Cryptococcus neoformans, Malassezia pachydermatis and Malassezia furfur was investigated. The MICs for the most effective derivatives were found in the range from 3.7 to 56.7 microM and were 2-3 times lower compared to the commercial fungicide bifonazol. The more efficient fenpropimorph derivatives were the piperidine derivative for C. albicans and the allylamine derivative for Cr. neoformans, M. pachydermatis and M. furfur. The inhibitor in the growth medium reduced the unsaturation index of the total lipid content in M. furfur and C. albicans.     

Malassezia pityrosporum pachydermatis (Weidman) Dodge 1935

Priority of the name Malassezia pachydermatis (Weidman) Dodge 1935 is indicated for the microorganism which has been called Pityrosporum pachydermatis Weidman 1925 and P. canis Gustafson 1955. M. pachydermatis is here further characterized in culture with information drawn from 2 recent isolates, in particular the presence of spiral grooves on the inner surface of the cell wall, good growth on Mycosel agar, rapid production of urease, and assimilation of glucose by the Wickerham method.     

Essential mycorrhizal partners of the endemic Chilean orchids Chloraea collicensis and C. gavilu

Orchids are obligate mycoheterotrophic plants, relying on fungal nutrient resources to grow for their entire life or until they develop into photosynthetic seedlings. In Chile, orchids are represented by 7 genera and 63 species, 27 of which are endemic. Some Chilean species are considered endangered or rare, but many are insufficiently known. This study aims to isolate, culture, and identify fungal species found in symbiosis with the endemic Chilean orchids Chloraea collicensis Kraenzl. and Chloraea gavilu Lindl. for their potential to be used in future conservation programs. Roots of both species of orchids were collected in the field and those presenting pelotons were firstly cultured in agar-water and thereafter sub-cultured in potato dextrose agar media. Fungal colony growth was measured under the dissecting microscope. Fungal isolates from C. gavilu showed a higher growth rate than isolates from C. collicensis and could be used as inoculum for seed germination in further studies. Isolated colonies showed morphological characteristics of the form genus Rhizoctonia and presented two nuclei per cell. The ITS-nrDNA sequences confirmed their morphological identification as species of Tulasnella.     

Subculture on potato dextrose agar as a complement to the broth microdilution assay for Malassezia pachydermatis

The main aim of this study was to verify the efficacy of subculture on potato dextrose agar (PDA) as a complement to the in vitro susceptibility test for Malassezia pachydermatis strains by a broth microdilution method, as well as to determine the MIC and MFC of azole derivatives, amphotericin B and caspofungin. The microdilution assay was performed in 96-well plates using a modified RPMI 1640 medium. The M. pachydermatis strains were resistant to caspofungin. All strains (n=50) had shown MIC values of <0.03, <0.03, 2.0, 4.0 and 4.0 mug/ml for itraconazole, ketoconazole, voriconazole, fluconazole and amphotericin B, respectively. Thus, the subculture on PDA improved the analysis of the in vitro antifungal susceptibility of M. pachydermatis.     

Distribution and characteristics of root-nodule bacteria isolated from Australian Acacia spp.

Root-nodule bacteria capable of nodulating local acacias were isolated from five climatically diverse and geographically widely separated localities in New South Wales. Strains showed marked geographic localization. Fast-growing isolates, culturally and serologically related to Rhizobium, were obtained from the arid zone but from no other area. Alpine isolates had particularly slow growth rates, with fifty percent taking longer than 10 days to form colonies on yeast mannitol agar. Strains from the rain-forest and coastal health areas had the characteristics of typical Bradyrhizobium. Most of the strains tested had a wide host range, nodulating members of both the Mimosaceae and the Fabaceae, although the extra-slow growing alpine isolates appeared specific for their original host. Isolates varied in their effectiveness with a third of strains failing to give significant weight increases in inoculated plants.     

Permanent mounts of fungus cultures grown on media optimal for production of characteristic structures

Summary 1. Dermatophyte cultures in plastic flasks were compared on four different media for total growth, sporulation, and pigment production. Czapek's agar favored more rapid and more abundant sporulation in most cultures than Sabouraud's dextrose agar, potato dextrose agar, or wort agar.2. A simple technique for embedding agar island cultures of fungi is described. These plastic-embedded cultures form permanent mounts useful for study of microscopic morphology.3. A relatively durable slant culture in a similar plastic flask is suggested as a companion for study of gross morphologic features of the cultures.     

A comparison of saline tolerance and sporulation in marine and clinical isolates of Allescheria boydii shear

Summary Two marine and 14 clinical isolates ofA. boydii and its byssoid phase,M. apiospermum, were compared with respect to their saline tolerance, morphological development, and sporulation on various commercial and compounded media. Isolates from sea-water and human infections were morphologically alike, and possessed similar saline tolerance. Most strains had salinity optima near 9, about the salinity of blood. Only marine isolates fruited well on Sabouraud's dextrose agar and other routine mycological media. On a medium containing wood and sea-water, and few soluble organics, 12 strains formed mature ascocarps or synnemata, or both. Low nutrient level media containing wood or cellulosic paper, with or without sea-water, were recommended for the propagation and conservation of sporulating cultures of many saprobic Ascomycetes and synnematous Fungi Imperfecti.     

Identification of atypical strains of Malassezia spp. from cattle and dog

Yeast species in the genus Malassezia are lipophilic with the exception of Malassezia pachydermatis. During a study of the occurrence of Malassezia species in the external ear of 964 cattle and 6 dogs in Minas Gerais, Brazil, six lipid-dependent isolates could not be identified to known species. Four isolates came from healthy cows, one from a cow with otitis, and one from a healthy dog. When tested with Tweens and Cremophor EL as single sources of lipids, the strains grew on all sources except Cremophor EL. None of the six strains hydrolyzed esculin, and all produced catalase. Pigment production from tryptophan was variable. Partial large subunit rRNA sequences were obtained for two isolates that remained viable in culture. The strain from the cow with otitis was identified as a lipid-dependent variant of M. pachydermatis, and the strain from the dog was an atypical variant of Malassezia furfur.     

Restriction Digest Patterns of Total DNA from Different Races of Fusarium oxysporum f. sp. pisi– an Improved Method for Race Classification

Seven isolates of Fusarium oxysporum f. sp. pisi were allocated to either race 2 or race 6 by their ability or inability to cause disease on the six host differentials New Season, New Era, Dark Skin Perfection, WSU 28, WSU 23 and Little Marvel. The isolates showed similar colony morphology on various solid media and their growth rates were inhibited to a similar extent by nystatin, cycloheximide and trichodermin when these compounds were included in potato dextrose agar. Total DNA waspurified from each isolate, digested separately with the restriction endonucleases EcoRI, EcoRV, Pstl, BamHl, SmaI and PvuII and the resulting DNA fragments separated by agarose gel electrophoresis. The patterns obtained were indicative of the presenceof repetitive DNA sequences. Moreover, the patterns obtained for the five race 2 isolates were identical for a given enzyme as were the patterns for the two race 6 isolates. Hence classification of isolates into races by their restrictive enzyme digestion, patterns coincided with that according to pathotypes and could be an improved, more reliable and reproducible method of race classification. An eighth isolate, originally classified as race 1, had lost its ability to cause disease on all six host differentials. The restriction digest patterns of its DNA were different from those of the DNA from both the race 2 and the race 6 isolates.     

Mode of cell growth of Malassezia (Pityrosporum) as revealed by using plasma membrane configurations as natural markers

The mode of cell growth of Malassezia was studied by freeze-fracture using the plasma membrane configurations of this organism as natural markers. The plasma membrane of the mature cell bodies of M. pachydermatis had a ring swelling, and on each side of the ring, one set of straight and spiral grooves and circumvallate bulgings. The cell always divided at the ring swelling (M. pachydermatis) or depression (M. furfur), soon followed by budding there. A new set of similar configurations formed on the bud. In all the 12 strains of Malassezia studied, the spiral grooves in the mother and bud parts were both left-handed but opposite in the direction of elongation. By comparing distances between the spiral grooves in short and long buds and in mothers, the bud tip was suggested as the major, and adjacent regions as the minor, sites of wall growth. Some characterizations of the plasma membrane invaginations, especially in relation to the mode of cell growth, were also described.     

Amino acid- or protein-dependent growth of Trichophyton mentagrophytes and Trichophyton rubrum

Culture conditions were examined for Trichophyton mentagrophytes and Trichophyton rubrum, which are major pathogens involved in dermatophytosis. They grew well in Sabouraud's dextrose broth or RPMI 1640. Growth in phosphate-buffered yeast nitrogen base supplemented with glucose was very slow, although growth improved significantly with the addition of amino acids or proteins to the medium. The fungi could also grow using human nail fragments as the only source of nutrition. Examination of proteases by substrate gel electrophoresis indicated that distinct sets of proteases are secreted from the dermatophytes in two different media, Sabouraud's dextrose broth and nail fragments. A protease inhibitor, phenylmethanesulfonyl fluoride, inhibited the growth of the fungi on nail fragments, but it did not inhibit their growth in Sabouraud's dextrose broth.     

细脚拟青霉田间分离菌株间的异核现象

本文报道细脚拟青霉(paecilomyces tenuipes)不同田间分离菌株单孢子后代间的异核现象。用来自荣园、菜地、水稻田三种生境的四个菌株(803、2801、1401和3101)的单孢子培养后代,在加有酵母膏和麦芽糖的改良萨氏培养基上进行配接实验,仅在803与1401两菌株间能形成异核体,其频率为13.5%。在配接实验中两亲和菌落交界处长出白色致密的菌丝簇组成的实线可推测为异核体。从来自菌丝簇组线的每个单菌丝尖端培养物中分离出30个以上的单孢子,井分别移接到 PDA 平板上。将来源于单孢子的菌落与两亲本菌落进行比较,有三个单菌丝尖端培养物(C_3、B_3、F_3)重现了两亲本类型或出现了新的菌落类型,从而异核现象得到证实。     

The genus Malassezia: old facts and new concepts

Lipophilic yeasts are being considered as major opportunistic pathogens for a very long time. Most of the yeasts show an absolute requirement for long fatty acid chains and specific procedures are required for their isolation, conservation and identification. For that reason, the history of the nomenclature used for the Malassezia genus is quite complex. Before 1996, only 3 species were recognized: Malassezia furfur, M. pachydermatis and M. sympodialis. To date, the genus is composed of one non lipid-dependent species (M. pachydermatis) and 12 lipid-dependent species. No doubt that additional new taxa will be described in close future. Very recently the genome and secretory proteome of two Malassezia species was described. This analysis demonstrated the presence of multiple secreted lipases to aid in harvesting host lipids. It also revealed the presence of mating-type genes, providing an indication that Malassezia yeasts may be capable of sex.     

Colony variation in Sinorhizobium meliloti inoculant strain U 45

A culture of Sinorhizobium meliloti strain U 45, maintained on yeast extract-mannitol (YM) agar, produced a mixture of Congo red-absorbing (R1) and non-absorbing (W1) colonies when grown on YM medium containing Congo red. The original freeze-dried (FD) culture formed gummy (G), white (W2) and small red (R2) colony types on the above medium. All colonies were stable except G, which segregated into G and W2-like types. Immune diffusion patterns of all colony types were identical. The W1 colony type dominated R1 when a 1:1 combination was sub-cultured on YM agar. The parent cultures and their variants exhibited a range of N₂-fixing effectiveness and competitiveness when inoculated onto two cultivars of Medicago sativa. Variant R2 from the FD culture was ineffective on both cultivars. Genomic DNA fingerprinting with insertion elements ISRm3 and ISRm2011-2 suggested that transposition of these elements was not a cause of variation, but a DNA band was absent in the profiles of two out of three W2-like colonies. Protein profile comparisons showed high similarity (r = 0.98) between the colony types when grown in YM broth. When grown on Tryptone-Yeast extract medium, variants from the FD and agar-maintained cultures formed separate clusters with r = 0.79. Polymerase chain reaction fingerprinting using repetitive, site-directed and arbitrary primers failed to differentiate the variants. The results emphasize the need to monitor culture variability to maintain the quality of legume inoculants.     

Neonatal sepsis by Malassezia furfur.

We report one case of neonatal sepsis caused by Malassezia furfur in an infant who had been in the Intensive Care Unit for 64 days. She had received prolonged therapy with intravenous fat emulsion. We used Sabouraud's medium with an overlay of sterile olive oil for the blood culture, because we had observed yeast forms in one smear of peripheral blood. M. furfur was isolated after three days of incubation. The patient recovered following removal of the port-a-cath and antifungal treatment, and had no further evidence of fungal infection. The skin colonization by the same yeast was demonstrated.