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Biosynthesis of ergothioneine and hercynine by fungi and Actinomycetales.

Unlike other bacteria, aerobic members of the order Actinomycetales show a close biochemical relationship to the fungi by their capacity to synthesize hercynine and ergothioneine. The myxomycete Physarum polycephalum, possessing the same synthetic ability, also shows this relationship. Contrariwise, the unusual position of yeasts as fungi is indicated by the inability of all yeastlike Ascomycetes and all except a few false yeasts to synthesize these two betaines.

Actinomycetales↗

Effect of substrate on the fatty acid composition of hydrocarbon-utilizing filamentous fungi.

The fatty acid pattern in hydrocarbon-utilizing filamentous fungi was determined after growth on acetate, propionate, n-alkanes (C(13) to C(15)), and alk-1-enes (C(14) to C(18)). The fatty acid profile of Cunninghamella elegans and Penicillium zonatum after growth on acetate shows a predominance of even-carbon fatty acids (C(16), C(18:1), C(18:2)), whereas cells grown on propionate showed significantly higher levels of odd-carbon fatty acids (C(15), C(17), C(17:1)). Growth on n-alkanes resulted in the incorporation of fatty acids homologous to the growth substrate. Cunninghamella elegans grown on the alk-1-enes from C(14) to C(18) incorporated the unsaturated substrate into cellular fatty acid after oxidation at the saturated end of the molecule. Regardless of substrate these fungi contain, predominantly, fatty acids 18 carbons in length.

Acetates↗

Production of extracellular ribonuclease by yeasts and yeastlike fungi, and its repression by orthophosphate in species of Cryptococcus and Tremella.

A strain of Cryptococcus laurentii and a haploid isolate of Tremella foliacea were shown to produce orthophosphate-repressible ribonuclease in liquid culture. Addition of as little as 1 mM K2HPO4, pH 7.0, completely repressed enzyme production by both fungi. The orthophosphate-repressible enzyme was not produced by other species of the two genera tested. These results, together with other findings, suggest a close phylogenetic relationship between Cryptococcus laurentii and Tremella foliacea. The ability of other yeasts and yeastlike fungi to hydrolyze ribonucleic acid in a solid test medium was assessed. Based on the limited number of organisms available for study, extracellular ribonuclease activity was found in species having close affinity to the Basidiomycetes and in yeasts classified in the ascomycetous genera, Endomycopsis, Hansenula, and Kluyveromyces. Other ascomycetous yeasts did not exhibit extracellular ribonuclease.

Ascomycota↗

FLUORESCENT-ANTIBODY STUDIES OF THE MYCELIAL FORM OF HISTOPLASMA CAPSULATUM AND MORPHOLOGICALLY SIMILAR FUNGI.

Kaufman, Leo (Communicable Disease Center, Atlanta, Ga.), and Brenda Brandt. Fluorescent-antibody studies of the mycelial form of Histoplasma capsulatum and morphologically similar fungi. J. Bacteriol. 87:120-126. 1964.-Fluorescent-antibody reagents were developed to differentiate the mycelial form of the pathogen, Histoplasma capsulatum, from several morphologically similar but saprophytic species of the genera Sepedonium and Chrysosporium. Cross-staining and adsorption procedures revealed antigenic relationships among certain species of these three genera and other fungi. The significance of these observations to the diagnostician and taxonomist is discussed.

Coloring Agents↗

BIOCHEMICAL CHANGES DURING THE GROWTH OF FUNGI. I. NITROGEN COMPOUNDS AND CARBOHYDRATE CHANGES IN PENICILLIUM ATROVENETUM.

Gottlieb, David (University of Illinois, Urbana), and James L. Van Etten. Biochemical changes during the growth of fungi. I. Nitrogen compounds and carbohydrate changes in Penicillium atrovenetum. J. Bacteriol. 88:114-121. 1964.-Changes in the biochemical constituents of cells were studied during the growth and development of Penicillium atrovenetum. Growth of the fungus, as measured by the dry weight, could be divided into four phases: lag, log, stationary, and death. The percentages of total nitrogen, cold trichloroacetic acid-soluble nitrogen, ribonucleic acid (RNA), and protein increased to a maximum during the lag phase, and subsequently decreased as the fungus aged. The percentage of deoxyribonucleic acid (DNA) was always slightly higher in the spores than in the mycelium. The DNA in the mycelium decreased in the lag phase, and then increased slightly to a plateau for the duration of the log phase, followed by a decrease to a constant percentage during the stationary and death phases. Carbohydrates were present in higher concentration in the mycelium than in the spores. The percentage of carbohydrates in the mycelium increased continually until it reached a maximum late in the log phase, and then decreased as the fungus entered the death phase. The results reported for this fungus are, in general, in agreement with those reported for other microorganisms. Namely, the percentages of enzyme-forming compounds, such as amino acids, nucleotides, RNA, and protein, were highest in the lag phase, whereas storage compounds such as carbohydrates increased to a maximum near the end of the log phase. The definition of log phase in fungi depends on the criteria that are used. If, instead of using the linear increase in dry weight to delimit this growth period, one uses the end of net protein, RNA, and DNA synthesis, a more realistic concept of growth emerges.

Biochemical Phenomena↗

BIOCHEMICAL CHANGES DURING THE GROWTH OF FUNGI. II. ERGOSTEROL AND FATTY ACIDS IN PENICILLIUM ATROVENETUM.

Van Etten, James L. (University of Illinois, Urbana), and David Gottlieb. Biochemical changes during the growth of fungi. II. Ergosterol and fatty acids in Penicillium atrovenetum. J. Bacteriol. 89:409-414. 1965.-Changes in the lipid constituents of Penicillium atrovenetum were studied during the growth and development of this fungus. The stages in development, as measured by the dry weight, were divided into four phases: lag, log, stationary, and death. The total fatty acids on a dry-weight basis increased from a minimum in the spores to a maximum near the end of the log phase of growth. The major fatty acids were palmitic, stearic, oleic, and linoleic. Myristic, pentadecanoic, palmitoleic, heptadecanoic, linolenic, arachidic, and heptadecenoic acids, together with two unidentified components, were also present in the fatty acid fraction. Compared to ungerminated spores, young mycelium contained a much lower percentage (on the basis of total fatty acids) of linoleic acid. There was a corresponding increase of oleic acid. Except for palmitic acid, which remained constant, the remaining fatty acids increased slightly. During subsequent growth of the fungus, linleic acid decreased, whereas the percentage of palmitic and stearic acids increased steadily from the lag phase to the end of the log phase. Ergosterol was the only sterol detected. The percentage of ergosterol, on a dry-weight basis, increased to a maximum at the start of the log phase and then steadily decreased. The following changes in lipids appear to be associated with the development and aging of fungi: (i) the presence of a relatively high content of nonsaponifiable lipid and ergosterol in the young mycelium, and their later decrease with age, and (ii) a shift from more unsaturated to less unsaturated fatty acids with age.

Ergocalciferols↗

Effects of time lapse between sputum collection and culturing on isolation of clinically significant fungi.

A retrospective study of 34,314 sputum specimens received by a reference laboratory over a 10-year period demonstrated that clinically significant fungi could be isolated even after long periods of delay between collecting and culturing. As a result of this study, it should be stressed that although immediate culturing for fungi is the ideal, specimens should not be rejected because of delays in transport.

Fungi↗

Detection of fungi in clinical specimens by phase-contrast microscopy.

During 1973 and 1974, the following fungi were detected in clinical specimens by using phase-contrast microscopy: Blastomyces dermatitidis, 5; Coccidioides immitis, 3; Cryptococcus neoformans, 11; other yeasts 918; dermatophytes, 863; Mucor species, 1; and Aspergillus fumigatus, 16. This technique allows rapid detection and, in many instances, immediate identification of fungi in clinical specimens.

Arthrodermataceae↗

Exoantigen test for differentiation of Exophiala jeanselmei and Wangiella dermatitidis isolates from other dematiaceous fungi.

Concentrated (25X) exoantigens of 105 isolates of pathogenic and saprophytic dematiaceous fungi and 3 isolates of Sporothrix schenckii were analyzed by the microimmunodiffusion method. The reagents used were nonadsorbed and adsorbed sera produced in New Zealand rabbits. One set of rabbits was immunized with soluble antigens of a 1-month-old culture of Exophiala jeanselmei (ATCC 34123), and the other set was immunized with soluble antigens from a culture of Wangiella dermatitidis (ATCC 28869). The reference antigens were 25X-concentrated exoantigens of the above cultures. This exoantigen test permitted the differentiation of E. jeanselmei and W. dermatitidis from one another as well as from other Exophiala species, Fonsecaea species, Phialophora species, Cladosporium species, Rhinocladiella species, and Sporothrix schenckii by presence or absence of lines of identity or of partial identity, or lines of nonidentity. Using adsorbed serum eliminated the problems with cross-reactivity seen with nonadsorbed serum. Thus, with an adsorbed serum as the reagent, it was possible to presumptively differentiate E. jeanselmei and W. dermatitidis from one another and from other dematiaceous fungi.

Antigens, Fungal↗

Comparative antigenic studies of species of Basidiobolus and other medically important fungi.

An immunodiffusion technique was used to evaluate the antigenic relationship of various pathogenic and saprobic Basidiobolus spp., Conidiobolus spp., isolates of the order Mucorales, and several other medically important nonzygomycetous fungi. The antiserum to Basidiobolus haptosporus shared two lines of identity, designated inner (N) and outer (Y), when tested against exoantigens of known strains of B. haptosporus and Basidiobolus ranarum as well as exoantigens of a human Nigerian isolate and several wild isolates tentatively identified in B. ranarum. Both bands were heat stable at 56 degrees C for 30 min. Exoantigens of strains of Basidiobolus meristosporus, Basidiobolus microsporus, Conidiobolus incongruus, Conidiobolus coronatus, and other wild isolates of Basidiobolus spp. tested formed only the N immunoprecipitin band. Exoantigens of 10 isolates from other taxa did not produce any cross-reactive precipitin line. B. meristosporus antiserum that was tested against exoantigens of Basidiobolus spp. and of Conidiobolus spp. developed only an N band without a Y band. These data suggest that B. haptosporus and B. ranarum are antigenically similar to each other and distinct from B. meristosporus. Basidiobolus spp. and Conidiobolus spp. share a common N immunoprecipitin band, which implies a taxonomic relationship between these two genera. The absence of lines of identity between Basidiobolus spp. and other fungi tested suggests that, antigenically, Basidiobolus is a distinct genus.

Antibodies, Fungal↗

Morphology and ultrastructure of fungi in extended-wear soft contact lenses.

Filamentous fungi of the genera Acremonium, Aspergillus, Alternaria, Cladosporium, Curvularia, and Fusarium penetrated the matrix of soft contact lenses both during normal usage and in laboratory studies. Growth of the fungi within the lens matrix increased with increasing water content of the lens. Hyphae within the lens were coiled. Some species penetrated completely through the lens within 96 h. More frequent cleaning and disinfection of extended-wear soft contact lenses is recommended.

Acremonium↗

New fluorescence assay for the quantitation of fungi.

Quantitative determination of fungal mass is easily achieved with a new procedure that detects particle epifluorescence. Fungi are detected after exposure to a fluorescent stain (Fungiqual; CIBA-GEIGY Corp., Summit, N.J.) by using a fluorescence particle concentration analyzer. This report describes a simple fluorescence method for quantitation of either yeast or mycelial forms of fungi. The nature of the staining reaction was studied, and a practical application of this procedure for determination of fungal susceptibility to an antifungal agent is presented.

Amphotericin B↗

Effect of cetylpyridinium chloride on pathogenic fungi and Nocardia asteroides in sputum.

The effect of cetylpyridinium chloride (CPC) on pathogenic fungi and Nocardia asteroides was studied. Sputa seeded with each of 11 organisms (Aspergillus flavus; Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Geotrichum candidum, Histoplasma capsulatum; Nocardia asteroides, Paracoccidioides brasiliensis, and Sporothrix schenckii) were treated with CPC and kept for 2, 5 and 9 days. The CPC reagent used (0.5% CPC and 0.5% sodium chloride) is one the Mycobacteriolgoy Branch at the Center for Disease Control added to sputa before shipping them to laboratories for recovery of mycobacteria. None of the organisms tested survived this treatment, and none was recovered on mycological or mycobacteriological media. Seeded sputa containing these organisms were also tested with a second CPC reagent (0.02% CPC and 0.5% sodium chloride) and held for 2, 5 and 9 days. A few colonies of A. flavus, A. fumigatus, and N. asteroides were recovered from these samples. Neither the morphology of the fungi nor their stainability by the fluorescent antibody method was affected by treatment with the reagent containing 0.5% CPC. However, the background material in smears from the 0.5% CPC-treated samples retained the conjugate, and this made weakly fluorescing organisms more difficult to detect. The 0.5% CPC treatment did not alter the morphology of N. asteroides or its ability to be stained with Kinyoun acid-fast stain.

Cetylpyridinium↗

Clinical impact of bacteria and fungi recovered only from broth cultures.

We prospectively evaluated 356 bacteria and fungi recovered from broth enrichment tubes from cultures with sterile direct plates to determine the clinical impact of isolates recovered only from broth cultures. These "broth only" isolates (BOI) were classified as contaminants or true on the basis of review of patient charts. True isolates were considered clinically relevant only if they altered or should have altered patient management. Of 356 BOI, 259 (73%) were considered contaminants (mostly coagulase-negative staphylococci and Propionibacterium spp.) and 97 (27%) were considered true. For individual microorganisms, 9 of 9 (100%) Staphylococcus aureus isolates, 13 of 13 (100%) members of the family Enterobacteriaceae, 10 of 12 (83%) fungi, 7 of 10 (70%) enterococci, 7 of 11 (64%) other gram-negative bacilli, 13 of 31 (45%) anaerobic bacteria, 10 of 24 (42%) streptococci, 22 of 140 (16%) coagulase-negative staphylococci, 6 of 92 (7%) Propionibacterium spp., and 0 of 14 (0%) diphtheroids and Bacillus spp. were classified as true. Eleven of 97 (11%) patients with true BOI had clinically relevant isolates. Fifty-nine of the 97 (61%) patients with true isolates already were on therapy, and no change was made because of the BOI. Six (6%) patients with contaminants received therapy for their BOI. We conclude that broth inoculated as an adjunct to direct plating seldom yields results that favorably alter patient management and could be omitted for most specimens without compromising patient care.

Adult↗

Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system.

Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates. PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length. We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes. No cross-reaction occurred when samples were tested against human and bacterial genomic DNA. We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections.

Aspergillus↗

Inoculum standardization for antifungal susceptibility testing of filamentous fungi pathogenic for humans.

Two methods of inoculum preparation for filamentous fungi were compared: counting with a hematocytometer and spectrophotometric adjustment. One hundred eighty-two filamentous fungi pathogenic for humans were used. Colony counts were done for all inoculum preparations. The agreement between the hematocytometer counts and the colony counts (CFU per milliliter) was 97.2%. The reproducibility between the hematocytometer counts and the colony counts by means of an intraclass correlation coefficient was 0.70. Pearson's correlation index for hematocytometer counts versus colony counts was 0.56, whereas that for optical density versus colony counts was 0.008. Both methods can be used for inoculum size adjustment. However, the use of the spectrophotometric method requires that each species be standardized separately.

Antifungal Agents↗

Comparison of the E-test with the NCCLS M38-P method for antifungal susceptibility testing of common and emerging pathogenic filamentous fungi.

The National Committee for Clinical Laboratory Standards (NCCLS) M38-P method describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds). The present study evaluated the in vitro fungistatic activities of itraconazole and amphotericin B by the E-test and the NCCLS M38-P microdilution method against 186 common and emerging pathogenic molds (123 isolates of Aspergillus spp. [five species], 16 isolates of Fusarium spp. [two species], 4 Paecilomyces lilacinus isolates, 5 Rhizopus arrhizus isolates, 15 Scedosporium spp., 18 dematiaceous fungi, and 5 Trichoderma longibrachiatum isolates). The agreement between the methods for amphotericin B MICs ranged from 70% for Fusarium solani to > or =90% for most of the other species after the first reading; agreement was dependent on both the incubation time and the species being evaluated. Major discrepancies between the amphotericin B MICs determined by the E-test and the NCCLS M38-P method were demonstrated for three of the five species of Aspergillus tested and the two species of Fusarium tested. This discrepancy was more marked after 48 h of incubation; the geometric mean MICs determined by the E-test increased between 24 and 48 h from between 1.39 and 3.3 microg/ml to between 5.2 and >8 microg/ml for Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans. The agreement between the itraconazole MICs determined by the E-test and the NCCLS M38-P method ranged from 83.3% for A. nidulans to > or =90% for all the other species tested; the overall agreement was higher (92.7%) than that for amphotericin B (87.9%). The agreement was less dependent on the incubation time. Clinical trials need to be conducted to establish the role of the results of either the E-test or the NCCLS M38-P method in vitro for molds with the two agents as predictors of clinical outcome.

Amphotericin B↗