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Critical assessment of blood culture techniques: analysis of recovery of obligate and facultative anaerobes, strict aerobic bacteria, and fungi in aerobic and anaerobic blood culture bottles.

Recent reports have documented a decrease in anaerobic bacteremias and have questioned the need for routine anaerobic blood cultures. At the same time, we and others have noted an increase in fungal bloodstream infections. In this two-part study, we first compared recoveries of obligate anaerobic bacteria with those of fungi over a 13-year period and then examined the recoveries of all bacteria and fungi in aerobic and anaerobic blood culture bottles during a 12-month period. During the 13-year period, the number of patients with anaerobic bacteremia remained relatively constant (average, 39 patients per year), while the incidence of fungemia steadily increased, from 12 patients in 1978 to 117 patients in 1990. Of the 1,090 anaerobic isolates, 55.1 and 90.2% were recovered in aerobic and anaerobic bottles, respectively, compared with 98.6 and 37.0% of the 2,582 fungi. During the 12-month period of evaluation, 2,980 bacteria and fungi were recovered in cultures collected from 1,555 patients. Overall, 21.1% more organisms were recovered in aerobic bottles than in anaerobic bottles, including significantly more Staphylococcus species; gram-positive aerobic bacilli; Escherichia, Enterobacter, Pseudomonas, Xanthomonas, and Acinetobacter species; miscellaneous gram-negative bacilli; and yeasts. Only anaerobic gram-negative bacilli and non-spore-forming gram-positive bacilli were isolated more commonly in anaerobic bottles. These data support the concepts that bacteremia caused by obligate anaerobic bacteria is decreasing relative to sepsis caused by other bacteria and fungi and that the routine use of unvented anaerobic blood culture bottles reduces the recovery of common aerobic bloodstream pathogens.

Bacteremia↗

Effect of increasing inoculum sizes of pathogenic filamentous fungi on MICs of antifungal agents by broth microdilution method.

Inoculum size is a critical variable in development of methods for antifungal susceptibility testing for filamentous fungi. In order to investigate the influence of different inoculum sizes on MICs of amphotericin B, 5-fluorocytosine, itraconazole, and miconazole, 32 clinical isolates (8 Aspergillus fumigatus, 8 Aspergillus flavus, 5 Rhizopus arrhizus, 8 Pseudallescheria boydii, and 3 Fusarium solani isolates) were studied by the broth microdilution method. Four inoculum sizes were studied: 1 x 10(2) to 5 x 10(2), 1 x 10(3) to 5 x 10(3), 1 x 10(4) to 5 x 10(4), and 1 x 10(5) to 5 x 10(5) CFU/ml. The National Committee for Clinical Laboratory Standards reference method for antifungal susceptibility testing in yeasts was modified and applied to filamentous fungi. The inoculum was spectrophotometrically adjusted, and all tests were performed in buffered medium (RPMI 1640) at pH 7.0 with incubation at 35 degrees C for 72 h. MICs were read at 24, 48, and 72 h. Amphotericin B showed a minimum effect of inoculum size on MICs for all species with the exception of P. boydii (P < 0.05). A significant effect of inoculum size on MICs was observed with 5-fluorocytosine, for which there was an increase of more than 10-fold in MICs against all Aspergillus spp. between inoculum concentrations of 10(2) and 10(4) CFU/ml (P < 0.001). For itraconazole, the results showed a more species-dependent increase of MICs, most strikingly for R. arrhizus and P. boydii. Miconazole, which was tested only with P. boydii, did not demonstrate a significant effect of inoculum size on MICs. In summary, the effect of inoculum size on MICs for filamentous fungi was dependent upon the organism and antifungal compound tested. Thus, among antifungal compounds, itraconazole and 5-fluorocytosine demonstrated significant inoculum effects, while amphotericin B and miconazole showed comparatively minimum inoculum effects against pathogenic filamentous fungi. Moreover, among filamentous fungi, P. boydii and R. arrhizus exhibited the greatest inoculum effect.

Amphotericin B↗

Growth and detection of filamentous fungi in the BacT/Alert blood culture system.

Little is known about the behaviour of filamentous fungi in most blood culture systems, despite their increasingly recognised role in infections of immunocompromised hosts. The ability of the BacT/Alert system (Organon Teknika, Durham, North Carolina, USA) to detect the growth of 19 such fungi was examined. Eleven species grew and were detected rapidly; two species did not grow. Six species grew slowly, and were generally only recovered with terminal subculture after prolonged incubation. The CO2 production graph for some of these fungi showed a slow but steady rise, insufficient to cause the apparatus to signal positive. These results show that the BacT/Alert system may miss some fungi, either because of no growth in the medium or undetected slow growth. The latter problem could be overcome by prolonged incubation and terminal subculture when fungal infection is considered likely. Alteration of the signalling mechanism might permit earlier detection of some slow growing fungi.

Blood↗

Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage.

Mycelial cultures of 64 isolates of 14 species of ectomycorrhizal fungi and 27 isolates of 15 species of plant pathogenic fungi were grown on agar medium in Petri dishes. Mycelial discs, 8 mm in diameter, were removed from the cultures and stored in sterile distilled water in test tubes at 5 degrees C. Sixty-four, 61, and 41 isolates of the symbiotic fungi were viable after 1, 2, and 3 years storage respectively. Only 19, 10, and 8 isolates of the pathogenic fungi were viable after 1, 2, and 3 years storage, respectively. Time in pure culture before water storage did not affect viability of any fungal species following water storage. After 3 years storage, four fungi (three symbionts and one pathogen) were tested and found to have retained their original growth rates and root-infecting abilities on pine seedlings. The same four isolates, however, maintained on agar slants at 5 degrees C and subcultured every 4 to 6 months, grew slower and did not infect as many feeder roots of pine as the water-stored isolates.

Cold Temperature↗

Lignin degrading system of white-rot fungi and its exploitation for dye decolorization.

With global attention and research now focused on looking for the abatement of pollution, white-rot fungi is one of the hopes of the future. The lignin-degrading ability of these fungi have been the focus of attention for many years and have been exploited for a wide array of human benefits. This review highlights the various enzymes produced by white-rot fungi for lignin degradation, namely laccases, peroxidases, aryl alcohol oxidase, glyoxal oxidase, and pyranose oxidase. Also discussed are the various radicals and low molecular weight compounds that are being produced by white-rot fungi and its role in lignin degradation. A brief summary on the developments in research of decolorization of dyes using white-rot fungi has been made.

Coloring Agents↗

Toxins of filamentous fungi.

Mycotoxins are low-molecular-weight secondary metabolites of fungi. The most significant mycotoxins are contaminants of agricultural commodities, foods and feeds. Fungi that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate (i.e. conditions favorable for fungal growth), they can also be found in temperate conditions. Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions and microbial, insect or other animal interactions. Although outbreaks of mycotoxicoses in humans have been documented, several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo. Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age and nutritional status. Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs. Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from the foods and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities. This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest. Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems. Current research is designed to develop natural biocontrol competitive fungi and to enhance host resistance against fungal growth or toxin production. These efforts could prevent toxin formation entirely. Rigorous programs for reducing the risk of human and animal exposure to contaminated foods and feed also include economically feasible and safe detoxification processes and dietary modifications. Although risk assessment has been made for some mycotoxins, additional, systematic epidemological data for human exposure is needed for establishing toxicological parameters for mycotoxins and the safe dose for humans. It is unreasonable to expect complete elimination of the mycotoxin problem. But multiple approaches will be needed to minimize the economic impact of the toxins on the entire agriculture industry and their harmfulness to human and animal health.

Aflatoxins↗

Current indoor allergen levels of fungi and cats, but not house dust mites, influence allergy and asthma in adults with high dust mite exposure.

We assessed the influence of current indoor levels of fungi, house dust mite allergen (Der p 1), and cat allergen (Fel d 1) on sensitization and asthma in adults. A total of 485 adults answered a questionnaire and had skin prick tests and lung function tests. Dust and air samples were collected from their bedrooms. The dust was analyzed for Der p 1, Fel d 1, and fungal biomass (ergosterol). Fungal propagules were measured in air samples. Current asthma was defined as having wheezed during the past 12 mo plus bronchial hyperreactivity (BHR) to methacholine. High exposure to total airborne fungi was associated with increased BHR, but perhaps paradoxically with a lower risk of being sensitized to fungi. Ergosterol levels in floor dust were a risk factor both for being sensitized to fungi and having wheezed within the last year. High Fel d 1 levels in floor dust were found to increase the risk of being sensitized to cats and in beds to increase the risk of current asthma. Although Der p 1 levels in homes were high, people exposed to high Der p 1 levels in floor dust were less likely to be sensitized to house dust mites or to have wheezed within the past year. Current indoor levels of fungi and Fel d 1, but not Der p 1, influenced sensitization and asthma in adults with high dust mite exposure.

Adult↗

Effect of fungi fermentation on organoleptic properties, energy content and in-vitro multienzyme digestibility of cassava products (flour & gari).

The present study sought to investigate the effect of fungi fermentation on the energy content, sensory quality and the digestibility (in vitro) of cassava products (flour and gari). The fungi fermented cassava products (gari and flour) were produced, by fermenting cassava mash with pure strains of some common saprophytes, namely, Aspergillus flavus, Aspergillus niger, Rhizopus oryzae and Saccharomyces spp (Baker's yeast and palm wine yeast) for 72 hrs before processing into cassava flour and gari, the forms in which cassava is popularly consumed in Nigeria. Parameters determined include energy (Bomb calorimetry), digestibility (in vitro) and sensory quality by trained taste panel. The results of the study indicated that fungi fermentation of the cassava mash significantly (P < 0.05) increased the acceptability of the colour, texture, aroma and taste of the "gari", with that of Rhizopus oryzae fermentation having the highest general acceptability. Furthermore, the results also indicated that fungi fermentation of cassava mash significantly increased (P < 0.05) the in vitro multienzyme protein digestibility of the cassava products. In view of this, fungi fermentation could be used to improve the sensory quality and protein digestibility of cassava products without any significant (P > 0.05) effect on the energy-giving role of cassava products.

Aspergillus flavus↗

Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria.

The association of arbuscular mycorrhizal (AM) fungi with plant roots is the oldest and ecologically most important symbiotic relationship between higher plants and microorganisms, yet the mechanism by which these fungi detect the presence of a plant host is poorly understood. Previous studies have shown that roots secrete a branching factor (BF) that strongly stimulates branching of hyphae during germination of the spores of AM fungi. In the BF of Lotus, a strigolactone was found to be the active molecule. Strigolactones are known as germination stimulants of the parasitic plants Striga and Orobanche. In this paper, we show that the BF of a monocotyledonous plant, Sorghum, also contains a strigolactone. Strigolactones strongly and rapidly stimulated cell proliferation of the AM fungus Gigaspora rosea at concentrations as low as 10(-13) M. This effect was not found with other sesquiterperne lactones known as germination stimulants of parasitic weeds. Within 1 h of treatment, the density of mitochondria in the fungal cells increased, and their shape and movement changed dramatically. Strigolactones stimulated spore germination of two other phylogenetically distant AM fungi, Glomus intraradices and Gl. claroideum. This was also associated with a rapid increase of mitochondrial density and respiration as shown with Gl. intraradices. We conclude that strigolactones are important rhizospheric plant signals involved in stimulating both the pre-symbiotic growth of AM fungi and the germination of parasitic plants.

Daucus carota↗

Biolistic transformation of arbuscular mycorrhizal fungi. Progress and perspectives.

Gene transfer systems have proved effective for the transformation of a range of organisms for both fundamental and applied studies. Biolistic transformation is a powerful method for the gene transfer into various organisms and tissues that have proved recalcitrant to more conventional means. For fungi, the biolistic approach is particularly effective where protoplasts are difficult to obtain and/or the organisms are difficult to culture. This is particularly applicable to arbuscular mycorrhizal (AM) fungi, being as they are obligate symbionts that can only be propagated in association with intact plants or root explants. Furthermore, these fungi are aseptate and protoplasts cannot be released. Recent advancements in gene transformation systems have enabled the use of biolistic technology to introduce foreign DNA linked to molecular markers into these fungi. In this review we discuss the development of transformation strategies for AM fungi by biolistics and highlight the areas of this technology which require further development for the stable transformation of these elusive organisms.

Biophysics↗

15N in symbiotic fungi and plants estimates nitrogen and carbon flux rates in Arctic tundra.

When soil nitrogen is in short supply, most terrestrial plants form symbioses with fungi (mycorrhizae): hyphae take up soil nitrogen, transport it into plant roots, and receive plant sugars in return. In ecosystems, the transfers within the pathway fractionate nitrogen isotopes so that the natural abundance of 15N in fungi differs from that in their host plants by as much as 12% per hundred. Here we present a new method to quantify carbon and nitrogen fluxes in the symbiosis based on the fractionation against 15N during transfer of nitrogen from fungi to plant roots. We tested this method, which is based on the mass balance of 15N, with data from arctic Alaska where the nitrogen cycle is well studied. Mycorrhizal fungi provided 61-86% of the nitrogen in plants; plants provided 8-17% of their photosynthetic carbon to the fungi for growth and respiration. This method of analysis avoids the disturbance of the soil-microbe-root relationship caused by collecting samples, mixing the soil, or changing substrate concentrations. This analytical technique also can be applied to other nitrogen-limited ecosystems, such as many temperate and boreal forests, to quantify the importance for terrestrial carbon and nitrogen cycling of nutrient transfers mediated by mycorrhizae at the plant-soil interface.

Arctic Regions↗

Direct invasion of bones by highly pathogenic fungi in an in vitro model and its ecological significance.

Animal bones after being devitalized at death are strongly resistant to wear and tear and remain in the soil or environment much longer than other organic components from dead animals. Yet over the course of time they seem to disappear and thus our ecological surroundings are not cluttered with bone remnants. Mechanical factors creating compression or friction and chemical factors like pH of the soil and surroundings must together have provided concerted degrading effects. Microorganisms in the soil also help in this process by utilizing the organic components of devitalized bones. Certain highly pathogenic fungi that have been collected from soil from time to time and many other environmental fungi may take part in the degrading of the bone remnants. In this study, several strains from the highly pathogenic dimorphic fungi Coccidioides immitis, Blastomyces dermatitidis, Histoplasma spp., Paracoccidioides brasiliensis and also some strains of dematiaceous fungi (Exophiala spp. and Foncecaea pedrosoi) were inoculated to dissected and devitalized murine long bones that had been placed on solidified water agar plates to see if they would survive, grow and invade the bones. After being kept for 12 weeks at 25 degrees C all the parts of the histological sections of these bones showed invasion by most of the strains used in this study, although the cortical component of the bony architecture seemed to be comparatively resistant to invasion. Their ability to grow and sporulate in the aforementioned nutrient-limiting condition hinted at a possible role of these fungi in the degradation of devitalized bones.

Animals↗

[Molecular taxonomy and identification of pathogenic fungi based on DNA sequence analysis].

Although approximately 80,000 fungi are known, less than 1% are associated with human infection. However, their taxonomy has long been insufficient. During the last decade, DNA sequence analysis was introduced to the taxonomy of pathogenic fungi. Taxonomic advances in the field of medical mycology are helping to identify the causative agents of infectious diseases accurately, facilitating diagnosis and treatment. For example, Malassezia furfur was long considered the major microflora in atopic dermatitis, yet recent studies have indicated that this is not the case, as M. furfur is taxonomically heterogeneous and consists of five species. DNA sequence analysis resolved its taxonomic heterogeneity. Similar examples can be seen in "Candida albicans and C. dubliniensis" and "Trichosporon cutaneum and T. asahii". DNA sequence analysis also enables accurate identification of fungi. At present, almost all pathogenic fungi can be identified by determining the D1/D2 26S rDNA and ITS region of rRNA gene. This paper describes the practical taxonomy and identification of pathogenic fungi based on DNA sequence analysis.

Fungi↗

Hydrolytic enzymes and ability of arbuscular mycorrhizal fungi to colonize roots.

The production of hydrolytic enzymes from external mycelia associated with roots and colonized soybean roots (Glycine max L.) inoculated with different arbuscular-mycorrhizal (AM) fungi of the genus GLOMUS:, and the possible relationship between these activities and the capacity of the AM fungi to colonize plant roots was studied. There were differences in root colonization and plant growth between the GLOMUS: strains, and also between two isolates of G. mosseae. Hydrolytic activities in the root and external mycelia associated with roots differed in the AM fungi tested. Correlations were only found between the endoxyloglucanase activity of the external mycelia associated with roots of the AM fungi tested and the percentage root colonization or plant growth. However, hydrolytic activities of roots colonized by the different endophytes correlated with those of external mycelia. The hydrolytic activities were not qualitatively different because the endoxyloglucanase from AM colonized roots and the external mycelia did not show a high degree of polymorphism in the different species of fungus tested. The possible role of the hydrolytic activity of external hyphae of AM fungi was discussed as a factor affecting fungal ability to colonize the root and influence plant growth.

Enzymes↗

Indoor air quality, fungi, and health. How do we stand?

OBJECTIVE: To equip medical practitioners with up-to-date scientific and medical information on the health effects of exposure to fungi in indoor air, clinical evaluation of these health problems, and possible preventive measures. QUALITY OF EVIDENCE: MEDLINE was searched from 1985 to 2000 using the MeSH words mould (mold), fungus, indoor air, and health effects. Nearly all studies were case reports, case-control studies, and cross-sectional studies. Evidence of an association between respiratory problems and the presence of fungi and dampness is strong. MAIN MESSAGE: Recent well designed studies and literature reviews indicate that exposure to dampness and fungi in indoor air brings on or exacerbates asthma and other respiratory complaints. More studies are required, however, before a definite conclusion on other potential effects of such exposure (such as systemic and long-term effects and pulmonary hemorrhage in infants) is possible. The various health problems that can result from exposure to dampness and fungi in indoor air make such exposure unacceptable from a public health perspective. Physicians are important in treating and preventing such problems; various resources are available to help them. CONCLUSION: Even though some scientific issues remain to be resolved concerning the health effects of exposure to dampness and fungi in indoor air, family physicians can identify potential problems and refer patients to appropriate resources.

Air Pollution, Indoor↗

Fungi isolated from asthmatic homes in the Taipei area.

Indoor fungi in Taipei were surveyed during 1969-1974 using Sabouraud's agar plates. No remarkable yearly variation of fungal incidence was observed. The fungal population was found to be different from home to home. The dominant members were Aspergillus, Penicillium, Mycelia sterila, Oospora, Rhodotorula, Hormodendrum and yeasts. The order of frequency of fungi found indoors was quite different from that of outdoor fungi. The fungal spores were more numerous during summer months, and this trend was more apparent for Aspergillus, Oospora, Rhodotorula, and Hormodendrum. The kind and the incidence of fungi found in asthmatic homes were not different from those detected in non-asthmatic homes, except that in asthmatic homes there were greater numbers of fungi.

Air Microbiology↗

[Comparative studies on the fatty acids contained in four species of medicinal plants from family Euphorbiaceae and their endophytic fungi].

OBJECTIVE: The relation of four species of medicinal plants from family Euphorbiaceae and their endophytic fungi was studied to find the source of active substances for developing new pharmaceutical resources. METHOD: The main fatty acids contained in Sapium sebiferum, Euphorbia pekinensis, Euphorbia helioscopia, Bischofia polycarpam and their 28 strains of endophytic fungi were compared and analysed by GC. RESULT: The main fatty acids of the plants are: alpha-linolenic acid, palmitic acid, linolenic acid and oleic acid. Linolenic acid, palmitic acid and oleic acid are the main fatty acids of the endophytic fungi. CONCLUSION: The fatty acids could be produced by the endophytic fungi, which could be used as a factor for identification. There are great differences at the contents of alpha-linolenic acid between the plants and their endophytic fungi, which were suggested to be related with the nutrition absorption and the relationship between the endophytes and the host plant.

Euphorbia↗

[Fungi growth in buildings].

The results of research into occurrence of fungi in buildings are hereby presented. Indoor air pollution balanced between 10(2)-10(3) cfu/m3. Examined building surfaces have been found significantly mouldy. Total number of fungi ranged from 3.28 x 10(5) to 9.75 x 10(10) cfu/100 cm2. The results of the research show the active development of this group of microorganisms. In rooms, over 30 fungi species belonging to 11 genera have been detected. The most frequently occurring ones were fungi genera: Cladosporium, Penicillium, Aspergillus and Acremonium. Among those, potentially toxinogenic species have been separated (Aspergillus ochraceus, Cladosporium cladosporioides, Penicillium chrysogenum, Penicillium notatum). Apart from micotoxins they have been found to produce huge amounts of conidia which can cause allergy in sensitive people. Hence, people should definitely avoid fungi in their environment.

Air Pollution, Indoor↗