Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca²⁺ concentrations in the mutant strain Aspergillus awamori 66A containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity of sporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 μM) stimulated spore germination: the number of germinating spores was 2–3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 μM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca²⁺ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 μM) were shown to cause an instantaneous increase in Ca²⁺ concentrations compared to the control and lower amphotericin concentration (2.5 μM). Ca²⁺ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.     

Antifungal pharmacodynamic characteristics of amphotericin B against Trichosporon asahii, using time-kill methodology

We determined the MIC of amphotericin B against 45 Trichosporon asahii isolates from various clinical and environmental sources, and used in vitro time-kill methods to characterize the relationship between amphotericin B concentrations and MIC for four representative T. asahii isolates. Amphotericin B had concentration-dependent antifungal activity. MICs ranged from 0.5 to 16 microg/ml, and most T. asahii isolates (76%, 34/45) were inhibited at safely achievable amphotericin B serum concentrations (< or = 2 microg/ml). However, 40% (18/45) of isolates were not killed at these concentrations (MFCs from 1.0 to 32 microg/ml). At concentrations > or = 2 x MIC, amphotericin B exhibited fungicidal activity (< 99.9% reduction in CFU) over a 12-hr time-period; the maximal effect was achieved at > or =4 x MIC. Susceptibility testing confirmed the resistance of T. asahii to amphotericin B, and in vitro pharmacodynamic results also suggest that amphotericin B is not suitable therapy for T. asahii infection.     

In vitro activity of fluconazole and amphotericin B against Candida inconspicua clinical isolates as determined by the time-kill method

Candida inconspicua is an emerging pathogen in immunocompromised patients possessing inherently decreased susceptibility to fluconazole. We determined the MICs and killing activity of fluconazole and amphotericin B against C. inconspicua clinical isolates as well as reference strain C. inconspicua ATCC 16783 for comparison. MICs were determined using the standard broth microdilution method. Killing rates were determined using time-kill methodology at 0.5-16 x MIC fluconazole and amphotericin B concentrations. Fluconazole and amphotericin B MIC values varied between 16-128 mg/l and 0.5-1 mg/l, respectively. In time kill-assays fluconazole showed fungistatic effect at 1-16 x MIC concentrations against all tested strains after 24 h-incubation, but became fungicidal after 48 h at 4-16 x MIC concentrations. The time necessary to achieve fungicidal endpoint at 1 mg/l amphotericin B concentration ranged from 2 to 24 h. Our in vitro results confirm the data that fluconazole is ineffective against C. inconspicua at the fluconazole serum concentration attainable in humans. Amphotericin B due to its rapid killing activity seems to be a good alternative for the treatment of infections caused by C. inconspicua.     

Selection of a Highly Virulent Fungal Isolate,Metarhizium anisopliae Ma TB 101, for Control of Taro Beetle,Papuana uninodis (Coleoptera: Scarabaeidae)

Fungal isolates (31 Metarhizium anisopliae var. anisopliae , five M. anisopliae var majus , three Beauveria bassiana and four B. brongniartii ) originating from a wide range of geographical locations, insect species and soil types were tested against Papuana uninodis (Coleoptera: Scarabaeidae). In a first test series, spores were applied topically to third-instar larvae and adults. The most effective strain against P. uninodis larvae and adults was Ma TB 101, a M. anisopliae isolate from adult P. woodlarkiana . For adults, strain Ma FI 384, a M. anisopliae from Popillia japonica , was almost equally effective. The 11 most effective isolates (nine M. anisopliae var. anisopliae , one B. brongniartii and one B. bassiana ) with LT values of less than 6 weeks in 50 adults and/or less than 4 weeks in larvae were tested for their efficacy against adults and larvae of P. uninodis by application of spores to soil (107 spores/g). Ma TB 101 was significantly more effective against both adults and larvae (LT ca. 10 days) than all other isolates (LT > 50 50 3 weeks). Two other M. anisopliae strains, Ma F 248 from soil and Ma FI 384 from P.japonica , were more effective than most isolates in adults. The latter three M. anisopliae isolates were tested in a concentration series against third-instar larvae and adults. Mortality was concentration related. Isolates Ma F 248 and Ma FI 384 did not achieve 50% mortality within the test period at concentrations lower than 107 spores/g of soil or feed. For concentrations of Ma TB 101 from 1 107 to 2 105 spores/g the LT ranged from 13 to 30 days in adults and 12 to 24 50 days in third-instar larvae. Accordingly, concentrations causing 50% mortality (LC ) for Ma 50 TB 101 were significantly lower than for the two other M. anisopliae isolates tested.     

In vitro antifungal susceptibility of clinical isolates of Cryptococcus neoformans var. neoformans and C. neoformans var. gattii

One of the differences observed between the two varieties of Cryptococcus neoformansis the greater difficulty to achieve an adequate therapeutical response in patients affected by C. neoformans var. gattii, an observation that has been validated in vitro only rarely. The aim of this work was to study the susceptibility patterns of 35 Colombian clinical isolates of C. neoformans, 20 of which belonged to the var. neoformans and 15 to the var. gattii. The minimal inhibitory concentration (MIC) was determined by broth microdilution, according to a modification of the methodology proposed by the National Committee for Clinical Laboratory Standards (NCCLS), using the breakpoints recently suggested by Nguyen et al. (Antimicrob Agents Chemother 1998; 42: 471-472). The antifungals tested were amphotericin B, fluconazole and itraconazole. Most of the isolates were susceptible to the three antimycotics tested regardless of the variety. Resistance to amphotericin B (MIC=2 microg/ml) was documented in two (10%) C. neoformans var. neoformans isolates; additionally, five (33%) C. neoformans var. gattii isolates felt in the category of fluconazole susceptible but dose dependent (MIC 16 microg/ml). In general, all C. neoformans var. gattii isolates proved susceptible only to the higher concentrations of the antifungals tested. For amphotericin B, seven (47%) isolates of this variety had MICs of 1 microg/ml, for fluconazole there were seven (47%) with MICs of 8 microg/ml and in the case of itraconazole, 10 isolates (66%) had MICs > 0.03 microg/ml. The data showed that although these isolates would be classified as susceptible, they actually require greater concentrations of the antifungals to be inhibited. This finding may well correlate both with the difficulty to attain therapeutic success in patients affected with C. neoformans var. gattii and with the need for more prolonged treatment courses in such cases.     

The effects of amphotericin B on the interaction of Candida albicans with fibroblast cultures

Sublethal amounts of amphotericin B inhibited the interaction of Candida albicans with cultured fibroblasts. Different C. albicans clinical isolates exhibited varying degrees of sensitivity to the drug, but those isolates that were the most infective in control cultures appeared to be the most resistant to amphotericin B mediated infection inhibition. Although amphotericin B inhibited germ tube formation at the sublethal concentration of 0.3 microgram/mL, lower concentrations inhibited infection without preventing germination. The extent of this latter activity varied with the isolate and amphotericin B concentration and appeared to be related to sublethal effects on germinated yeasts. While amphotericin B effectively prevented new fibroblast infection, it did not dissociate those yeasts which had established an infection before its addition.     

Stimulation of germination of unactivated Bacillus cereus spores by ammonia

Inclusion of ammonia in germinant mixtures containing L-alanine and inosine stimulated germination of unactivated Bacillus cereus spores at rates equal to those obtained using heat-activated spores without ammonia. D-Alanine had little effect on germination of heat-activated spores, but severely inhibited germination of unactivated spores in the presence of ammonia. Ammonia did not replace the requirement for either L-alanine or inosine: all three compounds were required for rapid germination. Kinetic analysis suggested that the functions of ammonia and L-alanine were more closely related than the functions of ammonia and inosine. With rate-saturating concentrations of L-alanine and inosine, germination rates showed saturation kinetics for ammonia with a Km for NH4Cl of 5 mM. Comparisons of the effects of salts, amines and pH on germination rates suggested that NH4OH rather than NH+4 was the rate-limiting form of ammonia. In comparisons of various strains of B. cereus, stimulation of germination by ammonia occurred in all cases, although spores of most soil isolates germinated more rapidly than B. cereus T spores in the absence of ammonia.     

Isoenzyme analysis of Babesia microti infections in humans

We used high-resolution polyacrylamide gradient gel electrophoresis (PGGE) to separate four babesial enzymes to aid in the identification of two Babesia microti isolates established in hamsters. The isolates were compared to two different hamsters passages of the "Gray" strain. All isoenzymes patterns from the two isolates and the "Gray" strain were similar except glucose phosphate isomerase (GPI) from one of the "Gray" strain passages. It showed a polymorphic GPI pattern as opposed to a monomorphic GPI pattern seen in the other "Gray" strain passage and the two isolates. The observed differences suggested that some population of B. microti are capable of having polymorphic GPI, that the "Gray" strain originally contained (and may still contain) a heterogeneous population of B. microti, and that the population possessing polymorphic GPI was selected over that with monomorphic GPI. This information was obtained by a PGGE method that eliminated hemoglobin from gels and allowed, for the first time, detection of babesial leucine amino peptidase (LAP) and isocitrate dehydrogenase (IDH). In addition, this method provided molecular weight estimations on babesial GPI, LAP, IDH, and glutamate dehydrogenase (GDH), and it proved useful in the identification and characterization of the B. microti isolates.     

Isoenzyme Analysis of Babesia microti Infections in Humans

ABSTRACT. We used high-resolution polyacrylamide gradient gel electrophoresis (PGGE) to separate four babesial enzymes to aid in the identification of two Babesia microti isolates established in hamsters. The isolates were compared to two different hamster passages of the "Gray" strain. All isoenzyme patterns from the two isolates and the "Gray" strain were similar except glucose phosphate isomerase (GPI) from one of the "Gray" strain passages. It showed a polymorphic GPI pattern as opposed to a monomorphic GPI pattern seen in the other "Gray" strain passage and the two isolates. The observed differences suggested (i) that some populations of B. microti are capable of having polymorphic GPI, (ii) that the "Gray" strain originally contained (and may still contain) a heterogenous population of B. microti , and (iii) that the population possessing polymorphic GPI was selected over that with monomorphic GPI. This information was obtained by a PGGE method that eliminated hemoglobin from gels and allowed, for the first time, detection of babesial leucine amino peptidase (LAP) and isocitrate dehydrogenase (IDH). In addition, this method provided molecular weight estimations on babesial GPI, LAP, IDH, and glutamate dehydrogenase (GDH), and it proved useful in the identification and characterization of the B. microti isolates.     

Clostridium perfringens spore germination: characterization of germinants and their receptors

Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of L-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in C-cpe isolates only; and (ii) L-alanine or L-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with L-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.     

In vitro activity of a liposomal nystatin formulation (Nyotran) against Cryptococcus neoformans

The in vitro antifungal activity of a new liposomal nystatin formulation (NISTL, Nyotran, Aronex Ltd., EE.UU.) was evaluated by a microdilution method with RPMI based on the M27A document of the National Committee for Clinical Laboratory Standards (NCCLS) against 22 isolates of Cryptococcus neoformans. This antifungal activity was compared with those of other seven antifungal agents, such as nystatin (NIST), amphotericin B deoxycholate, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B colloidal dispersion, fluconazole, and itraconazole. NISTL was more active in vitrothan NIST, showing MIC values 2-3 fold smaller in 90% of the isolates. The results obtained suggest that this new formulation would be very helpful for the treatment of cryptococcosis.     

Activity of Combined Antifungal Agents Against Multidrug-Resistant <Emphasis Type="Italic">Candida glabrata</Emphasis> Strains

In this study, we evaluated the in vitro activity of echinocandins, azoles, and amphotericin B alone and in combination against echinocandin/azole-sensitive and echinocandin/azole-resistant Candida glabrata isolates. Susceptibility tests were performed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute document M27-A3. The checkerboard method was used to evaluate the fractional inhibitory concentration index of the interactions. Cross-resistance was observed among echinocandins; 15% of the isolates resistant to caspofungin were also resistant to anidulafungin and micafungin. Synergistic activity was observed in 70% of resistant C. glabrata when anidulafungin was combined with voriconazole or posaconazole. Higher (85%) synergism was found in the combination of caspofungin and voriconazole. The combinations of caspofungin with fluconazole, posaconazole and amphotericin B, micafungin with fluconazole, posaconazole and voriconazole, and anidulafungin with amphotericin B showed indifferent activities for the majority of the isolates. Anidulafungin combined with fluconazole showed the same percentage of synergism and indifference (45%). Antagonism was detected in 50% of isolates when micafungin was combined with amphotericin B. Combinations of echinocandins and antifungal azoles have great potential for in vivo assays which are required to evaluate the efficacy of these combinations against multidrug-resistant C. glabrata strains.     

Lethal and mutagenic action of N-nitroso-N-methylbiuret on the producers of levorin, amphotericin and mycoheptin]

The lethal and mutagenic effect of N-nitrozo-N-methylbiuret (NMB) on the organisms producing levorin, amphotericin B and mycoheptin was studied. The mutagen effect depended on the dose, culture and physiological state of the spores. NMB had a low mutagenic effect on the levorin-producing organism characterized by high activity and genetic homogenicity with respect to the colony morphology and antibiotic production. As for the organisms producing amphotericin B and mycoheptin characterized by high genetic heterogenicity, significant variation of all the features studied was observed on their exposure to the mutagen. Inspite of diverse reaction of the organisms producing levorin, amphotericin B and mycoheptin to the effect of NMB mutants with increased antibiotic production were obtained from the three cultures. The lethal and mutagenic effect of NMB on the mycoheptin-producing organism depended on the process of the spore DNA replication. The spores during the DNA replication period were least sensitive to the lethal effect of the mutagen and most mutable with the respect to the colony morphology. For selection of highly active and stable strains exposure to NMB of the spores of the mycoheptin-producing organism during replication of DNA proved to be more effective than that of the spores during the lag-phase.     

Characteristics of a strain of C. albicans resistant to polyene antibiotics]

It was found that a resistant strain R2 of C. albicans obtained as a result of passages on media containing increasing concentrations of amphotericin B differed from the initial strain by its lower pathogenicity. Treatment of the infection caused by the resistant strain on modeling of candidiasis in mice was not successful. The decrease in the average life span of the mice infected with the resistant strain R2 and treated with amphotericin B was lower than that in the control animals and such indices of the disease as the levels of the kidney dissemination and the cell vegetation even increased under the effect of amphotericin B. The results of the study suggest that the resistant strain R2 of C. albicans depend on amphotericin B in the host. The data obtained emphasize the necessity of determinining the antibiotic sensitivity of C. albicans strains isolated from patients.     

In vitro susceptibility to antifungal agents of environmental Cryptococcus spp. isolated in the city of Ribeirão Preto, São Paulo, Brazil

Infections by Cryptococcus strains other than C. neoformans have been detected in immunocompromised patients. Of these strains, three are considered human pathogens: C. albidus, C. laurenttii, and C. uniguttulatus. This study deals with the in vitro susceptibility of Cryptococcus to drugs such as amphotericin B, itraconazole, fluconazole, and 5-fluorocytosine. Environmental Cryptococcus isolates (50) distributed as follows: C. neoformans var. neoformans (16), C. albidus (17), C. laurentii (14), and C. uniguttulatus (3) were evaluated by the micro and macrodilution techniques, according to EUCAST and NCCLS recommendations, respectively. Considering both methodologies the respective minimal inhibitory concentrations (MIC) were 0.125 and 2 microg/ml for amphotericin B, 0.06 and 8 microg/ml for itraconazole, and 0.5 and more than 64 microg/ml for fluconazole and 5-fluorocytosine. Agreement percentages for the two methodologies were 100% for amphotericin B and fluconazole for all the strains tested. For itraconazole, the agreement percentage was 81.3% in the C. neoformans strain and 100% for all the others. All species had a agreement percentage of 94.1 to 100% when susceptibility to 5-fluorocytosine was tested. It is concluded that environmental isolates of C. neoformans var. neoformans, C. albidus, C. laurentii, and C. uniguttulatus may show high MICs against certain drugs, suggesting in vitro primary resistance to the antifungals tested.     

Comparison of Different In Vitro Tests to Detect <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Not Susceptible to Amphotericin B

Infections due to Cryptococcus neoformans cause severe disease, mostly in AIDS patients. The antifungal drug recommended for the initial treatment of these infections is amphotericin B with or without flucytosine, but treatment failure occurs, associated with high mortality. Thus, antifungal susceptibility testing is needed. However, the in vitro susceptibility tests available for C. neoformans are not useful to detect isolates that are not susceptible to antifungal agents such as amphotericin B. The aims of the present study were: (1) to determine and compare the in vitro activity of amphotericin B against C. neoformans clinical isolates by using different dilution and diffusion methods; (2) to evaluate the concordance among the methods used and the reference method; (3) to evaluate which method could be the best to correlate with the clinical outcome. The reference method EDef 7.2 from the European Committee on Antimicrobial Susceptibility Testing and commercial Etest strips were used to determine the minimal inhibitory concentration against amphotericin B. curves, minimal fungicidal concentration, and a disk diffusion method were also developed to evaluate the cidal activity of amphotericin B. The time–kill curve assay showed correlation (p < 0.05) with clinical outcome, whereas EDef 7.2, minimal fungicidal concentration, Etest, and disk diffusion showed no correlation (p > 0.05). Thus, the time–kill curve assay could be a potential tool to guide a more efficient treatment when amphotericin B is used.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Variation of the sterols from Kluyveromyces lactis and from a resistant mutant by culture in the presence of sublethal doses of amphotericin B

A mutant strain of Kluyveromyces lactis resistant to amphotericin B and weakly to nystatin has been isolated from subcultures of the wild strain grown in the presence of sublethal doses of amphotericin B. The mutant and the wild strain were equally sensitive to pimaricin, filipin, and candicidin. The efficacy of fungizone was very low. In comparison with the wild strain the level of sterols was two times lower in the resistant strain but the composition of these sterols was about the same in the two strains. The action of sublethal doses of amphotericin B on the composition of the sterols was the same in these two yeasts and brought a 40% decrease of the total sterol level and a modification in their distribution. This variation cannot fully explain the resistance of the yeast but it may be associated to other changes of the membranes.     

Anfotericina B liposomal: farmacología clínica,farmacocinética y farmacodinamia

Liposomal amphotericin B is a lipid formulation of the antifungal drug amphotericin B with some distinguishing characteristics in its pharmacological behavior that entail some clinical differences of great interest. The significant improvement in the systemic and renal tolerability is one of them. This fact is related to the great stability of the liposome, promoted by its negative charge, the presence of cholesterol and the remarkable thermo-stability of the remaining lipids that compose it. In this situation, amphotericin B seems to be released from the liposome not spontaneously but when the liposome binds to the ergosterol in the fungal cell membrane. For this reason, there is almost no free amphotericin B in plasma or tissues, although it seems that its availability is greater when there is fungal infection. As a consequence, when the pharmacokinetic behavior is studied, the concentration and availability of liposomal amphotericin B are very high, and its volume of distribution is reduced in comparison with the other formulations.     

Candida albicans strain differentiation in complete denture wearers

Strain differentiation of 66 clinical isolates of Candida albicans obtained from healthy dentate and complete denture wearers was performed. Resistogram method based on differences in the resistance of C. albicans isolates to sodium selenite, boric acid, cetrimide, sodium periodate and silver nitrate was used for strain differentiation. Of the 32 potential strains that can be distinguished, 14 different resistogram strains of C. albicans were found among the 66 isolates tested. Strain-C--was the most predominant (24.3% of total isolates), while strain A-CDE was the least predominant (1.5%). The results showed no particular association of certain strains with Candida infections in complete denture wearers. Sensitivity to antifungal agents showed that isolates from different strains were most sensitive to amphotericin B and nystatin and least sensitive to miconazole.