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.     

Optimal Combination and Concentration of Antibiotics in Media for Isolation of Pathogenic Fungi and Nocardia asteroides

Strains of Blastomyces dermatitidis, Sporothrix schenckii, Histoplasma capsulatum, Cryptococcus neoformans, Nocardia asteroides, and Coccidioides immitis were tested for in vitro susceptibility to polymyxin, gentamicin, kanamycin, chloramphenicol, and neomycin at concentrations of 1, 2, 4, 8, and 16 mug/ml. Polymyxin was the most inhibitory and gentamicin was the least inhibitory of the five antibiotics. Two Histoplasma mycelial strains were partially inhibited by 2 and 8 mug of gentamicin per ml and showed at least a 2+ growth at the higher antibiotic concentration. Kanamycin and neomycin produced significant inhibition of N. asteroides but otherwise were noninhibitory. A combination of chloramphenicol and kanamycin, each at 16 mug/ml, and gentamicin, at 4 mug/ml, was noninhibitory to the strains tested except for N. asteroides. Chloramphenicol at 16 mug/ml was not inhibitory for N. asteroides. The results suggest that the optimal antibiotic combination to use in the isolation of fungi and higher bacteria is chloramphenicol, 16 mug/ml, and gentamicin, 4 mug/ml. Addition of sheep blood (5%) had no effect on antibiotic susceptibility of the organisms studied.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.     

In-vitro activity of 5-fluorocytosine against 1,021 Spanish clinical isolates of Candida and other medically important yeasts

The aim of this study was to determine the prevalence of primary resistance to 5-fluorocytosine (5FC) among clinical isolates of yeasts in Spain where this drug is not currently available for therapy. We have tested the in vitro activity of 5FC against 1,021 recent yeast clinical isolates, including 522 Candida albicans, 140 Candida parapsilosis, 68 Candida glabrata, 41 Candida dubliniensis, 50 Candida guilliermondii, 34 Candida tropicalis, 28 Candida krusei, 20 Candida famata, 11 Cryptococcus neoformans, 5 Cryptococcus albidus, 43 Rhodotorula spp., 24 Trichosporon spp., 5 Saccharomyces cerevisiae, 9 Pichia spp., and 21 isolates from other 11 yeast species. The MICs were determined by the ATB Fungus agar microdilution test (bioMerieux, France) and the following interpretive breakpoints were used: susceptible, > 4 microg/ml; intermediate, 8 to 16 microg/ml; resistant, > 32 microg/ml. 5FC was very active against Candida spp. and other medically important yeasts as 852 (83.4%) of the studied isolates were susceptible (MIC < 4 microg/ml). The species most susceptible to 5FC were C. dubliniensis (100%of isolates; MIC90, 0.25 microg/ml), C. famata (100% of isolates; MIC90, 0.25 microg/ml), C. guilliermondii (98%of isolates; MIC90, 0.25 microg/ml), C. glabrata (95.5% of isolates; MIC90, 0.25 microg/ml), and C. neoformans (90.9% of isolates; MIC90, 2 microg/ml). Primary resistance to 5FC was very uncommon, and a MIC > 32 microg/ml, indicator of in vitro resistance, was observed in 106 isolates (10.4%): 77 C. albicans (16.5% of isolates; MIC90, > 128 microg/ml), 9 C. parapsilosis (6.4% of isolates; MIC90, 8 microg/ml), 4 C. albidus (80% of isolates, MIC50, > 128 microg/ml), 3 C. glabrata (4.4% of isolates; MIC90, 0.25 microg/ml), 3 C. tropicalis (8.8% of isolates; MIC90, 4 microg/ml), 2 C. krusei (7.1% of isolates; MIC90, 8 microg/ml), 2 Rhodotorula spp. (4.6% of isolates, MIC90, 1 microg/ml), 8 Trichosporon spp. (33.3% of isolates; MIC90, 64 microg/ml), and 1 C. lipolytica (50% of isolates). Interestingly, most C. albicans (67 out of 77 isolates) resistant to 5FC were serotype B isolates.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Effect of Inoculum Size on In Vitro Susceptibility Testing with Lincomycin

There is disagreement in the literature as to whether lincomycin is primarily a bacteriostatic or a bactericidal agent against gram-positive cocci and also regarding the levels of activity of this agent against susceptible microorganisms. These questions were examined in a study of the effect of inoculum size on the results of tube dilution susceptibility determinations with lincomycin against 49 clinical isolates of Staphylococcus aureus and 25 strains of streptococci and pneumococci. Lincomycin was both highly active and bactericidal when tested against 40 strains of S. aureus with inocula containing a maximum of 10(4) cells per ml [median minimal inhibitory concentration (MIC), 0.78 mug/ml; median minimal bactericidal concentration (MBC), 1.56 mug/ml]. With inocula of 10(5) cells per ml, lincomycin was primarily bacteriostatic (median MIC, 1.56 mug/ml; median MBC, 12.5 mug/ml). There were further decreases in inhibitory levels and significant losses of bactericidal activity when inocula containing more than 10(7) cells were tested (median MIC, 3.13 mug/ml; median MBC > 100 mug/ml). Similar measurements with streptococci and pneumococci revealed a lesser effect of inoculum size. The mean MBC value for alpha-hemolytic streptococci increased from 0.40 to 1.05 mug/ml with an increase in inocula from 10(4) to 10(6) cells per ml, but without a marked increase in MIC values. Similar results were obtained for beta-hemolytic streptococci and pneumococci.     

Systemic Antifungal Activity of Pyrrolnitrin

The antifungal activity of pyrrolnitrin, previously shown to be effective against superficial infections, was evaluated against experimental systemic mycoses. Pyrrolnitrin was inhibitory in vitro at <0.78 to 100 μg/ml to Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, Sporotrichum schenckii, and Histoplasma capsulatum. Pyrrolnitrin activity was reduced about 90% in sera. After multiple subcutaneous doses of pyrrolnitrin at 20 mg/kg, activity was recovered in mouse blood and urine as well as kidney, liver, and brain homogenates. Multiple daily doses (50 mg/kg) of this antibiotic were effective in reducing by 74% the number of viable cells of C. albicans recovered from kidney homogenates. Multiple doses (15 mg/kg) resulted in a 74% reduction in the number of C. neoformans from brain homogenates. Pyrrolnitrin was ineffective in reducing the recovery of B. dermatitidis or H. capsulatum from liver or spleen homogenates of infected mice. When compared with amphotericin B, hamycin, 5-fluorocytosine, and saramycetin, this antibiotic was less effective. This study indicates that pyrrolnitrin would have limited usefulness as a systemic antifungal agent.     

Development of bovine in vitro-matured follicular oocytes activated with ethanol

Bovine follicular oocytes cultured in vitro for 30 hours were activated by ethanol and then the developmental ability of the oocytes was assessed. When the activated oocytes were treated with cytochalasin B at concentrations of 0, 1.25, 2.5, 5 and 7.5 mug/ml, the percentages of oocytes having two pronuclei were 8, 18, 54, 66 and 60%, respectively. The percentage of the activated oocytes with two pronuclei also increased with increased exposure time to cytochalasin B. When the activated oocytes were treated with cytochalasin B (5mug/ml) for 10 hours, 20% of them cleaved after 36 hours and 5% of them developed to the eight-cell stage after 60 hours of culture. These results indicated that bovine in vitro matured oocytes can be activated by ethanol and develop to the eight-cell stage following treatment with cytochalasin B.     

Inactivation of bovine herpesvirus-1 and bovine viral diarrhea virus in association with preimplantation bovine embryos using photosensitive agents

Hematoporphyrin (HP), hematoporphyrin derivative (HPD), and thiopyronine (TP) are photosensitive agents (PSA) that have a germicidal effect when they are activated by light: helium neon laser (He Ne ) light (HP, HPD), white light (HP, HPD), and yellow-green light (TP). Experiments were conducted with appropriate controls to determine the effect of photosensitive agents a) for inactivating bovine herpesvirus-1 (BHV-1; titre 10(6) TCID(50) /ml) and bovine viral diarrhea virus (BVDV; titre 10(6) TCID(50) /ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 10(6) TCID(50) /ml) or BVDV (titre 10(6) TCID(50) /ml); and c) on the in vitro development of embryos. Exposure to HP, HPD and TP followed by light irradiation inactivated BHV-1 and BVDV. Embryos exposed to BHV-I were disinfected by HP or HPD (5 mug/ml) in combination with He Ne light, or by HP or HPD (10 mug/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 mug/ml) followed by He Ne or white light irradiation. Exposure of embryos to light alone or to light and HP or HPD had no detrimental effect on their in vitro development; however, exposure of embryos to TP (5 mug/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 mug of HPD followed by He Ne light, or 10 mug/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.     

In Vitro Sensitivity of Torulopsis glabrata to Amphotericin B, 5-Fluorocytosine, and Clotrimazole (Bay 5097)

Thirty-five strains of Torulopsis glabrata were tested by a tube dilution method for their susceptibility to amphotericin B, 5-fluorocytosine, and clotrimazole (Bay 5097). Amphotericin B was the most active in vitro, inhibiting all strains at a concentration of 1 μg/ml and killing all strains at 2 μg/ml. 5-Fluorocytosine inhibited over 80% of strains at 0.24 μg/ml, but three strains required ≥7.8 μg/ml for killing. A concentration of 2 μg of clotrimazole per ml inhibited less than 50% of strains, and 8 μg/ml killed only 10% of strains. Most strains of T. glabrata were killed by therapeutically achievable concentrations of amphotericin B and 5-fluorocytosine, but not clotrimazole.     

Biochemical Effects of Oxolinic Acid on Proteus vulgaris

Oxolinic acid (1-ethyl-1,4-dihydro-6,7-methylenedioxy-4-oxo-3-quinolinecarboxylic acid) is an antimicrobial agent effective against a variety of gram-negative pathogens, including Proteus. With the exception of Staphylococcus aureus, oxolinic acid is inactive against gram-positive bacteria and against fungi. Our results suggest that oxolinic acid exerted its primary action on synthesis of deoxyribonucleic acid (DNA). The rate of thymidine-2-(14)C incorporation into DNA was significantly depressed in the presence of 0.1 mug of oxolinic acid per ml and was markedly inhibited at 1 mug/ml. No evidence of complexing with DNA was observed. Pulse labeling with radioactive precursors revealed that at levels approximating the minimal inhibitory concentration, oxolinic acid had no effect on rate of incorporation of (14)C-valine into protein, uracil-2-(14)C into ribonucleic acid, or sodium acetate-1-(14)C into lipid. Filamentous forms of P. vulgaris ATCC 881 were observed after in vitro exposure to subinhibitory levels of oxolinic acid. Concentrations of oxolinic acid in excess of the minimal inhibitory concentration (0.39 mug/ml) did not cause lysis of cells of P. vulgaris or leakage of cytoplasmic materials. Mg(++) ions diminished the in vitro activity of oxolinic acid, possibly through formation of Mg(++) chelates     

Effect of Nitrofurans and Chlortetracycline on Microorganisms Associated with Shrimp

Nitrofuran AF-2 displayed greater inhibitory effect than did nitrofuran Z when a mixed bacterial culture, including several proteolytic bacteria, isolated from shrimp was subjected to these compounds in vitro. Nitrofuran Z exhibited greater bactericidal properties than did chlortetracycline in all cultures used. Only 10 mug of nitrofuran AF-2 per ml was sufficient to inhibit the growth of mixed bacteria in nutrient broth, whereas 50 mug of nitrofuran Z per ml was necessary to accomplish the same inhibition. A 50-mug amount of chlortetracycline per ml displayed about the same inhibitory effect as either 10 mug of AF-2 per ml or 20 mug of Z per ml. The isolated proteolytic bacteria showed greater suppression of growth when subjected to AF-2 than when subjected to Z; however, both nitrofurans were effective in preventing growth. The addition of either 1 mug of AF-2 per ml or 5 mug of Z per ml to nutrient broth inhibited the growth of Achromobacter aquarmarinus, whereas chlortetracycline was less effective, requiring about 20 mug to suppress growth to the same degree.     

Mechanism for the pyridoxal neutralization of isoniazid action of Mycobacterium tuberculosis

In Sauton's synthetic liquid medium, 10 mug of pyridoxal per ml completely protected Mycobacterium tuberculosis (H37R(a)) from the effects of a minimal inhibitory concentration of isoniazid (0.01 mug/ml). (14)C-labeled isoniazid was employed to study the nature of this protective effect. Uptake of the drug by cells in a Sauton environment containing 0.01 mug of (14)C-isoniazid per ml was inhibited 20 to 40% by 10 mug of pyridoxal per ml during the early hours of drug exposure. A stronger inhibition of uptake resulted when labeled isoniazid and pyridoxal were increased to 0.1 mug/ml and 50 to 100 mug/ml, respectively. Further studies revealed that certain Sauton nutrients are required to achieve this effect. When l-asparagine or salts (MgSO(4) and ferric ammonium citrate) or both were deleted from the menstruum, pyridoxal did not inhibit isoniazid incorporation by the tubercle bacilli. Pyridoxal also failed to inhibit uptake when (NH(4))(2)SO(4) was substituted for l-asparagine. Growth experiments in Sauton's medium modified to contain (NH(4))(2)SO(4) instead of l-asparagine were consistent with the latter finding. Pyridoxal did not prevent isoniazid growth inhibition in this medium. It is postulated that a large excess of pyridoxal in Sauton's medium protects tubercle bacilli from the effects of isoniazid through formation of an extracellular complex involving drug, vitamin, and certain medium constituents, thereby reducing the level of isoniazid available to the cells.     

Antifungal and fungicidal activities of tea extract and catechin against Trichophyton

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antifungal and fungicidal activities against Trichophyton mentagrophytes, T. rubrum, Candida albicans and Cryptococcus neoformans. Tea extract (2.5%) inhibited completely the growth of both T. mentagrophytes and T. rubrum. EGCg at 2.5 mg/ml failed to inhibit their growth, whereas TF3 at 0.5 mg/ml inhibited the growth. EGCg (1mg/ml) showed no fungicidal activity against Trichophyton. TF3 (1mg/ml) killed Trichophyton by a long time contact (72-96 hrs). Tea extract showed a fungicidal activity against Trichophyton in a dose- and contact time-dependent manner. It did not inhibit the growth of C. albicans, but at a high concentration, inhibited slightly the growth of C. neoformans. It had no fungicidal activity against C. albicans or C. neoformans.     

Intravenous and Intrathecal Miconazole Therapy for Systemic Mycoses

Ten patients with systemic mycoses, including five with fungal meningitis, were treated with intravenously or intrathecally administered miconazole, or both. Minimal inhibitory concentrations of miconazole for clinical isolates of Coccidioides immitis, Cryptococcus neoformans and Candida albicans were less than 0.6 µg per ml. Except for pruritis of variable degrees, the drug was well tolerated both intravenously and intrathecally by all patients. No measurable impairment of renal, hepatic or bone marrow function was observed in patients after 4½ months of intravenous therapy. No hematological or biochemical abnormalities and no evidence of recurrent coccidioidal osteomyelitis were observed in 16 months of follow-up in our first patient treated with this drug. Miconazole is apparently an effective antifungal drug of low toxicity and is a potentially useful agent for treatment of human systemic mycoses.     

大黄酚体外抗白念珠菌生物膜作用的研究

目的研究大黄酚对体外白念珠菌生物膜的影响。方法采用XTT减低法评价大黄酚对白念珠菌的生物膜及黏附性的影响;镜下观察该药对白念珠菌生物膜的形态学影响;细胞毒试验检测该药的毒副作用。结果大黄酚对白念珠菌生物膜的SMIC50、SMIC80分别为125、1000μg/ml;100μg／ml及1000μg/ml含量浓度的大黄酚对自念珠菌的早期黏附及菌丝生长有抑制作用;大黄酚对人细胞毒性较弱。结论大黄酚对体外白念珠菌生物膜有较强的抑制作用。     

Effect of synthetic antifungal drugs on the adherence of Candida albicans to the mouth mucosa epithelium in vitro

Ten Candida albicans strains were tested for their adherence to buccal epithelial cells in vitro in the presence of synthetic antimycotic drugs as 5-fluorocytosine, clotrimazole and ketoconazole. The drugs when applied in therapeutic concentrations inhibited adherence stronger than when applied in subinhibitory concentrations although in both situations the inhibition was statistically significant in comparison to control experiments without these drugs (p less than 0.01). The highest inhibition of adherence was seen with 5-fluorocytosine and the weakest with clotrimazole. Out of imidazole derived drugs strong inhibition of adherence was achieved with ketoconazole.     

Antifungal effect of silver nanoparticles on dermatophytes

Spherical silver nanoparticles (nano-Ag) were synthesized and their antifungal effects on fungal pathogens of the skin were investigated. Nano-Ag showed potent activity against clinical isolates and ATCC strains of Trichophyton mentagrophytes and Candida species (IC80, 1-7 mug/ml). The activity of nano-Ag was comparable to that of amphotericin B, but superior to that of fluconazole (amphotericin B IC80, 1-5 mug/ml; fluconazole IC80, 10- 30 mug/ml). Additionally, we investigated their effects on the dimorphism of Candida albicans. The results showed nano-Ag exerted activity on the mycelia. Thus, the present study indicates nano-Ag may have considerable antifungal activity, deserving further investigation for clinical applications.     

FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.     

Bacteriostatic action of progesterone on staphylococci and other microorganisms

Yotis, William (Loyola University, Hines, Ill.), and Ronald Stanke. Bacteriostatic action of progesterone on staphylococci and other microorganisms. J. Bacteriol. 92:1285-1289. 1966.-Progesterone has been examined in vitro for antibacterial activity against 10 microorganisms. Turbidimetric and manometric techniques were used to assay the antibacterial activity of progesterone. The organisms tested consisted of Staphylococcus aureus, S. epidermidis, Gaffkya tetragena, Bacillus subtilis, Listeria monocytogenes, Candida albicans, Escherichia coli, Aerobacter aerogenes, Salmonella paratyphi, and Proteus vulgaris. Antibacterial action was shown by progesterone only against the gram-positive microorganisms when they were grown in tryptic soy broth containing 10 to 20 mug of progesterone per ml. Pregnenolone, 4-pregnen-20beta-ol-3-one, and 5alpha-pregnane also possessed antistaphylococcal properties, whereas pregnanolone, pregnandione, 11alpha-hydroxyprogesterone, and 17alpha-hydroxyprogesterone did not. The bacteriostatic action of progesterone on staphylococci was exerted primarily during the first 8 hr of incubation, and it was reduced in the presence of oxygen. In the presence of 20 mug of progesterone per ml, there was significant reduction in the oxidation by resting staphylococcal suspensions or utilization by staphylococci of pyruvate as an energy source during growth.