Molds were isolated from moldy cheese trimmings and both nonmoldy and moldy samples of domestic and imported retail samples of cheese. The majority of molds found were Penicillium species. A small percentage (about 4 p. 100) of the molds encountered were species known to be capable of producing recognized mycotoxins (P. cyclopium, P. viridicatum, A. flavus and A. ochraceus). Most of the molds isolated has psychrotrophic properties and were capable of growing at 5 degrees C. Sorbates were found to be partially effective in delaying the growth of molds obtained from cheese. However, 65--75 p. 100 of the mold isolates were capable of initiating growth in the presence of 0.3 p. 100 potassium sorbate within 7 days at 5 degrees C. Pimaricin, on the other hand, prevented the growth of all the mold isolates at 12 and 5 degrees C and of 96 p. 100 of the isolates at 25 degrees C for at least 7 days.

Natamycin (pimaricin) (E235) is an antifungal that can be used as an antibiotic to treat most fungus infections. It has been globally used in a variety of foods and beverages. In the present study, the effects of natamycin on chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) formation in human lymphocytes cells were investigated. The human lymphocytes were treated with 13, 18, 23, and 28 microg/mL of natamycin for 24 and 48 h. Natamycin induced the SCE frequency at the highest concentration for 48 h only; however, it induced the structural CA and MN frequency at all concentrations when compared to control and at all concentrations, except the lowest concentration (13 microg/mL), when compared to solvent control. Natamycin showed a cytotoxic effect by decreasing the replication index, mitotic index, and nuclear division index (NDI), especially at the highest concentrations for two treatment periods.

The pea phytoalexin pisatin, at its inhibitory concentration, was shown to have two distinct inhibitory effects on amoebae of the cellular slime mould Dictyostelium discoideum. One effect was cytolytic and was demonstrable even in non-growing cells whereas the second effect was observed only under conditions favourable to growth. Pretreatment with a sublethal concentration of pisatin induced the amoebae to acquire resistance to both these effects. Mutations in nysC that alter membrane sterols and confer resistance to the polyene antibiotics nystatin and pimaricin blocked resistance to the growth-associated inhibitory effect but did not affect acquisition of resistance to the cytolytic effect. The nysB sunD double mutant HK412 displayed a partially constitutive resistance to the cytolytic effect but, like the nysC mutants, was blocked in the acquisition of resistance to the growth-associated inhibitory effect. Pistatin-treated cells incubated in pisatin-free medium lost their ability to grow on pisatin-containing medium much more rapidly than they lost resistance to the cytolytic effect of pisatin. These results suggest that the induction of pisatin resistance may involve the turning-on of independent resistance mechanisms against the two inhibitory effects of pisatin. This could account for our inability to isolate pisatin-resistant mutants in a single step. The Neurospora crassa erg1 and erg3 mutants that have altered membrane sterols and are nystatin resistant displayed sensitivity to pisatin. The pisatin-sensitivity phenotype of the erg mutants was used in selections to identify complementing plasmids from an ordered Neurospora genomic library.(ABSTRACT TRUNCATED AT 250 WORDS)

Results of treatment of 151 cases of vaginitis in patients attending a leukorrhea clinic were studied. The incidence of each type of vaginitis is recorded. Analysis of results of treatment with six compounds currently used in the therapy of vaginitis indicated that acetarsol or pimaricin compounds, with their broader spectra of activity, appeared to be most useful, prior to establishment of a definite diagnosis by means of cultures. Chlordantoin is an effective antifungal agent and is associated with a high percentage of "culture cures". Resistant cases should be investigated for diabetes mellitus, and many are aided by a low carbohydrate diet. Metronidazole was used only for resistant cases of trichomoniasis, with a cure rate of over 80% when both partners were treated simultaneously. Triple-sulfa vaginal cream was effective in over 80% of patients with nonspecific vaginitis; no cases of resistant bacterial infections were encountered. Dienestrol cream was effective in relieving the symptoms of atrophic vaginitis.

Aspergillosis was induced experimentally in hatching chick embryos by dipping them in water containing spores of Aspergillus fumigatus or A. flavus. The addition to the dip water of antifungal compounds prevented the development of this syndrome. Of the compounds studied, amphotericin B was most effective, followed by pimaricin and nystatin. Sorbic acid was without effect.

Five polyene antibiotics were compared for their effects on colony formation of either Chinese hamster V79 or Saccharomyces cerevisiae cells. A 10 to 40 times higher concentration of amphotericin B (heptaene) or nystatin (degenerated heptaene) was necessary to inhibit colony formation of hamster cells than that needed to inhibit colony formation of yeast cells. In contrast, colony formation of both hamster and yeast cells was inhibited to the same extent by similar concentrations of filipin (pentaene), pentamycin (pentaene), or pimaricin (tetraene). The five polyene antibiotics were also compared for their effects on colony formation of either V79 or S. cerevisiae cells when combined with a nonpolyene antibiotic, fusidic acid or bleomycin A2. Amphotericin B or nystatin could augment the cytocidal effect of fusidic acid but not that of bleomycin A2, whereas pentamycin or pimaricin could augment the cytocidal effect of both fusidic acid and bleomycin A2 against hamster and yeast cells. Filipin was found to enhance the action of fusidic acid and bleomycin upon growth of mammalian cells, whereas the pentaene polyene significantly potentiated the action of fusidic acid, but not that of bleomycin A2, against S. cerevisiae. It was therefore suggested that these polyene antibiotics be classified into two groups: group 1 (pimaricin, pentamycin, and filipin) and group 2 (amphotericin B and nystatin).

Carbon magnetic resonance establishes conclusively that six polyene macrolide antibiotics containing keto groups (the heptaene amphotericin B, the tetraene-diene nystatin A1, and the tetraenes tetrin A, tetrin B, pimaricin, and lucensomycin) exist in the hemiketal form in solution. Their spectra all contain a hemiketal carbon's absorption near 97 ppm but lack a keto carbon's absorption near 210 ppm. The non-polyenic macrolide erythromycin, on the other hand, exists in the keto form.

OBJECTIVE

An increase in the frequency of fungal infections is related with progress in mycology and decreased susceptibility of fungal strains to commonly used antifungal agents. Diabetes and pregnancy are two independent factors believed to be responsible for an increased risk of mycoses.

OBJECTIVE

The aim of the study was to assess the susceptibility of fungal strains isolated from pregnant women with diabetes as well as healthy pregnant women to ten antifungal agents.

METHODS

In the study 106 diabetic pregnant women and 102 healthy pregnant women were included. Susceptibility of the fungal strains was assessed in vitro by disk diffusion test.

RESULTS

Fungal strains were detected in 190 (30.4%) out of 624 samples obtained from vagina, rectum and oral cavity of 208 women. Fungi were found in 42.1% of pregnant women with diabetes and in 41.5% of health pregnant patients. Strains isolated from the diabetic women showed the highest susceptibility to pimaricin (34.4%), nystatin (31.3%) and tioconazole (31.3%) while those from healthy pregnant women were mostly susceptible to itraconazole (59.6%) and miconazole (53.2%). The comparison of the susceptibility of fungi to antifungal agents revealed that the strains isolated form healthy women were significantly more susceptible to clotrimazole (p=0.003), itraconazole (p<0.001) and miconazole (p=0.001). No difference was found in susceptibility to pimaricin (p=0.54), nystatin (p=0.75), amphotericine B (p=0.84), ketoconazole (p=0.123) and fluconazole (p=0.61) between those two groups of fungal strains.

CONCLUSIONS

Significant differences in susceptibility of fungi isolated from pregnant diabetic women to clotrimazole, itraconazole and miconazole suggest that diabetes has influence on resistance of fungal strains to some antifungal agents.

BACKGROUND

Fusarium species are common soil saprophytes and plant pathogens. Members of the genus have been frequently reported as etiologic agents of opportunistic infections in humans and animals. We report six cases of confirmed or suspected onychomycosis caused by members of the genus Fusarium (F. solani and F. oxysporum species complexes).

METHODS

The isolates were identified by rDNA ITS sequencing analysis. The EMBL accession numbers for the ITS are HE974453-HE974458. A disk diffusion method was used for in vitro susceptibility testing. Comparison of disks (ITEST) and Neo-Sensitabs tablets (Rosco) on a different media at two different temperatures (25 °C and 35 °C) was made.

RESULTS

Six strains of Fusarium spp. (4 strains of F. solani and 2 strains of F. oxysporum) were isolated from patients with confirmed or suspected onychomycosis. Natamycin (pimaricin) was the only antifungal effective in vitro in all isolates tested. Variable susceptibility of the isolates was detected in amphotericin B, econazole and terbinafine. The remaining antifungals tested were not effective. The results varied depending on the culture medium and temperature for nystatin and econazole disks and amphotericin B and terbinafin tablets.

CONCLUSIONS

It is important to adhere to recommended methods when testing in vitro susceptibility to antifungals in moulds. An incubation temperature of 35 °C is important for obtaining valid results in amphotericin B tablets (and probably also terbinafine ones). Determination of multidrug-resistant Fusarium spp. in onychomycosis make the choice of therapy difficult. Good clinical effect was recorded with nail plate ablation and subsequent local econazole therapy.

OBJECTIVE

Aim of this study was to ascertain the prevalence of differrent candida strains and their sensitivity to different antimycotic treatment in women with vulvovaginal candidosis.

METHODS

Prospective study.

METHODS

Department of Obstetrics and Gynecology, 1st Faculty of Medicine and the General Faculty Hospital, Charles University, Prague. UKLB, Clinical microbiology and ATB center, 1st Faculty of Medicine and the General Faculty Hospital, Charles University, Prague. Department of Obstetrics and Gynecology, Hospital Milosrdných bratrí, Brno.

METHODS

124 women with symptomatic vulvovaginal candidosis were included in the study between January and October 2003. After complete clinical exam, vaginal pH, wet prep, the cultures were taken for special fungal examination.

RESULTS

In 92% of patient was confirmed infection with C. albicans, from topical antifugal agents were most sensitive to nystatin and pimaricin (100% cases), from oral antifugal agents to fluconasol, itraconasol and ketokonasol in 97-98% cases. C. nonalbicans strains were confirmed in 8% cases of vaginitis and there were similiar sensitivity to topical antifugal agents as in C. albicans strains. To oral antifugal agents there were in 10% resistance to fluconasol and in 20% to itraconasol.

CONCLUSIONS

In this study we did not confirmed the higher prevalence of C. nonalbicans strains in vulvovaginal candidiasis, in literature the prevalence is 10-15%. We did not confirmed the increasing resistance to antifugal agents.

OBJECTIVE

Prolonged use of topical antifungal agents may compromise corneal epithelial integrity. Here, we used an in vitro model of human stratified corneal epithelium to compare the ocular toxicity profiles of 4 different antifungal eye drops.

METHODS

Human corneal epithelial cell sheets were cultured in a serum-free medium containing 0.1% micafungin, 1% voriconazole, 5% pimaricin, 0.1% amphotericin B, or controls (saline or 5% glucose). Cell viability and barrier function were measured by WST-1 assay and carboxyfluorescein permeability assay, respectively. Cell migration was measured on a wound healing assay.

RESULTS

WST-1 assay and carboxyfluorescein permeability assay revealed that amphotericin B was the most toxic drug, followed by pimaricin, micafungin, and voriconazole. Cell migration on a wound healing assay was decreased in the following order, amphotericin B, pimaricin, micafungin, and voriconazole.

CONCLUSIONS

Topical micafungin and voriconazole appeared to be the least toxic to the corneal epithelium. Drug prescription should consider not only fungal species and susceptibility but also ocular toxicity and stage of treatment.

Exposure of washed cells of Saccharomyces cerevisiae C-299 to inhibitory concentrations of pimaricin decreased resistance to lethal temperatures and to freezing and thawing. Varying the pH of the recovery medium had little effect on the decrease in heat resistance, but addition of peptone or yeast extract resulted in a partial recovery of apparent heat resistance. Addition of peptone or calcium ion to the heating menstruum did not affect the reduction in resistance caused by pimaricin. Varying the composition of the recovery medium similarly affected the resistance to freezing the thawing of pimaricin-exposed and unexposed cells. Sensitivity to ultraviolet light was apparently not affected by pimaricin either by preliminary exposure or when irradiated in the presence of pimaricin. It is suggested that pimaricin lowers the heat resistance of S. cerevisiae by depleting essential nutrients of the cells through a disruption of permeability, and that heat-damaged cells cannot resynthesize these nutrients. It is also suggested that spoilage of acid foods by S. cerevisiae may be retarded by a combination of mild heat treatment and the addition of pimaricin.

In a 10 years old girl sustaining a corrosive injury of the lower leg from sulphuric acid, in the region of a skin transplantation colonization with Aspergillus fumigatus (Fresenium) and Aspergillus niger (van Tieghem) took place. This infection endangered the attempt of transplantation and the saving of the foot. Treatment by medication with nystatin (moronal) and canesten (clotrimazol) were ineffective. Pimaricin (pimafucin, natamycin) quickly erradicated the mycotic infection and secured an undisturbed progress for the transplantation. Additionally the epidemiology of infections by Aspergillus is briefly discussed.

The prior condition for the application of pimaricin is its heat stability (up to 80 degrees C) and the low penetration (around 2.6 mm), so that it is available for the effect on the cheese surface for a long period. With 13 different aflatoxin forming moulds, the inhibition on the mycel development and thereby on the aflatoxin formation was tested; the different strains were inhibited to various degrees. The effect on cheese was not definite, because the native cheese surface remained free of mould for 8 weeks after pimaricin treatment, whereas cheese slices dipped into pimaricin solution were covered with moulds very soon. The aflatoxin formation itself is only inhibited, if the growth of the moulds is inhibited. Therefore the cheeses have to be treated very early before strong growth of the moulds has started. The aflatoxin formation, however only starts at a certain growth period of the moulds, but at a not completed inhibition, a reduced aflatoxin formation has to be taken into consideration.

The sensitivity to natamycin of molds and yeasts isolated in cheese warehouses where natamycin has been used for various periods was determined. After several years of continuous use of natamycin no natamycin-insensitive molds and yeasts were found. In 26 strains of molds isolated in cheese warehouses it was not possible under laboratory conditions to decrease the sensitivity for natamycin. Besides the sensitivity of fungi to natamycin, production of mycotoxins by the isolated molds was investigated. Strains of Penicillium viridicatum , Aspergillus versicolor , and Penicillium cyclopium isolated in the warehouses produced, under experimental conditions, ochratoxin A, sterigmatocystin, and penicillic acid, respectively.

A glucose-yeast extract-salt medium containing 0, 5, 7.5, 10, 15 or 20 micrograms pimaricin/ml with an initial pH of 3.5 or 5.5 was inoculated with Aspergillus parasiticus WB 108 and incubated at 15 degrees or 28 degrees C. The pH, weight of mycelium and amount of aflatoxin produced were determined after 3, 7, and 10 days and after 14, 21, and 30 days when incubation was at 28 degrees or 15 degrees C, respectively. Increasing the concentration of pimaricin in the medium with an initial pH of 5.5 decreased the amounts of aflatoxin B1 and G1 produced after 3 days of incubation. When the initial pH of the medium was 3.5, no growth or toxin production occurred after 3 days of incubation in the medium containing 7.5 micrograms or more of pimaricin/ml. The presence of 20 micrograms of pimaricin/ml inhibited growth and toxin production after 7 days of incubation. When cultures were incubated at 15 degrees C, there was a lag phase which extended from 9 to 16 days, and the amounts of aflatoxin produced decreased with an increasing concentration of pimaricin. Pimaricin did not completely inhibit the growth and aflatoxin production by A. parasiticus. Pimaricin, in combination with a low pH, low temperature or 4% or 6% NaCl, initially caused slow mycelial growth and low toxin production, but the mold overcame the inhibitory effects and produced substantial amounts of mycelium and toxin.

OBJECTIVE

To investigate the causative fungi of fungal keratitis in Japan and their drug susceptibility.

METHODS

Identification and antifungal susceptibility test for 8 drugs (micafungin, amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, miconazole and pimaricin) were performed using isolated fungi from patients with fungal keratitis treated at 27 facilities in Japan between November 1, 2011 and October 31, 2013.

RESULTS

Fungal strains were detected in 72 (50.7%) out of 142 samples. The major isolates were Fusarium spp. (18), Candida parapsilosis (12), C. albicans (11) and Alternaria spp. (6), in all, fungi of 31 species were identified by gene analysis. In the yeast-like fungi, susceptibility rates were evident for more than 80% in voriconazole, pimaricin, flucytosine, micafungin, amphotericin B and fluconazole. In filamentous fungi, the susceptibility rate was less than 50% except for PMR (90%). Fusarium spp., which were susceptible to amphotericin B and pimaricin, showed lower susceptibility rates compared with other genera.

CONCLUSIONS

Although various genera and species of fungi cause fungal keratitis, the obtained drug susceptibility data in this study demonstrates the different susceptibility patterns among the major isolates (Fusarium spp., C. parapsilosis, C. albicans and other groups). This is important evidence useful for fungal keratitis treatment.

Strains of Saccharomyces cerevisiae FL200 capable of growing on a solid medium containing a mixture of polyene macrolide antibiotics (nystatin, 40 micrograms/ml, amphotericin B, 40 micrograms/ml, pimaricin, 150 micrograms/ml and RP9971 antibiotic, 10 micrograms/ml) have been isolated after successive selection steps. The mutant strains, PR13 and PRC24, are 10 to 100 times more resistant than the polyene macrolide antibiotics. When 4% Tween 80 is added to the medium, resistance to these antifungal drugs is further increased. In addition, strain PRC24, derived from strain PR13, is resistant to a non-polyene macrolide antifungal antibiotic, cycloheximide. In contrast, PR13 and PRC24 are both highly susceptible to a large range of compounds, including non-polyenic antifungal, antitumor and antibacterial agents. These particular characteristics make these strains useful for the rapid detection of antifungal compounds of the polyene macrolide and cycloheximide types, as well as for the recognition of antimitotic substances.

The standard Pythium selective medium PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar), was modified by replacing PCNB and pimaricin with other antifungal agents. Several antifungal agents such as fluazinam, miconazole, 2,4,5,6-tetrachloroisophthalonitrile (TPN), iminoctadine triacetate, tolclofos-methyl, captan, and nystatin, initially were screened for effects on Pythium growth. Based on these results, the following three media were developed: PARF (pimaricin + ampicillin + rifampicin + fluazinam agar), NARF (nystatin + ampicillin + rifampicin + fluazinam agar), and NARM (nystatin + ampicillin + rifampicin + miconazole agar). New media were comparable with PARP on yield of naturally occurring Pythium spp. from two different types of soil using the soil-dilution plating technique. PARF and NARF were significantly better than PARP on inhibition of non-pythiaceous microbes on the soil-dilution plates, but were significantly lower than PARP on the rate of mycelial growth of six of eight isolates belonging to seven species of Pythium. NARM was equivalent to PARP on inhibition of non-pythiaceous microbes except for Fusarium oxysporum, and was significantly better than PARP on rate of mycelial growth of five of eight isolates of Pythium.

Polyene antibiotics, including amphotericin, nystatin, pimaricin, and tetramycin, are important antifungal agents. Increasing the production of polyenes and generation of their improved analogues based on the biosynthetic pathway engineering has aroused wide concern in application researches. Herein, tetramycin and nystatin, both of which share most of acyl-CoA precursors, are produced by Streptomyces hygrospinosus var. beijingensis CGMCC 4.1123. Thus, the intracellular malonyl-CoA is found to be insufficient for PKSs (polyketide synthases) extension of tetramycin by quantitative analysis in this wild-type strain. To circumvent this problem and increase tetramycin titer, the acyl-CoA competing biosynthetic gene cluster (BGC) of nystatin was disrupted, and the biosynthetic genes of malonyl-CoA from S. coelicolor M145 were integrated and overexpressed in nys-disruption mutant strain (SY02). Moreover, in order to specifically accumulate tetramycin B from A, two copies of tetrK and a copy of tetrF were introduced, resulting in elevating tetramycin B fermentration titer by 122% to 865 ± 8 mg/L than the wild type. In this optimized strain, a new tetramycin derivative, 12-decarboxy-12-methyl tetramycin B, was generated with a titer of 371 ± 26 mg/L through inactivation of a P450 monooxygenase gene tetrG. Compared with tetramycin B, the new compound exhibited higher antifungal activity against Saccharomyces cerevisiae and Rhodotorula glutinis, but lower hemolytic toxicity to erythrocyte. This research provided a good example of employing biosynthetic engineering strategies for fermentation titer improvement of polyene and development of the derivatives for medicinal applications.