The efficacy of natamycin (pimaricin) was studied and compared with the standard amphotericin B, using a reproducible model of keratomycosis from Candida albicans in the rabbit. C. albicans DSM 70010 (2.5 X 10(5) cells) was injected into the corneal stroma of both eyes. All eyes developed a corneal ulcer without pretreatment with immunosuppressive agents. Forty-eight hours after inoculation, the animals were divided into five groups: I (10 eyes) and II (12 eyes) receiving natamycin drops, 1%; groups III (12 eyes) and IV (12 eyes) receiving natamycin, 2.5%; the control group (14 eyes) receiving 0.9% NaCl. For better penetration of the drug, the epithelium of groups I and III was removed every 3rd day. Half of the control group was debrided as well. In a further series, the penetration of natamycin into the cornea and aqueous humor was studied. There was no difference between the treated eyes and the control eyes concerning complications (descemetocele or perforation). It is concluded that natamycin (pimaricin) is inferior to amphotericin B and not effective in controlling experimental keratomycosis.

The sensitivity against 5 antimycotics was examined with 353 yeast strains of 13 species from clinical material. The tests were carried out by means of the disc diffusion test on Bacto yeast morphology agar under standardized conditions. 33 (6.3%) of the isolates were resistant against 5-fluorocytosine, 11 (2.1%) strains showed no sensitivity against the imidazolyl antimycotics clotrimazole or miconazole. The resistant strains belonged to the species Candida albicans and Candida tropicalis. All of the isolates were sensitive to the polyene antimycotics nystatin and pimaricin (natamycin).

ABSTRACT A microbioassay was developed for the discovery of compounds that inhibit Phytophthora spp. This assay uses a 96-well format for high-throughput capability and a standardized method for quantitation of initial zoospore concentrations for maximum reproducibility. Zoospore suspensions were quantifiable between 0.7 and 1.5 x 10(5) zoospores per ml using percent transmittance (620 nm). Subsequent growth of mycelia was monitored by measuring optical density (620 nm) at 24-h intervals for 96 h. Full- and half-strength preparations of each of three media (V8 broth, Roswell Park Memorial Institute mycological broth [RPMI], and mineral salts medium) and four zoospore concentrations (10, 100, 1,000, and 10,000 zoospores per ml) were evaluated. Both full- and half-strength RPMI were identified as suitable synthetic media for growing P. nicotianae, and 1,000 zoospores per ml was established as the optimum initial concentration. The assay was used to determine effective concentration values for 50% growth reduction (EC(50)) for seven commercial antifungal compounds (azoxystrobin, fosetyl-aluminum, etridiazole, metalaxyl, pentachloronitrobenzene, pimaricin, and propamocarb). These EC(50) values were compared with those obtained by measuring linear growth of mycelia on fungicide-amended medium. The microbioassay proved to be a rapid, reproducible, and efficient method for testing the efficacy of compounds that inhibit spore germination in P. nicotianae and should be effective for other species of Phytophthora as well. The assay requires relatively small amounts of a test compound and is suitable for the evaluation of natural product samples.

The potentiation of the antiviral activity of acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] by polyene macrolide antibiotics has been studied as a function of the macrolide structure. The 12 polyenes chosen for this study represented the major structural groups of these antibiotics and induced in mammalian cells repairable membrane alterations or irreversible cell damage. The potentiating activity of the polyene macrolides was determined based on the differential decrease of in vitro production of infectious virions in the presence of acyclovir alone or in combination with the polyene. Pseudorabies virus, a representative herpesvirus susceptible to acyclovir, was replicated in BHK-21 cells grown in serum-free medium to avoid the interference of serum factors in the polyene macrolide-cell interaction. The potentiation activity of the polyene antibiotics was concentration dependent. The enhancement of the antiviral activity of acyclovir was observed at polyene concentrations which had no direct effect on pseudorabies virus replication in BHK-21 cells. The optimal potentiating concentrations of polyenes were 2 to 15 times lower than that inducing 50% of potassium efflux from BHK-21 cells. The highest potentiating activity was observed for the methyl ester of the trimethylammonium derivative of aureofacin B, which reduced the pseudorabies virus titer by two orders of magnitude. Potentiation by polyene macrolides appeared to coincide with the K+-dependent membrane repair process. The acyclovir potentiating activity was associated with polyene macrolide antibiotics having a large and rigid macrolide ring (amphotericin B and aureofacin). Polyene antibiotics with small and rigid (pimaricin and filipin) or large but flexible (nystatin A1 and lienomycin) macrolide rings showed no potentiation of the antiviral effect of acyclovir.

The sensitivity of Saccharomyces cerevisiae spores and vegetative cells to various antimicrobial compounds was compared. Sulphur dioxide, benzoic acid, potassium sorbate, salicylic acid, nystatin, actidione and pimaricin were tested. Generally, the Saccharomyces spores were more resistant than the corresponding vegetative cells. It was also observed that this greater resistance shown by the spores varied with the antimicrobial compound used. Only potassium sorbate was not selective and killed both vegetative cells and spores at about the same rate.

To investigate the molecular mechanisms that interplay between oxygen metabolism and secondary metabolism in Streptomyces natalensis, we compared the transcriptomes of the strains CAM.02 (ΔsodF), pimaricin under-producer phenotype, and CAM.04 (ΔahpCD), pimaricin over-producer phenotype, with that of the wild type at late exponential and stationary growth phases. Microarray data interpretation was supported by characterization of the mutant strains regarding enzymatic activities, phosphate uptake, oxygen consumption and pimaricin production.Both mutant strains presented a delay in the transcription activation of the PhoRP system and pimaricin biosynthetic gene cluster that correlated with the delayed inorganic phosphate (Pi) depletion in the medium and late onset of pimaricin production, respectively. The carbon flux of both mutants was also altered: a re-direction from glycolysis to the pentose phosphate pathway (PPP) in early exponential phase followed by a transcriptional activation of both pathways in subsequent growth phases was observed. Mutant behavior diverged at the respiratory chain/tricarboxylic acid cycle (TCA) and the branched chain amino acid (BCAA) metabolism. CAM.02 (ΔsodF) presented an impaired TCA cycle and an inhibition of the BCAA biosynthesis and degradation pathways. Conversely, CAM.04 (ΔahpCD) presented a global activation of BCAA metabolism.The results highlight the cellular NADPH/NADH ratio and the availability of biosynthetic precursors via the BCAA metabolism as the main pimaricin biosynthetic bottlenecks under oxidative stress conditions. Furthermore, new evidences are provided regarding a crosstalk between phosphate metabolism and oxidative stress in Streptomyces.

An improved general method for the preparation of methyl esters of polyene antibiotics is discussed. Using this method methyl esters of pimaricin, nystatin, eurocidin, hamycin, hamycins A and B, aureofungin, partricins A and B, candimycin, candicidin, and amphotericin B have been prepared and their physical properties are reported. The biological activities of hamycins A and B and their methyl esters are also described.

Sequence analysis of ketosynthase domain amplicons from Streptomyces bicolor NBRC 12746(T) revealed the presence of previously unreported type I polyketide synthases (PKS-I) genes. The clustering of these genes with the reference PKS-1 sequences suggested the possibility to produce a polyene compound similar to pimaricin. Thus, the cultured sample from NBRC 12746(T) was analyzed for the production of polyene compounds. The strain produced an antifungal compound which displayed the UV absorption spectrum of tetraene macrolides. The structure determination based on the spectroscopic analysis of the purified compound resulted in the identification of a novel pimaricin analog JBIR-13 (1). This study therefore strongly suggested that a careful analysis of PKS-I genes can provide valuable information in the search of novel bioactive compounds within a class predicted from phylogenetic analysis.

Cytotoxicity and membrane permeability alterations induced by the polyene macrolide antibiotics filipin (FIL) and pimaricin (PIM) have been compared in parental intraspecific and interspecific somatic cell hybrids. B82 (mouse) and B1 (hamster) cells were found to be more resistant than RAG (mouse) parental cells to both polyene macrolides as indicated by 24-hour survival, 72-hour viability, and growth rate. Analysis of both intraspecific and interspecific somatic cell hybrids indicated that polyene macrolide resistance was being expressed even in the presence of the polyene macrolide-sensitive (RAG) genome. Where one of the two parental cell types is relatively polyene macrolide resistant, the use of specific polyene macrolides may prove efficacious as half-selective agents in cell hybridization.

Before 1950 no reliable or safe therapy existed for systemic and invasive mycoses, and only traditional and empirical topical preparations were available for dermatomycoses. Two distinct eras of rapid progress in antifungal therapy followed: first, in the 1950's came the introduction of the polyenes, nystatin and pimaricin for cutaneous, vaginal and intestinal candidiasis, and amphotericin B for the treatment of severe systemic mycoses. The second phase saw the successful introduction and clinical use of 5-fluorocytosine and several imidazole derivatives some twenty years later, at a time when the vast increase in iatrogenic systemic mycoses caused by opportunistic fungi had created an urgent and pressing need for new agents in addition to those still effective.

An experimental Keratitis study of Aspergillus fumigatus was performed in 130 rabbits divided into 12 groups of ten animals each. Three antifungal drugs (myconazole, amphotericin B and pimaricin) were tested using two procedures (topical drops and subconjunctival injections) and two different concentrations (500 and 10 000 times the MIC). In each case, the drugs were applied every 3 h starting 14 h after inoculation. Miconazole was useful at 10 mg/ml concentration by topical drops and subconjunctival injections, but was less useful at 5 mg/ml. Amphotericin B was useful at 5 mg/ml concentration by topical drops and less useful at 2 mg/ml. No differences were found between the two concentrations by subconjunctival administration. Pimaricin was useful by topical drops at 50 mg/ml concentration and less useful at 10 mg/ml as well as by subconjunctival injections.

Using histological, immunohistological and electron microscopical methods, it could be demonstrated that the course of experimental Candida albicans keratitis in mice invlves three phases: a traumatically caused alternative phase is followed by a granulocytic exsudative phase and finally by a proliferative phase. The experiments were followed over 41 days. Fungal elements could be detected only during the first 4 days after infection. When topically treated with hydrocortisone, the exsudation of leucocytes was diminished and delayed and the cellular proliferation was reduced considerably in the challenged corneal stroma. A lower number of Candida organisms was found in the cornea when treatment with hydrocortisone was combined with the antifungal drug pimaricin. When treated with pimaricin only however, fungal growth and inflammation were least apparent.

A quick and simple affinity chromatography method for gauging the interaction of polyene antifungal agents with sterols has been developed. The required affinity columns are prepared from a standard C-18 reverse-phase HPLC column by injecting a measured quantity of sterol under conditions where it is completely retained. After the assay, the sterol is eluted with a less polar solvent and the column reused. By comparing the elution volume of a polyene injected onto the sterol-free column (Ve) with that of the polyene injected onto the sterol-doped column (V), an association constant (Ka) for the polyene-sterol complex was determined. Association constants of different amphotericin B-sterol and pimaricin-sterol complexes were determined and correlated with the polyene's ability to induce membrane permeability and its antifungal properties. This procedure provides a new tool for screening polyene macrolides for antifungal therapy.

ABCA1 belongs to the A class of ABC transporter, which is absent in yeast. ABCA1 elicits lipid translocation at the plasma membrane through yet elusive processes. We successfully expressed the mouse Abca1 gene in Saccharomyces cerevisiae. The cloned ABCA1 distributed at the yeast plasma membrane in stable discrete domains that we name MCA (membrane cluster containing ABCA1) and that do not overlap with the previously identified punctate structures MCC (membrane cluster containing Can1p) and MCP (membrane cluster containing Pma1p). By comparison with a nonfunctional mutant, we demonstrated that ABCA1 elicits specific phenotypes in response to compounds known to interact with membrane lipids, such as papuamide B, amphotericin B and pimaricin. The sensitivity of these novel phenotypes to the genetic modification of the membrane lipid composition was studied by the introduction of the cho1 and lcb1-100 mutations involved respectively in phosphatidylserine or sphingolipid biosynthesis in yeast cells. The results, corroborated by the analysis of equivalent mammalian mutant cell lines, demonstrate that membrane composition, in particular its phosphatidylserine content, influences the function of the transporter. We thus have reconstituted in yeast the essential functions associated to the expression of ABCA1 in mammals and characterized new physiological phenotypes prone to genetic analysis. This article is a part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

A cryptic plasmid, pSNA1, has been identified in the pimaricin-producing Streptomyces natalensis strain ATCC 27448. pSNA1 has been mapped with restriction endonucleases and its complete nucleotide sequence was determined. The circular DNA molecule is 9367 bp in length and has a 71.3% G+C content. Its estimated copy number is 30. Analysis of the sequence and codon preferences indicated that pSNA1 contains seven open reading frames [encoding peptides larger than 90 amino acid (aa) residues], ORF 1 to ORF 7, located on both strands of pSNA1. ORF 3 codes for a protein (476 aa) that shows high sequence similarity to replication-associated proteins in Streptomyces plasmids known to replicate via the rolling circle mechanism. Accumulation of single-strand intermediates further indicates that pSNA1 replicates via the rolling circle replication model. ORF 1 encodes a polypeptide of 246 aa that shares homology with KorA proteins encoded by other streptomycete plasmids. ORF 4 (SpdA) codes for a protein (161 aa) possibly involved in intramycelial plasmid transfer. Protein encoded by ORF 2 (309 aa) shares homology with a Streptomyces protein (SpdB2) also involved in plasmid spreading.

Tandem mass spectrometry combined with Fourier transform ion cyclotron resonance (FT-ICR) has been the basis for rationalizing the fragmentation mechanisms of anti-fungal macrolides nystatin A(1), amphotericin B and pimaricin. The positive ion mass spectra were not informative, however, the dissociation of deprotonated molecules led to structurally significant ring-opened fragments. Using this approach of tandem FT-ICR mass spectrometry and electrospray ionisation coupled with high-performance liquid -chromatography (HPLC), 11 macrolide natural analogues or degradation products were characterised in the nystatin mixture.

A serial dilution and a disc method were used for evaluation of susceptibility of 50 dermatophyte strains belonging to the species Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis and Epidermophyton floccosum. Following drugs were investigated: griseofulvin, pimaricin, clotrimoxazole, miconazole, ketoconazole, biphonazole and naftifin. Application of two methods of testing resulted in high convergence of determinations. Naftifin was most effective and pimaricin least active. Among imidazole drugs, relatively high activity against dermatophytes was exhibited by clotrimoxazole and ketoconazole. Strain of Trichophyton rubrum were more susceptible to antifungal drugs than strains of Trichophyton mentagrophytes.

Pimaricin is an important polyene antifungal antibiotic that binds ergosterol and extracts it from fungal membranes. In previous work, two pimaricin derivatives (1 and 2) with improved pharmacological activities and another derivative (3) that showed no antifungal activity were produced by the mutant strain of Streptomyces chattanoogensis, in which the P450 monooxygenase gene scnG has been inactivated. Furthermore, inactivation of the DH12 dehydratase domain of the pimaricin polyketide synthases (PKSs) resulted in specific accumulation of the undesired metabolite 3, suggesting that improvement of the corresponding dehydratase activity may reduce or eliminate the accumulation of 3. Accordingly, the DH12-KR12 didomain within the pimaricin PKS was swapped with the fully active DH11-KR11 didomain. As predicted, the mutant was not able to produce 3 but accumulated 1 and 2 in high yields. Moreover, the effect of the flanking linker regions on domain swapping was evaluated. It was found that retention of the DH12-KR12 linker regions was more critical for the processivity of hybrid PKSs. Subsequently, high-yield production of 1 or 2 was obtained by overexpressing the scnD gene and its partner scnF and by disrupting the scnD gene, respectively. To our knowledge, this is the first report on the elimination of a polyketide undesired metabolite along with overproduction of desired product by improving the catalytic efficiency of a DH domain using a domain swapping technology.

Mild acid treatment of natamycin (IV) results in biologically inactive aponatamycin (VI), an amphoteric substance with some natamycin-like chemical and physical properties. Aponatamycin contains one natamycin- and one natamycinolide-moiety. More drastic acid degradation of natamycin eliminates the aminosugar under formation of the dimer (VII) of the hypothetical aglycone of natamycin, natamycinolide (V) as well as a non-ionic compound, the dimer of the 12-decarboxy-11-anhydro analogue of natamycinolide.

We investigated the inhibitory effects of antibacterial, biocidal, and antifungal agents against Fusarium spp. Seven Fusarium spp: four F. falciforme (Fusarium solani species complex), one Fusarium spp, one Fusarium spp. (Fusarium incarnatum-equiseti species complex), and one F. napiforme (Gibberella fujikuroi species complex), isolated from eyes with fungal keratitis were used in this study. Their susceptibility to antibacterial agents: flomoxef, imipenem, gatifloxacin, levofloxacin, moxifloxacin, gentamicin, tobramycin, and Tobracin® (contained 3,000 μg/ml of tobramycin and 25 μg/ml of benzalkonium chloride (BAK), a biocidal agent: BAK, and antifungal agents: amphotericin B, pimaricin (natamycin), fluconazole, itraconazole, miconazole, voriconazole, and micafungin, was determined by broth microdilution tests. The half-maximal inhibitory concentration (IC50), 100% inhibitory concentration (IC100), and minimum inhibitory concentration (MIC) against the Fusarium isolates were determined. BAK had the highest activity against the Fusarium spp. except for the antifungal agents. Three fluoroquinolones and two aminoglycosides had inhibitory effects against the Fusarium spp. at relatively high concentrations. Tobracin® had a higher inhibitory effect against Fusarium spp. than tobramycin alone. Amphotericin B had the highest inhibitory effect against the Fusarium spp, although it had different degrees of activity against each isolate. Our findings showed that fluoroquinolones, aminoglycosides, and BAK had some degree of inhibitory effect against the seven Fusarium isolates, although these agents had considerably lower effect than amphotericin B. However, the inhibitory effects of amphotericin B against the Fusarium spp. varied for the different isolates. Further studies for more effective medications against Fusarium, such as different combinations of antibacterial, biocidal, and antifungal agents are needed.

The fractional change in the corrected fluorescence of pimaricin or filipin in the presence of a limiting amount of sterol and a competing polyene antibiotic has been used to estimate the relative affinity of amphotericin B, nystatin, filipin, and pimaricin for stigmasterol and for cholesterol. The relative affinities for cholesterol were filipin greater than amphotericin B greater than pimaricin greater than nystatin, while the relative affinities for stigmasterol were filipin greater than pimaricin greater than amphotericin B greater than nystatin. The data indicate that pimaricin and filipin can both interact simultaneously with about 30% of the cholesterol or stigmasterol. However, the stoichiometry of filipin and pimaricin alone for cholesterol and for stigmasterol in dilute aqueous solutions is 1 : 1. In the experiments which indicated both pimaricin and filipin interact with the same sterol molecule changes in corrected fluorescence and the absorbance spectra were monitored; and these criteria indicated that both pimaricin and filipin had interacted with the sterols. Light-scattering measurements indicate large aggregates were not formed. Although the data shows in dilute aqueous solutions the stoichiometry of filipin and/or pimaricin for sterols is 1 : 1, in more complex solutions, other combinations or interactions are indicated especially for pimaricin.

Frequency of occurrence of fungal species distinct from C. albicans isolated from vaginal mucosa and their sensitivity to antimycotic chemotherapeutics was determined. Material consisted of 452 fungal strains isolated from vagina from patients suffering from afflictions within genital area. Fungal strains isolated belonged to 13 genera. Fungi distinct from C. albicans constituted 27.1% of all the strains. Fungi the most frequently isolated from vagina belonged to following genera: T. glabrata 35.2% C. krusei 18.4% C. pseudotropicalis 15.2% S. cerevisiae 10.4%. In the majority of cases of vaginal infections caused by fungi distinct from C. albicans, Lactobacillus sp. was present and normal pH values of vaginal content 3/4 with variable number of leucocytes were observed. Evaluation of sensitivity to antimycotic drugs of fungal strains was performed by agar dilution technique. In this study the following chemotherapeutics were assayed: nystatin, pimaricin, amphotericin B, flucytosine, clotrimazole, miconazole and ketoconazole. It is worth to underline resistance of T. glabrata and S. cerevisiae to clotrimazole and ketoconazole. Moreover, resistance of strains belonging to genera C. krusei and C. pseudotropicalis to amphotericin B and C. krusei strains to nystatin and flucytosine was noted.