The aim of this study was to determine the sensitivity of Aspergillus niger strains isolated from birds to available antifungal drugs using different in vitro assays--classical disk diffusion, Etest and broth microdilution NCCLS/CLSI M 38-A. The study material consisted of about 2.000 swabs and samples from different species of birds. A. niger (n=10) was accounted for 6.81% of the total pool of strains isolated. Determinations were made for 13 antifungal drugs using the disk diffusion method. The A. niger exhibited high susceptibility to enilconazole, terbinafine, voriconazole, tioconazole and ketoconazole, low susceptibility to clotrimazole, miconazole and nystatin, and resistance to amphotericin B, itraconazole, pimaricin, fluconazole and 5-fluorocytosine. Minimum inhibitory concentration (MIC) was determined for 9 antifungal drugs using the micromethod of duplicate serial dilutions in a liquid medium. A. niger strains were most susceptible to enilconazole and voriconazole. MIC ranged from 0.0625 to 0.5 microg/ml for enilconazole, with MIC90-0.5 microg/ml and MIC50-0.125 microg/ml. The corresponding values for voriconazole were 0.25-1 microg/ml, 1 microg/ml and 0.5 microg/ml. MIC for amphotericin B and terbinafine ranged from 0.5 to 4 microg/ml, while the values for the remaining drugs were highly varied. MIC was measured by the gradient diffusion method using Etest for 5 antifungal drugs: amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole. By far the highest susceptibility was obtained in the case of voriconazole, with MIC ranging from 0.0625 to 1 microg/ml. MIC for amphotericin B ranged from 0.25 to 4 microg/ml, for itraconazole and ketoconazole ranging from 0.5 to 16 microg/ml. Methods available for this purpose are not always applicable in field conditions. The present results indicate that the Etest technique, due to its high percentage of agreement with the M 38-A microdilution method, should find application in medical and veterinary practice.

Polyene antifungal antibiotics produce various degrees of membrane damage in sheep erythrocytes in vitro. Mediocidin, filipin, amphotericin B, and candicidin were found to result in greater damage than nystatin, pimaricin, and amphotericin B methyl ester. The degree of sensitivity of the cells varied by 100-fold for mediocidin verus amphotericin B methyl ester as measured by curves of hemoglobin release versus drug concentration. In erythrocytes prelabeled with radioactive chromium, release of the isotope through polyene-damaged cell membranes was found to occur at lower drug concentrations than measurable hemoglobin release, and the percentage of isotope released at the highest drug dose was consistently greater than the percentage of hemoglobin released. Thus, the isotope assay is a more sensitive indicator of polyene-induced membrane damage in the test system. These significant differences in release of molecules through polyene-induced membrane lesions indicate the complex nature of the binding and further interactions of this class of drugs with the plasma membrane.

Previous studies from our laboratory have shown that Chinese hamster V79 cells mutated to high level resistance to amphotericin B have a lower cellular level of cholesterol, the target molecule for the polyene antibiotic. Two amphotericin B-resistant (AMBR) mutants were each hybridized to their parental amphotericin B-sensitive (AMBS) V79 cells. All the hybrids derived from AMBR/AMBS fusions were as sensitive to polyene antibiotics (amphotericin B, filipin, and pimaricin) as AMBS cells or AMBS/AMBS hybrids. The AMBR/AMBS hybrids were found to contain cholesterol per phospholipids that is comparable to those in AMBS or AMBS/AMBS. The analysis of hybrids formed between mutant and wild-type cells thus indicated that resistance to amphotericin B is a recessive marker, and that the cellular level of cholesterol is compensated in the AMBS/AMBR hybrids. Hybrids of AMBR and AMBR cells were all resistant, so that the three AMBR mutants all fell into a single complementation group.

Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT(0) in the nystatin PKS loading module NysA with the propionate-specific AT(1) from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT(0) domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT(0) and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT(0) domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of a rimA-deficient mutant of the rimocidin/CE-108 producer Streptomyces diastaticus. Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT(0) domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS of Streptomyces noursei.

Pregnancy creates conditions which favour the development and persistence of candidiasis (moniliasis). The resultant vaginitis is often refractory to treatment. A new fungicidal antibiotic, pimaricine (Pimafucine), was studied in 91 women with vaginal moniliasis, all of whom were pregnant. Fifty-six (63%) were cured on the basis of negative cultures and 28 became symptomfree in spite of positive cultures. In this series, 94.3% benefited from treatment.

Fungicides inhibiting sterol biosynthesis belong to chemically distinct classes such as imidazole, morpholine, pyridine, pyrimidine and triazole derivatives. Incubation of mycelium of Aspergillus nidulans for 90 min with representatives of these fungicides induced an efflux activity which prevented accumulation of fenarimol, a pyrimidine derivative, into the mycelium. Induction of this efflux activity reduced the fungitoxicity of fenarimol. Addition of oligomycin to mycelium in which fenarimol-efflux activity was induced immediately increased the uptake of fenarimol, indicating that the efflux activity is energy-dependent. Subsequent disruption of membrane permeability with sodium lauryl sulphate instantaneously caused leakage of fenarimol from the mycelium into the medium. The ability to induce fenarimol efflux is rather specific for inhibitors of sterol biosynthesis: except for pimaricin, fungicides with unrelated mechanisms of action did not have this ability.

Clinical findings and treatment of keratomycosis caused by Beauveria bassiana, an entomopathogenic filamentous fungus, are described for an 80-year-old woman, who was referred to the hospital for ocular pain and redness on the 9th day after an ocular injury caused by the frame of her glasses. She had a long history of recurrent diabetic iritis and continuously used topical antibiotics and corticosteroids. At her first visit, a slit-lamp examination indicated a corneal ulcer confined within the superficial stromal layer, along with a slight infiltration and edema. Only a very few inflammatory cells were seen in the anterior chamber. Direct microscopic examination of corneal scrapings revealed septate fungal hyphae with zig-zag rachis and budding that was subsequently identified as B. bassiana by slide culture. Topical voriconazole with miconazole, pimaricin and oral itraconazole were effective and the lesion disappeared leaving only a mild scar at 2 months. The sensitivity of B. bassiana to various antimycotic agents was confirmed by broth microdilution, agar dilution with the Clinical Laboratory Standard Institute standard, and a disk method using topically applied concentrations. B. bassiana, which exhibits a characteristic appearance in smears and causes superficial keratomycosis, is sensitive to voriconazole with miconazole, pimaricin, and itraconazole.

OBJECTIVE

To report the isolation of Pestalotiopsis clavispora from the cornea of a patient with recurrent keratitis.

METHODS

A 73-year-old male gardener presented with conjunctival injection and an oval infiltrate with feathery margins in the temporal half of the cornea in the right eye. His ocular history in the right eye included cataract surgery, five episodes of herpes simplex keratitis, three glaucoma surgeries, and bullous keratopathy. He had been treated with corticosteroids for years. Light microscopy of corneal scrapings revealed a filamentous fungus, and fungal keratitis was diagnosed. Treatment with topical voriconazole and pimaricin ointment was commenced. One month later, the infiltrate resolved. The antifungal agents were discontinued 7 months later, and keratitis relapsed 4 days after the discontinuation. The fungus was isolated and identified by molecular techniques as P. clavispora. Based on the results of antifungal susceptibility testing, treatment with topical and intravenous micafungin was initiated. The corneal infiltrate resolved 1 month after the relapse.

CONCLUSIONS

Molecular identification of the pathogen, and antifungal susceptibility testing, are useful in treating patients with fungal keratitis caused by a rare human pathogen.

The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimRSARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding.IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM The expression of this gene is controlled by the Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator PimR, which binds a series of heptameric direct repeats in its promoter region. The structure and importance of such repeats in protein binding, transcriptional activation, and polyene production have been investigated. These findings should provide important clues to understand the regulatory machinery that modulates antibiotic biosynthesis in Streptomyces and open new possibilities for the manipulation of metabolite production. The presence of PimR orthologues encoded by gene clusters for different secondary metabolites and the conservation of their operators suggest that the improvements observed in the activation of pimaricin biosynthesis by Streptomyces natalensis could be extrapolated to the production of different compounds by other species.

Pimaricin is an important antifungal antibiotic for antifungal therapy and prevention of mould contamination in the food industry. In this study, three new pimaricin derivatives, 12-decarboxy-12-methyl pimaricin (1), 4,5-desepoxy-12-decarboxy-12-methyl pimaricin (2), and 2-hydro-3-hydroxy-4,5-desepoxy-12-decarboxy-12-methyl pimaricin (3), were generated through the inactivation of P450 monooxygenase gene scnG in Streptomyces chattanoogensis L10. Compared with pimaricin, 1 displayed a twofold increase in antifungal activity against Candida albicans ATCC 14053 and a 4.5-fold decrease in cytotoxicity with erythrocytes, and 2 had comparable antifungal activity and reduced cytotoxicity, whereas 3 showed nearly no antifungal and hemolytic activities. Genetic and biochemical analyses proved that 1 is converted from 2 by P450 monooxygenase ScnD. Therefore, the overexpression of scnD in scnG-null strain eliminated the accumulation of 2 and improved the yield of 1 by 20 %. Conversely, scnG/scnD double mutation abolished the production of 1 and improved the yield of 2 to 2.3-fold. These results indicate that the pimaricin derivatives with improved pharmacological properties obtained by genetic engineering can be further developed into antifungal agents for potential clinical application.

Complete and independent tetramycin and nystatin gene clusters containing varying lengths of type I polyketide synthase (PKS) genes were isolated from Streptomyces ahygroscopicus, a producer of tetramycin (a tetraene) in large amounts and nystatin A1 (a heptaene) in small amounts. Tetramycin was similar to pimaricin, and nystatin A1 was similar to amphotericin. All these polyene macrolide antibiotics possessed the same macrolactone ring biosynthesized from coenzyme A precursors by PKSs but had different number of atoms in the macrolactone ring and side groups. Because tetramycin and nystatin shared limited coenzyme A precursors in the same producer organism, blocking the consumption of precursors in tetramycin pathway may increase the coenzyme A pool. Thus, we genetically manipulated the tetramycin PKS to enhance nystatin production. The type I PKS ttmS1 gene mutant abolished production of tetramycin and had a beneficial effect on the production of nystatin A1. For the mutant, the yield of nystatin A1 was increased by 10-fold compared to that of the wild-type. Thus, deletion of the tetramycin pathway redirected precursor metabolic fluxes and provided an easy genetic approach to manipulate organisms and to increase production levels of a precise target.

The aim of this study was to elucidate in vitro antiamoebic activity of antimicrobial agents at short exposure times similar to those used for actual treatment against Acanthamoeba strains isolated from patients with keratitis.

The 5 clinical Acanthamoeba isolated in Japan were used in this study. Identification of genotypes for the Acanthamoeba isolates was performed using partial 18S ribosomal DNA (rDNA), including the ASA.S1 region sequences. Fluconazole, miconazole, itraconazole, voriconazole, amphotericin B, natamycin (pimaricin), and micafungin (antifungal agents), and chlorhexidine (a biguanide disinfectant), and sulfamethoxazole and paromomycin (antibacterial agents) were used to determine the antiamoebic activity against Acanthamoeba, which were determined by 50% and 90% growth inhibitory concentrations (IC50 and IC90) following exposing to each drug at 25°C for 7 days and 12 h.

Among the tested antimicrobial agents, natamycin strongly inhibited the growth of all Acanthamoeba isolates at low concentration in both the 7-day (IC90 = 4.1 μg/mL) and 12-h (IC90 = 11.6 μg/mL) assays. Additionally, sulfamethoxazole exhibited strong antiamoebic activity (IC90 = 9.8 μg/mL) at low concentration in the 7-day assay.

Our findings showed that natamycin ophthalmic solution might be an effective agent against Acanthamoeba keratitis. Additionally, frequent administration of sulfamethoxazole ophthalmic solution or systemic sulfamethoxazole-trimethoprim is also considered as an effective treatment for Acanthamoeba keratitis.

The ERG6 gene encodes an S-adenosylmethionine dependent sterol C-24 methyltransferase in the ergosterol biosynthetic pathway. In this work we report the results of functional analysis of the Kluyveromyces lactis ERG6 gene. We cloned the KlERG6 gene, which was able to complement the erg6Δ mutation in both K. lactis and Saccharomyces cerevisiae. The lack of ergosterol in the Klerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthesis pathway as well as the KlPDR5 gene encoding an ABC transporter. The Klerg6Δ mutation resulted in reduced cell susceptibility to amphotericin B, nystatin and pimaricin and increased susceptibility to azole antifungals, fluphenazine, terbinafine, brefeldin A and caffeine. The susceptibility phenotype was suppressed by the KlPDR16 gene encoding one of the phosphatidylinositol transfer proteins belonging to the Sec14 family. Decreased activity of KlPdr5p in Klerg6Δ mutant (measured as the ability to efflux rhodamine 6G) together with increased amount of KlPDR5 mRNA suggest that the zymosterol which accumulates in the Klerg6Δ mutant may not fully compensate for ergosterol in the membrane targeting of efflux pumps. These results point to the fact that defects in sterol transmethylation appear to cause a multitude of physiological effects in K. lactis cells. Copyright © 2016 John Wiley & Sons, Ltd.

Corneal infection due to the species Aspergillus flavus is rare and often destructive. This report describes a case of Aspergillus flavus corneal ulcer that failed to respond to amphotericin B, pimaricin, and clotrimazole. The eye subsequently became phthisical. The rational approach to the management of fungal corneal infection is to culture or perform sensitivity tests on the isolated organism. Early surgical intervention should be done if the infection fails to respond to medical treatment, and if the disease process extends deep into the stromal tissue and anterior chamber. Fungal malignant glaucoma requires excisional keratoplasty, lens extraction, and anterior vitrectomy.

The pimaricin-inducing (PI) factor, produced by Streptomyces natalensis is a proposed pheromone with a peculiar vicinal diamine structure. The first synthesis of this molecule is reported. It features oxidative dimerization of an aci-nitro anion derived from tris(hydroxymethyl)nitromethane and disproportionation catalyst-facilitated hydrogenation of the resulting vicinal tertiary dinitro compound. As the synthesis requires only four steps with no chromatographic separations, it provides a convenient route to prepare PI factor for biological studies and industrial applications.

The objective of this study was to describe 2 cases of keratitis caused by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) that occurred in disposable contact lens users, which were successfully treated with topical voriconazole. Case 1 was a healthy 44-year-old woman, who wore weekly disposable contact lenses and had developed a superficial corneal infection in her right eye. For diagnosis, corneal scraping and molecular identification of the cultured pathogen were performed. A corneal smear revealed the presence of fungi. The pathogen was identified as P. lilacinum by traditional morphological identification of fungal culture, and this identification was confirmed by DNA sequencing of the ribosomal internal transcribed spacer (ITS) sequence. Therapy with topical fluconazole, topical pimaricin (natamycin), and oral itraconazole were ineffective. Topical voriconazole showed a significant effect, and the keratitis was successfully treated. Case 2 was a 43-year-old woman with bilateral recurrent peripheral corneal ulcers by meibomian gland dysfunction, who used therapeutic bandage contact lenses on her left eye. However, a corneal abscess with hypopyon occurred in the eye after 3 months. The microbial smear examination showed the presence of fungi and the fungal culture, and the DNA sequence of ITS region revealed that the causative agent was P. lilacinum. The susceptibility testing against antifungal agents showed that voriconazole was effective. The lesion improved gradually by topical voriconazole. As a conclusion, P. lilacinum keratitis can occur in disposable soft contact lens wearer. Early and accurate detection of the pathogenic organism is essential. Topical voriconazole was effective against P. lilacinum keratitis.

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.

Eight beagles were experimentally inoculated intraotally with Malassezia pachydermatis to induce acute otitis externa. Three or 4 days after the inoculation, the animals showed the symptoms of otitis externa. All ear canals were erythematous and the dogs were shaking their heads. A large number of M. pachydermatis was noticed in exudate taken from every ear canal. Clinical signs of otitis externa were reduced after treatment with 0.1 ml (per canal) of 1% pimaricin suspension twice a day for 3 days. The amount of exudate decreased gradually and 12 of the 16 ear swabs examined, thereafter, were found to be negative for M. pachydermatis within 10 days. No side effects were observed in all the treated cases. These results suggested that M. pachydermatis could induce the canine otitis externa, and that pimaricin is effective agent for M. pachydermatis infection in ear canals.