Paecilomyces lilacinus was cultured from an ulcerated corneal transplant, sections of which, following surgical removal, revealed dense mycelial growth throughout the corneal remnant and penetrating Descemet's membrane. PAS-stained sections showed, in addition to the hyphae, numerous small, ovoid elements consistent with conidia of P. lilacinus. Infection apparently had spread to the transplant from the recipient's cornea. The fungal isolate proved resistant, in vitro, to amphotericin B, 5-fluorocytosine, and pimaricin, but sensitive to ketoconazole and moderately sensitive to miconazole. Following a second transplant and intensive antifungal therapy, the infection appears to have been eliminated.

The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by 51Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.

Production of pimaricin by Streptomyces natalensis ATCC 27448 is elicited by the PI-factor, an autoinducer secreted by the producer strain during the rapid growth phase. Exogenous PI-factor restored pimaricin production in a mutant strain npi287 defective in PI-factor biosynthesis. During purification of the PI-factor, a second pimaricin-inducing fraction different from PI-factor was isolated from the culture broth of wild-type S. natalensis ATCC 27448. After purification by HPLC and analysis by MS and NMR, this active fraction was shown to contain glycerol and lactic acid. Pure glycerol restored pimaricin production in liquid cultures of the autoinducer-defective npi287 mutant. A similar effect was exerted by ethylene glycol, 1,2-propanediol and 1,3-propanediol but not by higher polyalcohols or by glycerol acetate or glycerol lactate esters. Glycerol stimulated (30-270 %) the production of six different polyene macrolide antibiotics by their respective producer strains. Addition of glycerol to the inducer-defective npi287 strain restored pimaricin production but did not result in extracellular or intracellular accumulation of PI-factor. Exogenously added PI-factor was internalized by the cells in the presence of glycerol, and a mixture of both PI-factor and glycerol produced a slightly higher inducing effect on pimaricin production than PI-factor alone. In summary, glycerol, ethylene glycol and propanediol exert a bypass of the PI-factor inducing effect on pimaricin biosynthesis.

[reaction: see text] New derivatives of Amphotericin B (AmB) were synthesized through a double reductive alkylation of the mycosamine. These derivatives of AmB displayed superior antifungal activity against Saccharomyces cerevisiae wild-type strain and especially in the case of an AmB-resistant Candida albicans strain. Moreover, these compounds are potential drug candidates because of significantly reduced hemotoxicity compared to AmB. Furthermore, the same mycosamine modification can be applied to other polyene macrolides such as Nystatin and Pimaricin to improve their antifungal activity.

Cholesterol oxidases (3beta-hydroxysterol oxidases; EC 1.1.3.6), serve as catalysts for the initial step in the degradation of cholesterol, and probably other natural sterols, that are used as carbon sources for growth of different bacteria. Because of their suitability for attacking cholesterol they have been widely used for the quantification of cholesterol in clinical and food specimens. Cholesterol oxidase has also found application as a probe for membrane structure, as an insecticide, and has been implicated in bacterial pathogenesis. Recently, we have found that a Streptomyces cholesterol oxidase is required for the biosynthesis of the antifungal polyene pimaricin, apparently acting as an antifungal sensor. Here we describe our current understanding of these fascinating enzymes.

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the Streptomyces antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. The PimR SARP domain (PimR(SARP)) was expressed in Escherichia coli as a glutathione S-transferase (GST)-fused protein. Electrophoretic mobility shift assays showed that GST-PimR(SARP) binds a single target, the intergenic region between the regulatory genes pimR and pimMs in the pimaricin cluster. The PimR(SARP)-binding site was investigated by DNaseI protection studies, revealing that it contains three heptameric direct repeats adjusting to the consensus 5'-CGGCAAG-3'. Transcription start points of pimM and pimR promoters were identified by 5'-RACE, revealing that unlike other SARPs, PimR(SARP) does not interact with the -35 region of its target promoter. Quantitative transcriptional analysis of these regulatory genes on mutants on each of them has allowed the identification of the pimM promoter as the transcriptional target for PimR. Furthermore, the constitutive expression of pimM restored pimaricin production in a pimaricin-deficient strain carrying a deletion mutant of pimR. These results reveal that PimR exerts its positive effect on pimaricin production by controlling pimM expression level, a regulator whose gene product activates transcription from eight different promoters of pimaricin structural genes directly.

The polyene antibiotics, a category that includes nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very promising antifungal polyketide compounds and are typically produced by soil actinomycetes. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly similar cytochrome P450 hydroxylase (CYP) genes. Using polyene CYP-specific PCR screening with several actinomycete genomic DNAs, Pseudonocardia autotrophica was determined to contain a unique polyene-specific CYP gene. Genomic DNA library screening using the polyene-specific CYP gene probe identified a positive cosmid clone, which contained a DNA fragment of approximately 34.5 kb. The complete sequencing of this DNA fragment revealed a total of seven complete and two incomplete open reading frames, which were found to be highly similar, but still unique, when compared to previously known polyene biosynthetic genes. These results suggest that the polyene-specific screening approach may constitute an efficient method for the isolation of potentially valuable cryptic polyene biosynthetic gene clusters from various rare actinomycetes.

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.

Pimaricin (natamycin) is a small polyene macrolide antibiotic used worldwide. This efficient antimycotic and antiprotozoal agent, produced by several soil bacterial species of the genus Streptomyces, has found application in human therapy, in the food and beverage industries and as pesticide. It displays a broad spectrum of activity, targeting ergosterol but bearing a particular mode of action different to other polyene macrolides. The biosynthesis of this only antifungal agent with a GRAS status has been thoroughly studied, which has permitted the manipulation of producers to engineer the biosynthetic gene clusters in order to generate several analogues. Regulation of its production has been largely unveiled, constituting a model for other polyenes and setting the leads for optimizing the production of these valuable compounds. This review describes and discusses the molecular genetics, uses, mode of action, analogue generation, regulation and strategies for increasing pimaricin production yields.

OBJECTIVE

To report a case of fungal keratitis caused by Colletotrichum gloeosporioides, which is a rare pathogen in humans.

METHODS

An 80-year-old woman developed fungal keratitis after having sustained a traumatic injury during field work. The patient was initially examined by slit-lamp biomicroscopy and the Heidelberg Retina Tomograph II-Rostock Cornea Module (HRT II-RCM). Corneal scrapings were collected and submitted for laboratory investigations.

RESULTS

Many septate, hyphae-like interlocking and branching white lines were observed in the area of the infiltrate by HRT II-RCM. A tentative diagnosis of fungal keratitis was made, and the patient was treated with systemic and topical voriconazole and pimaricin ophthalmic ointment. The infectious focus resolved within 2 weeks, and there were no signs of a recurrence after 3 months of treatment with the antifungal agents. The culture of the corneal scraping grew C. gloeosporioides.

CONCLUSIONS

HRT II-RCM was useful in detecting filamentous fungi in the cornea. The treatment with voriconazole and pimaricin was effective in the treatment of C. gloeosporioides keratitis.

In a 15-year-old boy a culture-proved keratitis after a corneal perforation healed without antifungal agents after corneal suturing and application of tissue glue. Eight months later a posterior corneal abscess developed. Diagnostic and therapeutic penetrating keratoplasty was performed when the lesion failed to respond to pimaricin. Cultures were positive for Acremonium potronii, the same fungus isolated from the original corneal laceration eight months previously. To our acknowledge, this is the first case report of a central corneal ulcer or abscess due to this specific organism.

The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations greater than or equal to 10 micrograms/ml, filipin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a Ki value of about 0.13 mM. the effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at approximately 50 micrograms/ml. Our findings suggest the existence of binding sites (sterols?) on the chitosome that are selective for certain polyenes and that play a role in the operation of chitin synthase. Because the minimal growth inhibitory concentrations of AmB or nystatin are lower than the concentrations that inhibit chitin synthase in vitro, the possibility of chitosomal chitin synthase being a primary target for the antifungal action of these polyenes seems unlikely.

From cell-free extracts of Streptomyces RGU5.3, a tailoring activity of pimaricin, leading to the biosynthesis of its natural carboxamide derivative AB-400, was recently identified. The two polyene macrolides, pimaricin and AB-400, were produced in almost equal quantities and can be detected in the fermentation broth of the producer strain. This report concerns the isolation and partial characterization of the gene, polyene carboxamide synthase (pcsB), responsible for the bioconversion. The gene encoded an asparagine synthase-like protein, belonging to the type II glutamine amidotransferase family, and was named pcsB. The fermentation broth of a recombinant strain carrying the engineered pcsB gene under the control of the inducible tipA promoter within an integrative vector produces the carboxamide AB-400 as the main polyene macrolide.

Six mutants of Cryptococcus neoformans resistant to nystatin and pimaricin and three mutants resistant to amphotericin B were isolated by ultraviolet irradiation techniques from two wild-type strains. The major sterols of the wild-type strains were Delta(7)-ergosten-3beta-ol and ergosterol. All six mutants resistant to nystatin and pimaricin showed either loss of ergosterol and concurrent production of Delta(7, 22)-ergostadien-3beta-ol and Delta(7)-ergosten-3beta-ol, or loss of both the wild-type sterols, with production of Delta(8(9))-ergosten-3beta-ol and Delta(5, 8(9), 22)-ergostatrien-3beta-ol. The mutants producing Delta(7, 22)-ergostadien-3beta-ol and Delta(7)-ergosten-3beta-ol showed relatively low levels of resistance to nystatin and pimaricin, whereas the mutants producing Delta(8(9))-ergosten-3beta-ol and Delta(5, 8(0), 22)-ergostatrien-3beta-ol showed a high level of resistance to either drug. Although highly resistant to amphotericin B, however, the three mutants produced sterol compositions identical to those of the wild types, indicating that the strains acquired resistance other than by alteration of the membrane sterols. The mutants producing Delta(8(9)) and Delta(5, 8(9), 22) sterols were not virulent for mice, showed reduced growth rates at 25 C, and failed to grow at 37 C. The other mutants showed a slightly reduced rate of growth both at 25 and 37 C, and the virulence in mice was slightly reduced in comparison with that of the wild types. These comparisons were on gross observations and were not statistically analyzed.

Medical treatment failure necessitated surgery in nine cases of fungal keratitis. Therapeutic surgery eliminated fungal infection in seven cases, and useful vision was retained in five out of six penetrating keratoplasties. In three cases Natamycin (Pimaricin) therapy rendered fungi non-viable, but two were demonstrable by histopathology. These results suggest that antifungal treatment should be applied for as long as possible before therapeutic surgery in order to improve the final visual outcome.

Control of permeation of bleomycin A2, a well-known antitumor antibiotic, in combination with various polyene macrolide antibiotics was analyzed in cultured Chinese hamster cells in vitro. Three polyene antibiotics, filipin, pentamycin, and pimaricin, were found to enhance the action of bleomycin A2 remarkably, while amphotericin B or nystatin could not. Although DNA synthesis and colony-forming activity of polyene-sensitive Chinese hamster V79 cells were synergistically inhibited by the combination of filipin and bleomycin A2, in a polyene-resistant subline (AMBR-1) derived from V79, they were only slightly affected in the presence of both drugs. The cellular uptake of [14C]bleomycin A2 by V79 was enhanced 2- to 4-fold in the presence of increasing doses of filipin or pentamycin, but not in the presence of amphotericin B. The treatment of V79 cells with filipin for 20 to 30 min was enought to block DNA synthesis almost completely when combined with 20 microgram belomycin A2 per ml. The pretreatment of the hamster cells with 6 microgram filipin per ml for 60 min continued to enhance the inhibitory action by bleomycin A2 of DNA synthesis up to 5 hr after the removal of filipin from the cultured medium.

To report successful medical management of Pythium insidiosum keratitis using an antibiotic combination of minocycline, linezolid, and chloramphenicol.

A 20-year-old Japanese man was referred for visual disturbance, hyperemia, and discharge from his right eye. Slit-lamp examination revealed a paracentral corneal hyphate ulcer. His visual acuity was 20/28. Smear examination of corneal scrapings revealed a filamentous fungus. Pimaricin ointment four times a day and voriconazole eye drops hourly were initially prescribed. Although intravenous liposomal amphotericin B 100 mg was added, the corneal infiltrates and ulcer worsened. The possibility of P. insidiosum keratitis was considered, and in vitro antifungal susceptibility testing were performed based on the disc diffusion method. The inhibition zones around each antibiotic disc revealed that the pathogen was susceptible to minocycline, linezolid, and chloramphenicol. Therefore, minocycline ointment four times a day, chloramphenicol eye drops hourly, and linezolid 1200 mg orally per day were also administered. Eventually, sequencing of ribosomal DNA confirmed the pathogen to be P. insidiosum. The triple regimen dramatically improved the patient's keratitis. Therapeutic penetrating keratoplasty for corneal perforation was successfully performed, and his visual acuity recovered from 20/2000 to 20/25.

We have encountered a case of P. insidiosum keratitis that responded to a combination of minocycline, linezolid, and chloramphenicol. This triple combination should be considered in patients with P. insidiosum keratitis.

A 53-year-old woman was found to have an ulcer on her successfully transplanted corneal graft. Many fungal elements were observed in the smear of the ulcerated tissue, and Alternaria alternata was cultured. The ulcer was treated with pimaricin and thimerosol topically, and 5-fluorocytocin (5FC) generally and healed to scar after two months. The ulcer did not invade the host cornea, but remained in the donner cornea in all clinical course. The MIC of five drugs on the isolated strain was following; thimerosal 0.0063, pimaricin 2.0, amphotericin B 3.2, aystatin 6.3 and 5FC 100.0 micrograms/ml each.

We propose here a delivery drug-polymer system using poly(N-isopropylacrylamide) (PNIPA) nanohydrogels that enables pimaricin to be protected from hostile environments and allows the controlled release of the antifungal through environmental stimuli. We synthesized 2 nanohydrogels, 1 with 100% N-isopropylacrylamide (PNIPA(5)) and 1 with 80% N-isopropylacrylamide copolymerized and 20% acrylic acid (PNIPA-20AA(5)). Both were then, loaded with a pimaricin aqueous solution. The pimaricin release profiles of these 2 nanohydrogels were considerably different: PNIPA(5) released 10% and PNIPA-20AA(5) released 30% with respect to the free pimaricin release. Moreover, the diffusion experiments showed that pimaricin was released from the PNIPA-20AA(5) nanohydrogel for up to 3 times longer than free pimaricin. Therefore, incorporating acrylic acid as comonomer into the PNIPA nanohydrogel resulted in a slower but more continuous release of pimaricin. The highest pimaricin levels were reached when the most hydrophilic nanohydrogel was used. The bioassay results showed that the pimaricin-nanohydrogel system was highly effective in inhibiting the growth of the indicator strain in conditions of thermal abuse. The spoilage in acidified samples stored under fluorescent lighting was reduced by 80.94% ± 33.02% in samples treated with a pimaricin-loaded nanohydrogel, but only by 19.91% ± 6.68% in samples treated with free pimaricin. Therefore, 2 conclusions emerge from this study. One is that the nanohydrogel delivery system could impede the degradation of pimaricin. The other is that the inhibitory effect of the antifungal on yeast growth is more pronounced when it is added included into the nanohydrogel to the food, especially in an acidic environment.

CONCLUSIONS

This article presents relevant results on the use of nanohydrogels in food packaging. Nanohydrogels could provide protection so that the pimaricin remains active for a longer time. They also allow the controlled release of pimaricin, which thus regulates the unnecessary presence of the antifungal in the food.

BACKGROUND

Malassezia species rarely cause keratitis, and, thus, the clinical manifestations of Malassezia keratitis are not well known.

METHODS

We report the clinical findings in a 70-year-old woman who complained of pain in her left eye. Slit-lamp biomicroscopy showed a corneal ulcer with irregular infiltration, which resembled keratitis caused by filamentous fungi. KOH Parker ink stain of a corneal scraping showed mold hyphae and yeast, but cultures on Sabouraud's glucose agar plates and blood agar plates were negative. Treatment with antibacterial agents failed.

METHODS

Polymerase chain reaction (PCR) and DNA typing of the fungal gene between the internal transcribed spacer 2 (ITS2) and the 5.8S ribosomal DNA of the scraping were performed. PCR amplified a band with a sequence that was 99% homologous with Malassezia restricta. Antifungal agents, topical 5% pimaricin ointment and 0.2% miconazole, and oral itraconazole 150 mg/day, were applied, and the corneal ulcer disappeared within 5 weeks.

CONCLUSIONS

Malassezia restricta can cause keratitis, and the clinical findings resemble keratitis caused by filamentous fungi. PCR and DNA typing of ITS2 and 5.8S ribosomal DNA are valuable techniques for detecting and identifying Malassezia species. Proper identification of Malassezia keratitis permits a prompt and successful treatment by antifungal agents.

The effect of four polyene antibiotics, Candicidin, Etruscomycin, Filipin and Pimaricin upon mouse lymphocytes was studied. Polyene antibiotics are known to form aqueous pores in the cell membranes inducing known anion or cation selective fluxes. Candicidin was capable of inducing marked DNA-synthesis and polyclonal antibody production when added to normal spleen cells. Etruscomycin and Pimaricin showed a weak inconsistent DNA synthetic stimulatory effect, whereas Filipin was found to be totally uneffective. The stimulating property of Candicidin was also demonstrated on spleen cells from nude mice whereas there was no effect on cortisone resistant thymocytes or spleen cells passed through a nylon fibre column. Thus we conclude that Candicidin is a PBA for mouse lymphocytes. We have previously reported that the two anionselective polyenes, Nystatin and Amphotericin B, are polyclonal B-cell activators for mouse lymphocytes and in this paper the possible mechanism of triggering is further discussed.