Antifungal Activity of Capric Acid, Nystatin, and Fluconazole and Their In Vitro Interactions Against Candida Isolates from Neonatal Oral Thrush.

Due to the increasing resistance of various Candida species to azole drugs, particularly fluconazole, it would be of significant importance to look for alternative therapies. The aim of this study was to investigate the antifungal activity of capric acid and its in vitro interactions with nystatin and fluconazole against Candida isolates. A total of 40 Candida isolates (C. albicans, 36; C. kefyr, 2; C. tropicalis, 1; C. glabrata, 1) collected from the oral cavity of neonates with oropharyngeal candidiasis and a reference strain of C. albicans (ATCC 10231) were used in this study. Antifungal activity of capric acid and two comparator antifungal drugs, namely fluconazole and nystatin, was tested according to CLSI M27-A3/M60 method. The in vitro interaction between capric acid with fluconazole and nystatin was determined following a checkerboard method and results were interpreted using fractional inhibitory concentration index. Nystatin had the lowest minimum inhibitory concentrations (range, 0.125-8 µg/mL; geometric mean [GM], 0.6229 µg/mL) followed by fluconazole (range, 0.5-16 µg/mL; GM, 1.9011 µg/mL) and capric acid (range, 128-2,048 µg/mL; GM, 835.9756 µg/mL). When tested in combination, capric acid with fluconazole demonstrated synergistic, indifferent, and antagonistic interactions in 3 (7.317%), 24 (58.536%), and 14 (34.146%) cases, respectively. For combination of capric acid with nystatin, synergistic, indifferent, and antagonistic interactions were observed in 1 (2.439%), 19 (46.341%), and 21 (51.219%) cases, respectively. All cases of synergistic interactions were against resistant or susceptible dose-dependent isolates. Fluconazole, nystatin, and capric acid seem to be more effective when they are used alone compared with their combination. However, their combination might be effective on resistant isolates.

Evaluation of nystatin isolated from Streptomyces griseus SDX-4 against the ciliate, Ichthyophthirius multifiliis.

The present study was conducted to evaluate the in vitro and in vivo antiparasitic efficacy of active compounds from the bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis. Bioassay-guided fractionation and isolation of compounds with antiparasitic activity were performed on n-butanol extract of S. griseus yielding a pure bioactive compound, nystatin (Nys), identified by comparing spectral data (EI-MS, (1)H NMR, and (13)C NMR) with literature values. Results from in vitro antiparasitic assays revealed that Nys could be 100% effective against I. multifiliis theronts and encysted tomonts at the concentration of 6.0 mg L(-1), with the median effective concentration (EC50) values of 3.1 and 2.8 mg L(-1) for theronts and encysted tomonts (4 h), respectively. Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the gold fish treated with Nys was markedly lower than the control group at 10 days after exposed to theronts (p < 0.05). In the control group, 85.7% mortality was observed owing to heavy I. multifiliis infection at 10 days after the exposure. On the other hand, only 23.8% mortality owing to parasite infection was recorded in the groups treated with the Nys (4.0 and 6.0 mg L(-1)). In addition, our results showed that the survival and reproduction of I. multifiliis tomont exited from the fish were significantly reduced after treated with the 6.0 mg L(-1) Nys. The median lethal dose (LD50) of Nys for goldfish was 16.8 mg L(-1). This study firstly demonstrated that Nys has potent antiparasitic efficacy against I. multifiliis, and it can be a good candidate drug for chemotherapy and control of I. multifiliis infections.

Novel Nystatin A1 derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis.

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A1, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A1 had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A1 was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C. albicans and tissue cell viability after RHOE infection, Nystatin A1 derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A1. Furthermore, compared with Nystatin A1, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.

Isolating antifungals from fungus-growing ant symbionts using a genome-guided chemistry approach.

We describe methods used to isolate and identify antifungal compounds from actinomycete strains associated with the leaf-cutter ant Acromyrmex octospinosus. These ants use antibiotics produced by symbiotic actinomycete bacteria to protect themselves and their fungal cultivar against bacterial and fungal infections. The fungal cultivar serves as the sole food source for the ant colony, which can number up to tens of thousands of individuals. We describe how we isolate bacteria from leaf-cutter ants collected in Trinidad and analyze the antifungal compounds made by two of these strains (Pseudonocardia and Streptomyces spp.), using a combination of genome analysis, mutagenesis, and chemical isolation. These methods should be generalizable to a wide variety of insect-symbiont situations. Although more time consuming than traditional activity-guided fractionation methods, this approach provides a powerful technique for unlocking the complete biosynthetic potential of individual strains and for avoiding the problems of rediscovery of known compounds. We describe the discovery of a novel nystatin compound, named nystatin P1, and identification of the biosynthetic pathway for antimycins, compounds that were first described more than 60 years ago. We also report that disruption of two known antifungal pathways in a single Streptomyces strain has revealed a third, and likely novel, antifungal plus four more pathways with unknown products. This validates our approach, which clearly has the potential to identify numerous new compounds, even from well-characterized actinomycete strains.

Multivariate analysis of nystatin and metronidazole in a semi-solid matrix by means of diffuse reflectance NIR spectroscopy and PLS regression.

A multivariate method of analysis of nystatin and metronidazole in a semi-solid matrix, based on diffuse reflectance NIR measurements and partial least squares regression, is reported. The product, a vaginal cream used in the antifungal and antibacterial treatment, is usually, quantitatively analyzed through microbiological tests (nystatin) and HPLC technique (metronidazole), according to pharmacopeial procedures. However, near infrared spectroscopy has demonstrated to be a valuable tool for content determination, given the rapidity and scope of the method. In the present study, it was successfully applied in the prediction of nystatin (even in low concentrations, ca. 0.3-0.4%, w/w, which is around 100,000 IU/5g) and metronidazole contents, as demonstrated by some figures of merit, namely linearity, precision (mean and repeatability) and accuracy.

[Comparative analysis of in vitro antifungal activity and in vivo acute toxicity of the nystatin analogue S44HP produced via genetic engineering].

New polyene macrolide S44HP was purified from the culture of recombinant Streptomyces noursei strain with engineered nystatin polyketide synthase. S44HP, nystatin (NYS), and amphotericin B (Amph-B) were tested against 19 clinical fungal isolates in agar diffusion assay, which demonstrated clear differences in antifungal activities of these antibiotics. Sodium deoxycholate suspensions of all three antibiotics were subjected to acute toxicity studies in vivo upon intravenous administration in mice. NYS exhibited the lowest acute toxicity in mice in these experiments, while both Amph-B and S44HP were shown to be 4 times more toxic as judged from the LD50 values. While the acute toxicity of S44HP was higher than that of Amph-B, the data analysis revealed a significantly increased LD10 to LD50 dose interval for S44HP compared to Amph-B. The data revealed structural features of polyene macrolides, which might have an impact on both the activity and toxicity profiles of these antibiotics. These results represent the first example of preclinical evaluation of an "engineered" polyene macrolide, and can be valuable for rational design of novel antifungal drugs with improved pharmacological properties.

Batch control system project for a pharmaceutical plant.

This paper describes a case study of a control system design for a batch pharmaceutical process. The ISA standard S88.01 batch control models and terminology were used as the main guidelines for the implementation. As the S88 is not a guide for how to apply the definitions/structures, etc., one of the main goals of our work was to create a methodology for decomposition of functional requirements in terms of S88 models and structures. This methodology was tested on a real problem, described in the case study. Also presented are some remarks on project methodology and Food and Drug Administration validation.

Differential antifungal activity of isomeric forms of nystatin.

When nystatin is placed in RPMI and other biological fluids, there is loss of pure nystatin, with the development of two distinguishable chromatographic peaks, 1 and 2. Peak 1 appears identical to commercially prepared nystatin. By nuclear magnetic resonance (NMR) and mass spectral analysis, peak 2 appears to be an isomer of peak 1. The isomers are quantitatively and fully interconvertible. Formation of peak 2 is accelerated at a pH of >7.0 and ultimately reaches a near 55:45 (peak 1/peak 2 ratio) mixture. We sought to determine the relative activities of peaks 1 and 2 against Candida spp. Peak 2 consistently showed higher MICs when it was the predominant form during the experiment. Time-kill analyses showed that peak 2 required > or =8 x the concentration of peak 1 to produce a modest and delayed killing effect, which was never of the same magnitude as that produced by peak 1. In both types of assays, the activity of peak 2 corresponded with intra-assay formation of peak 1. Both MIC measurements and time-kill analysis suggest that peak 2 has considerably less activity, if any at all, against Candida spp. Peak 2 may serve as a reservoir for peak 1.

Methods for the recovery and purification of polyene antifungals.

Despite the development of newer antifungal drugs, the polyene antifungals continue to be the most potent broad-spectrum fungicides available for clinical use. The incidence and severity of fungal infections are on the rise, underscoring the need for new and more effective antifungal drugs. Thus, the search for new polyene antifungals is ongoing. The limited solubility, polymorphic character, and inherent chemical instability of these compounds make their economical recovery and purification from mass culture challenging problems in biotechnology. This article provides a comprehensive review of the methods that have been developed for the recovery and purification of amphotericin B and nystatin, the two most important polyenes currently in clinical use.

Self-association of the polyene antibiotic nystatin in dipalmitoylphosphatidylcholine vesicles: a time-resolved fluorescence study.

The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, both in gel (T = 21 degrees C) and in liquid-crystalline (T = 45 degrees C) phases, was studied by steady-state and time-resolved fluorescence measurements by taking advantage of the intrinsic tetraene fluorophore present in this antibiotic. It was shown that Nystatin aggregates in aqueous solution with a critical concentration of 3 microM. The enhancement in the fluorescence intensity of the antibiotic was applied to study the membrane binding of Nystatin, and it was shown that the antibiotic had an almost fivefold higher partition coefficient for the vesicles in a gel (P = (1.4 +/- 0.1) x 10(3)) than in a liquid-crystalline phase (P = (2.9 +/- 0.1) x 10(2)). Moreover, a time-resolved fluorescence study was used to examine Nystatin aggregation in the membrane. The emission decay kinetics of Nystatin was described by three and two exponentials in the lipid membrane at 21 degrees C and 45 degrees C, respectively. Nystatin mean fluorescence lifetime is concentration-dependent in gel phase lipids, increasing steeply from 11 to 33 ns at an antibiotic concentration of 5-6 microM, but the fluorescence decay parameters of Nystatin were unvarying with the antibiotic concentration in fluid lipids. These results provide evidence for the formation of strongly fluorescent antibiotic aggregates in gel-phase membrane, an interpretation that is at variance with a previous study. However, no antibiotic self-association was detected in a liquid-crystalline lipid bilayer within the antibiotic concentration range studied (0-14 microM).

Tetrafungin, a new polyene macrolide antibiotic. II. Taxonomy of the producing organism and comparison with nystatin by means of high performance liquid chromatography.

Tetrafungin, a new polyene macrolide antibiotic, is produced by a Streptomyces strain identified as a new subspecies of Streptomyces albulus and named Streptomyces albulus subsp. tetrafungini. Tetrafungin and nystatin have been investigated and compared by HPLC. It has been demonstrated that tetrafungin and nystatin differ qualitatively in, at least, one component, and quantitatively in their relative amounts of common components.

[Dimethyl sulfoxide extraction of nystatin from mycelium]. / Ekstraktsiia nistatina dimetilsul'fosidom iz mitseliia.

Solubility of nystatin in dimethylsulfoxide (DMSO) and its water solutions was studied. It was found that the capacity of DMSO with respect to nystatin was at least 40 times higher than that of the known extracting agents. DMSO is recommended for extraction of nystatin from dry mycelium. The optimal conditions for extraction of nystatin and its recovery from the extract phases were determined. Nystatin isolated with this method meets the specification requirements.

[Method of partitioning polyene macrolide antibiotics]. / Sposob razdeleniia polienovykh makrolidnykh antibiotikov.

Levorin and mycoheptin, polyenic antibiotics were separated into 3 and 7 heptaenic components respectively by the method of disc electrophoresis in 7.5 per cent polyacryl amide gel prepared with tris-HCl buffer, pH 8.9. It was shown that nystatin had different composition as dependent on the organism producing it. The results of electrophoresis confirmed the data of the counter-current distribution.