Antifungal activity of magnoflorine against <Emphasis Type="Italic">Candida</Emphasis> strains

Candida albicans is a major invasive pathogen, and the development of strains resistant to conventional antifungal agents has been reported in recent years. We evaluated the antifungal activity of 44 compounds against Candida strains. Magnoflorine showed the highest growth inhibitory activity of the tested Candida strains, with a minimum inhibitory concentration (MIC) of 50 μg/mL based on microdilution antifungal susceptibility testing. Disk diffusion assay confirmed the antifungal activity of magnoflorine and revealed that this activity was stable over 3 days compared to those of berberine and cinnamaldehyde. Cytotoxicity testing showed that magnoflorine could potentially be used in a clinical setting because it didn’t have any toxicity to HaCaT cells even in 200 μg/mL of treatment. Magnoflorine at 50 μg/mL inhibited 55.91?±?7.17% of alpha-glucosidase activity which is required for normal cell wall composition and virulence of Candida albicans. Magnoflorine also reduced the formation of C. albicans’ biofilm. Combined treatment with magnoflorine and miconazole decreased the amount of miconazole required to kill various Candida albicans. Therefore, magnoflorine is a good candidate lead compound for novel antifungal agents.     

Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing

Subtilosin, the cyclic lantibiotic protein produced by Bacillus subtilis KATMIRA1933, targets the surface receptor and electrostatically binds to the bacterial cell membrane. In this study, subtilosin was purified using ammonium sulfate ((NH₄)₂SO₄) precipitation and purified via column chromatography. Subtilosin’s antibacterial minimum and sub-minimum inhibitory concentrations (MIC and sub-MIC) and anti-biofilm activity (biofilm prevention) were established. Subtilosin was evaluated as a quorum sensing (QS) inhibitor in Gram-positive bacteria using Fe(III) reduction assay. In Gram-negative bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter. The results showed that Gardnerella vaginalis was more sensitive to subtilosin with MIC of 6.25 μg/mL when compared to Listeria monocytogenes (125 μg/mL). The lowest concentration of subtilosin, at which more than 90% of G. vaginalis biofilm was inhibited without effecting the growth of planktonic cells, was 0.78 μg/mL. About 80% of L. monocytogenes and more than 60% of Escherichia coli biofilm was inhibited when 15.1 μg/mL of subtilosin was applied. Subtilosin with 7.8–125 μg/mL showed a significant reduction in violacein production without any inhibitory effect on the growth of C. violaceum. Subtilosin at 3 and 4 μg/mL reduced the level of Autoinducer-2 (AI-2) production in G. vaginalis. However, subtilosin did not influence AI-2 production by L. monocytogenes at sub-MICs of 0.95–15.1 μg/mL. To our knowledge, this is the first report exploring the relationship between biofilm prevention and quorum sensing inhibition in G. vaginalis using subtilosin as a quorum sensing inhibitor.     

Clinical and Microbiological Investigation of Fungemia from Four Hospitals in China

In this study, fungemia cases from four tertiary hospitals located in Shanghai and Anhui province in China from March 2012 to December 2013 were enrolled to investigate clinical features, species distribution, antifungal susceptibility and strain relatedness. During the study period, 137 non-duplicate cases and their corresponding isolates were collected. Six different genera of fungi were identified, of which Candida spp. were the most common (126/137, 91.97 %), with C. albicans predominating (48/137, 35.03 %). The non-Candida fungi rate reached 8.03 % (11/137), and Pichia spp. was the most common (5/137, 3.65 %). Compared with C. albicans, non-C. albicans fungi-associated fungemia was more likely in younger (p = 0.004) and male patients (χ ² = 6.2618, p = 0.0123) and patients from ICUs (χ ² = 6.3783, p = 0.0116). Overall, the susceptible/WT rates of common Candida spp. to fluconazole, itraconazole, voriconazole, flucytosine, amphotericin B and caspofungin were 74.63, 92.31, 93.16, 96.58, 100 and 95.69 %, respectively. C. tropicalis and C. guilliermondii had a low susceptibility to fluconazole: 79.95 and 77.78 %, respectively. No isolates were resistant/WT to caspofungin, but C. parapsilosis and C. guilliermondii had high MIC₉₀ values; 1 and 2 mg/L, respectively. In terms of genotyping, MLST was taken for C. glabrata and C. tropicalis, while microsatellite marker analysis was used for C. albicans and C. parapsilosis. C. glabrata was predominantly clone ST7, accounting for 75 %, while the other isolates showed genetic diversity. Considering the increased proportion of non-C. albicans fungi and the presence of endemic clones of C. glabrata, it is essential to undertake additional surveillance of fungemia.     

Nimesulide inhibits pathogenic fungi: PGE2-dependent mechanisms

Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 μg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 μg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.     

Characterization of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) lectin for biological activity

Lectins are proteins that are subject of intense investigations. Information on lectin from chickpea (Cicer arietinum L.) with respect to its biological activities are very limited. In this study, we purified lectin from the seeds of chickpea employing DEAE-cellulose and SP-Sephadex ion exchange chromatography and identified its molecular subunit mass as 35 kDa. The free radical scavenging activity of lectin measured by the DPPH assay has IC₅₀ of 0.88 µg/mL. Lectin exerted antifungal activity against Candida krusei, Fusarium oxysporium oxysporium, Saccharomyces cerevisiae and Candida albicans, while antibacterial activity against E. coli, B. subtilis, S. marcescens and P. aeruginosa. The minimum inhibitory concentrations were 200, 240, 160 and 140 µg for C. krusei, F. oxysporium, S. cerevisiae and C. albicans respectively. Lectin was further examined for its antiproliferative potential against cancerous cell line. The cell viability assay indicated a high inhibition activity on Ishikawa, HepG2, MCF-7 and MDA-MB-231 with IC₅₀ value of 46.67, 44.20, 53.58 and 37.46?µg/mL respectively. These results can provide a background for future research into the benefits of chickpea lectin to pharmacological perspective.     

Geographic distribution of phylogenetic species of the <Emphasis Type="Italic">Fusarium graminearum</Emphasis> species complex and their 8-ketotrichothecene chemotypes on wheat spikes in Iran

Isolates of the Fusarium graminearum species complex (FGSC, n = 446) were collected from wheat spikes from northern and western regions of Iran with a history of Fusarium head blight (FHB) occurrences. The trichothecene mycotoxin genotypes/chemotypes, the associated phylogenetic species, and geographical distribution of these isolates were analyzed. Two phylogenetic species, Fusarium asiaticum and F. graminearum, were identified and were found to belong to sequence characterized amplified region (SCAR) groups V and I. Isolates from F. asiaticum species lineage 6 were within SCAR group V, whereas F. graminearum species lineage 7 were of SCAR group I. Of the 446 isolates assayed, 274 were F. asiaticum species predominantly of the nivalenol (NIV) genotype, while other isolates were either deoxynivalenol (DON) plus 3-acetyldeoxynivalenol (3-AcDON) or DON plus 15-acetyldeoxynivalenol (15-AcDON) genotype. Based on Tri7 gene sequences, a new subpopulation of 15-AcDON producers was observed among F. asiaticum strains in which 11-bp repeats were absent in the Tri7 sequences. The trichothecene chemotype was confirmed and quantified by high-performance liquid chromatography (HPLC) in 46 FGSC isolates. Isolates produced NIV (33.4–108.2 μg/g) and DON (64.7–473.6 μg/g) plus either 3-AcDON (51.4–142.4 μg/g) or 15-AcDON (24.1–99.3 μg/g). Among FGSC isolates, F. asiaticum produced the highest levels of trichothecenes. Using BIOCLIM based on the climate data of 20-year during 1994–2014, modelling geographical distribution of FGSC showed that F. asiaticum was restricted to warmer and humid areas with a median value of mean annual temperature of about 17.5 °C and annual rainfall of 658 mm, respectively (P < 0.05). In contrast, F. graminearum (only 15-AcDON producers) was restricted to cooler and drier areas, with a median value of the mean annual temperature of 14.4 °C and an annual rainfall of 384 mm, respectively (P < 0.05). Based on climate parameters at anthesis, the recorded distribution of F. graminearum and F. asiaticum was similar to that based on BIOCLIM parameters. Therefore, geographic differences on the wheat-growing areas in Iran have had a significant effect on distribution of FGSC and their trichothecene chemotypes.     

Comparison of Killing Activity of Micafungin Against Six <Emphasis Type="Italic">Candida</Emphasis> Species Isolated from Peritoneal and Pleural Cavities in RPMI-1640, 10 and 30% Serum

Currently echinocandins are recommended in Candida peritonitis and pleuritis. We determined micafungin killing rates (k values) at therapeutic concentrations (0.25–2 mg/L) in RPMI-1640 with and without 10 and 30% serum mimicking in vivo conditions against six Candida species isolated from peritoneal and pleural fluid. In RPMI-1640, micafungin was fungicidal against C. glabrata, C. krusei and C. kefyr within 2.27?±?10.68, 2.69?±?10.29 and 3.10?±?4.41 h, respectively, while was fungistatic against C. albicans, C. tropicalis and C. parapsilosis. In 10% serum, ≥?0.25, ≥?0.5, ≥?0.5 and ≥?1 mg/L micafungin produced positive k values (killing) for all C. albicans, C. glabrata, C. kefyr and C. krusei, respectively. In 30% serum, 2 mg/L micafungin produced killing against all C. albicans, C. glabrata and C. kefyr isolates, but was ineffective against C. krusei, C. parapsilosis and 2 of 3 C. tropicalis. Micafungin exposure should be increased against non-albicans species to eradicate fungi from peritoneal and pleural cavities.     

Genetic Diversity and Antifungal Susceptibility of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> Sensu Stricto Isolated from Bloodstream Infections in Turkish Patients

Candida parapsilosis sensu stricto is an emerging cause of hospital-acquired Candida infections, predominantly in southern Europe, South America, and Asia. We investigated the genetic diversity and antifungal susceptibility profile of 170 independent C. parapsilosis sensu stricto strains obtained from patients with candidemia who were treated at the Ege University Hospital in Izmir, Turkey, between 2006 and 2014. The identity of each strain was confirmed via PCR amplification and digestion of the secondary alcohol dehydrogenase-encoding gene. The 24-h geometric mean minimum inhibitory concentrations of the antifungal agents, in increasing order, were as follows: posaconazole, 0.10 µg/mL; voriconazole, 0.21 µg/mL; caspofungin, 0.38 µg/mL; amphotericin B, 0.61 µg/mL; anidulafungin, 0.68 µg/mL; and fluconazole, 2.95 µg/mL. Microsatellite genotyping of the isolates (using fluorescently labeled primers and a panel of four different short-nucleotide repeat fragments) identified 25, 17, 17, and 8 different allelic genotypes at the CP6, B5, CP4, and CP1 locus, respectively. Posaconazole, caspofungin, and amphotericin B showed the greatest in vitro activity of the tested systemic azole, echinocandin, and polyene agents, respectively, and the observed antifungal susceptibility of the isolates was shown to be independent of their isolation source. We obtained a combined discriminatory power of 0.99 with a total of 130 genotypes for 170 isolates tested. Finally, microsatellite profiling analysis confirmed the presence of identical genotype between separate isolates, supporting that effective surveillance and infection-prevention programs are essential to limit the impact of C. parapsilosis sensu stricto on hospitalized patients’ health.     

Factors Influencing Non-<Emphasis Type="Italic">albicans</Emphasis> Candidemia: A Case–Case–Control Study

The study identified factors predisposing to non-albicans candidemia with special interest to prior antimicrobial treatment. A retrospective, case–case–control study was performed at the University Hospital of Heraklion, Greece, from November 2007 through September 2011 including adult patients. The study had three groups. The first included 58 patients with non-albicans candidemia, the second 48 with C. albicans candidemia, while the third (control) 104 without candidemia. Each of the two candidemia groups was compared with the control using multivariate logistic regression model. The mean (SD) age of the non-albicans, the albicans and the control patients was 67 (12), 67 (18) and 59 (19) years, respectively. The most common non-albicans Candida spp. isolated were C. parapsilosis in 19 patients (33%), C. glabrata in 17 (29%) and C. tropicalis in 15 (26%). Independent risk factors for non-albicans candidemia were prior treatment with quinolones (p < 0.001), b-lactam-b-lactamase inhibitors (p = 0.011) and presence of central venous catheter (p = 0.05), while for C. albicans candidemia were prior treatment with quinolones (p < 0.001), carbapenems (p = 0.003) along with cardiac disease (p < 0.001). Neither duration of hospitalization nor in-hospital mortality [41% for the non-albicans vs 29% for C. albicans group (p = 0.192)] was significantly different between the two candidemia groups. The study reveals the role of antimicrobial exposure as a risk factor for candidemia caused by different species. Prior treatment with b-lactam-b-lactamase inhibitors was associated with non-albicans, while with carbapenems with C. albicans candidemia. Prior use of quinolones was associated with candidemia in general.     

In vitro anticancer properties of selected <Emphasis Type="Italic">Eucalyptus</Emphasis> species

In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 μg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI₅₀ values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 μg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC₅₀ values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 μg/mL) and fruit (64.66 ± 15.97 μg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 μg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.     

Zinc oxide nanoparticle inhibits the biofilm formation of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Biofilms are structured consortia of microbial cells that grow on living and non living surfaces and surround themselves with secreted polymers. Infections with bacterial biofilms have emerged as a foremost public health concern because biofilm growing cells can be highly resistant to both antibiotics and host immune defenses. Zinc oxide nanoparticles have been reported as a potential antimicrobial agent, thus, in the current study, we have evaluated the antimicrobial as well as antibiofilm activity of zinc oxide nanoparticles against the bacterium Streptococcus pneumoniae which is a significant cause of disease. Zinc oxide nanoparticles showed strong antimicrobial activity against S. pneumoniae, with an MIC value of 40 μg/ml. Biofilm inhibition of S. pneumoniae was also evaluated by performing a series of experiments such as crystal violet assay, microscopic observation, protein count, EPS secretion etc. using sub-MIC concentrations (3, 6 and 12 µg/ml) of zinc oxide nanoparticles. The results showed that the sub-MIC doses of zinc oxide nanoparticles exhibited significant anti-biofilm activity against S. pneumoniae, with maximum biofilm attenuation found at 12 μg/ml. Taken together, the results indicate that zinc oxide nanoparticles can be considered as a potential agent for the inhibition of microbial biofilms.     

In Vitro Antimicrobial Activity and Downregulation of Virulence Gene Expression on <Emphasis Type="Italic">Helicobacter pylori</Emphasis> by Reuterin

Helicobacter pylori is an infectious agent commonly associated with gastrointestinal diseases. The use of probiotics to treat this infection has been documented, however, their potential antimicrobial metabolites have not yet been investigated. In the present study, the effect of reuterin produced by Lactobacillus reuteri on H. pylori growth and virulence gene expression was evaluated. It was observed that reuterin caused significant (P < 0.05) H. pylori growth inhibition at concentrations from 0.08 to 20.48 mM, with minimal inhibitory concentrations (MICs) of 20.48 mM for H. pylori ATCC700824 and 10.24 mM for H. pylori ATCC43504. In a reuterin bacterial killing assay, it was observed that half of the MIC value for H. pylori (ATCC700824) significantly (P < 0.01) reduced colony numbers from 5.65 ± 0.35 to 3.78 ± 0.35 Log₁₀ CFU/mL after 12 h of treatment and then increased them to 5.25 ± 0.23 Log₁₀ CFU/mL at 24 h; at its MIC value (20.48 mM), reuterin abrogated (P < 0.01) H. pylori (ATCC700824) growth after 20 h of culture. In addition, reuterin significantly (P < 0.01) reduced H. pylori (ATCC 43504) colony numbers from 5.65 ± 0.35 to 4.1 ± 0.12 Log10 CFU/mL from 12 to 24 h of treatment and abrogated its growth at its MIC value (10.24 mM), after 20 h of treatment. Reuterin did not alter normal human gastric Hs738.St/Int cell viability at the concentrations tested for H. pylori strains. Furthermore, 10 μM reuterin was shown to significantly (P < 0.01) reduce mRNA relative expression levels of H. pylori virulence genes vacA and flaA at 3 h post-treatment, whose effect was higher at 6 h post-treatment, as measured by RT-qPCR. The observed direct antimicrobial effect and the downregulation of expression of virulence genes on H. pylori by reuterin may contribute to the understanding of the mechanisms of action of probiotics against H. pylori.     

Comparative Pharmaceutical Evaluation of Candesartan and Candesartan Cilexetil: Physicochemical Properties, <Emphasis Type="Italic">In Vitro</Emphasis> Dissolution and <Emphasis Type="Italic">Ex Vivo In Vivo</Emphasis> Studies

The aim of the present work is to answer the question is it possible to replace the ester prodrug candesartan cilexetil (CC) by its active metabolite candesartan (C) to bypass the in vivo variable effect of esterase enzymes. A comparative physicochemical evaluation was conducted through solubility, dissolution, and stability studies; additionally, ex vivo permeation and in vivo studies were assessed. C demonstrated higher solubility over CC at alkaline pH. Moreover, dissolution testing using the pharmacopeial method showed better release profile of C even in the absence of surfactant in the testing medium. Both drugs demonstrated a slight degradation in acidic pH after short-term stability. Instead, shifting to alkaline pH of 6.5 and 7.4 showed superiority of C solution stability compared to CC solution. The ex vivo permeation results demonstrated that the parent compound C has a significant (P < 0.05) enhanced permeation compared to its prodrug from CC, that agreed with in vivo results in which C suspension reached significantly (P < 0.05) higher C _max of 1.39 ± 0.59 μg/mL at T _max of 0.66 ± 0.11 h, while CC suspension reached C _max of 0.47 ± 0.22 μg/mL at T _max of 2.00 ± 0.27 h, a lag period of 40 min is needed prior to detection of any absorbed CC in plasma. Those findings are not in agreement with the previously reported rationale on the prodrug formation owing to the poor permeability of the parent compound, suggesting the possibility of marketing the parent drug candesartan for clinical use similarly to azilsartan and its prodrug.     

The Mycotoxin Zearalenone Hinders <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Formation and Hyphal Morphogenesis

Yeast–mold mycobiota inhabit several natural ecosystems, in which symbiotic relationships drive strategic pathoadaptation. Mycotoxins are metabolites produced by diverse mycotoxigenic fungi as a defense against yeasts, though at times yeasts secrete enzymes that degrade, detoxify, or bio-transform mycotoxins. The present study is focused on the in vitro inhibitory effects of zearalenone (ZEN), a F2 mycotoxin produced by several Fusarium and Gibberella species, on different microbial strains. ZEN exhibited no effect on the planktonic growth or biofilms of several Gram positive and negative bacteria at the tested concentrations. Remarkably, Candida albicans biofilm formation and hyphal morphogenesis were significantly inhibited when treated with 100 µg/mL of ZEN. Likewise, ZEN proficiently disrupted pre-formed C. albicans biofilms without disturbing planktonic cells. Furthermore, these inhibitions were confirmed by crystal violet staining and XTT reduction assays and by confocal and scanning electron microscopy. In an in vivo model, ZEN significantly suppressed C. albicans infection in the nematode Caenorhabditis elegans. The study reports the in vitro antibiofilm efficacy of ZEN against C. albicans strains, and suggests mycotoxigenic fungi participate in asymmetric competitive interactions, such as, amensalism or antibiosis, rather than commensal interactions with C. albicans, whereby mycotoxins secreted by fungi destroy C. albicans biofilms.     

Genotyping and Persistence of <Emphasis Type="Italic">Candida albicans</Emphasis> from Pregnant Women with Vulvovaginal Candidiasis

Objective

To study Candida albicans genotypes using RAPD and their susceptibility to fluconazole in healthy pregnant women and in vulvovaginal candidiasis (VVC) patients after topical treatment with clotrimazole.

Methods

Vaginal swabs were collected at t = 0 and t = 1 (1 month later) in pregnant women (control group, n = 33), and before (t = 0), at 1 month (t = 1) and at 2 months (t = 2) after clotrimazole treatment in pregnant women with VVC.

Results

Candida albicans was isolated in 30% of healthy pregnant women and 80% of patients with VVC. A high genetic heterogeneity was observed in C. albicans genotypes between individuals. In patients with VVC, topical antifungal treatment with clotrimazole was clinically effective, but only in a 62% C. albicans was eradicated. In patients in which C. albicans was not eradicated, this microorganism persisted for 1 or 2 months after the antifungal treatment. The persistent colonies were not associated with a specific genotype, but they were associated with higher MICs in comparison with colonies isolated from the control group.

Conclusions

Therapy with topical clotrimazole, despite a good clinical outcome, could not eradicate completely C. albicans allowing the persistence of genotypes, with higher MICs to fluconazole. More studies with higher number of patients are needed to validate this preliminary finding.

     

Resistance Mechanism in a Terbinafine-Resistant Strain of <Emphasis Type="Italic">Microsporum canis</Emphasis>

To clarify the terbinafine (TRF) resistance mechanism in a TRF-resistant strain of Microsporum canis, the expression of the pleiotropic drug resistance (PDR1), multidrug resistance (MDR1), MDR2 and MDR4 genes were investigated by real-time quantitative PCR (RT-qPCR) analysis, given the known interaction of the corresponding proteins with antifungals and with the efflux blocker FK506. The expression of the PDR1, MDR1, MDR2 and MDR4 genes was 2–4 times higher in the TRF-resistant strain grown in the presence of 0.14 µg/mL of TRF than in TRF-susceptible strains cultured in the absence of TRF. The TRF-resistant strain exhibited MICs of > 32 µg/mL for TRF alone; this resistance was attenuated to an MIC of 8 µg/mL in the presence of FK506, indicating that the TRF inhibitory concentration index value was < 0.75. The additive effect of the efflux blocker FK506 on TRF resistance was detected in the TRF-resistant strain. These results indicated that the TRF resistance in this strain reflects overexpression of genes encoding ABC transporter proteins.     

Getting the Jump on the Development of Bullfrog Oil Microemulsions: a Nanocarrier for Amphotericin B Intended for Antifungal Treatment

Amphotericin B (AmB), a potent antifungal drug, presents physicochemical characteristics that impair the development of suitable dosage forms. In order to overcome the AmB insolubility, several lipid carriers such as microemulsions have been developed. In this context, the bullfrog oil stands out as an eligible oily phase component, since its cholesterol composition may favor the AmB incorporation. Thus, the aim of this study was to develop a microemulsion based on bullfrog oil containing AmB. Moreover, its thermal stability, antifungal activity, and cytotoxicity in vitro were evaluated. The microemulsion formulation was produced using the pseudo-ternary phase diagram (PTPD) approach and the AmB was incorporated based on the pH variation technique. The antifungal activity was evaluated by determination of minimal inhibitory concentration (MIC) against different species of Candida spp. and Trichosporon asahii. The bullfrog oil microemulsion, stabilized with 16.8% of a surfactant blend, presented an average droplet size of 26.50?±?0.14 nm and a polydispersity index of 0.167?±?0.006. This system was able to entrap AmB up to 2 mg mL^?1. The use of bullfrog oil as oily phase allowed an improvement of the thermal stability of the system. The MIC assay results revealed a growth inhibition for different strains of Candida spp. and were able to enhance the activity of AmB against T. asahii. The microemulsion was also able to reduce the AmB toxicity. Finally, the developed microemulsion showed to be a suitable system to incorporate AmB, improving the system’s thermal stability, increasing the antifungal activity, and reducing the toxicity of this drug.     

Methods of <Emphasis Type="BoldItalic">Candida dubliniensis</Emphasis> identification and its occurrence in human clinical material

Candida dubliniensis was reported as a new species in 1995. This species is often misidentified as Candida albicans. The aims of this work were to determine the occurrence of C. dubliniensis in various clinical materials, to evaluate several ways to identify it and to examine the genetic variability of isolates. Among 7706 isolates originally identified as C. albicans, 237 were identified as C. dubliniensis (3.1%). Most of the C. dubliniensis isolates were obtained from the upper and lower respiratory tract (61.4 and 22.9%). Five phenotypic methods including latex agglutination were used (cultivation on CHROMagar Candida, on Staib agar, at 42 °C and in medium with 6.5% NaCl), but only cultivation on the medium with an increased concentration of NaCl and latex agglutination gave reliable results. Species-specific polymerase chain reaction was used as the confirmation method. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided less reliable results. In fact, 78.9% of C. dubliniensis isolates had scores above 1.7. However, the rest of them (21.1%) were also identified as C. dubliniensis even when the scores were lower than 1.7. Divergences among C. dubliniensis strains were evaluated by means of pulsed-field gel electrophoresis. Eighty-six selected C. dubliniensis isolates showed a 69.6% level of similarity. The results of this study expand the knowledge of the incidence, means of identification and genotypic divergence of C. dubliniensis isolates.     

Bioactivity,Safety, and Efficacy of Amphotericin B Nanomicellar Aerosols Using Sodium Deoxycholate Sulfate as the Lipid Carrier

We report nanomicelles of amphotericin B (AmB) using various molar ratios of AmB and sodium deoxycholate sulfate (SDCS) for inhalation with improved stability, solubility, bioactivity, and safety. The particle sizes of all aerosolized formulations are expressed as mass median aerodynamic diameter (0.9–1.6 μm), fine particle fraction (70.3–86.5%), and geometric standard deviation (1.4–2.1) which indicated their sizes are appropriate for use as an inhaler. In vitro cytotoxicity studies conducted using respiratory and kidney cell lines demonstrated that the marketed Fungizone^® was toxic to macrophage and embryonic kidney cells and cell viability decreased from 96 to 48% and from 97 to 67%, respectively when the AmB equivalent concentration was increased from 1 to 16 μg/mL. However, AmB-SDCS formulations showed no evidence of toxicity even up to 8 μg/mL compared to Fungizone^®. Minimum inhibitory and fungicidal concentrations were significantly reduced against Cryptococcus neoformans, and Candida albicans. Also, antileishmanial activity significantly improved for AmB-SDCS formulations. There was an evidence of phagocytosis of the AmB-SDCS formulation by alveolar macrophages NR 8383. Molecular modeling studies suggested the role of hydrogen bonding in stabilization of the AmB-SDCS complex. This study indicated that AmB-SDCS nanomicelles can be used to design a safe and cost-effective AmB for inhalation.

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