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.

     

A Case Report of Penile Infection Caused by Fluconazole- and Terbinafine-Resistant <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida albicans is the most common pathogen that causes balanoposthitis. It often causes recurrence of symptoms probably due to its antifungal resistance. A significant number of balanitis Candida albicans isolates are resistant to azole and terbinafine antifungal agents in vitro. However, balanoposthitis caused by fluconazole- and terbinafine-resistant Candida albicans has rarely been reported. Here, we describe a case of a recurrent penile infection caused by fluconazole- and terbinafine-resistant Candida albicans, as well as the treatments administered to this patient. The isolate from the patient was tested for drug susceptibility in vitro. It was sensitive to itraconazole, voriconazole, clotrimazole and amphotericin B, but not to terbinafine and fluconazole. Thus, oral itraconazole was administrated to this patient with resistant Candida albicans penile infection. The symptoms were improved, and mycological examination result was negative. Follow-up treatment of this patient for 3 months showed no recurrence.     

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.     

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.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

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.     

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.     

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.     

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.     

Clinical and economic impact of the introduction of a nucleic acid amplification assay for <Emphasis Type="Italic">Clostridium difficile</Emphasis>

Background

The clinical outcomes and cost implications of a diagnostic shift from an EIA- to PCR-based assay for Clostridium difficile infection (CDI) have not been completely described in the literature.

Methods

The impact of the PCR-based assay on the incidence and duration of CDI therapy was compared to the EIA assay for patients with a negative CDI diagnostic result. Secondary clinical and economic outcomes were also evaluated. Independent predictors of receipt of antibiotic therapy were assessed via logistic regression.

Results

141 EIA and 140 PCR patients were included. Significantly more patients were started or continued on anti-CDI antibiotic therapy after a known negative assay result in the EIA group (26 patients vs. 8 patients, P = 0.002). Duration of antibiotic therapy after a known negative result was significantly shorter in the PCR group (1 vs. 4 days, P = 0.029) and a 23% reduction in the number of tests obtained per patient was observed (1.41 ± 0.86 vs. 1.82 ± 1.35, P = 0.007). The over fourfold difference in per-test cost of the EIA assay ($8.33 vs. $42.86, P < 0.0001) was offset by the overall medication costs required for the increased treatment in the EIA group ($546.60 vs. $188.96, P = 0.191). Utilization of the EIA-based CDI assay was associated with increased odds of CDI treatment after a negative test (aOR 4.71, 95% CI 1.93–11.46, P = 0.001).

Conclusion

The transition from an EIA to PCR-based assay for diagnosing CDI resulted in a significant decrease in the number of patients treated and the duration of treatment in response to a negative test result. This significant decrease in treatment resulted in decreased costs offsetting the utilization of a more expensive molecular test for patients with a negative CDI diagnostic result.

     

Synthesis,characterization and evaluation of antimicrobial and cytotoxic activities of biogenic silver nanoparticles synthesized from <Emphasis Type="Italic">Streptomyces xinghaiensis</Emphasis> OF1 strain

We report synthesis of silver nanoparticles (AgNPs) from Streptomyces xinghaiensis OF1 strain, which were characterised by UV–Vis and Fourier transform infrared spectroscopy, Zeta sizer, Nano tracking analyser, and Transmission electron microscopy. The antimicrobial activity of AgNPs alone, and in combination with antibiotics was evaluated against bacteria, namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis, and yeasts viz., Candida albicans and Malassezia furfur by using micro-dilution method. The minimum inhibitory concentration (MIC) and minimum biocidal concentration of AgNPs against bacterial and yeast strains were determined. Synergistic effect of AgNPs in combination with antibacterial and antifungal antibiotics was determined by FIC index. In addition, MTT assay was performed to study cytotoxicity of AgNPs alone and in combination with antibiotics against mouse fibroblasts and HeLa cell line. Biogenic AgNPs were stable, spherical, small, polydispersed and capped with organic compounds. The variable antimicrobial activity of AgNPs was observed against tested bacteria and yeasts. The lowest MIC (16 µg ml^?1) of AgNPs was found against P. aeruginosa, followed by C. albicans and M. furfur (both 32 µg ml^?1), B. subtilis and E. coli (both 64 µg ml^?1), and then S. aureus and Klebsiella pneumoniae (256 µg ml^?1). The high synergistic effect of antibiotics in combination with AgNPs against tested strains was found. The in vitro cytotoxicity of AgNPs against mouse fibroblasts and cancer HeLa cell lines revealed a dose dependent potential. The IC₅₀ value of AgNPs was found in concentrations of 4 and 3.8 µg ml^?1, respectively. Combination of AgNPs and antibiotics significantly decreased concentrations of both antimicrobials used and retained their high antibacterial and antifungal activity. The synthesis of AgNPs using S. xinghaiensis OF1 strain is an eco-friendly, cheap and nontoxic method. The antimicrobial activity of AgNPs could result from their small size. Remarkable synergistic effect of antibiotics and AgNPs offer their valuable potential in nanomedicine for clinical application as a combined therapy in the future.     

Virulence Factors in <Emphasis Type="Italic">Candida albicans</Emphasis> and <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilms Mediated by Farnesol

The aim of this study was to evaluate the effect of farnesol on the production of acids and hydrolytic enzymes by biofilms of Streptococcus mutans and Candida albicans. The present study also evaluated the time-kill curve and the effect of farnesol on matrix composition and structure of single-species and dual-species biofilms. Farnesol, at subinhibitory concentrations, showed a significant reduction in S. mutans biofilm acid production, but did not alter C. albicans hydrolytic enzyme production. The number of cultivable cells of both microorganisms was significantly reduced after 8 h of contact with farnesol. Extracellular matrix protein content was reduced for biofilms formed in the presence of farnesol. In addition, confocal laser scanning and scanning electron microscopy displayed structural alterations in all biofilms treated with farnesol, which included reduction in viable cells and extracellular matrix. In conclusion, farnesol showed favorable properties controlling some virulence factors of S. mutans and C. albicans biofilms. These findings should stimulate further studies using this quorum-sensing molecule, combined with other drugs, to prevent or treat biofilm-associated oral diseases.     

Up-Regulation of Antimicrobial Peptides Gallerimycin and Galiomicin in <Emphasis Type="Italic">Galleria mellonella</Emphasis> Infected with <Emphasis Type="Italic">Candida</Emphasis> Yeasts Displaying Different Virulence Traits

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p?=?0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.     

Phylogenetic Diversity and In Vitro Susceptibility Profiles of Human Pathogenic Members of the <Emphasis Type="Italic">Fusarium fujikuroi</Emphasis> Species Complex Isolated from South India

Availability of molecular methods, gene sequencing, and phylogenetic species recognition have led to rare fungi being recognized as opportunistic pathogens. Fungal keratitis and onychomycosis are fairly common mycoses in the tropics, especially among outdoor workers and enthusiasts. The frequently isolated etiological agents belong to genera Candida, Aspergillus, and Fusarium. Within the genus Fusarium, known to be recalcitrant to prolonged antifungal treatment and associated with poor outcome, members of the Fusarium solani species complex are reported to be most common, followed by members of the Fusarium oxysporum SC and the Fusarium fujikuroi SC (FFSC). Morphological differentiation among the various members is ineffective most times. In the present study, we describe different species of the FFSC isolated from clinical specimen in south India. All twelve isolates were characterized up to species level by nucleic acid sequencing and phylogenetic analysis. The molecular targets chosen were partial regions of the internal transcribed spacer rDNA region, the panfungal marker and translation elongation factor-1α gene, the marker of choice for Fusarium speciation. Phylogenetic analysis was executed using the Molecular Evolutionary Genetics Analysis software (MEGA7). In vitro susceptibility testing against amphotericin B, voriconazole, posaconazole, natamycin, and caspofungin diacetate was performed following the CLSI M38-A2 guidelines for broth microdilution method. The twelve isolates of the FFSC were F. verticillioides (n = 4), F. sacchari (n = 3), F. proliferatum (n = 2), F. thapsinum (n = 1), F. andiyazi (n = 1), and F. pseudocircinatum (n = 1). To the best of our knowledge, this is the first report of F. andiyazi from India and of F. pseudocircinatum as a human pathogen worldwide. Natamycin and voriconazole were found to be most active agents followed by amphotericin B. Elderly outdoor workers figured more among the patients and must be recommended protective eye wear.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Clinical strains of <Emphasis Type="Italic">Lactobacillus</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> and protect <Emphasis Type="Italic">Galleria mellonella</Emphasis> against experimental candidiasis

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.     

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 μg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.5 μg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.015 μg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 μg/mL and EUCAST was 0.06 μg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 μg/mL and EUCAST was 0.25 μg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 μg/mL and EUCAST varied from 0.5 to 1 μg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.     

Hyporientalin A,an anti-<Emphasis Type="Italic">Candida</Emphasis> peptaibol from a marine <Emphasis Type="Italic">Trichoderma orientale</Emphasis>

A Trichoderma orientale strain LSBA1 was isolated from the Mediterranean marine sponge Cymbaxinella damicornis. The crude extract of T. orientale mycelium showed inhibitory activity against growth of Gram-positive and Gram-negative bacteria as well as clinical isolates of Candida albicans. Purification of the anti-Candida component was performed using a combination of open silica gel-60 column and reverse phase high performance liquid chromatography. The active compound called hyporientalin A has been identified as a peptaibol analogue of longibrachin-A-II using mass spectrometry. It exhibited fungicidal activity against clinical isolates of C. albicans with minimal inhibitory concentrations (MICs) ranging from 2.49 to 19.66 µM, comparable to that of the antifungal agent amphotericin B. Our data support the use of hyporientalin A as a promising new and efficient antifungal drug in the treatment of candidiasis while controlling toxicity.     

Parasitism Rate of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> to <Emphasis Type="Italic">Ephestia kuehniella</Emphasis> Larvae: Residual Effect of Deltamethrin,Fenvalerate and Azadirachtin

Sublethal concentrations of chemical insecticides may cause changes in some behavioral characteristics of natural enemies such as functional responses. The residual effect of three synthetic insecticides including deltamethrin, fenvalerate and azadirachtin were studied on functional response of Habrobracon hebetor Say to Ephestia kuehniella Zeller larvae. Seven host densities (2, 4, 8, 16, 32, 64 and 96) were used during a 24 h period. The resulting data were appropriately fit to Type II functional response models in all treatments: (1) control (0.0916 h^?1; and T _h  = 0.2011 h); (2) deltamethrin (a = 0.0839 h^?1; and T _h  = 0.3560 h); (3) fenvalerate (a = 0.0808 h^?1 and T _h  = 0.3623 h); and (4) azadirachtin (a = 0.0900 h^?1 and T _h  = 0.2042 h). Maximum theoretical parasitism rate (T/T _h ) was 119.34 estimated for control wasps. There was no significant difference between the values of attack rates (a and a + D _a ) in all treatments while the handling time was statistically affected in female wasps treated with fenvalerate. Our findings will be useful in safe application of these insecticides in pest management programmes.