Inactivation of Candida biofilms by non-thermal plasma and its enhancement for fungistatic effect of antifungal drugs

We investigated the antifungal effect of non-thermal plasma, as well as its combination with common antifungal drugs, against Candida biofilms. A direct current atmospheric pressure He/O₂ (2%) plasma microjet (PMJ) was used to treat Candida biofilms in a 96-well plate. Inactivation efficacies of the biofilms were evaluated by XTT assay and counting colony forming units (CFUs). Morphological properties of the biofilms were evaluated by Scanning Electron Microscope (SEM). The sessile minimal inhibitory concentrations (SMICs) of fluconazole, amphotericin B, and caspofungin for the biofilms were also tested. Electron Spin Resonance (ESR) spectroscopy was used to detect the reactive oxygen species (ROS) generated directly and indirectly by PMJ. The Candida biofilms were completely inactivated after 1 min PMJ treatment, where severely deformed fungal elements were observed in SEM images. The SMICs of the tested antifungal drugs for the plasma-treated biofilms were decreased by 2–6 folds of dilution, compared to those of the untreated controls. ROS such as hydroxyl radical (^•OH), superoxide anion radical (^•O₂ ^-) and singlet molecular oxygen (¹O₂) were detected by ESR. We hence conclude that He/O₂ (2%) plasma alone, as well as in combination with common antifungal drugs, is able to inactivate Candida biofilms rapidly. The generation of ROS is believed to be one of the underlying mechanisms for the fungicidal activity of plasma.     

In vitro activity of juglone (5-hydroxy-1,4-naphthoquinone) against both fluconazole-resistant and susceptible Candida isolates

BackgroundThe rise in antifungal resistance and drug class limitations are causing higher morbidity and mortality rates all over the world. This issue highlights the urgent need for new and improved antifungal drugs with a novel target.AimsIn order to evaluate whether juglone can be served as an alternative antifungal to cure drug-resistant Candida infections, we studied the in vitro susceptibility of juglone against fluconazole-susceptible and -resistance Candida isolates, alone and in combination.MethodsAntifungal susceptibility testing was performed according to the CLSI (Clinical and Laboratory Standards Institute) guidelines.ResultsJuglone exhibited the highest minimal inhibitory concentration (MIC) values, followed by fluconazole and nystatin. Voriconazole showed significantly better antifungal activity than juglone, fluconazole, and nystatin, with MIC₅₀ and MIC₉₀ of 0.031 and 0.5 μg/mL. There were significant differences in MICs of fluconazole (p < 0.001) and juglone (p < 0.0003) between Candida albicans and the rest of the species. Combination of juglone with fluconazole revealed insignificant effects against fluconazole-susceptible and -resistant Candida isolates. Juglone increased the antifungal activity of fluconazole; however, no synergism effects were observed for any combination, and only an insignificant effect was found against all tested Candida species.ConclusionsAlthough obtaining new antifungal drugs is a critical point, a completely novel approach should be implemented.     

Actividad de la micafungina contra las biopelículas de Candida

BackgroundMost recalcitrant infections are associated to colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial therapy.AimTo describe the antifungal of micafungin against fungal biofilms based in the scientific and medical literature of recent years.MethodsWe have done a bibliographic retrieval using the scientific terms “micafungin”, “activity”, “biofilm”, “Candida”, “Aspergillus”, “fungi”, “mycos”*, susceptibility, in PubMed/Medline from the National Library of Medicine from 2006 to 2009.ResultsMost current antifungal agents (amphotericin B and fluconazole) and the new azole antifungals have no activity against fungal biofilms. However, micafungin and the rest of echinocandins are very active against Candida albicans, Candida dubliniensis, Candida glabrata, and Candida krusei biofilms but their activities are variable and less strong against Candida tropicalis and Candida parapsilosis biofilms. Moreover, they have not activities against the biofilms of Cryptococcus y Trichosporon.ConclusionsThe activity of micafungin against Candida biofilms gives more strength to its therapeutic indication for candidaemia and invasive candidiasis associated to catheter, prosthesis and other biomedical devices.     

In Vitro Effect of Amphotericin B on Candida albicans,Candida glabrata and Candida parapsilosis Biofilm Formation

Candida spp. biofilm is considered highly resistant to conventional antifungals. The aim of this study was to investigate the in vitro effect of amphotericin B on Candida spp. biofilms at different stages of maturation. We investigated the activity of amphotericin B against 78 clinical isolates of Candida spp., representing three species, growing as planktonic and sessile cells, by a widely accepted broth microdilution method. The in vitro effect on sessile cell viability was evaluated by MTT reduction assay. All examined strains were susceptible to amphotericin B when grown as free-living cells. At the early stages of biofilm maturation 96.7–100.0 % strains, depending on species, displayed amphotericin B sessile minimal inhibitory concentration (SMIC) ≤1 μg/mL. Mature Candida spp. biofilm of 32.1–90.0 % strains displayed amphotericin B SMIC ≤1 μg/mL. Based on these results, amphotericin B displays species- and strain-depending activity against Candida spp. biofilms.     

Candida albicans and Non-C. albicans Candida Species: Comparison of Biofilm Production and Metabolic Activity in Biofilms, and Putative Virulence Properties of Isolates from Hospital Environments and Infections

Candida albicans and, more recently, non-C. albicans Candida spp. are considered the most frequent fungi in hospitals. This study analyzed Candida spp. isolates and compared the frequency of different species, that is, C. albicans and non-C. albicans Candida spp., and the origins of isolates, that is, from hospital environments or infections. Yeast virulence factors were evaluated based on biofilm production and metabolic activity. Hemolysin production and the antifungal susceptibility profiles of isolates were also evaluated. Candida spp. were highly prevalent in samples collected from hospital environments, which may provide a reservoir for continuous infections with these yeasts. There were no differences in the biofilm productivity levels and metabolic activities of the environmental and clinical isolates, although the metabolic activities of non-C. albicans Candida spp. biofilms were greater than those of the C. albicans biofilms (p < 0.05). Clinical samples had higher hemolysin production (p < 0.05) and lower susceptibility to fluconazole (p < 0.05). Non-C. albicans Candida spp. predominated in samples collected from hospital environments and infections (p < 0.05). These species had a lower susceptibility to fluconazole and amphotericin B, and their biofilms had higher metabolic activities than those produced by C. albicans, which may explain the increased incidence of fungal infections with these yeasts during recent years.     

In vitro interactions between anidulafungin and nonsteroidal anti-inflammatory drugs on biofilms of Candida spp.

Candida spp. are responsible for many biomaterial-related infections; they give rise to infective pathologies typically associated with biofilm formation. We recently reported that the echinocandin anidulafungin (ANF) showed a strong in vitro activity against both planktonic and biofilms cells. Herein, we report the antifungal activities of ANF alone and in association with some non-steroidal anti-inflammatory drugs (NSAIDs) against nine Candida strain biofilms: four Candida albicans, two Candida glabrata and three Candida guilliermondii. The activity of ANF was assessed using an in vitro microbiological model relevant for clinical practice. ANF proved oneself to be active against biofilms cells, and a clear-cut synergism was found against Candida species biofilms when ANF was used in combination with three NSAIDs: aspirin, diclofenac, ibuprofen. The positive synergism against Candida spp. of ANF in association with aspirin or the other NSAIDs proved to be a very effective antifungal treatment (FICI <0.5). These results may provide the starting point for new combination therapies of ANF with NSAIDs against Candida biofilm pathologies.     

Yeasts isolated from nosocomial urinary infections: Antifungal susceptibility and biofilm production

BackgroundUrinary Candida infections in the hospital environment are frequent and need to be better understood.AimsTo compare the results of antifungal susceptibility profiles of yeasts isolated from patients with urinary infections obtained by broth microdilution method (BM) and by disk diffusion (DD), and also evaluate the capacity of these yeasts to form biofilms.MethodsOnly yeasts obtained from pure urine cultures with counts higher than 10⁵ colony-forming units per milliliter, without bacteria development, of symptomatic patients were included. The isolates were identified by classical methods and the antifungal susceptibility tests were performed with the following drugs: amphotericin B, ketoconazole, fluconazole, itraconazole, voriconazole and caspofungin. The biofilm studies were carried out in polystyrene microtitration plates.ResultsNinety-five yeasts isolates were analyzed, including 40 Candida albicans, 31 Candida glabrata, 24 Candida tropicalis. In general, the majority of the isolates were susceptible to the tested drugs but some resistance was observed, especially against fluconazole. Great variability in the antifungal susceptibility results was observed with the different tested drugs and a few discrepancies were observed between both methods. We suggest that in case of DD resistance this result should be confirmed by BM, the standard method. C. tropicalis isolates showed high biofilm production (91.7%) compared to C. albicans (82.5%) and C. glabrata (61.3%), with statistical significance (p = 0.0129).ConclusionsCandiduria in critical patients requires major attention and a better control. The different susceptibility results obtained in this study showed the need to identify yeasts up to the species level, especially in patients with urinary tract infection. The development of techniques of antifungal susceptibility tests can help the clinicians in the empiric treatment of candiduria.     

Sensitization of Candida albicans biofilms to various antifungal drugs by cyclosporine A

Background

Biofilms formed by Candida albicans are resistant towards most of the available antifungal drugs. Therefore, infections associated with Candida biofilms are considered as a threat to immunocompromised patients. Combinatorial drug therapy may be a good strategy to combat C. albicans biofilms.

Methods

Combinations of five antifungal drugs- fluconazole (FLC), voriconazole (VOR), caspofungin (CSP), amphotericin B (AmB) and nystatin (NYT) with cyclosporine A (CSA) were tested in vitro against planktonic and biofilm growth of C. albicans. Standard broth micro dilution method was used to study planktonic growth, while biofilms were studied in an in vitro biofilm model. A chequerboard format was used to determine fractional inhibitory concentration indices (FICI) of combination effects. Biofilm growth was analyzed using XTT-metabolic assay.

Results

MICs of various antifungal drugs for planktonic growth of C. albicans were lowered in combination with CSA by 2 to 16 fold. Activity against biofilm development with FIC indices of 0.26, 0.28, 0.31 and 0.25 indicated synergistic interactions between FLC-CSA, VOR-CSA, CSP-CSA and AmB-CSA, respectively. Increase in efficacy of the drugs FLC, VOR and CSP against mature biofilms after addition of 62.5 μg/ml of CSA was evident with FIC indices 0.06, 0.14 and 0.37, respectively.

Conclusions

The combinations with CSA resulted in increased susceptibility of biofilms to antifungal drugs. Combination of antifungal drugs with CSA would be an effective prophylactic and therapeutic strategy against biofilm associated C. albicans infections.     

Isolation of Candida in the blood cultures of patients admitted to the Emergency Room in a tertiary care hospital

BackgroundIn Spain, data of candidemia are limited to surveys conducted in specific areas or tertiary care centers. Also, in recent years, attention has shifted toward episodes of candidemia in non-ICU wards.AimsWe reviewed the cases of Candida isolates recovered from the blood of patients admitted to the Emergency Room (ER) in our tertiary care hospital.MethodsThe patients selected for this study had an isolation of Candida in the blood culture. All data were collected retrospectively from the clinical records of a 11-year period.ResultsCandida albicans and other species of the genus were present in 10 and 18 patients, respectively. The patients did not present different clinical features in comparison with other reports of hospitalized patients. All patients had several risk factors for candidemia. Only two patients had received previous antifungal therapy before admission. All the isolates of C. albicans, Candida glabrata and the only isolate of Candida tropicalis were susceptible to all the antifungal agents tested. Only one isolate of Candida parapsilosis was susceptible dose-dependent to fluconazole, and the only isolate of Candida metapsilosis was resistant to fluconazole.ConclusionsIt is essential to evaluate the risk factors, underlying conditions and clinical features in non-hospitalized patients in order to determine whether an empirical treatment for candidemia is appropriate.     

Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species (ROS) and disruption of cell wall against Candida albicans

In the present work, we investigated the antifungal activity of two de novo designed, antimicrobial peptides VS2 and VS3, incorporating unnatural amino acid α,β-dehydrophenylalanine (ΔPhe). We observed that the low-hemolytic peptides could irreversibly inhibit the growth of various Candida species and multidrug resistance strains at MIC₈₀ values ranging from 15.62 μM to 250 μM. Synergy experiments showed that MIC₈₀ of the peptides was drastically reduced in combination with an antifungal drug fluconazole. The dye PI uptake assay was used to demonstrate peptide induced cell membrane permeabilization. Intracellular localization of the FITC-labeled peptides in Candida albicans was studied by confocal microscopy and FACS. Killing kinetics, PI uptake assay, and the intracellular presence of FITC-peptides suggested that growth inhibition is not solely a consequence of increased membrane permeabilization. We showed that entry of the peptide in Candida cells resulted in accumulation of reactive oxygen species (ROS) leading to cell necrosis. Morphological alteration in Candida cells caused by the peptides was visualized by electron microscopy. We propose that de novo designed VS2 and VS3 peptides have multiple detrimental effects on target fungi, which ultimately result in cell wall disruption and killing. Therefore, these peptides represent a good template for further design and development as antifungal agents.     

Comparative Study of the Effects of Fluconazole and Voriconazole on <Emphasis Type="Italic">Candida glabrata</Emphasis>, <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida rugosa</Emphasis> Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC₅₀ of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.     

Biofilm formation by <Emphasis Type="Italic">Candida albicans</Emphasis> isolated from intrauterine devices

Our survey revealed that infected intrauterine devices (IUDs) recovered from patients suffering from reproductive tract infections (RTIs) were tainted with Candida biofilm composed of a single or multiple species. Scanning electron microscopy (SEM) analysis of C. albicans biofilm topography showed that it consists of a dense network of mono- or multilayer of cells embedded within the matrix of extracellular polymeric substances (EPS). Confocal scanning laser microscopy (CSLM) and atomic force microscopy (AFM) images depicted that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements with EPS distributed in the cell-surface periphery or at cell-cell interface. Biochemical analysis showed that EPS produced by C. albicans biofilm contained significantly reduced total carbohydrate (40%), protein (5%) and enhanced amount of hexosamine (4%) in contrast to its planktonic counterparts. The in vitro activity of antifungal agents amphotericin B, nystatin, fluconazole and chlorhexidine against pre-formed C. albicans biofilm, assessed using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay revealed increased resistance of these infectious biofilm (50% reduction in metabolic activity at a concentration of 8, 16, 64, 128 μg/ml respectively) in comparison to its planktonic form.     

Clinical Implications of <Emphasis Type="Italic">Candida</Emphasis> Biofilms

Purpose of Review

In this review, we will assess what is currently known about Candida biofilms, its epidemiology and impact on clinical practice, and the biology underlying biofilm-related antifungal drug resistance. We also review potential antifungal lock therapies and discuss what avenues are available for future discoveries for the prevention and eradication of fungal biofilms.

Recent Findings

There is a multitude of in vitro tests of agents against Candida biofilms, but many of these studies have not demonstrated their utility against other Candida species and/or their efficacy in in vivo systems. This makes it particularly difficult to translate these findings for use in the clinical setting.

Summary

Research has helped us to understand the complexity of biofilms and its inherent antifungal resistance. The extracellular matrix remains a significant barrier for most antifungal therapy, and thus it stands to reason that agents directed at disrupting this protective barrier could be useful in antifungal lock therapies.

     

D319 induced antifungal effects through ROS-mediated apoptosis and inhibited isocitrate lyase in Candida albicans

BackgroundCandida albicans (C. albicans) is an opportunistic pathogen that can cause superficial and life-threatening systemic infections in immunocompromised patients. However, the available clinically antifungals are limited. Therefore, the development of effective antifungal agents and therapies is urgently needed. Quinoline type of compounds were reported to possess potent anti-fungal effect. A series of quinoline derivatives were synthesized. Moreover their inhibitory activities and mechanisms on C. albicans were evaluated in this study.MethodsThe structure of D319 was identified by extensive spectroscopic analysis. The antifungal activity of D319 on C. albicans was evaluated using conventional methods, including the inhibition zone diameters with filter paper, Clinical Laboratory Standard Institute (CLSI) broth microdilution method in vitro, and in a murine model in vivo. Flow cytometry, fluorescence microscopy, western blot, knockout mutant and revertant strain techniques, and molecular modeling were applied to explore the mechanism of action of D319 in anti-Candida.ResultsD319 exhibited potent anti-Candida activity with Minimum Inhibitory Concentration value of 2.5 μg/mL in vitro. D319 significantly improved survival rate and reduced fungal burden compared to vehicle control in a murine model in vivo. The treatment of C. albicans with D319 resulted in fungal apoptosis through reactive oxygen species (ROS) accumulation in C. albicans. Furthermore, D319 inhibited the glyoxylate enzyme isocitrate lyase (ICL) of C. albicans, which was also confirmed by docking analysis.ConclusionsQuinoline compound D319 exhibited strong anti-Candida activities in vitro and in vivo models through inhibiting ICL activity and ROS accumulation in C. albicans.General significanceThis study showed that compound D319 as a novel isocitrate lyase inhibitor, would be a promising anti-Candida lead compound, which provided a potential application of this type of compounds in fighting clinical fungal infections. Furthermore, this study also supported ICL as a potential target for anti-Candida drug discovery.     

The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance

BackgroundFor many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species.AimsIn this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole.MethodsScreening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing.ResultsWe identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p.ConclusionThe L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.     

Utilidad clínica de la micafungina en el tratamiento de las candidiasis invasoras en el paciente crítico

BackgroundOver the last 30 years a significant increase of Candida spp. invasive disease has been observed in non-neutropenic critical ill patients. Both fluconazole and amphotericin B have been considered first line treatment for invasive (proven and probable) Candida spp. disease, although the mortality rate is still high.ObjectivesTo review the current data on the use of micafungin for the treatment of Candida invasive disease in critical ill patients.MethodsThe pharmacologic, mycological and clinical properties of micafungin are reviewed based on current published data. The use and efficacy of micafungin for the treatment of Candida invasive disease in critical ill patients is discussed.Results and conclusionsTo reduce the rate of mortality more effective antifungals and pre-emptive treatment strategies are currently warranted. Candins achieve better results for the treatment of invasive Candida disease in non-neutropenic critical ill patients. Micafungin has a good safety profile (similar to fluconazole). Micafungin is a first line drug for the treatment of invasive Candida disease and may be used as a pre- emptive approach followed by a de-escalating strategy with azoles.     

Treatment of Invasive Candida Infections in the Neonatal Intensive Care Unit

Invasive Candida infections are a leading cause of morbidity and mortality in the neonatal intensive care unit (NICU). Extremely preterm and very low birth weight infants are at the highest risk of infection. There are currently no antifungal agents that have FDA-labeling for the treatment of invasive candidiasis in the neonatal population. Based on the current IDSA guidelines, amphotericin and fluconazole are considered first-line options for neonatal candidiasis. The newer antifungal agents (i.e., echinocandins and voriconazole) are currently considered second-line or salvage therapy; however, evidence supporting their use is emerging. This review focuses on the supporting evidence for the selection of antifungal agents for treatment of invasive Candida infections in the NICU.     

Isavuconazole and Nine Comparator Antifungal Susceptibility Profiles for Common and Uncommon Candida Species Collected in 2012: Application of New CLSI Clinical Breakpoints and Epidemiological Cutoff Values

The in vitro activity of isavuconazole and nine antifungal comparator agents was assessed using reference broth microdilution methods against 1,421 common and uncommon species of Candida from a 2012 global survey. Isolates were identified using CHROMagar, biochemical methods and sequencing of ITS and/or 28S regions. Candida spp. were classified as either susceptible or resistant and as wild type (WT) or non-WT using CLSI clinical breakpoints or epidemiological cutoff values, respectively, for the antifungal agents. Isolates included 1,421 organisms from 21 different species of Candida. Among Candida spp., resistance to all 10 tested antifungal agents was low (0.0–7.9 %). The vast majority of each species of Candida, with the exception of Candida glabrata, Candida krusei, and Candida guilliermondii (modal MICs of 0.5 µg/ml), were inhibited by ≤0.12 µg/ml of isavuconazole (99.0 %; range 94.3 % [Candida tropicalis] to 100.0 % [Candida lusitaniae and Candida dubliniensis]). C. glabrata, C. krusei, and C. guilliermondii were largely inhibited by ≤1 µg/ml of isavuconazole (89.7, 96.9 and 92.8 %, respectively). Decreased susceptibility to isavuconazole was most prominent with C. glabrata where the modal MIC for isavuconazole was 0.5 µg/ml for those strains that were SDD to fluconazole or WT to voriconazole, and was 4 µg/ml for those that were either resistant or non-WT to fluconazole or voriconazole, respectively. In conclusion, these data document the activity of isavuconazole and generally the low resistance levels to the available antifungal agents in a large, contemporary (2012), global collection of molecularly characterized species of Candida.     

Niazirin from Moringa oleifera Lam. attenuates high glucose-induced oxidative stress through PKCζ/Nox4 pathway

BackgroundDiabetic complications-coronary atherosclerosis is closely related to the increased reactive oxygen species (ROS) induced by hyperglycemia. ROS are reported to induce the abnormal proliferation of vascular smooth muscle cells (VSMCs) under high glucose conditions. Leaf and seed extracts from Moringa oleifera are found to exhibit antioxidant activity. However, few studies are evaluating the antioxidant activities of chemical compounds isolated from the M. oleifera especially in cardiovascular field.PurposeThe aim of this study is to explore the antioxidative effect during hyperglycemia of niazirin from M. oleifera.Study designA cell model was applied.MethodsAfter the taking the in vitro antioxidant experiment including ferric reducing antioxidant power (FRAP), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Cell viability was carried out using high glucose-induced VSMCs model. ROS production was tested by 2′,7′-dichlorofluorescein diacetate (DCF-DA) assay. The protein kinase C zeta (PKCζ) and NADPH oxidase 4 (Nox 4) expression in vitro and in vivo were measured by western blot analysis.ResultsNiazirin showed good free radical scavenging activity. Niazirin significantly attenuated the proliferation of high glucose-induced VSMCs. Furthermore, it could decrease the ROS and malondialdehyde (MDA) productions, while increased total antioxidant capacity (T-AOC), superoxide dismutase (SOD) as well as glutathione peroxidase (GPx) levels in high glucose-induced VSMCs and streptozotocin-induced mice. In addition, niazirin could eliminate the high glucose-induced PKCζ activation, indicated by Thr410 phosphorylation and inhibition of the Nox4 protein expression in vitro and in vivo.ConclusionNiazirin from M. oleifera exhibited notably antioxidant activities and could be utilized as a potential natural antioxidant in preventing diabetic atherosclerosis.     

Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD₆₀₀; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.