FLC-resistant Strains Research Articles

Antifungal properties of abnormal cannabinoid derivatives: Disruption of biofilm formation and gene expression in Candida species

Abnormal cannabinoids (including comp 3) are a class of synthetic lipid compounds with non-psychoactive properties and regioisomer configurations, but distinct from traditional cannabinoids since they do not interact with the established CB1 and CB2 receptors. Previous research showed the cardioprotective and anti-inflammatory potentials of comp 3 and more recently its antimicrobial effect on methicillin-resistant Staphylococcus aureus (MRSA). Given the escalating challenges posed by Candida infections and the rise of antifungal drug resistance, the exploration of novel therapeutic avenues is crucial. This study aimed to assess the anti-Candida properties of newly synthesized AbnCBD derivatives. AbnCBD derivatives were synthesized by acid catalysis-induced coupling and further derivatized. We evaluated the potential of the AbnCBD derivatives to inhibit the growth stages of various Candida species. By in vitro colorimetric assays and in vivo mice experiments, we have shown that AbnCBD derivatives induce differential inhibition of Candida growth. The AbnCBD derivatives, especially comp 3, comp 10, and comp 9 significantly reduced the growth of C. albicans, including FLC-resistant strains, and of C. tropicalis and C. parapsilosis but not of C auris compared to their controls (FLC and 0.5 % DMSO). Comp 3 also disrupted C. albicans biofilm formation and eradicated mature biofilms. Notably, other derivatives of AbnCBD disrupted the biofilm formation and maturation of C. albicans but did not affect yeast growth. In a murine model of VVC, comp 3 demonstrated significant fungal clearance and reduced C. albicans burden compared to vehicle and FLC controls. These findings highlight the potential of AbnCBDs as promising antifungal agents against Candida infections.

Mechanism and bioinformatics analysis of the effect of berberine-enhanced fluconazole against drug-resistant Candida albicans

Biofilms produced by Candida albicans present a challenge in treatment with antifungal drug. Enhancing the sensitivity to fluconazole (FLC) is a reasonable method for treating FLC-resistant species. Moreover, several lines of evidence have demonstrated that berberine (BBR) can have antimicrobial effects. The aim of this study was to clarify the underlying mechanism of these effects. We conducted a comparative study of the inhibition of FLC-resistant strain growth by FLC treatment alone, BBR treatment alone, and the synergistic effect of combined FLC and BBR treatment. Twenty-four isolated strains showed distinct biofilm formation capabilities. The antifungal effect of combined FLC and BBR treatment in terms of the growth and biofilm formation of Candida albicans species was determined via checkerboard, time-kill, and fluorescence microscopy assays. The synergistic effect of BBR and FLC downregulated the expression of the efflux pump genes CDR1 and MDR, the hyphal gene HWP1, and the adhesion gene ALS3; however, the gene expression of the transcriptional repressor TUP1 was upregulated following treatment with this drug combination. Furthermore, the addition of BBR led to a marked reduction in cell surface hydrophobicity. To identify resistance-related genes and virulence factors through genome-wide sequencing analysis, we investigated the inhibition of related resistance gene expression by the combination of BBR and FLC, as well as the associated signaling pathways and metabolic pathways. The KEGG metabolic map showed that the metabolic genes in this strain are mainly involved in amino acid and carbon metabolism. The metabolic pathway map showed that several ergosterol (ERG) genes were involved in the synthesis of cell membrane sterols, which may be related to drug resistance. In this study, BBR + FLC combination treatment upregulated the expression of the ERG1, ERG3, ERG4, ERG5, ERG24, and ERG25 genes and downregulated the expression of the ERG6 and ERG9 genes compared with fluconazole treatment alone (p < 0.05).

Overcoming Drug Resistance in a Clinical C. albicans Strain Using Photoactivated Curcumin as an Adjuvant.

The limited antifungal drugs available and the rise of multidrug-resistant Candida species have made the efforts to improve antifungal therapies paramount. To this end, our research focused on the effect of a combined treatment between chemical and photodynamic therapy (PDT) towards a fluconazole-resistant clinical Candida albicans strain. The co-treatment of PDT and curcumin in various doses with fluconazole (FLC) had an inhibitory effect on the growth of the FLC-resistant hospital strain of C. albicans in both difusimetric and broth microdilution methods. The proliferation of the cells was inhibited in the presence of curcumin at 3.125 µM and FLC at 41 µM concentrations. The possible involvement of oxidative stress was analyzed by adding menadione and glutathione as a prooxidant and antioxidant, respectively. In addition, we examined the photoactivated curcumin effect on efflux pumps, a mechanism often linked to drug resistance. Nile Red accumulation assays were used to evaluate efflux pumps activity through fluorescence microscopy and spectrofluorometry. The results showed that photoactivated curcumin at 3.125 µM inhibited the transport of the fluorescent substrate that cells usually expel, indicating its potential in combating drug resistance. Overall, the findings suggest that curcumin, particularly when combined with PDT, can effectively inhibit the growth of FLC-resistant C. albicans, addressing the challenge of yeast resistance to azole antifungals through upregulating multidrug transporters.

Study on the mechanisms of action of berberine combined with fluconazole against fluconazole-resistant strains of Talaromyces marneffei

Talaromyces (Penicillium) marneffei (T. marneffei) is a thermally dimorphic fungus that can cause opportunistic systemic mycoses. Our previous study demonstrated that concomitant use of berberine (BBR) and fluconazole (FLC) showed a synergistic action against FLC-resistant T. marneffei (B4) in vitro. In this paper, we tried to figure out the antifungal mechanisms of BBR and FLC in T. marneffei FLC-resistant. In the microdilution test, the minimum inhibitory concentration (MIC) of FLC was 256 μg/ml before FLC and BBR combination, and was 8 μg/ml after combination, the partial inhibitory concentration index (FICI) of B4 was 0.28. After the treatments of BBR and FLC, the studies revealed that (i) increase reactive oxygen species (ROS), (ii) reduce ergosterol content, (iii) destroy the integrity of cell wall and membrane, (iv) decrease the expression of genes AtrF, MDR1, PMFCZ, and Cyp51B however ABC1 and MFS change are not obvious. These results confirmed that BBR has antifungal effect on T. marneffei, and the combination with FLC can restore the susceptibility of FLC-resistant strains to FLC, and the reduction of ergosterol content and the down-regulation of gene expression of AtrF, Mdr1, PMFCZ, and Cyp51B are the mechanisms of the antifungal effect after the combination, which provides a theoretical basis for the application of BBR in the treatment of Talaromycosis and opens up new ideas for treatment of Talaromycosis.

Broadening antifungal spectrum and improving metabolic stablity based on a scaffold strategy: Design, synthesis, and evaluation of novel 4-phenyl-4,5-dihydrooxazole derivatives as potent fungistatic and fungicidal reagents

5-phenylthiophene derivatives exhibited excellent antifungal activity against Candida albicans, Candida tropicalis and Cryptococcus neoformans. However, optimal compound 7 was inactive against Aspergillus fumigatus and unstable in human liver microsomes in vitro with a half-life of 18.6 min. To discover antifungal agents with a broad spectrum and improve the metabolic properties of the compounds, the scaffold hopping strategy was adopted and a series of 4-phenyl-4,5-dihydrooxazole derivatives were designed and synthesized. It was especially encouraging that compound 22a displayed significant antifungal activities against eight susceptible strains and seven FLC-resistant strains. Furthermore, the potent compound 22a could prevent the formation of fungalbiofilms and displayed satisfactory fungicidal activity. In addition, the metabolic stability of compound 22a was improved significantly, with the half-life of 70.5 min. Compound 22a was almost nontoxic to mammalian A549, MCF-7, HepG2, and 293T cells. Moreover, pharmacokinetic studies in SD rats showed that compound 22a exhibited pharmacokinetic properties with a bioavailability of 15.22% and a half-life of 4.44 h, indicating that compound 22a is worthy of further study.

Synergistic Effect of Pseudolaric Acid B with Fluconazole Against Resistant Isolates and Biofilm of Candida tropicalis.

PurposeCandida tropicalis (C. tropicalis) has emerged as an important fungal pathogen due to its increasing resistance to conventional antifungal agents, especially fluconazole (FLC). Pseudolaric acid B (PAB), a herbal-originated diterpene acid from Pseudolarix kaempferi Gordon, has been reported to possess inhibitory activity against fungus. The present study aims to investigate the antifungal effect of PAB alone and in combination with FLC on planktonic and biofilm cells of C. tropicalis.MethodsThe antifungal activity of PAB against planktonic isolates was evaluated alone and in combination with FLC using the chequerboard microdilution method and growth curve assay. The anti-biofilm effects were quantified by tetrazolium (XTT) reduction assay, which were further confirmed by scanning electron microscopy (SEM) and fluorescent microscope to observe morphological changes of biofilm treated with PAB and FLC.ResultsIt was revealed that PAB alone exhibited similar inhibitory activity against FLC-resistant and FLC-susceptible strains with median MIC ranging from 8 to 16 µg/mL. When administered in combination, synergism was observed in all (13/13) FLC-resistant and (2/9) FLC-susceptible strains with FICI ranging from 0.070 to 0.375. Moreover, the concomitant use of PAB and FLC exhibited a strong dose-dependent synergistic inhibitory effect on the early and mature biofilm, eliminating more than 80% biofilm formation. SEM found that PAB, different from azoles, could significantly inhibit spore germination and destroy the cell integrity causing cell deformation, swelling, collapse and outer membrane perforation.ConclusionPAB was highly active against FLC-resistant isolates and biofilm of C. tropicalis, particularly when combined with FLC. These findings suggest that PAB may have potential as a novel antifungal agent with different targets from azole drugs.

Oxidative stress induced by piperine leads to apoptosis in Candida albicans.

Piperine inhibit Candida albicans growth (planktonic and biofilm) significantly in our study. Piperine exhibits excellent synergistic potential with fluconazole The proteome analysis suggests that piperine induced membrane damage leads to oxidative stress followed by cell cycle arrest and apoptosis.

Fluconazole induces ROS in Cryptococcus neoformans and contributes to DNA damage in vitro.

Pathogenic basidiomycetous yeast, Cryptococcus neoformans, causes fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug commonly administered to treat cryptococcosis. Unfortunately, FLC-resistant strains characterized by various degree of chromosomal instability were isolated from clinical patients. Importantly, the underlying mechanisms that lead to chromosomal instability in FLC-treated C. neoformans remain elusive. Previous studies in fungal and mammalian cells link chromosomal instability to the reactive oxygen species (ROS). This study provides the evidence that exposure of C. neoformans to FLC induces accumulation of intracellular ROS, which correlates with plasma membrane damage. FLC caused transcription changes of oxidative stress related genes encoding superoxide dismutase (SOD1), catalase (CAT3), and thioredoxin reductase (TRR1). Strikingly, FLC contributed to an increase of the DNA damage in vitro, when complexed with iron or copper in the presence of hydrogen peroxide. Strains with isogenic deletion of copper response protein metallothionein were more susceptible to FLC. Addition of ascorbic acid (AA), an anti-oxidant at 10 mM, reduced the inhibitory effects of FLC. Consistent with potential effects of FLC on DNA integrity and chromosomal segregation, FLC treatment led to elevated transcription of RAD54 and repression of cohesin-encoding gene SCC1. We propose that FLC forms complexes with metals and contributes to elevated ROS, which may lead to chromosomal instability in C. neoformans.

Tetrandrine enhances the antifungal activity of fluconazole in a murine model of disseminated candidiasis

BackgroundTetrandrine (TET), a bis-benzylisoquinoline alkaloid isolated from the Chinese medicinal herb Stephaniae tetrandrae, has a long history in Chinese clinical applications as an anti-inflammatory or anti-arrhythmic agent in the treatment of diverse diseases. In our previous study, TET exhibited the synergisitic action on azoles against pathogenic fungi. PurposeIn the current study, we examined whether TET can enhance the antifungal activity of FLC against disseminated candidiasis in mice. MethodsBALB/c mice were inoculated intravenously with FLC-sensitive or FLC-resistant strains of Candida albicans, randomized and treated intraperitoneally with different doses of TET and/or FLC daily for 7 days. The treatment effectiveness, fungal burdens and the levels of the IFN-γ, IL-10, TGF-β1 and IL-17A are determined in serum by ELISA and in the kidney by Real-time RT-PCR methods. ResultsWe found that treatment with 45, 30 and 15 mg/kg of TET, enhanced the antifungal activities of a sub-critical dose (0.4 or 5 mg/kg) and minimal dose (0.8 or 10 mg/kg) of FLC against FLC-sensitive and FLC-resistant (respectively) infected mice. In the resistant strains the resistance mechanisms included MDR1 overexpression-and CDR1/CDR2 overexpression. Furthermore, when animals were treated with a sub-high dose (1.6–3.2 and 20–30 mg/kg) of FLC in the presence of fixed amounts of TET at 45, 30 and 15 mg/kg, the therapeutic doses of FLC could be substantially reduced in all strains tested. The findings in infected animal are consistent with the conclusion that TET exerts a synergistic effect on FLC against C. albicans by fractional inhibitory concentration index (FICI) and time-killing test in vitro. ConclusionIn summary, our data indicate that TET will enhance the antifungal activity of FLC against C. albicans infection in disseminated mice model.

Evaluation of four calcium channel blockers as fluconazole resistance inhibitors in Candida glabrata.

The aim of this study was to evaluate the ability of four calcium channel blockers (CCBs), namely verapamil, diltiazem, nicardipine (NIC) and nifedipine (NIF), to enhance the susceptibility of Candida glabrata strains to fluconazole (FLC). Synergistic antifungal effects of the CCBs with FLC were examined by the chequerboard method, and fractional inhibitory concentration indices (FICIs) were determined. The time-kill curve method was used for the most promising combination to further evaluate the synergetic effects. NIC showed an additive effect with FLC against FLC-resistant and FLC-susceptible-dose-dependent strains (DSY 565 and CBS 138) known to express efflux pumps, but not against FLC-susceptible strains. NIF exhibited an additive effect with FLC both by the chequerboard method (0.5<FICI<1) and time-kill curves (<2 log10 CFU/mL decrease in viable count). In addition, NIF had its own antifungal effect consistently against most of the strains used in this study, with minimum inhibitory concentrations (MICs) of 8μg/mL. NIC showed an additive effect with FLC against FLC-resistant C. glabrata strains, most probably via efflux pump inhibition as demonstrated selectively in FLC-resistant strains with known efflux pumps. NIF displayed a promising antifungal effect alone as well as an additive effect with FLC against most of the strains.

Involvement of mitochondrial aerobic respiratory activity in efflux-mediated resistance of C. albicans to fluconazole

Reduced intracellular accumulation of drugs mediated by efflux pump is one of the most critical mechanisms governing fluconazole (FLC) resistance in Candida albicans (C.albicans). Besides, mitochondrial aerobic respiration plays a major role in C.albicans metabolism. However, it is unclear whether mitochondrial aerobic respiration is involved with efflux-mediated resistance of C.albicans to azole. We measured key parameters of energy conversion, including the activity of respiratory chain complexes I, III and V (CI, CIII and CV), and reactive oxygen species (ROS) in two C.albicans strains (FLC-susceptible strain CA-1S and FLC-resistant strain CA-16R) obtained from a single parental source. Additionally, we quantified intracellular ATP levels and mitochondrial membrane potential (ΔΨm), which has critical effect on energy transport. Our analyses revealed a higher ATP level and ΔΨm in CA-16R compared with CA-1S (P<0.05), and a higher ATP level and ΔΨm in Sc5314S (FLC-susceptible strain) compared with Sc5314R (FLC-resistant strain). CI and CV activity increased in CA-16R, activity of CI, CIII and CV increased in Sc5314R. Additionally, ROS decreased in CA-16R and Sc5314R compared with their respective susceptible counterparts. Our data suggest that mitochondrial aerobic respiratory metabolism might be directly associated with the efflux-mediated resistance of C.albicans to azole. C.albicans strains might enhance the activity of efflux pumps and therefore decrease sensitivity to FLC through alteration of mitochondrial aerobic respiratory metabolism, by increased ATP production and decreased ROS generation.

Employment of methylene blue irradiated with laser light source in photodynamic inactivation of biofilm formed by Candida albicans strain resistant to fluconazole.

A promising approach for the eradication of biofilm formed by the yeast Candida albicans seems to be photodynamic inactivation (PDI). This work presents a use of methylene blue (MB, 1mM) irradiated with a red laser (output power 190mW/cm2, wavelength 660nm) for the eradication of a biofilm formed by the fluconazole-resistant (FLC-resistant) strain C. albicans CY 1123 compared to the standard strain C. albicans SC5314. The periods of irradiation corresponded to the fluence of 15, 23 and 57J/cm2. Effectiveness of PDI was evident with following percentage of survived biofilm cells: 24.57, 23.46, and 22.29% for SC5314 and 40.28, 17.91, and 5.89% for CY 1123, respectively, compared to the samples without irradiation. Light and confocal laser scanning microscopy confirmed the effectiveness of PDI. However, the morphological form of C. albicans seems to play an important role as well, since prolonged duration of irradiation did not increase efficiency of PDI on C. albicans SC5314. An experiment with the yeast-to-hyphae transition revealed that the FLC-resistant strain expressed a markedly reduced capacity to form hyphae compared to SC5314. We summarized that PDI was effective on biofilm formed by the FLC-resistant strain, but resistance most likely did not play significant role in PDI. Additionally, we observed differences in susceptibility to PDI between biofilms composed of the mycelia and only of the yeasts, and finally, the employment of a laser in PDI enabled a decreasing period of irradiation while maintaining the high effectiveness of PDI.

VT-1598 Inhibits the in vitro Growth of Mucosal Candida Isolates and Protects Against Oropharyngeal Candidiasis in IL-17 Deficient Mice

Background Patients with chronic mucocutaneous candidiasis (CMC) often develop azole-resistant Candida infections, making treatment difficult due to lack of oral antifungal drug options. VT-1598 is a novel broad-spectrum fungal CYP51 inhibitor designed for exquisite selectivity for the fungal target versus human CYP enzymes to circumvent classic azole side effects like drug–drug interactions. We report the efficacy of VT-1598 in the treatment of oral Candida infection (including by azole-resistant strains).Methods The in vitro MIC values of 28 Candida species isolated from patients with CMC due to AIRE mutations were tested against VT-1598 and fluconazole (FLC), using CLSI broth microdilution M27-S4. Plasma VT-1598 levels were measured using LC–MS/MS with electrospray ionization. Tongue fungal load was determined in IL-17 deficient Act1- /- mice following sublingual C. albicans infection and once-daily oral treatment for 4 days with 25 mg/kg FLC or 3.2, 8, and 20 mg/kg VT-1598 starting 18 hours post-infection.ResultsAmong 28 Candida isolates tested (22 C. albicans, three C. glabrata, and one each of C. utilis, C. dubiliensis, and C. krusei), 10 (36%) were not susceptible to FLC, based on CLSI breakpoints (>4 mg/ml). Remarkably, all 28 isolates were highly susceptible to VT-1598 (MIC50 and MIC90, 0.06 and 0.125 mg/ml, respectively). Oral administration of VT-1598 led to mean drug levels in mouse plasma (2.0, 3.0, and 11 mg/ml at the low, mid, and high doses, respectively) that were higher than the MIC values. In vivo, VT-1598 was significantly more effective, compared with FLC, against FLC-susceptible and -resistant C. albicans, and led to elimination of fungal growth even at the lowest tested dose (3.2 mg/kg). After a 10-day washout period from the last dose, mice treated with VT-1598 did not have mucosal fungal growth, while mice treated with FLC had tongue fungal loads similar to vehicle control.Conclusion VT-1598 shows in vitro activity against mucosally derived Candida, including FLC-resistant strains. In vivo, VT-1598 achieves high plasma concentrations and eliminates viable C. albicans, even at low doses and after an extended washout period. These data indicate that VT-1598 may be a significantly improved treatment option for patients with CMC.Disclosures C. M. Yates, Viamet Pharmaceuticals, Inc.: Employee, Salary. O. J. Cohen, Viamet Pharmaceuticals, Inc.: Employee, Salary. R. J. Schotzinger, Viamet Pharmaceuticals, Inc.: Employee, Salary. E. P. Garvey, Viamet Pharmaceuticals, Inc.: Employee, Salary. M. Lionakis, Viamet Pharmaceuticals: Research support (CRADA), Research support

Synergistic anti-candidal activity and mode of action of Mentha piperita essential oil and its major components

Context: Mentha piperita L. (Lamiaceae) has been used in folk medicine since antiquity. Its essential oil (mint EO) and major bioactive components have antimicrobial properties but their mechanism of action is still not clear.Objective: The present work aims to elucidate M. piperita’s anti-Candida activity and mode of action.Materials and methods: Chemical constituents of mint EO were identified by GC-MS by injecting 0.1 ml sample in a splitless mode. MIC was determined by the broth dilution method. Synergy with fluconazole (FLC) was evaluated by checkerboard assay and FICI. Mid log phase cells harvested from YPD media were used for proton extrusion measurement and the rate of glucose-induced H+ efflux gives PM-ATPase activity. Cell membrane integrity was estimated by total ergosterol content and scanning microscopy at respective MIC and sub-MIC values. In vitro hemolytic activity was performed to rule out possible cytotoxicity of the test compounds.Results: The MIC value of mint EO, carvone, menthol, and menthone was 225, 248, 500, and 4200 µg/ml, respectively. At their respective MICs, these compounds showed 47, 42, 35, and 29% decrease in PM-ATPase activity besides showing synergy with FLC. In case of FLC-resistant strains, the decrease in H+ efflux was by 52, 48, 32, and 30%, a trend similar to the susceptible cases. Exposed Candida cells showed a 100% decrease in the ergosterol content, cell membrane breakage, and alterations in morphology.Discussion and conclusion: Our studies suggest that mint EO and its lead compounds exert antifungal activity by reducing ergosterol levels, inhibiting PM-ATPase leading to intracellular acidification, and ultimately cell death. Our results suggest that mint EO and its constituents are potential antifungal agents and need to be further investigated.

Effects of fluconazole treatment of mice infected with fluconazole-susceptible and -resistant Candida tropicalis on fungal cell surface hydrophobicity, adhesion and biofilm formation

Background: The incidence of Candida tropicalis less susceptible to fluconazole (FLC) has been reported in many parts of the world. Objectives: The aim of this study was to examine the changes of putative virulence attributes of Candida tropicalis accompanying the development of resistance to FLC in vitro and in vivo. Materials and Methods: A FLC-resistant strain (FLC-R) was obtained after sequential exposure of a clinical isolate FLC-sensitive (FLC-S) to increasing concentrations of the antifungal. The course of infection by both strains was analyzed in BALB/c mice. Analyses of gene expression were performed by real-time polymerase chain reaction PCR. The cell surface hydrophobicity, adhesion and biofilm formation were also determined. Results: Development of resistance to FLC could be observed after 15 days of subculture in azole-containing medium. Overexpression of MDR1 and ERG11 genes were observed in FLC-R, and this strain exhibited enhanced virulence in mice, as assessed by the mortality rate. All mice challenged with the FLC-R died and FLC-treatment caused earlier death in mice infected with this strain. All animals challenged with FLC-S survived the experiment, regardless of FLC-treatment. Overall, FLC-R derivatives strains were significantly more hydrophobic than FLC-S strains and showed greater adherence and higher capacity to form biofilm on polystyrene surface. Conclusions: The expression of virulence factors was higher in FLC-R-C. tropicalis and it was enhanced after FLC-exposure. These data alert us to the importance of identifying microorganisms that show resistance to the antifungals to establish an appropriate management of candidiasis therapy.

Synergistic mechanism for tetrandrine on fluconazole against Candida albicans through the mitochondrial aerobic respiratory metabolism pathway.

We found that tetrandrine (TET) can reverse the resistance of Candida albicans to fluconazole (FLC) and that this interaction is associated with the inhibition of drug efflux pumps. Mitochondrial aerobic respiration, which plays a major role in C. albicans metabolism, is the primary source of ATP for cellular processes, including the activation of efflux pumps. However, it was unclear if TET exerts its synergistic action against C. albicans via its impact on the mitochondrial aerobic respiratory metabolism. To investigate this mechanism, we examined the impact of FLC in the presence or absence of TET on two C. albicans strains obtained from a single parental source (FLC-sensitive strain CA-1 and FLC-resistant strain CA-16). We analysed key measures of energy generation and conversion, including the activity of respiration chain complexes I and III (CI and CIII), ATP synthase (CV) activity, and the generation of reactive oxygen species (ROS), and studied intracellular ATP levels and the mitochondrial membrane potential (ΔΨm), which has a critical impact on energy transport. Mitochondrial morphology was observed by confocal microscopy. Our functional analyses revealed that, compared with strains treated only with FLC, TET+FLC increased the ATP levels and decreased ΔΨm in CA-1, but decreased ATP levels and increased ΔΨm in CA-16 (P<0.05). Additionally, CI, CIII and CV activity decreased by 23-48%. The production of ROS increased by two- to threefold and mitochondrial morphology was altered in both strains. Our data suggested that TET impacted mitochondrial aerobic respiratory metabolism by influencing the generation and transport of ATP, reducing the utilization of ATP, and resulting in the inhibition of drug efflux pump activity. This activity contributed to the synergistic action of TET on FLC against C. albicans.

Alcohol dehydrogenase I expression correlates with CDR1, CDR2 and FLU1 expression in Candida albicans from patients with vulvovaginal candidiasis

Background The most critical mechanism governing drug resistance in Candida albicans (C. albicans) involves efflux pumps, the functionality of which largely depends on energy metabolism. Alcohol dehydrogenase I (ADH1) plays an important role in intracellular energy metabolism. The aim of this study was to explore the relationship between ADH1 and drug resistance in C. albicans. Methods Twenty clinical C. albicans samples isolated from individual patients diagnosed with vulvovaginal candidiasis, and two C. albicans strains obtained from a single parental source (the fluconazole (FLC)-sensitive strain CA-1sand the FLC-resistant strain CA-16R) were included in our study. In accordance with the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines, we used the microdilution method to examine the FLC minimum inhibitory concentrations (MICs) and real-time reverse transcription polymerase chain reaction (RT-PCR) to measure the mRNA expression levels of ADH1 and the azole resistance genes CDR1, CDR2, MDR1, FLU1 and ERG11 in all the isolates. Results A highly significant positive correlation between the mRNA levels of ADH1 and the MICs (rs =0.921, P=0.000), as well as positive correlations between the mRNA level of ADH1 and those of CDR1, CDR2 and FLU1 (rs of 0.704, 0.772 and 0.779, respectively, P &lt;0.01), were observed in the 20 clinical C. albicans samples. The relative expression of ADH1 was upregulated 10.63- to 17.61-fold in all of the drug-resistant isolates. No correlations were found between the mRNA levels of ADH1 and those of MDR1 or ERG11(P &gt;0.05). The mRNA levels of the examined drug resistance genes were higher in the CA-16R strain than in CA-1S, and the mRNA levels of ADH1 in CA-16R were 11.64-fold higher than those in CA-1S(P &lt;0.05). Conclusions These results suggest that high levels of ADH1 transcription are implicated in FLC resistance in C. albicans and that the mRNA expression levels of ADH1 are positively correlated with those of CDR1, CDR2 and FLU1.

Molecular mechanisms underlying the tetrandrine-mediated reversal of the fluconazole resistance of Candida albicans

Context: Our previous study demonstrated that tetrandrine (TET) could reverse the resistance of Candida albicans to fluconazole.Objective: The aim of this study was to investigate the molecular mechanism underlying this action.Materials and methods: Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was performed to compare the expression levels of the drug resistance genes CDR1, CDR2, MDR1, FLU1 and ERG11 in fluconazole-sensitive CA-3 and resistant CA-16 cells that were either treated with FLC and/or TET or left as untreated controls. In addition, intracellular ATP levels were measured using an ATP assay kit, and the expression level of the energy metabolism gene ADH1 was measured by real-time RT-PCR.Results: Compared with FLC/TET-free conditions, FLC + TET treatment strains showed statistically different (p < 0.05) expression of CDR1 and CDR2 (increased in the FLC-sensitive strains, while decreased in the FLC-resistant strains), MDR1 (increased in the FLC-resistant strains), FLU1 and ERG11 (increased in the FLC-sensitive strains), ADH1 (decreased in both the FLC-sensitive and the FLC-resistant strains). And also, the FLC + TET treatment decreased the intracellular ATP levels in both the FLC-sensitive and the FLC-resistant strains (p < 0.05).Discussion and conclusion: These results suggest that changes in the expression levels of the drug resistance genes CDR1 and CDR2, the cellular ATP supply and the expression level of the energy metabolism gene ADH1 contribute to the TET-mediated reversal of the fluconazole resistance of C. albicans.

The synergy of honokiol and fluconazole against clinical isolates of azole-resistant Candida albicans

To evaluate the interaction of fluconazole (FLC) and honokiol (HNK) in vitro and vivo against azole-resistant (azole-R) clinical isolates of Candida albicans. A checkerboard microdilution method was used to study the in vitro interaction of FLC and HNK in 24 azole-R clinical isolates of C. albicans. In vivo antifungal activity was performed to further analyse the interaction between FLC and HNK. In the in vitro study, synergism was observed in all 24 FLC-resistant strains tested as determined by fractional inhibitory concentration index (FICI), and in 22 strains by Delta E models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed by using the time-killing test for the selected strain C. albicans YL371, which shows strong susceptible to the combination of HNK and FLC. In the in vivo study, the mice with candidiasis were treated successfully by a combination therapy of HNK with FLC, the results showed a decrease of the colony forming unit in infected and treated animals compared to the controls, at the conditions of the treatment used in this study. Synergistic activity of HNK and FLC against clinical isolates of FLC-resistant C. albicans was observed in vitro and in vivo. This report might provide a potential therapeutic method to overcome the problem of drug-resistance in C. albicans.

In vitro activity of isavuconazole against 140 reference fungal strains and 165 clinically isolated yeasts from Japan

The in vitro susceptibilities of 140 laboratory reference strains of fungi, including type strains, and 165 clinical yeast isolates from Japan towards isavuconazole compared with fluconazole (FLC), itraconazole (ITC), voriconazole and amphotericin B were measured. Broth microdilution methods based on Clinical and Laboratory Standards Institute (CLSI) methods were used for yeasts, and RPMI-MOPS medium semi-solidified with 0.2% low-melting-point agarose based on CLSI guidelines was used for moulds. The range of isavuconazole minimum inhibitory concentrations (MICs) was 0.0004–0.21 mg/L for Candida albicans, 0.0036–0.4 mg/L for Candida glabrata, 0.023–0.058 mg/L for Candida krusei, 0.0026–0.032 mg/L for Cryptococcus neoformans, 0.1–0.39 mg/L for Aspergillus fumigatus and 0.2–0.39 mg/L for Aspergillus terreus. Isavuconazole was as active as ITC against the dimorphic true pathogenic fungi, with a range of MICs from <0.0004 mg/L to 0.0063 mg/L for Blastomyces dermatitidis and Histoplasma capsulatum. It was also active against uncommon dematiaceous fungi such as Exophiala spp. and Phialophora spp. as well as against dermatophytic species. Isavuconazole showed very good in vitro antifungal activity with a broad spectrum, including against FLC-resistant Candida spp., Aspergillus spp. and uncommon opportunistic fungal species. This is the first report of the in vitro susceptibility of Japanese clinical yeast isolates to isavuconazole. No cross-resistance was found to isavuconazole amongst FLC-resistant strains.