Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles.
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ABSTRACT:
SUBMITTER: Youngsaye W
PROVIDER: S-EPMC3386803 | biostudies-literature | 2012 May
REPOSITORIES: biostudies-literature
Publications
Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles.
Youngsaye Willmen W Dockendorff Chris C Vincent Benjamin B Hartland Cathy L CL Bittker Joshua A JA Dandapani Sivaraman S Palmer Michelle M Whitesell Luke L Lindquist Susan S Schreiber Stuart L SL Munoz Benito B
Bioorganic & medicinal chemistry letters 20120222 9
Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have identified an indole derivative that sensitizes strains demonstrating resistance to fluconazole. This tetracycle (3, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in Saccharomyces cerevisiae models, and may be a useful probe to uncover alternative resistance pathways. ...[more]
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Project description:The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria.
| S-EPMC3287054 | biostudies-literature
Project description:Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen Candida albicans. Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1, CDR2, and MDR1. In the present study, a collection of unmatched clinical isolates of C. albicans was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of CDR1 and CDR2 correlates with resistance, suggesting that the two genes may be coregulated. MDR1 overexpression was observed infrequently in some resistant isolates. Overexpression of FLU1, an efflux pump gene related to MDR1, did not correlate with resistance, nor did overexpression of ERG11. Limited analysis of the ERG11 gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in ERG11 associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.
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Project description:We investigated the azole resistance mechanisms and clinical features of fluconazole-nonsusceptible (FNS) isolates of Candida tropicalis recovered from Korean surveillance cultures in comparison with fluconazole-less-susceptible (FLS) isolates. Thirty-five clinical isolates of C. tropicalis, comprising 9 FNS (fluconazole MIC, 4 to 64 ?g/ml), 12 FLS (MIC, 1 to 2 ?g/ml), and 14 control (MIC, 0.125 to 0.5 ?g/ml) isolates, were assessed. CDR1, MDR1, and ERG11 expression was quantified, and the ERG11 and UPC2 genes were sequenced. Clinical features of 16 patients with FNS or FLS bloodstream isolates were analyzed. Both FNS and FLS isolates had >10-fold higher mean expression levels of CDR1, MDR1, and ERG11 genes than control isolates (P values of <0.02 for all). When FNS and FLS isolates were compared, FNS isolates had 3.4-fold higher mean ERG11 expression levels than FLS isolates (P = 0.004), but there were no differences in those of CDR1 or MDR1 Of all 35 isolates, 4 (2 FNS and 2 FLS) and 28 (8 FNS, 11 FLS, and 9 control) isolates exhibited amino acid substitutions in Erg11p and Upc2p, respectively. Both FNS and FLS bloodstream isolates were associated with azole therapeutic failure (3/4 versus 4/7) or uncleared fungemia (4/6 versus 4/10), but FNS isolates were identified more frequently from patients with previous azole exposure (6/6 versus 3/10; P = 0.011) and immunosuppression (6/6 versus 3/10; P = 0.011). These results reveal that the majority of FNS C. tropicalis isolates show overexpression of CDR1, MDR1, and ERG11 genes, and fungemia develops after azole exposure in patients with immunosuppression.
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Project description:Previous studies have revealed that Candida albicans isolates involved in chronic vulvovaginal candidosis (cVVC) phenotypically express less virulent traits than clinical isolates involved in sporadic infections. In this study, we aimed to further explore this finding by studying the behaviour of those same clinical isolates in in-vitro models of infection. Eighteen clinical Candida albicans isolates were collected from women suffering sporadic (eight isolates) or chronic infections (ten isolates). Adhesion to HeLa cells (human cervical cancer epithelial cell line) and resistance to phagocytosis by RAW 264.7 cells (murine macrophages cell line) were tested in-vitro. In addition, phenotypic expression of virulence factors related with either adhesion or resistance to phagocytosis was tested in-vitro. Results indicated that yeast isolates involved in sporadic infection adhered in a higher proportion of HeLa cells than those of chronic infections, which was related with their ability to produce biofilm (p < 0.05). The ability to evade phagocytosis was related to an elevated production of proteases (p < 0.05) by chronic isolates, while sporadic isolates’ resistance to phagocytosis was related to a higher hydrophobicity of cell walls (p < 0.05). We conclude that the evasion of macrophage-mediated phagocytosis related to the production of proteases might be an important factor involved in the recurrence of vulvovaginal candidosis infection.
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Project description:The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1 and Tac1, which result in constitutive overexpression of multidrug efflux pumps, and Upc2, which result in constitutive overexpression of ergosterol biosynthesis genes. However, the deregulated gene expression that is caused by hyperactive forms of these transcription factors also reduces the fitness of the cells in the absence of the drug. To investigate whether fluconazole-resistant clinical C. albicans isolates have overcome the fitness costs of drug resistance, we assessed the relative fitness of C. albicans isolates containing resistance mutations in these transcription factors in competition with matched drug-susceptible isolates from the same patients. Most of the fluconazole-resistant isolates were outcompeted by the corresponding drug-susceptible isolates when grown in rich medium without fluconazole. On the other hand, some resistant isolates with gain-of-function mutations in MRR1 did not exhibit reduced fitness under these conditions. In a mouse model of disseminated candidiasis, three out of four tested fluconazole-resistant clinical isolates did not exhibit a significant fitness defect. However, all four fluconazole-resistant isolates were outcompeted by the matched susceptible isolates in a mouse model of gastrointestinal colonization, demonstrating that the effects of drug resistance on in vivo fitness depend on the host niche. Collectively, our results indicate that the fitness costs of drug resistance in C. albicans are not easily remediated, especially when proper control of gene expression is required for successful adaptation to life within a mammalian host.
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Project description:Commonly found colonizing the human microbiota, Candida albicans is a microorganism known for its ability to cause infections, mainly in the vulvovaginal region, and is responsible for 85% to 90% of vulvovaginal candidiasis (VVC) cases. The development of drug resistance in C. albicans isolates after long-term therapy with fluconazole is an important complication to solve and new therapeutic strategies are required to target this organism and its pathogenicity. In the present study, phenyllactic acid (PLA) an important broad-spectrum antimicrobial compound was investigated for its antifungal and antivirulence activities against clinical isolates of C. albicans. Previously characterized strains of C. albicans isolates from women with VVC and C. albicans ATCC90028 were used to evaluate the antimicrobial and time dependent killing assay activity of PLA showing a MIC 7.5 mg mL-1 and a complete reduction of viable Candida cells detected by killing kinetics after 4 h of treatment with PLA. Additionally, PLA significantly reduced the biomass and the metabolic activity of C. albicans biofilms and impaired biofilm formation also with changes in ERG11, ALS3, and HWP1 genes expression as detected by qPCR. PLA eradicated pre-formed biofilms as showed also with confocal laser scanning microscopy (CLSM) observations. Furthermore, the compound prolonged the survival rate of Galleria mellonella infected by C. albicans isolates. These results indicate that PLA is a promising candidate as novel and safe antifungal agents for the treatment of vulvovaginal candidiasis.
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Project description:Abstract Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objective To determine the profile of fluconazole-resistant Candida isolates (FRCS) and the proportion of erg11 gene mutation in fluconazole-resistant C. albicans (FRCA) isolates Methods After getting approval from ethics the study was conducted on 150 isolates of FRCS obtained from patients admitted to our tertiary care hospital from September 2019 to December 2021. All the isolates were revived onto Sabouraud dextrose agar (SDA) and identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Fluconazole-resistance was detected by VITEK 2 automated system. Deoxyribonucleic acid (DNA) of FRCA isolates were extracted by QIAamp DNA Mini kit (Qiagen-51 304, Hilden, Germany) as per manufacturer's instruction. A single primer pair targeting the nucleotide sequence from 1067 to 1576 bp region of erg11 gene was used for amplification as per Xu et al. with minor modification and 500 bp polymerase chain reaction (PCR) products were observed by gel electrophoresis. A positive clinical isolate of FRCA was used as a positive control. The PCR products were purified and subjected to Sanger sequencing with an ABI PRISM DNA analyzer (Applied Biosystems) and mutations were detected by using Mega (version 7) software by comparing with the published GenBank sequence AF153844.1 of C. albicans strain ATCC 28516. Categorical variables were expressed in percentages and continuous variables were expressed in mean (average). Results Out of the total of 150 FRCS, the most common fluconazole-resistant agent of candidiasis was C. auris, which was isolated from 60 (40%) cases followed by 24 (16%) isolates of C. glabrata, 19 (12.7%) isolates of C. krusei, 18 (12%) isolates of C. parapsilosis, 17 (11.3%) isolates of C. albicans, 10 (6.7%) isolates of C. tropicalis, and only two (1.3%) isolates of C. guilliermondii. Among these 17 FRCA isolates, 9 isolates were obtained from genital swabs, 5 from blood, and a single isolate each from the perianal swab, ear swab, and sputum. A total of 12 (70.6%) out of 17 FRCA isolates yielded 500 bp region in erg11 gene. Gel electrophoresis image of PCR products of erg11 gene is depicted in Figure 1. Sequencing of these products detected a single missense mutation A1309G (V437I) in one FRCA isolate. Most of the FRCA isolates had an average of 4 silent mutations (observed in 6 isolates) and a single isolate had 6 silent mutations. A total of 8 different silent mutations were observed among FRCA isolates T1296C (83.3%), C1203T (66.7%), A1440G (66.7%), C1302T (50%), T1470C (50%), T1140C (25%), T1110C (16.7%), and T1284C (8.3%). Conclusion C. auris was the most common fluconazole resistant isolate observed, followed by C. glabrata, C. krusei, C. parapsilosis, C. albicans, C. tropicalis, and C. guilliermondii. There were many silent mutations observed in erg11 gene of FRCA isolates and detected only a single missense mutation (V437I). Fluconazole-resistant in FRCA isolates may be due to mechanisms other than the studied one in our region.
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Project description:Candida albicans is both a member of the healthy human microbiome and a major pathogen in immunocompromised individuals. Infections are typically treated with azole inhibitors of ergosterol biosynthesis often leading to drug resistance. Studies in clinical isolates have implicated multiple mechanisms in resistance, but have focused on large-scale aberrations or candidate genes, and do not comprehensively chart the genetic basis of adaptation. Here, we leveraged next-generation sequencing to analyze 43 isolates from 11 oral candidiasis patients. We detected newly selected mutations, including single-nucleotide polymorphisms (SNPs), copy-number variations and loss-of-heterozygosity (LOH) events. LOH events were commonly associated with acquired resistance, and SNPs in 240 genes may be related to host adaptation. Conversely, most aneuploidies were transient and did not correlate with drug resistance. Our analysis also shows that isolates also varied in adherence, filamentation, and virulence. Our work reveals new molecular mechanisms underlying the evolution of drug resistance and host adaptation.
| S-EPMC4383195 | biostudies-literature
Project description:In this study, we found that ambroxol hydrochloride (128 μg/mL) exhibits synergistic antifungal effects in combination with fluconazole (2 μg/mL) against resistant planktonic Candida albicans (C. albicans) cells. This combination also exhibited synergistic effects against resistant C. albicans biofilms in different stages (4, 8, and 12 h) according to the microdilution method. In vitro data were further confirmed by the success of this combination in treating Galleria mellonella infected by resistant C. albicans. With respect to the synergistic mechanism, our result revealed that ambroxol hydrochloride has an effect on the drug transporters of resistant C. albicans, increasing the uptake and decreasing the efflux of rhodamine 6G, a fluorescent alternate of fluconazole. This is the first study to investigate the in vitro and in vivo antifungal effects, as well as the possible synergistic mechanism of ambroxol hydrochloride in combination with fluconazole against resistant C. albicans. The results show the potential role for this drug combination as a therapeutic alternative to treat resistant C. albicans and provide insights into the development of antifungal targets and new antifungal agents.
| S-EPMC5383865 | biostudies-literature
Project description:It was found in our previous study that berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. The aim of the present study was to clarify how BBR and FLC worked synergistically and the underlying mechanism. Antifungal time-kill curves indicated that the synergistic effect of the two drugs was BBR dose dependent rather than FLC dose dependent. In addition, we found that BBR accumulated in C. albicans cells, especially in the nucleus, and resulted in cell cycle arrest and significant change in the transcription of cell cycle-related genes. Besides BBR, other DNA intercalators, including methylene blue, sanguinarine, and acridine orange, were all found to synergize with FLC against FLC-resistant C. albicans. Detection of intracellular BBR accumulation by fluorescence measurement showed that FLC played a role in increasing intracellular BBR concentration, probably due to its effect in disrupting the fungal cell membrane. Similar to the case with FLC, other antifungal agents acting on the cell membrane were able to synergize with BBR. Interestingly, we found that the efflux of intracellular BBR was FLC independent but strongly glucose dependent and associated with the drug efflux pump Cdr2p. These results suggest that BBR plays a major antifungal role in the synergism of FLC and BBR, while FLC plays a role in increasing the intracellular BBR concentration.
| S-EPMC3837902 | biostudies-literature