Candida Parapsilosis Biofilms Research Articles

Fluconazole is not inferior than caspofungin, micafungin or amphotericin B in the presence of 50% human serum against Candida albicans and Candida parapsilosis biofilms.

Biofilm formation is a relevant risk factor for mortality in candidemia. Data about serum-based susceptibility testing against Candida biofilms are scant; therefore, the activity of fluconazole, amphotericin B, caspofungin and micafungin was determined against Candida albicans and C. parapsilosis biofilms with or without 50% human serum using XTT-based assays. Serum caused a remarkable adverse effect regarding biofilm structure for both species. Additionally, the ratio of nonviable cells increased for C. parapsilosis biofilms, as confirmed by fluorescent microscopy and flow cytometry. Despite impaired biofilm development, traditionally biofilm-active antifungals, surprisingly, showed decreased activity against C. albicans biofilms in serum at concentrations ranging from 0.5 to 1mg/l and from 0.015 to 1mg/l for amphotericin B and echinocandins, respectively (P < .01-.05). However, C. parapsilosis showed higher susceptibility to these antifungals due to reduced biofilm mass and the fungicidal effect of serum at concentrations ranging from 0.015 to 1mg/l and from 0.015 to 512mg/l for amphotericin B and echinocandins, respectively (P < .01-.05). Fluconazole exerted better antifungal activity in serum than traditionally biofilm-active antifungals against both examined biofilms. For fluconazole, significant differences were observed in susceptibility between serum-treated and serum-free biofilms at concentrations ranging from 0.015 to 8mg/l and from 0.03 to 512mg/l for C. albicans and C. parapsilosis isolates, respectively (P < .01-.05). The high antifungal activity of fluconazole in 50% serum both against C. albicans and C. parapsilosis biofilms supports the utility of fluconazole prophylaxis to reduce the risk of catheter-associated fungal infections.

Pharmacodynamic and Immunomodulatory Effects of Micafungin on Host Responses against Biofilms of Candida parapsilosis in Comparison to Those of Candida albicans

Micafungin (MFG) demonstrates potent activity against biofilms of Candida albicans and Candida parapsilosis, the most frequent opportunistic fungal pathogens. Little is known about its immunopharmacologic effect on antibiofilm activity of phagocytic cells following exposure to Candida biofilms. In this study, we investigated the effects of MFG on human neutrophil-mediated damage of C. albicans and C. parapsilosis biofilms by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] and the potential mechanisms underlying the immunomodulatory MFG activities on cultured monocyte-derived THP-1 cells in response to these biofilms by reverse transcription-PCR and sandwich and multiplex enzyme-linked immunosorbent assay. Preexposure of C. albicans to subinhibitory MFG concentrations significantly enhanced neutrophil-mediated biofilm damage, an effect that appears to be species specific since a comparable effect was not observed with drug-pretreated C. parapsilosis biofilms. Human THP-1 cells responded to both Candida biofilms through Toll-like receptor 2 (TLR2) and TLR4 upregulation, modest TLR6 involvement, and enhanced NLRP3 activation, whereas the signal was relayed to the nucleus via NF-κB p65 activation. MFG caused 2- to 3-fold lower TLR2 and TLR4 mRNA levels than those caused by either organism. C. albicans biofilms induced a robust proinflammatory response, whereas C. parapsilosis biofilms either alone or in the presence of MFG caused increased interleukin-1β (IL-1β) production, but small amounts of IL-8, IL-23, and tumor necrosis factor alpha. In conclusion, MFG may condition THP-1 cells toward an inflammatory response through TLR2/TLR4 recruitment. Inflammatory signals observed with C. albicans biofilms are considerably reduced upon exposure of THP-1 cells to C. parapsilosis biofilms, possibly enhancing fungal survival and increasing biofilm pathogenicity.

Relevance of antifungal penetration in biofilm-associated resistance of Candida albicans and non-albicans Candida species

The role of penetration limitation in Candida biofilm-associated antifungal resistance remains unclear. Most of the previous work has been done on Candida albicans, although non-albicans (NAC) species are also implicated in invasive candidiasis and the biofilm matrix has been shown to vary amongst different species. Only a few studies have evaluated clinical isolates. This study aimed to determine the relevance of penetration limitation in the antifungal resistance of biofilms formed by C. albicans and NAC clinical isolates, using an agar disk diffusion assay. The penetration of posaconazole and amphotericin B through the biofilms was significantly reduced. Fluconazole, voriconazole and caspofungin showed a superior penetration capacity in C. albicans, Candida tropicalis and Candida parapsilosis biofilms, but exhibited inter-species and strain/isolate variation. Candida krusei biofilms were the most resilient to antifungal permeation. All of the antifungal drugs failed to kill the biofilm cells, independent of penetration, suggesting that the other factors contribute markedly to the recalcitrance of the biofilms.

In vitro activity of micafungin against biofilms of Candida albicans, Candida glabrata, and Candida parapsilosis at different stages of maturation

Candida spp. is able to form a biofilm, which is considered resistant to the majority of antifungals used in medicine. The aim of this study was to evaluate the in vitro activity of micafungin against Candida spp. biofilms at different stages of their maturation (2, 6, and 24h). We assessed the inhibitory effect of micafungin against 78 clinical isolates of Candida spp., growing as planktonic or sessile cells, by widely recommended broth microdilution method. The in vitro effect on sessile cells viability was evaluated by colorimetric reduction assay. All examined strains were susceptible or intermediate to micafungin when growing as planktonic cells. At the early stages of biofilm maturation, from 11 (39.3%) to 20 (100%), tested strains, depending on the species, exhibited sessile minimal inhibitory concentrations (SMICs) of micafungin at ≤2mg/L. For 24-h-old Candida spp. biofilms, from 3 (10.7%) to 20 (100%) of the tested strains displayed SMICs of micafungin at ≤2mg/L. Our findings confirm that micafungin exhibits high potential anti-Candida-biofilm activity. However, this effect does not comprise all Candida species and strains. All strains were susceptible or intermediate to micafungin when growing as planktonic cells, but for biofilms, micafungin displays species- and strain-specific activity. Paradoxical growth of C. albicans and C. parapsilosis was observed. Antifungal susceptibility testing of Candida spp. biofilms would be the best solution, but to date, no reference method is available. The strongest antibiofilm activity of micafungin is observed at early stages of biofilm formation. Possibly, micafungin could be considered as an effective agent for prevention of biofilm-associated candidiasis, especially catheter-related candidaemia.

Activity of exogenous tyrosol in combination with caspofungin and micafungin against Candida parapsilosis sessile cells.

Fungal quorum-sensing molecules may have an inhibitory effect as adjuvant against Candida biofilms. Therefore, invitro activity of caspofungin and micafungin was evaluated against Candida parapsilosis biofilms in the presence of tyrosol. Interactions were assessed using fractional inhibitory concentration index (FICI) determination, metabolic activity-based time-kill experiments and scanning electron microscopy (SEM). Tyrosol caused 1-16-fold and 2-32-fold decrease in median caspofungin and micafungin MICs respectively. Based on FICI, synergy was observed in isolates 27001 and 17820 with caspofungin and 27001 with micafungin. In time-kill experiments, the metabolic activity reduction was higher for micafungin compared to caspofungin at 24h especially when used in 64 and 256mgl-1 concentrations. In the case of micafungin, the 256mgl-1 +1mmoll-1 combination caused significantly higher decrease in metabolic activity compared to the corresponding concentration alone (256mgl-1 ) at 24h (P<0·05). SEM confirmed the higher killing effect of tested echinocandins with tyrosol. Considerable metabolic activity reduction and cell damage was detected for combinations especially in the case of micafungin. Our findings support the development of new alternative therapeutic strategies against C. parapsilosis biofilms.

Effect of caspofungin and micafungin in combination with farnesol against Candida parapsilosis biofilms

The in vitro activities of caspofungin and micafungin were determined with and without farnesol against Candida parapsilosis biofilms. Drug interactions were examined using the XTT colorimetric assay-based broth microdilution chequerboard method. Drug–drug interactions were assessed utilising the FICI, Bliss independence models and time–kill experiments. Median sessile MICs of five C. parapsilosis clinical isolates ranged between 32–256mg/L, 16–512mg/L and >300μM for caspofungin, micafungin and farnesol, respectively. Median MICs for caspofungin and micafungin in combination with farnesol showed 8–64- and 4–64-fold decreases, respectively. Paradoxical growth noticed with both echinocandins was eliminated by farnesol. Based on FICIs for sessile clinical isolates, synergism was observed for caspofungin (range of median FICIs, 0.155–0.5) and micafungin (range of median FICIs, 0.093–0.5). Concordantly, MacSynergy analysis and global fitting of non-linear regression based on a Bliss independence models also showed synergism for caspofungin and micafungin. In line with FICI findings and the Bliss independence model, synergistic interactions were confirmed by time–kill experiments. The metabolic activity of fungal cells was significantly inhibited by caspofungin+farnesol at all three tested combinations (4mg/L+75μM, 8mg/L+75μM and 16mg/L+75μM) between 3 and 24h compared with the control (P<0.05–0.001). Significant inhibition was observed for micafungin+farnesol between 3 and 12h (P<0.001) but not at 24h. Despite the favourable effect of farnesol in combination with echinocandins, further in vivo studies are needed to confirm its therapeutic advantage in catheter-associated infections caused by C. parapsilosis.

Synergistic effects of tea catechin epigallocatechin gallate and antimycotics against oral Candida species

ObjectiveEpigallocatechin gallate (EGCG), the major antimicrobial tea polyphenol, has been reported to inhibit the growth of Candida albicans planktonic cells and enhance the antifungal activity of antimycotics. We hypothesised that synergism exists between EGCG and conventional antimycotics against biofilms of Candida species. DesignThe minimal inhibitory concentrations (MIC) of EGCG, miconazole, fluconazole and amphotericin B against planktonic cells and the sessile MIC (SMIC) against biofilms of Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Candida kefyr and Candida krusei were determined by a microdilution method. For assessment of biofilm metabolic activity, the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was used. The interactions between EGCG and antimycotics were evaluated by checkerboard microdilution assay and determined by fractional inhibitory concentration index (FIC). ResultsSynergism was observed between EGCG and miconazole, fluconazole or amphotericin B against most test planktonic and biofilm cells of Candida species (FIC≤0.5). All biofilm cells were significantly more resistant to EGCG and antimycotics (20–3200 times higher) compared with their planktonic counterparts. ConclusionsWe conclude that EGCG enhances the antifungal effects of miconazole, fluconazole and amphotericin B. Combined treatment with EGCG may lower the dosages of antimycotics, thus preventing adverse effects and the emergence of drug-resistant oral Candida species.

Micafungin at physiological serum concentrations shows antifungal activity against Candida albicans and Candida parapsilosis biofilms.

We assessed the in vitro activity of micafungin against preformed Candida biofilms by measuring the concentration of drug causing the most fungal damage and inhibition of regrowth. We studied 37 biofilm-producing Candida spp. strains from blood cultures. We showed that micafungin was active against planktonic and sessile forms of Candida albicans strains and moderately active against Candida parapsilosis sessile cells. Concentrations of micafungin above 2 μg/ml were sufficiently high to inactivate regrowth of Candida sessile cells.

Candida Species: New Insights into Biofilm Formation

Biofilms of Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis are associated with high indices of hospital morbidity and mortality. Major factors involved in the formation and growth of Candida biofilms are the chemical composition of the medical implant and the cell wall adhesins responsible for mediating Candida-Candida, Candida-human host cell and Candida-medical device adhesion. Strategies for elucidating the mechanisms that regulate the formation of Candida biofilms combine tools from biology, chemistry, nanoscience, material science and physics. This review proposes the use of new technologies, such as synchrotron radiation, to study the mechanisms of biofilm formation. In the future, this information is expected to facilitate the design of new materials and antifungal compounds that can eradicate nosocomial Candida infections due to biofilm formation on medical implants. This will reduce dissemination of candidiasis and hopefully improve the quality of life of patients.

Could liposomal amphotericin B (L-AMB) lock solutions be useful to inhibit Candida spp. biofilms on silicone biomaterials?

Candida infections associated with catheters remain difficult to manage. Antifungal lock strategies could be a therapeutic option when the device is difficult to remove or in combination with systemic treatment to increase efficacy. This study deals with the antibiofilm potential of liposomal amphotericin B (L-AMB) used as a lock solution to inhibit Candida albicans, Candida glabrata and Candida parapsilosis biofilms in vitro. Biofilms aged 12 h and 5 days were formed on silicone catheters. L-AMB (200 or 1000 mg/L) was added to biofilms and catheters were incubated for 4, 12 or 24 h at 37°C. L-AMB was then removed by washing. The metabolic activity of yeasts was assessed by the XTT method up to 48 h after the end of the locks to evaluate the persistence of the antibiofilm activity. Controls without antifungal were used as references to calculate the inhibition percentages induced by L-AMB lock solutions. L-AMB (200 and 1000 mg/L) inhibited, for up to 48 h, C. albicans and C. glabrata biofilms by >70%, regardless of the lock duration. The activity of L-AMB (200 mg/L) against C. parapsilosis mature biofilms was lower and less sustained, especially for 4 h locks. L-AMB (1000 mg/L) lock solutions strongly inhibited Candida spp. in young and mature biofilms for up to 48 h after the end of the lock. However, overall eradication of the biofilm was not obtained using 1000 mg/L L-AMB as a single lock. These results suggest the usefulness of systemic treatment combined with an L-AMB lock to control Candida spp. biofilms associated with catheters.

Additive antifungal activity of anidulafungin and human neutrophils against Candida parapsilosis biofilms

To investigate the activities of two newer triazoles and two echinocandins combined with human phagocytes against Candida parapsilosis biofilms. An in vitro model of C. parapsilosis biofilms was used. Biofilms were grown on silicone elastomer discs in 96-well plates at 37°C for 72 h. Biofilms or planktonic cells were incubated with voriconazole, posaconazole, caspofungin or anidulafungin, at clinically relevant concentrations, and human phagocytes (neutrophils or monocytes) alone or in combination with each of the antifungal agents for a further 22 h. Fungal damage induced by antifungal agents and/or phagocytes was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide] metabolic assay. Each of the antifungal agents alone and in combination with human phagocytes induced less damage against C. parapsilosis biofilms compared with planktonic cells. No antagonistic interactions between antifungal agents and phagocytes were found. Furthermore, anidulafungin, but not caspofungin, and neutrophils exerted additive activity against C. parapsilosis biofilms. Besides a lack of antagonistic interactions between newer antifungal agents and phagocytes, anidulafungin exerts additive immunopharmacological activity against C. parapsilosis biofilms.

Demonstration of Antibiofilm and Antifungal Efficacy of Chitosan against Candidal Biofilms, Using an In Vivo Central Venous Catheter Model

Candida species are a major cause of catheter infections. Using a central venous catheter Candida albicans biofilm model, we demonstrated that chitosan, a polymer isolated from crustacean exoskeletons, inhibits candidal biofilm formation in vivo. Furthermore, chitosan statistically significantly decreased both the metabolic activity of the biofilms and the cell viability of C. albicans and Candida parapsilosis biofilms in vitro. In addition, confocal and scanning electron microscopic examination demonstrated that chitosan penetrates candidal biofilms and damages fungal cells. Importantly, the concentrations of chitosan that were used to evaluate fungal biofilm susceptibility were not toxic to human endothelial cells. Chitosan should be considered for the prevention or treatment of fungal biofilms on central venous catheters and perhaps other medical devices.

In vitro activity of terpenes against Candida biofilms

The antibiofilm activity of 10 terpenes was tested in vitro against three Candida species by 24-h treatment of biofilms aged 1–5 days. Treatment of 24-h-old Candida albicans biofilms with carvacrol, geraniol or thymol (0.06%) resulted in >80% inhibition. Carvacrol (0.03%) inhibition was ≥75% independent of the age of the C. albicans biofilm. Carvacrol (0.125%) inhibition was >75% against Candida glabrata and Candida parapsilosis biofilms. Geraniol (≥0.125%) and thymol (0.06% or 0.125%) inhibition was >75% against C. parapsilosis biofilms whatever their age. This study demonstrates the antibiofilm activity of terpenes and points out the exceptional efficiency of carvacrol, geraniol and thymol, which could represent candidates in the treatment of candidiasis associated with medical devices.

Anti-metabolic activity of caspofungin against Candida albicans and Candida parapsilosis biofilms

Candidiasis can be associated with the formation of biofilms on bioprosthetic surfaces and the intrinsic resistance of Candida albicans biofilms to the most commonly used antifungal agents has been demonstrated. In this study, we report on the antifungal activity of caspofungin at two different concentrations, on C. albicans and Candida parapsilosis biofilms with different ages of maturation. Fifteen strains of C. albicans (10 strains susceptible to fluconazole in vitro and five strains resistant to this antifungal agent) and six strains of C. parapsilosis (all were susceptible to fluconazole in vitro) were studied. The antifungal activity of caspofungin was assessed by looking for a significant inhibition of the metabolic activity of yeasts within biofilms. Biofilms of Candida were produced in vitro, on silicone catheters. Caspofungin used at MIC did not modify the metabolic activity of C. albicans, whatever the maturation age of the biofilms. The same concentration of caspofungin significantly reduced the metabolism (P<or=0.001) of 25% (biofilms of 48 h) to 50% (biofilms of 2 h) of the C. parapsilosis yeasts. The use of a therapeutic concentration of caspofungin (2 mg/L) significantly decreased (P<or=0.001) the metabolism of all the strains of C. albicans and C. parapsilosis tested, independently of the biofilm maturation age. This potent antifungal activity of caspofungin on C. albicans biofilms was observed independently of the yeast susceptibility to fluconazole. This study demonstrated that caspofungin used at MIC was not sufficient to reduce C. albicans biofilms, but it suggested an activity on C. parapsilosis biofilms depending on their maturation age. This study also indicated that caspofungin used at 2 mg/L could be a good candidate in the prevention of candidiasis associated with silicone medical devices. Our results also suggested that fluconazole resistance of yeasts did not affect caspofungin activity.