Renátó Kovács

In vivo studies with a Candida tropicalis isolate exhibiting paradoxical growth in vitro in the presence of high concentration of caspofungin

We investigated the activity of caspofungin against a Candida tropicalis clinical isolate showing paradoxical growth in vitro. BALB/c mice immunosuppressed by cyclophosphamide were infected intraperitoneally using 107 CFU/mouse. Caspofungin was administered intraperitoneally once daily for 5 days or as a single dose using the following doses: 0.12, 0.25, 1, 2, 3, 5, and 15 mg/kg. The single dose of caspofungin was effective only at 5 and 15 mg/kg concentrations (100% survival). Five-day caspofungin treatment led to 100% survival at doses of 1 mg/kg or higher. Caspofungin treatment significantly decreased the number of viable yeasts in the peritoneal lavage samples as well as in the infected abscesses at doses 1, 3, 5, and 15 mg/kg caspofungin as compared to the untreated control (P<0.001 in all cases), and even to the group treated with 0.12 mg/kg caspofungin (P<0.05 in all cases). At 2 mg/kg caspofungin dose, sterilization of the internal organs was reproducibly incomplete, suggesting that the role of paradoxical growth in the late clinical failure cannot be excluded.

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 32-256 mg/L, 16-512 mg/L and >300 μM 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 (4 mg/L+75 μM, 8 mg/L+75 μM and 16 mg/L+75 μM) between 3 and 24 h 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 24 h. 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.

Killing rates exerted by caspofungin in 50 % serum and its correlation with in vivo efficacy in a neutropenic murine model against Candida krusei and Candida inconspicua.

Killing rates (K) of 1-32 µg ml(-1) caspofungin were determined in RPMI-1640 and in 50 % serum using time-kill methodology against three Candida krusei (MICs of all three isolates 0.25 µg ml(-1) in RPMI-1640 and 2 µg ml(-1) in serum) and three Candida inconspicua clinical isolates (MIC ranges 0.06-0.12 µg ml(-1) in RPMI-1640 and 0.25-0.5 µg ml(-1) in serum), against C. krusei ATCC 6258 and against one C. krusei isolate that was resistant to echinocandins (MIC 8 µg ml(-1) in RPMI-1640 and 32 µg ml(-1) in serum). In RPMI-1640, the highest mean K values were observed at 4 (-1.05 h(-1)) and 16 (-0.27 h(-1)) μg ml(-1) caspofungin for C. krusei and C. inconspicua clinical isolates, respectively. In 50 % serum, mean K value ranges at 1-32 and 4-32 µg ml(-1) concentrations for C. inconspicua and C. krusei were -1.12 to -1.44 and -0.42 to -0.57 h(-1), respectively. While K values against C. krusei in RPMI-1640 and 50 % serum were comparable, serum significantly increased the killing rate against C. inconspicua (P<0.0003 for all tested concentrations). In a neutropenic murine model, daily caspofungin at 1, 2, 3, 5 and 15 mg kg(-1) significantly decreased the fungal tissue burden of C. inconspicua in the kidneys (P<0.05-0.001). Against C. krusei, doses of 3, 5 and 15 mg kg(-1) caspofungin were effective (P<0.05-0.01). All effective doses were comparably efficacious for both species. Only the highest 15 mg kg(-1) caspofungin dose was effective even against the echinocandin-resistant C. krusei isolate. In 50 % serum, killing was concentration independent at effective concentrations (≥4 and ≥1 µg ml(-1) for C. krusei and C. inconspicua, respectively), suggesting that the efficacy of dose escalation is questionable. These in vitro results were also supported by the murine model.

Effect of 50% human serum on the killing activity of micafungin against eight Candida species using time–kill methodology

Micafungin activity was determined against 24 wild-type clinical isolates and 5 American Type Culture Collection strains belonging to 8 Candida species in RPMI-1640 with and without 50% serum using broth microdilution and time-kill methodology. MIC values increased from 4- to 128-folds in 50% serum for all Candida species. Micafungin was not fungicidal against C. albicans, C. tropicalis, and against 2 of 3 C. metapsilosis at ≥0.25, 1, and 1 μg/mL, respectively, after 48 h with 50% serum, showing good fungistatic activity. Fungicidal activity at ≥2, 4, and 32 μg/mL was noticed against C. glabrata, C. inconspicua, and C. krusei isolates, respectively. Micafungin at 8-32 μg/mL showed fungistatic activity against C. parapsilosis and C. orthopsilosis. Serum decreased the in vitro activity of micafungin. With serum binding of echinocandins taken into account, safely fungistatic or fungicidal concentrations seem to require elevated doses against some Candida species, including C. parapsilosis, C. orthopsilosis, and C. krusei.

The in vitro and in vivo efficacy of fluconazole in combination with farnesol against Candida albicans isolates using a murine vulvovaginitis model

Farnesol is a quorum-sensing molecule that inhibits biofilm formation in Candida albicans. Previous in vitro data suggest that, in combination with certain antifungals, farnesol may have an adjuvant anti-biofilm agent. However, the in vivo efficacy of farnesol is very questionable. Therefore, the in vitro and in vivo activity of fluconazole combined with farnesol was evaluated against C. albicans biofilms using fractional inhibitory concentration index (FICI) determination, time-kill experiments and a murine vulvovaginitis model. The median biofilm MICs of fluconazole-sensitive C. albicans isolates ranged between 4 -> 512 mg/L and 150–300 μM for fluconazole and farnesol, respectively. These values were 512 -> 512 mg/L and > 300 μM for fluconazole-resistant clinical isolates. Farnesol decreased the median MICs of fluconazole by 2-64-fold for biofilms. Based on FICI, synergistic interaction was observed only in the case of the sessile SC5314 reference strain (FICIs: 0.16–0.27). In time-kill studies, only the 512 mg/L fluconazole and 512 mg/L fluconazole + 75 μM farnesol reduced biofilm mass significantly at each time point in the case of all isolates. The combination reduced the metabolic activity of biofilms for all isolates in a concentration- and time-dependent manner. Our findings revealed that farnesol alone was not protective in a murine vulvovaginitis model. Farnesol was not beneficial in combination with fluconazole for fluconazole-susceptible isolates, but partially increased fluconazole activity against one fluconazole-resistant isolate, but not the other one.

Dose escalation studies with caspofungin against Candida glabrata

Echinocandins are recommended as first-line agents against invasive fungal infections caused by Candida glabrata, which still carry a high mortality rate. Dose escalation of echinocandins has been suggested to improve the clinical outcome against C. glabrata. To address this possibility, we performed in vitro and in vivo experiments with caspofungin against four WT C. glabrata clinical isolates, a drug-susceptible ATCC 90030 reference strain and two echinocandin-resistant strains with known FKS mutations. MIC values for the clinical isolates in RPMI 1640 were ≤ 0.03 mg l(-1 ) but increased to 0.125-0.25 mg l(-1 )in RPMI 1640+50% serum. In RPMI 1640+50% serum, the replication of C. glabrata was weaker than in RPMI 1640.Caspofungin in RPMI 1640 at 1 and 4 mg l(-1) showed a fungicidal effect within 7 h against three of the four clinical isolates but was only fungistatic at 16 and 32 mg l(-1) (paradoxically decreased killing activity). In RPMI 1640+50% serum, caspofungin at ≥ 1 mg l(-1) was rapidly fungicidal (within 3.31 h) against three of the four isolates. In a profoundly neutropenic murine model, all caspofungin doses (1, 2, 3, 5 and 20 mg kg(-1) daily) decreased the fungal tissue burdens significantly (P < 0.05-0.001) without statistical differences between doses, but the mean fungal tissue burdens never fell below 105 cells (g tissue)(-1). The echinocandin-resistant strains were highly virulent in animal models and all doses were ineffective. These results confirm the clinical experience that caspofungin dose escalation does not improve efficacy.

Killing Rates of Caspofungin in 50 Percent Serum Correlate with Caspofungin Efficacy Against Candida albicans in a Neutropenic Murine Model.

Previous studies suggested that caspofungin dose escalation against Candida species is more beneficial than currently used lower daily doses. Thus, we determined in vitro and in vivo activity of caspofungin against six wild-type C. albicans clinical isolates, the ATCC 10231 strain and an echinocandin resistant strain. MIC ranges of clinical isolates in RPMI-1640 with and without 50% serum were 0.125-0.25 and 0.015-0.06 mg/L, respectively. Two and three isolates showed paradoxical growth in MIC and time-kill tests, respectively, in RPMI-1640 but not in 50% serum. Caspofungin killing rate (k) in RPMI-1640 at 1 mg/L was higher than at 16 and 32 mg/L for all isolates (p<0.001). Killing rates for five of six isolates were concentration independent between 1-32 mg/L in 50% serum (p>0.05 for all comparisons), but for one isolate k value at 32 mg/L was significantly lower than at 1-16 mg/L. Although k values at 1-32 mg/L showed a great variability in 50% serum (the lowest and highest k value ranges were 0.085-0.109 and 0.882-0.985 1/h, respectively), daily 3, 5 and 15 mg/kg caspofungin was effective in a neutropenic murine model against all isolates, without significant differences between the effective doses. This study confirms that paradoxical growth does not affect the in vivo efficacy of caspofungin. We demonstrated that dose escalation did not increase the efficacy of caspofungin against C. albicans either in vitro or in vivo. These results are in concordance with the clinical experience that efficacy of echinocandins does not increase at larger doses.

Betamethasone augments the antifungal effect of menadione--towards a novel anti-Candida albicans combination therapy.

The fluorinated glucocorticoid betamethasone stimulated both the extracellular phospholipase production and hypha formation of the opportunistic human pathogen Candida albicans and also decreased the efficiency of the polyene antimycotics amphotericin B and nystatin against C. albicans in a dose‐dependent manner. Importantly, betamethasone increased synergistically the anti‐Candida activity of the oxidative stress generating agent menadione, which may be exploited in future combination therapies to prevent or cure C. albicans infections, in the field of dermatology.

Commercial strain-derived clinical Saccharomyces cerevisiae can evolve new phenotypes without higher pathogenicity

SCOPE Saccharomyces cerevisiae is one of the most important microbes in food industry, but there is growing evidence on its potential pathogenicity as well. Its status as a member of human mycobiome is still not fully understood. METHODS AND RESULTS In this study, we characterize clinical S. cerevisiae isolates from Hungarian hospitals along with commercial baking and probiotic strains, and determine their phenotypic parameters, virulence factors, interactions with human macrophages, and pathogenicity. Four of the clinical isolates could be traced back to commercial strains based on genetic fingerprinting. Our observations indicate that the commercial-derived clinical isolates have evolved new phenotypes and show similar, or in two cases, significantly decreased pathogenicity. Furthermore, immunological experiments revealed that the variability in human primary macrophage activation after coincubation with yeasts is largely donor and not isolate dependent. CONCLUSION Isolates in this study offer an interesting insight into the potential microevolution of probiotic and food strains in human hosts. These commensal yeasts display various changes in their phenotypes, indicating that the colonization of the host does not necessarily impose a selective pressure toward higher virulence/pathogenicity.

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, in vitro activity of caspofungin and micafungin was evaluated against Candida parapsilosis biofilms in the presence of tyrosol.