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Monitoring of Fluconazole and Caspofungin Activity against In Vivo Candida glabrata Biofilms by Bioluminescence Imaging.

ABSTRACT: Candida glabrata can attach to various medical implants and forms thick biofilms despite its inability to switch from yeast to hyphae. The current in vivo C. glabrata biofilm models only provide limited information about colonization and infection and usually require animal sacrifice. To gain real-time information from individual BALB/c mice, we developed a noninvasive imaging technique to visualize C. glabrata biofilms in catheter fragments that were subcutaneously implanted on the back of mice. Bioluminescent C. glabrata reporter strains (luc _OPT 7/2/4 and luc _OPT 8/1/4), free of auxotrophic markers, expressing a codon-optimized firefly luciferase were generated. A murine subcutaneous model was used to follow real-time in vivo biofilm formation in the presence and absence of fluconazole and caspofungin. The fungal load in biofilms was quantified by CFU counts and by bioluminescence imaging (BLI). C. glabrata biofilms formed within the first 24 h, as documented by the increased number of device-associated cells and elevated bioluminescent signal compared with adhesion at the time of implant. The in vivo model allowed monitoring of the antibiofilm activity of caspofungin against C. glabrata biofilms through bioluminescent imaging from day four after the initiation of treatment. Contrarily, signals emitted from biofilms implanted in fluconazole-treated mice were similar to the light emitted from control-treated mice. This study gives insights into the real-time development of C. glabrata biofilms under in vivo conditions. BLI proved to be a dynamic, noninvasive, and sensitive tool to monitor continuous biofilm formation and activity of antifungal agents against C. glabrata biofilms formed on abiotic surfaces in vivo.

SUBMITTER: Persyn A

PROVIDER: S-EPMC6355587 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Monitoring of Fluconazole and Caspofungin Activity against In Vivo Candida glabrata Biofilms by Bioluminescence Imaging.

Persyn Aranka A Rogiers Ona O Brock Matthias M Vande Velde Greetje G Lamkanfi Mohamed M Jacobsen Ilse D ID Himmelreich Uwe U Lagrou Katrien K Van Dijck Patrick P Kucharíková Soňa S

Antimicrobial agents and chemotherapy 20190129 2

Candida glabrata can attach to various medical implants and forms thick biofilms despite its inability to switch from yeast to hyphae. The current in vivoC. glabrata biofilm models only provide limited information about colonization and infection and usually require animal sacrifice. To gain real-time information from individual BALB/c mice, we developed a noninvasive imaging technique to visualize C. glabrata biofilms in catheter fragments that were subcutaneously im ...[more]

PMID: 30420485

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Project description:Background and purposeAlthough the mechanism of action for echinocandins is known, the physiological mechanisms by which these antifungal agents cause cell death via the classical apoptotic pathways are not well-defined yet. Regarding this, the present study aimed to evaluate the mechanisms of caspofungin-induced Candida glabrata cell death.Materials and methodsFor the purpose of the study, the minimum inhibitory concentration (MIC) of caspofungin against C. glabrata (ATCC 90030) was determined using the broth microdilution reference method (CLSI M27-A2 and M27-S4). The annexin V and propidium iodide staining was performed to determine the way through which caspofungin acts against C. glabrata (i.e., through the induction of apoptosis and/or necrosis). Additionally, the possible effect of caspofungin on inducing the expression of two apoptotic genes, namely MCA1 and NUC, was studied using the real-time polymerase chain reaction assay.ResultsAccording to the obtained MIC value (0.5 µg/mL), C. glabrata, exposed to 0.25, 0.5, and 1 µg/mL of caspofungin, exhibited the features of late apoptosis/necrosis after 18 h of incubation. Furthermore, the use of 0.25, 0.5, and 1 µg/ml caspofungin induced apoptosis (early/late) in 14.67%, 17.04%, and 15.89% of the cells, respectively. The results showed a significant difference between the percentages of early-apoptotic cells at the three concentrations (P<0.05). In addition, the rate of necrosis was significantly greater than that of apoptosis in response to caspofungin. Accordingly, necrosis occurred in 71.26%, 71.26%, and 61.26% of the cells at the caspofungin concentrations of 0.25, 0.5, and 1 µg/mL, respectively (P<0.05). The analysis of the data in the REST software demonstrated a significant increase in the expression of MCA1 and NUC1 genes (P<0.05).ConclusionAs the findings of the present study indicated, caspofungin promoted both necrosis and apoptosis of C. glabrata cells at concentrations higher than or equal to the MIC value.

Project description:Candida glabrata causes persistent infections in patients treated with fluconazole and often acquires resistance following exposure to the drug. Here we found that clinical strains of C. glabrata exhibit cell-to-cell variation in drug response (heteroresistance). We used population analysis profiling (PAP) to assess fluconazole heteroresistance (FLC(HR)) and to ask if it is a binary trait or a continuous phenotype. Thirty (57.6%) of 52 fluconazole-sensitive clinical C. glabrata isolates met accepted dichotomous criteria for FLC(HR) However, quantitative grading of FLC(HR) by using the area under the PAP curve (AUC) revealed a continuous distribution across a wide range of values, suggesting that all isolates exhibit some degree of heteroresistance. The AUC correlated with rhodamine 6G efflux and was associated with upregulation of the CDR1 and PDH1 genes, encoding ATP-binding cassette (ABC) transmembrane transporters, implying that HetR populations exhibit higher levels of drug efflux. Highly FLC(HR) C. glabrata was recovered more frequently than nonheteroresistant C. glabrata from hematogenously infected immunocompetent mice following treatment with high-dose fluconazole (45.8% versus 15%, P = 0.029). Phylogenetic analysis revealed some phenotypic clustering but also variations in FLC(HR) within clonal groups, suggesting both genetic and epigenetic determinants of heteroresistance. Collectively, these results establish heteroresistance to fluconazole as a graded phenotype associated with ABC transporter upregulation and fluconazole efflux. Heteroresistance may explain the propensity of C. glabrata for persistent infection and the emergence of breakthrough resistance to fluconazole. Heteroresistance refers to variability in the response to a drug within a clonal cell population. This phenomenon may have crucial importance for the way we look at antimicrobial resistance, as heteroresistant strains are not detected by standard laboratory susceptibility testing and may be associated with failure of antimicrobial therapy. We describe for the first time heteroresistance to fluconazole in C. glabrata, a finding that may explain the propensity of this pathogen to acquire resistance following exposure to fluconazole and to persist despite treatment. We found that, rather than being a binary all-or-none trait, heteroresistance was a continuously distributed phenotype associated with increased expression of genes that encode energy-dependent drug efflux transporters. Moreover, we show that heteroresistance is associated with failure of fluconazole to clear infection with C. glabrata Together, these findings provide an empirical framework for determining and quantifying heteroresistance in C. glabrata.

Dataset Information

Monitoring of Fluconazole and Caspofungin Activity against In Vivo Candida glabrata Biofilms by Bioluminescence Imaging.

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Monitoring of Fluconazole and Caspofungin Activity against <i>In Vivo</i> Candida glabrata Biofilms by Bioluminescence Imaging.

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