Features of antifungal therapy during long-lasting infectious process: a clinical case of fungal keratitis and profile of antifungal sensitivity based on assessing biofilm formation

Abstract

Among infectious diseases, opportunistic mycoses hold a special place. There has been accumulating a lot of evidence regarding the clinical and epidemiological aspects of infection caused by Fusarium spp., which global incidence rate among microbial keratitis ranges from 2 to 40% depending on the geographical location of the country. Colonizing mucous membranes, fungi can exist not only in the form of plankton, but form biofilms after surface attachment, which leads to elevated resistance to multiple antifungal agents. Here we describe a clinical case of fungal keratitis due to Fusarium solani by determining profile of the antifungal sensitivity for isolated fungal strains, by taking into account their potential for biofilm formation. We used an F. solani culture isolated from the patient as well as F. solani test culture obtained from the Russian National Collection of Microorganisms. While determining the sensitivity of fungal planktonic cultures to antifungal agents from the azole group (fluconazole, voriconazole), amphotericin B and terbinafine, it was revealed that antimycotics amphotericin B and voriconazole exerted a marked antifungal activity against clinical isolate, whereas the plankton F. solani test culture was more sensitive to all groups of antifungal agents. Due to a long-lasting progressive course of the infectious process and the high biofilm-forming ability of the clinical strain F. solani, the activity of antifungal agents on biofilm cells was modeled and examined in vitro. It was shown that regarding to the fungal biofilms, value of the minimally inhibitory concentration exceeded those for planktonic cultures by 100-fold. The mechanisms of action for antifungal agents on vital parameters of fungal cell structures were analyzed by using confocal laser scanning microscopy after staining samples with propidium iodide and acridine orange for 15 min to detect changes between intact and damaged cell surface. It was found that within the biofilm fungal cells preserved viability even after exposure to high concentrations of antifungals. In addition, despite the fungicidal drug activity at substantial concentrations acting on the biofilm cell membrane, the cell nuclei remained viable. Owing to the presence ot the mechanism of resistance in mycelial fungi shown in the study, it is necessary to take into account and investigate characteristics of biofilms in terms of drug sensitivity that will allow to optimize a choice of antimicrobial therapy.