Effect of probiotic cell free extracts on antifungal drug resistance and virulence expression in Candida albicans

Abstract

Introduction Candidiasis caused by multidrug resistant Candida species continues to be difficult to eradicate and as such it results in life-threatening clinical failures. Moreover, existing antifungal drugs are toxic, inefficient and their misappropriation promotes drug resistance. The use of whole live probiotic bacteria has gained a lot of interest in the treatment of candidiasis; however, it is often associated with high risk of sepsis. Therefore, there is an urgent need of developing new strategies and safer methods for the development of novel antifungal drugs using these beneficial probiotic strains. One such strategy is to use probiotic cell free extracts (CFEs) to target virulence traits which play an important role in pathogenesis of opportunistic microorganisms. The other strategy to combat drug resistance is to inhibit the drug efflux pumps, which are major sources of drug resistance in Candida albicans. With this background, we studied the antifungal effects of probiotic CFEs from three probiotic strains against fluconazole susceptible and resistant C. albicans. The most active CFE was then assessed against different virulence traits and reversal of drug resistance in C. albicans. Method and Materials In this study, one laboratory strain along with ten clinical isolates of C. albicans were used. All these isolates were stored at -80 ºC in the Department of Clinical Microbiology and Infectious Diseases, University of the Witwatersrand, South Africa. The probiotic cultures were obtained from the culture collection of the Department of Biotechnology and Food Technology, Tshwane University of Technology, South Africa. Identification of C. albicans was carried out using germ tube assay and the API 20C AUX kit. To prepare CFEs, probiotic cultures (L. rhamnosus, L. acidophilus V and B. animalis subspecies lactis) were extracted with ethyl acetate. Minimum Inhibitory Concentrations (MIC) and Minimum Fungicidal Concentrations (MFC) of all the CFEs were determined using the microtiter plate dilution assay following CLSI recommended guidelines. The effect of the most active CFE (L. rhamnosus) on yeast to hyphae transition, production of proteinases and phospholipases were studied using standard techniques. The effect of L. rhamnosus CFE on drug efflux pumps was also investigated using a standard technique. Real time PCR was used to determine the effect of L. rhamnosus CFE on expression of virulence and drug efflux pump genes in C. albicans. The results were analyzed using Student’s two sample t-test and one-way ANOVA. Result All the isolates were positive for germ tube assays and API profile confirmed identity as C. albicans. The MIC of the three probiotic CFEs ranged from 84.5 to 338 mg/ml. On preliminary screening of the three CFEs, L. rhamnosus showed lowest MIC of 84.5 mg/ml. MFC was not determined as growth was observed on all agar plates. L. rhamnosus CFE reduced formation of hyphae in C. albicans by 87 % at a concentration of 84.5 mg/ml. The length of the hyphae was subsequently reduced in CFE treated cells compared to untreated cells. L. rhamnosus CFE exhibited an inhibitory effect on production of proteinases and phospholipases at concentrations of 84.5 and 42.25 mg/ml. L. rhamnosus CFE also showed inhibitory effect on drug efflux pumps. Gene expression analysis in the presence of L. rhamnosus CFE showed down regulation of virulence and drug efflux pump genes at a concentration of 84.5 mg/ml with varying fold changes. Conclusion L. rhamnosus CFE portrayed moderate anticandidal activity against different C. albicans isolates. At inhibitory and subinhibitory concentrations, L. rhamnosus CFE significantly inhibited virulence traits and drug efflux pump in both susceptible and resistant C. albicans isolates. The study also demonstrated that the effect on virulence traits and efflux pumps is at the molecular level by observing the downregulation of virulence and drug efflux pump genes after C. albicans were treated with L. rhamnosus CFE. These findings advocate that L. rhamnosus CFE has a potential to be used for therapeutic purposes to treat Candida infections and to overcome drug resistance. This study also laid a foundation for further research in this area to test more probiotic strains as antifungal and anti-virulent agents in different fungal pathogens.