Datucin

With the increasing prevalence of fungal infections coupled with emerging drug resistance, there is an urgent need for new and effective antifungal agents. Here we report the antifungal activities of 19 diverse halogenated quinoline (HQ) small molecules against Candida albicans and Cryptococcus neoformans. Four HQ analogues inhibited C. albicans growth with a minimum inhibitory concentration (MIC) of 100 nM, whilst 16 analogues effectively inhibited C. neoformans at MICs of 50-780 nM. Remarkably, two HQ analogues eradicated mature C. albicans and C. neoformans biofilms [minimum biofilm eradication concentration (MBEC) = 6.25-62.5 µM]. Several active HQs were found to penetrate into fungal cells, whilst one inactive analogue was unable to, suggesting that HQs elicit their antifungal activities through an intracellular mode of action. HQs are a promising class of small molecules that may be useful in future antifungal treatments.

The radish defensin RsAFP2 was previously characterized as a peptide with potent antifungal activity against several plant pathogenic fungi and human pathogens, including Candida albicans. RsAFP2 induces apoptosis and impairs the yeast-to-hypha transition in C. albicans. As the yeast-to-hypha transition is considered important for progression to mature biofilms, we analyzed the potential antibiofilm activity of recombinant (r)RsAFP2, heterologously expressed in Pichia pastoris, against C. albicans biofilms. We found that rRsAFP2 prevents C. albicans biofilm formation with a BIC-2 (i.e., the minimal rRsAFP2 concentration that inhibits biofilm formation by 50% as compared to control treatment) of 1.65 ± 0.40 mg/mL. Moreover, biofilm-specific synergistic effects were observed between rRsAFP2 doses as low as 2.5 μg/mL to 10 μg/mL and the antimycotics caspofungin and amphotericin B, pointing to the potential of RsAFP2 as a novel antibiofilm compound. In addition, we characterized the solution structure of rRsAFP2 and compared it to that of RsAFP1, another defensin present in radish seeds. These peptides have similar amino acid sequences, except for two amino acids, but rRsAFP2 is more potent than RsAFP1 against planktonic and biofilm cultures. Interestingly, as in case of rRsAFP2, also RsAFP1 acts synergistically with caspofungin against C. albicans biofilms in a comparable low dose range as rRsAFP2. A structural comparison of both defensins via NMR analysis revealed that also rRsAFP2 adopts the typical cysteine-stabilized αβ-motif of plant defensins, however, no structural differences were found between these peptides that might result in their differential antifungal/antibiofilm potency. This further suggests that the conserved structure of RsAFP1 and rRsAFP2 bears the potential to synergize with antimycotics against C. albicans biofilms.

Microdilution method that determines the minimum inhibitory concentrations (MIC) of antifungal agents against Candida spp. is still the only method used in laboratories for both biofilm and planktonic forms. However, it was determined in several studies that there were susceptibility differences between the biofilm and planktonic forms of the same microorganism. The aims of this study were the determination of in vitro susceptibilities of planktonic and biofilm forms of Candida strains against antifungal agents, the comparison of the data obtained from planktonic and biofilm forms and the evaluation of two different methods used for the detection of susceptibilities of biofilm forms. Candida albicans ATCC 10231, Candida parapsilosis ATCC 90028 and Candida krusei ATCC 6258 were used as reference strains together with clinical isolates of one of each C.albicans, C.parapsilosis and Candida tropicalis. Microdilution method was used to determine the susceptibilities of planktonic forms of the strains according to CLSI M27-A3 standards, and MIC values of fluconazole, itraconazole, flucytosine, amphotericin B and nystatin were determined. For the detection of antifungal susceptibilities of Candida spp. biofilm forms, Calgary biofilm method (CBM) and BioTimer assay (BTA) were used, and minimum biofilm eradication concentration (MBEC) and minimum biofilm inhibition concentration (MBIC) values of the same antifungals were determined. The difference between MIC and CBM-MBEC, CBM-MBEC and BTA-MBEC, CBM-MBEC and BTAMBIC values were found statistically significant (p < 0.05). In general CBM-MBEC values were found to be higher than MIC values. However, MBEC values were not always very reliable since the exact number of the microorganisms in biofilm can not be determined. BTA-MBIC values were also generally lower than the MBEC values and higher than the MIC values. Statistically significant difference between two methods was determined only for the MBEC values of flucytosine (p= 0.002) and itraconazole (p = 0.025). For flucytosine (p = 0.001) and itraconazole (p = 0.001), there was also a significant difference between CBM-MBEC and BTA-MBIC values, however, the difference was not significant (p > 0.05) for the other antifungal agents. These findings supported that antifungal susceptibilities of biofilm forming Candida strains should also be investigated. However, MBEC and MBIC of the antifungal agents should not always be expected to be higher than the MIC values since the mechanism of action of the specific antifungal agents and the first inoculum concentration of the microorganisms might differ.