Abstract

Background: Control of fungi is problematic, since effective methods for intervening fungal infection or contamination, especially for those resistant to drugs/fungicides, are often very limited. Generally regarded as safe (GRAS) agents, such as natural compounds or their structural analogs could be potential sources of sustainable antifungal agents. As a proof of concept, antifungal efficacy of twenty-one compounds (mostly GRAS) and six conventional preservatives was investigated against Aspergillus brasiliensis, one of the challenge microbes for preservative evaluation. In particular, efficacy of octylgallate (OG) and 4-isopropyl- 3-methylphenol (4I3M) was evaluated for synergism as well as for overcoming fludioxonil (phenylpyrrole fungicide) tolerance of Aspergillus mutants.

Findings: Out of twenty-one compounds examined (Clinical Laboratory Standards Institute Protocol), five compounds showed the highest antifungal activity, viz., OG > benzaldehyde > nonylgallate > 2-hydroxy-4-methylbenzaldehyde > carvacrol (higher to lower activity), where OG and benzaldehyde exhibited fungicidality. For formulation purpose, synergism was further investigated between OG (showing the highest activity) and conventional preservatives. 4I3M was the most responsive preservative to OG, where antifungal efficacy of 4I3M was augmented four-fold with OG + 4I3M combination. Model yeast bioassay showed vacuolar and antioxidant mutants were highly susceptible to 4I3M. 4I3M further overcame fludioxonil tolerance of oxidative signaling mutants of Aspergillus. Of note, while 4I3M and its natural derivatives (thymol, carvacrol) were compatible with two out of three commercial surfactants explored, the remaining surfactant antagonized antifungal activity of 4I3M and its derivatives.

Conclusions: OG (GRAS agent) and 4I3M (conventional preservative), alone or in combination, effectively prevented fungal growth, where vacuolar detoxification and/or antioxidant system were important for fungal survival against 4I3M. While 4I3M overcame fludioxonil tolerance of Aspergillus mutants, types of surfactants co-applied were crucial for achieving optimum activity of antifungal compounds. Accordingly, comprehensive characterization of compound-compound or compound-surfactant specificity would be required further when developing sustainable antifungals.

Keywords: Antifungal, Aspergillus, fludioxonil resistance, 4-Isopropyl-3-methylphenol, octylgallate, surfactant compatibility, sustainability, yeast