# Antimicrobial Efficacy of Edible Mushroom Extracts: Assessment of Fungal Resistance
## 3 Results and Discussion  
### Antifungal Activity of T. camphoratus Methanol Extract (TcM)  
The antimicrobial or health effects of \"methanol\" extracts from different mushrooms have been investigated elsewhere [13; 24; 43]. To determine the efficacy of antifungal compounds, it is preferred to perform standard broth microdilution bioassays in microtiter plates according to the protocols outlined by the Clinical Laboratory Standards Institute (CLSI) M38-A [44] or by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); definitive document EDef 7.2 [45] for filamentous fungal or yeast pathogens. In these cases, minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of compounds can be determined including compound interactions [41; 46; 47]. However, the standard microdilution bioassays could not be used in our investigation with mushroom extracts since: (a) the final density of the mushroom extracts prepared in the delivery vehicles was high, thus hampering the serial dilution of the extracts in microtiter liquid medium; (b) the turbidity of the food matrices (fruit juices; see Section 3.2) was also high, so that monitoring the level of visible fungal growth for the determination of MICs was obscured; and (c) one of the test (risk assessment) compounds, SA, is volatile so it needs to be remotely applied from the SA-saturated membrane filter placed on solid agar plates (see Section 3.3). Therefore, we pursued the determination of antimicrobial activity of mushroom extracts on a solid agar medium by monitoring zones of inhibition or fungal radial growth. In Figure 1, a zone of inhibition bioassay showed that only the methanol extract of _T. camphoratus_ (TcM) possessed potent antifungal activity when tested against _A. fumigatus_ AF293 and _C. albicans_ ATCC 10231, the causative agents of human aspergilllossis and candidiasis, respectively. Since invasive aspergilllossis and candidiasis can also be acquired via contaminated foods, beverages, or dietary supplements, there exists a need to find novel antifungal therapies to combat these threats to our food and environmental safety [1]. In _A. fumigatus_, the antifungal activity of TcM increased with the concentration of TcM (Figure 1). The level of antifungal activity was steady in _C. albicans_ up to 10% of TcM. The results indicate that a \"compound-strain relationship\" exists for the differential antifungal activity of TcM, where the filamentous fungal pathogen _A. fumigatus_ was more susceptible to TcM compared to the yeast pathogen _C. albicans_. The results also showed that the water extract of _T. camphoratus_ (TcW) did not exhibit any antifungal activity against _A. fumigatus_ or _C. albicans_. Moreover, neither of the _A. blazei_ and _G. lucidum_ extracts exhibited any antifungal activity against test fungi (data not shown). Differential antifungal susceptibility between yeast and filamentous fungal pathogens has been previously observed. For instance, the benzoic derivative 3,5-dimethoxybenzaldehyde (3,5-DMBA) exhibited potent antifungal activity (average MIC: 1.17 mM) against filamentous fungi such as _Aspergillus_ and _Penicillium_ spp. [41]. In contrast, 3,5-DMBA did not show any antifungal activity against any of the yeast strains tested including _C. albicans_, _C. brucei_, _C. tropicalis_, or _Cryptococcus neoformans_ at up to 6.4 mM [46]. It can be postulated that the tested yeast pathogens possess an intrinsic capability to detoxify 3,5-DMBA. The mechanism of detoxification could be exclusion of toxic compounds via transporters/efflux pumps, vacuolar sequestration, enzymatic degradation/transformation, and other possibilities. A similar detoxification mechanism may also govern the steady response of _C. albicans_ against TcM. Figure 1: Antifungal activity of methanol extracts of _T. camphoratus_ (TcM) (0.625% to 10.0%). (**a**) Incremental increase of TcM activity against _A. fumigatus_ AF293, proportional to the TcM concentrations. (**b**) A steady antifungal activity of TcM against _C.