Influence of different susceptibility testing methods and media on determination of the relevant fluconazole minimum inhibitory concentrations for heavy trailing Candida isolates with low-high phenotype

Abstract

We investigated the incidence of trailing growth with fluconazole in 101 clinical Candida isolates (49 C. albicans and 52 C. tropicalis) and tried to establish the convenient susceptibility testing method and medium for fluconazole minimum inhibitory concentration (MIC) determination. MICs were determined by CLSI M27-A2 broth microdilution (BMD) and Etest methods on RPMI-1640 agar supplemented with 2% glucose (RPG) and on Mueller-Hinton agar supplemented with 2% glucose and 0.5 μg ml(-1) methylene blue (GMB). BMD and Etest MICs were read at 24 and 48 h, and susceptibility categories were compared. All isolates were determined as susceptible with BMD, Etest-RPG and Etest-GMB at 24 h. While all isolates were interpreted as susceptible at 48 h on Etest-RPG and Etest-GMB, one C. albicans isolate was interpreted as susceptible-dose dependent (S-DD) and two C. tropicalis isolates were interpreted as resistant with BMD. On Etest-RPG, trailing growth caused widespread microcolonies within the inhibition zone and resulted in confusion in MIC determination. On Etest-GMB, because of the nearly absence of microcolonies within the zone of inhibition, MICs were evaluated more easily. We conclude that, for the determination of fluconazole MICs of trailing Candida isolates, the Etest method has an advantage over BMD and can be used along with this reference method. Moreover, GMB appears more beneficial than RPG for the fluconazole Etest.

Novel Cell Wall Antifungals Reveal a Special Synergistic Activity in pbr1 Mutants Resistant to the Glucan Synthesis Antifungals Papulacandins and Echinocandins

“…The type of medium i. e. composition, incubation times and temperature can influence the growth and detectability of fungi from the clinical samples and the MIC values of the tested strains (Als et al, 2010; Cruz et al, 2013; Balouiri et al, 2016). The Sabouraud and YEPD media are effective and most useful as media for subculture (Washington et al, 2006).…”

Impact of tolerance to fluconazole on treatment response in Candida albicans bloodstream infection

“…22 Genetic resistance is mediated in C albicans by gainof-function mutations in the transcription factor Upc2, resulting in constitutive upregulation of Erg11, 23 and transcription factors are associated with mortality rates at least as high as those of more resistant species, such as C glabrata. 24,25 18,[27][28][29][30][31][32] Trailing growth was shown to be associated with upregulation of efflux transporter expression, 30,33 but not with azole resistance as determined using sterol quantitation. 28 No association was found between trailing and response to fluconazole treatment in mice.…”

Clearing the FoG: Antifungal tolerance is a subpopulation effect that is distinct from resistance and is associated with persistent candidemia

“…Here, we measured susceptibility and tolerance in a broad range of clinical isolates spanning the major C. albicans clades 62-64 and asked how these parameters influence fungal infection outcomes. We analyzed disk diffusion assays using diskImageR , and quantified the radius (RAD) of the zone of inhibition, a parameter that relates to the MIC 65 , and the fraction of growth (FoG) within the zone of inhibition, a parameter that measures tolerance 19,38,50,66-68 . We found that a range of adjuvant drugs, used in combination with FLC, increased drug cidality by reducing FoG and not MIC and were effective both in vitro and in vivo at inhibiting strains with high (and not low) FoG levels.…”