Effect of sequential laboratory passaging on virulence factors traits and antifungal susceptibility variation in Candida albicans clinical isolates

The virulence of Candida albicans is associated with phenotypic plasticity and hydrolytic enzyme production. However, routine laboratory subculturing represents a large and understudied variable that has the potential to diminish research reproducibility and diagnostic accuracy. In this study, we investigated the effect of serial passages on key virulence factors and antifungal susceptibility variation changing clinical isolates. Thirty clinical C. albicans isolates, pre-selected for enzyme production were subjected to ten sequential in vitro passages. Enzymatic activities of phospholipase, proteinase and esterase were quantified at each passage. Phenotypic stability was assessed on each passages by the colony morphology, and fluconazole susceptibility was performed at passages 1, 5 and 10 by the standard broth microdilution method. Serial passaging induced significant variation, isolate-dependent instability. Dynamic phenotypic switching between different colony morphologies were realized. Enzyme stability was factor-specific: while proteinase activities showed significant fluctuation, phospholipase and esterase were more stable. Crucially, fluconazole susceptibility and the minimum inhibitory concentration (MIC) changed in both directions; two resistant isolates reverted to susceptible, and three susceptible isolates acquired resistance and majority of the isolates revealed MIC variation. Routine laboratory passaging can fundamentally and unpredictably alter critical pathogenic traits in C. albicans, including phenotype, enzyme production, and antifungal resistance. Such plasticity challenges the reliability of data generated from high-passage strains. These findings emphasize the need to use low-passage clinical isolates in research and diagnostics as a way of obtaining results that truly reflect the in vivo virulence potential of the pathogen.