Judith Berman

Disk diffusion assays (DDAs) are widely used to determine antimicrobial susceptibility, which is measured as the size of the zone of inhibition (ZOI) after 24 h of growth, and is inversely proportional to the minimal inhibitory concentration (MIC). DDAs can also be used to measure the level of antifungal drug tolerance, defined as the degree to which a subpopulation of cells grows slowly at drug concentrations above the MIC. Tolerance occurs via one or more mechanisms that involve stress responses and that are distinguishable from mechanisms of drug resistance. Measurements of antifungal drug tolerance are generally more sensitive to growth parameters, such as temperature and pH. Here, we investigated parameters used for executing DDAs and asked if they affect the reproducibility of fluconazole susceptibility and tolerance measurements using Candida albicans isolates. Specifically, we tested drug format, media composition, and inoculum parameters. Drug format had little effect on DDA measurements, whereas media composition did affect assay outcomes. In general, inoculum density affected the ability to distinguish between the levels of drug susceptibility and tolerance in different strains. Of note, fluconazole tolerance measurements were much more sensitive than susceptibility measurements to the medium properties. Finally, we recommend conditions that accurately and reproducibly measure fluconazole susceptibility and tolerance levels in C. albicans isolates. IMPORTANCE: Disk diffusion assays are a popular method for testing antimicrobial responses because they are inexpensive and easy to perform. However, experimental factors that influence assay measurements, especially factors that affect antifungal drug tolerance, have not been standardized; antifungal drug tolerance is defined as the ability of a subpopulation of cells to grow slowly at drug concentrations that inhibit growth of the remaining population. Here, we examined how modifying details of the drug format, media properties, and the initial cell number affects the ability to measure resistance and tolerance levels. We provide specific recommendations for performing reliable and robust disk diffusion assays that report both drug susceptibility and tolerance levels.

Antimicrobial resistance and tolerance pose escalating global health threats, necessitating reproducible and accessible tools for antimicrobial susceptibility testing (AST). While disk diffusion assays (DDAs) and Epsilometer tests (Etests) are widely used, there are limited open-source tools to analyze them. We present J-AST , a free, open-source, web-based platform for analyzing both DDAs and Etests. It provides automated and interactive annotation of regions of interest and metadata management, and quantifies microbial resistance and tolerance. J-AST outputs correlate strongly with those of existing tools, and equivalent DDA and Etest results correlate strongly with each other. The automated MIC detection achieved >90% agreement with manual readouts. J-AST is deployable both as desktop software and cloud service, unifies automated analysis with interactive review, and advances both fundamental research and clinical AST workflows.

Quiescence, defined as the reversible exit from mitotic division and proliferative growth, is the predominant state of all microbes. Despite its prevalence, the properties and consequences of quiescence in Candida albicans , an opportunistic fungal pathogen, remain largely unexplored. In this study, we characterized the morphological, molecular, and biophysical properties of quiescent C. albicans cells and assessed the effects of quiescence on antifungal drug efficacy. Quiescent cells that were induced via carbon starvation in rich and minimal media underwent distinct morphological changes upon entry into quiescence; this included an increase in cell buoyant density, altered fluidity of the cytoplasm and nucleus, and remodeling of mitochondria. Most C. albicans cells arrested in an unbudded G1/G0 state, although a significant fraction of cells had budded morphologies and 4N DNA content, indicating that they arrested at other cell cycle phases. Both budded and unbudded quiescent cells efficiently re-entered the cell cycle upon nutrient replenishment, with time-to-quiescence exit varying depending on the total nutritional quality of the medium. Quiescence was associated with large-scale gene expression remodeling, including downregulation of ribosomal biogenesis genes and upregulation of autophagy and stress response pathways. Notably, a greater proportion of quiescent cells than proliferative cells survived exposure to the commonly used antifungal drugs micafungin, caspofungin, and amphotericin B in genetically diverse strains. Thus, quiescence is a distinct cellular state with important implications for antifungal drug efficacy in C. albicans . Author Summary: We show that Candida albicans , a common fungal pathogen, can enter a reversible, non-dividing state when starved of carbon. Starved cells become smaller and denser, reorganize their mitochondria, change how densely packed the inside of the cell and its nucleus are, and switch on stress-protection and internal recycling programs while reducing protein synthesis activity. Most cells have ceased to actively divide, but many retained budded shapes and could restart growth when nutrients returned; the timing of recovery depended on the nutritional environment in which quiescence was initiated. Critically, quiescent cells from laboratory and clinical strains exhibited greater survival than proliferative cells when exposed to widely used fungicidal drugs including micafungin, caspofungin, and amphotericin B. These findings indicate that quiescence is an active, adaptive physiological state that helps Candida albicans survive hostile environmental conditions such as temperature stress and drug exposure. Accounting for the metabolic state of fungal cells in diagnostics and drug development may improve treatment outcomes.

Treatment failure rates for fungal infections cannot be explained simply by increased rates of drug resistance. Antifungal drug tolerance, the ability of a susceptible isolate to grow in the presence of inhibitory drug concentrations, relies upon a broad set of stress response pathways and can contribute to antifungal drug treatment failures. As the physical properties of the cytoplasm are critical for diverse cellular processes, we investigated whether cytoplasmic mesoscale fluidity is linked to antifungal tolerance, taking advantage of a fluorescent microrheological probe. Here, we show either using fluconazole, a common drug that inhibits ergosterol biosynthesis, or mutants in the ergosterol biosynthesis pathway, that cytoplasmic fluidity decreased and could be reversed upon drug removal. Reducing ribosome concentration decreased drug tolerance and restored cytoplasmic fluidity, highlighting a link between tolerance and cytoplasmic crowding and/or viscosity. However, growth in fluconazole did not increase ribosome concentrations; rather growth in fluconazole increased the number of dormant, hibernating ribosomes, cytoplasmic protein concentration, viscosity and condensate formation. Specifically, growth in fluconazole resulted in a substantial increase in processing bodies (P-bodies), whose presence correlated with azole tolerance. Furthermore, we found a substantial increase in cell-cell heterogeneity in all biophysical, biochemical and molecular outputs analyzed. Together, our results reveal that changes in the physical properties of the cytoplasm occur in response to antifungal drug and suggest that these changes, as well as increased cell-cell heterogeneity, are crucial for survival in fungistatic drugs.

Drug resistance is the chief cause of treatment failure for therapies targeting chronic and infectious diseases. Whether the emergence of resistance is accelerated by environmental exposure to low levels of therapeutics remains controversial. Here, we report a non-genetic mechanism of stress adaptation that promotes heritable resistance to the widely used antifungal drug fluconazole. In the human fungal pathogen Candida albicans, transient exposure to subtherapeutic fluconazole doses induces a protective response that we term para-resistance. Like conventional resistance mechanisms, para-resistance is heritable. However, it does not arise from genetic mutations and can revert spontaneously. Systematic analyses of para-resistant isolates suggest that its key regulators include the stress-activated MAP kinase Hog1, the histone deacetylase subunit Snt1, the chromatin regulator Rap1, and the Sko1 transcriptional factor. Notably, molecules that disrupt biomolecular condensation and prion propagation – crucial for the inheritance of protein assemblies – block the induction of para-resistance, whereas inhibiting histone deacetylases facilitates its induction. We find that para-resistance is common in clinical isolates and, remarkably, passage through the mammalian gut triggers its acquisition, compromising fluconazole’s therapeutic efficacy. Our work defines a pervasive, prion-like epigenetic mechanism of stress adaptation and highlights potential strategies to mitigate the rapid emergence of drug resistance.

Antifungal therapies frequently fail, resulting in persistent infections caused by the important opportunistic pathogen, Candida albicans , which is highly tolerant to azole drugs. Tolerance arises from a subpopulation of cells that survive prolonged drug exposure, yet the underlying mechanisms remain poorly defined. Here, transcription factor mutant library screens identified Mnl1 as a key repressor of tolerance. Loss of MNL1 elevated cAMP levels and activated the PKA pathway, linking Mnl1 to growth control and stress responses. Additionally, mnl1 cells exhibited altered sphingolipid composition, implicating Mnl1 in regulating membrane permeability, a key determinant of drug efflux and membrane integrity. Increased tolerance in mnl1 cells was also linked to increased mitochondrial function. Thus, Mnl1 regulates tolerance through coordinated control of sphingolipid metabolism, mitochondrial activity, and PKA signaling, highlighting that tolerant cells adopt a distinct physiological state primed to withstand antifungal stress.

Introduction. Fungal infections are increasingly concerning, particularly in immunocompromised patients. These patients often suffer from comorbidities and receive multiple, non-antifungal medications. Gap Statement. The effects of these co-administered medications on fungal cells – and their potential to influence antifungal drug efficacy – are poorly understood. Aim. This study investigates non-antifungal medications commonly administered in parallel to antifungals and evaluates their impact on fungal susceptibility. Methodology. We systematically reviewed clinical guidelines to identify non-antifungal medications frequently co-prescribed with antifungals. Focusing on Candida albicans, the most prevalent fungal pathogen, we examined whether the presence of these drugs influences antifungal responses of C. albicans. First, we tested the selected compounds together with antifungals in combination assays. Interactions were then characterized using checkerboard assays, and the impact on antifungal resistance and tolerance was evaluated through disc diffusion assays. To further explore these effects in vivo, the influence of selected antagonistic interactions on treatment efficacy was assessed using a Galleria mellonella model of disseminated candidiasis. Results. From 119 medications used to manage 40 conditions linked to a high risk of fungal infections, we identified 34 compounds that altered the effectiveness of the antifungals fluconazole (FLC) and/or anidulafungin. Most of these compounds reduced or antagonized antifungal efficacy, often due to increased resistance or tolerance. Validation in a G. mellonella infection model confirmed that compounds antagonistic to FLC, including loperamide, estradiol and levothyroxine, interfere with antifungal treatment efficacy in this in vivo model. Conclusion. Our findings highlight that medications frequently used by patients at risk for fungal infections can inadvertently increase fungal pathogen drug tolerance or resistance. We suggest that drugs targeting non-fungal conditions yet affecting fungal pathogens might represent an underestimated factor contributing to rising antifungal resistance and tolerance.

Advances in deep learning and AlphaFold2 have enabled the large-scale prediction of protein structures across species, opening avenues for studying protein function and evolution¹. Here we analyse 11,269 predicted and experimentally determined enzyme structures that catalyse 361 metabolic reactions across 225 pathways to investigate metabolic evolution over 400 million years in the Saccharomycotina subphylum². By linking sequence divergence in structurally conserved regions to a variety of metabolic properties of the enzymes, we reveal that metabolism shapes structural evolution across multiple scales, from species-wide metabolic specialization to network organization and the molecular properties of the enzymes. Although positively selected residues are distributed across various structural elements, enzyme evolution is constrained by reaction mechanisms, interactions with metal ions and inhibitors, metabolic flux variability and biosynthetic cost. Our findings uncover hierarchical patterns of structural evolution, in which structural context dictates amino acid substitution rates, with surface residues evolving most rapidly and small-molecule-binding sites evolving under selective constraints without cost optimization. By integrating structural biology with evolutionary genomics, we establish a model in which enzyme evolution is intrinsically governed by catalytic function and shaped by metabolic niche, network architecture, cost and molecular interactions.

Phenotypic heterogeneity in genetically clonal populations facilitates cellular adaptation to adverse environmental conditions while enabling a return to the basal physiological state. It also plays a crucial role in pathogenicity and the acquisition of drug resistance in unicellular organisms and cancer cells, yet the exact contributing factors remain elusive. In this review, we outline the current state of understanding concerning the contribution of phenotypic heterogeneity to fungal pathogenesis and antifungal drug resistance.

Antifungal drug resistance represents a serious global health threat, necessitating new treatment strategies. Here we investigated collateral sensitivity (CS), in which resistance to one drug increases sensitivity to another, and cross-resistance (XR), in which one drug resistance mechanism reduces susceptibility to multiple drugs, since CS and XR dynamics can guide treatment design to impede resistance development, but have not been systematically explored in pathogenic fungi. We used experimental evolution and mathematical modelling of Candida auris population dynamics during cyclic and combined drug exposures and found that especially CS-based drug cycling can effectively prevent the emergence of drug resistance. In addition, we found that a CS-based treatment switch can actively select against or eradicate resistant sub-populations, highlighting the potential to consider CS in therapeutic decision-making upon resistance detection. Furthermore, we show that some CS trends are robust among different strains and resistance mechanisms. Overall, these findings provide a promising direction for improved antifungal treatment approaches.