Journal of Microbiological Methods

Measuring the growth rate of filamentous fungi is an essential phenotype assay in fungal biology, enabling the comparison of nutrient-related fitness metrics across various isolates, species and genera. Conventional methods are time consuming and labor intensive, which prohibits the adaptation and implementation of high-throughput phenotyping. Here, we suggest a high-throughput methodological pipeline to study fungal growth on solid media combining the use of 24-well plates, an automated image acquisition system, and human assisted deep learning analysis of acquired images. Training a deep learning model through an iterative process - with continuous feedback and corrective annotations - enabled the development of a satisfying model that automatically segments pixels belonging to either fungus or background within a few hours. We evaluated this deep learning model by applying it to two test sets: First, a set of 336 images was used to validate the results by comparison with manual measurements. We demonstrate that the automated segmentation approach provides robust estimation of fungal growth not significantly different to manually segmented data. Second, a larger test set consisting of 2,016 images was used to illustrate the scalability of the model. After training the model for less than two hours, the deep learning model segmented the entire image data set automatically within minutes. The presented method is easily scalable and adjustable to other fungi and growth morphologies, due to the interactive training. Moreover, by combining 24-well plates and automatic image acquisition, measurements can be sped up as growth is detected across a smaller surface area than a standard six or nine cm diameter petri dish. The proposed methodological pipeline thus offers a new tool for estimating fungal growth rates, which can accelerate measurements, reduce bias, and increase throughput.

Bacteria increase in biomass and divide, but determining precisely when cell division completes is technically challenging. To aid time-lapse imaging and cell-cycle tracking, we set out to identify a protein in Bacillus subtilis, which when fused with a fluorophore would cause the membrane to fluoresce in a manner that was constitutive, uniform, and bright. A forward genetic transposon-based approach combined with fluorescence-activated cell sorting was used to identify a fluorescent fusion to the glucose PTS transport transmembrane protein PtsG with all desired properties. Moreover, PtsG-GFP was constitutive and neutral to growth under all conditions tested and also labeled membranes during sporulation. We used PtsG-GFP to track cell growth in microfluidic channels and determine when cytokinesis occurred, defined as when fluorescence reached a local maximum at the division plane. Simultaneous imaging with a compatible fluorescent fusion to the cell division protein FtsZ indicated that FtsZ peak intensity occurred midway through septum constriction and that Z-ring recycling coincided with cytokinesis. We conclude that PtsG-GFP is a powerful tool for membrane imaging and cell cycle tracking. As such, we provide constructs with fluorophores that emit across the visible spectrum and antibiotic resistance cassettes to facilitate deployment in B. subtilis. IMPORTANCEBacterial cells are fully divided when new membrane separates the cytoplasm of each daughter. Reproducibly staining of bacterial membranes with exogenous labels for fluorescence microscopy can be challenging, particularly during chemostatic growth in microfluidic devices. Here, we report that fusion of a fluorescent protein to the glucose transport protein PtsG causes the membrane of Bacillus subtilis to give off bright and even fluorescence under a variety of conditions. We use PtsG-GFP to operationally define when cytokinesis occurs during growth, and we note that a fluorescent PtsG fusion would likely make fluorescent staining of the membrane more facile theoretically in any organism.

Chromosome copy number variation (CNV) is a major contributor to genome plasticity and adaptation in Candida albicans, a leading fungal pathogen of humans. Aneuploidy, defined as deviations from the normal diploid chromosome set, rapidly alters gene dosage, enabling tolerance to host-imposed and antifungal stress. Accurate detection and quantification of chromosomal copy number changes are thus essential to dissect the mechanisms by which C. albicans adapts and evolves. Here, we describe the development, optimization, and validation of a six-color, 16-plex droplet digital PCR assay for simultaneous quantification of all C. albicans chromosome arms in a single reaction. Each target is detected by a unique dual-color or single-color combination of probes, enabling high-order multiplexing through binary fluorescence encoding. Following optimization of probe concentrations, PCR cycling parameters, genomic DNA extraction and pre-treatment with restriction enzymes, the assay provides accurate, reproducible chromosome-level copy number estimates that correlate closely with WGS results across euploid and aneuploid isolates. Compared to whole-genome sequencing, the assay is rapid, cost-effective, and scalable, requiring minimal DNA input and allowing high-throughput analysis of large isolate collections. The 16-plex assay thus provides a platform for dissecting genome instability and adaptive evolution in C. albicans. Article SummaryWe developed and validated a 16-plex droplet digital PCR assay that estimates chromosome dosage across the entire genome of the human fungal pathogen C. albicans in a single reaction. The assay uses six fluorescent colors and unique color combinations to track one marker on each chromosome arm, enabling rapid detection of aneuploidy (extra or missing chromosomes). Results closely matched whole-genome sequencing for isolates with simple aneuploid forms and detected low-frequency trisomic clones in mixed populations. With optimized DNA preparation, this method provides a practical tool for screening genome instability in research and clinical settings.

This dual-center study evaluated the impact of artificial intelligence (AI) on urine culture turnaround times in Canadian diagnostic laboratories employing full microbiology laboratory automation. Data were collected before and after the implementation of PhenoMATRIX (PM), an AI-based software designed to support culture sorting and result interpretation. In both a low-volume tertiary care hospital and a high-volume community laboratory, PM reduced the time to final culture reporting, with decreases of approximately 1.5 hours and 3.9 hours, respectively. Implementation of PM+, which automatically releases defined results to patient charts, further improved turnaround time. These findings indicate that microbiology laboratories with full laboratory automation can achieve further improvements in turnaround time by integrating AI-culture assessment and results release.

Accurate quantification of fungi is important for a myriad of applications but remains challenging. Previously, we demonstrated that an approach called the adenine-HPLC method can quantify bacteria, including those with aggregating properties that are difficult to quantify using conventional methods, by measuring cellular adenine derived from DNA and converting the adenine amount to genome copy number, without being influenced by cell morphology. However, in this study, when this adenine-HPLC method was applied to the quantification of budding yeast as a model fungus, accurate measurement proved impossible. This limitation was attributed to adenine release from other adenine-containing biomolecules, such as RNA and ATP, and we therefore developed a method that suppresses adenine release from these molecules. This method involves reducing the temperature of the acid treatment and prewashing the cells before acid treatment. In addition, we incorporated a process that corrects for the naturally occurring free adenine level as background during total adenine measurement. The improved adenine-HPLC method based on these modifications enables accurate quantification of budding yeast using genomic DNA content in whole cells as the quantification unit.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other Brassica crops. Polymerase chain reaction (PCR), particularly probe-based quantitative PCR (qPCR), is widely used for the detection of P. brassicae in soil samples. To improve consistency in clubroot detection while maintaining efficiency, diagnostic laboratories would benefit from adopting a single, highly efficient qPCR system for routine testing. In this study, we analyzed the primer and probe sequences of all published PCR and qPCR systems for P. brassicae detection. Based on these analyses, three independently developed probe-based qPCR systems were selected and their performance was evaluated using synthesized target DNA (gBlock). One probe-based qPCR system exhibiting superior sensitivity on gBlock was subsequently evaluated on P. brassicae genomic DNA. This system consistently detected DNA equivalent to four resting spores per reaction, corresponding to a soil sample containing 1,000 spores per g soil when the DNA extraction protocol was considered as a component of the qPCR system. The sensitivity of the system was further validated using DNA extracted from soil samples collected from multiple locations across Alberta, where P. brassicae was detected at levels below those associated with visible clubroot symptoms. Based on these results, we recommend this qPCR system for routine clubroot diagnostics in laboratories across Canada.

AbstractEmergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterisation of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of H. capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences, to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall (1,3)-glucan by targeting the transcripts of the -glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus. ImportanceEmergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognised in recent years and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (1) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (2) methods for knocking down gene expression in E. africanus, and (3) the permanent disruption of genes through CRISPR/Cas9 gene editing.

BackgroundThe environment in which a fungus grows can directly influence their development, transmission, and pathogenic potential. This environment encompasses factors like nutrient availability, biotic and abiotic stressors, as well as host-derived chemical cues. In fungal pathogens, where conidia act as the infectious agents, the environment impacts the quantity and quality of these spores, thereby aOecting their ability to infect and kill hosts. In the present study, we investigated the effect of host-derived medium types on various phenotypes, including spore production, growth rate, and virulence in two entomopathogenic fungi, Metarhizium acridum and Metarhizium brunneum. Three medium types derived from insect material were compared to a standard laboratory medium. ResultsConidia produced on the insect-derived media exhibited enhanced sporulation and reduced time to sporulation, while conidial germination and maximum growth rate were comparable across medium types, suggesting that some of the medium-induced phenotypic effects were transient. Notably, conidia derived from two of the insect medium types demonstrated higher virulence, indicating that host-derived cues may prime virulence. ConclusionThese results highlight that the composition of growth substrates can regulate fungal reproductive strategies and virulence, with implications for developing high-throughput phenotyping and for the biotechnological optimization of mass production and efficacy of entomopathogenic fungi in biological control applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/711814v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@189013eorg.highwire.dtl.DTLVardef@1b0cedborg.highwire.dtl.DTLVardef@dccb4eorg.highwire.dtl.DTLVardef@1a77895_HPS_FORMAT_FIGEXP M_FIG C_FIG

The study presented here shows Biofilm quantification in microtiter plates in strains of Candida auris and Candida albicans evaluated by means of Crystal violet, MTT, ATP-Luminescence and NBTZ/BCIP assays. The results showed significant differences in biofilm formation between Candida auris and Candida albicans but also within Candida auris outbreak strains in contrast to Candida auris DSM 21092 reference strain.

PurposeDiphtheria is caused by toxigenic strains of the Corynebacterium diphtheriae complex, mainly Corynebacterium diphtheriae and C. ulcerans. The diagnosis of diphtheria relies on detecting the diphtheria toxin (DT), for which Englers method of Eleks immunoprecipitation test is the gold standard. A recent optimization of Englers method was proposed by Melnikov and colleagues, showing higher sensitivity for C. ulcerans. The goal of our study was to test and adapt this optimized method, and to re-analyze apparent non-toxigenic tox gene bearing (NTTB) isolates from our collection. MethodsWe included 48 C. ulcerans, C. ramonii and C. diphtheriae isolates previously categorized as NTTB but for which no genetic explanation was found for the lack of DT expression. DT production was tested using Melnikovs method with further modifications made by us: i) increasing the antitoxin concentration; ii) using 5{degrees}C as the incubation temperature after 24h; and iii) modifying the layout of control and test strains on agar plates. Results35 of 38 C. ulcerans, 3 C. ramonii and 8 of 10 C. diphtheriae were found to be toxigenic. No genetic explanation was found regarding two non-toxigenic isolates (1 C. diphtheriae and 1 C. ulcerans), whereas for one C. diphtheriae, IS1132 was detected upstream of the tox gene. ConclusionOur modified implementation of Melnikovs Elek test improved our ability to detect diphtheria toxin production. Most isolates previously considered as NTTB but with no genetic explanation, were shown to be toxigenic using the novel method.

Enumeration of the heterotrophic plate counts of culturable microorganisms (HPC) is used for the assessment of water quality before, during and after the drinking water treatment processes and monitoring of integrity of the drinking water distribution systems. Any substantial change in HPC is a warning of potential contamination, treatment failure, or intrusion. This study produced data for international standardization of Reasoners 2 Agar (R2A) method. Interlaboratory studies were applied to find out which inoculation technique, spread plate or pour plate, is more applicable, and what is the acceptable transport and storage time between the sampling and initiation of the analysis. Based on the produced interlaboratory data, spread plate inoculation followed by incubation for 7 days at 22 {degrees}C was selected as the standard method, and it is recommended to analyse samples as soon as possible, but the samples may be kept at (5 {+/-} 3) {degrees}C for up to 24 hours after the sampling prior to examination. Performance characteristics of the method were determined in a single laboratory according to ISO 13843:2017 by using process water from waterworks, bottled water, chlorinated and non-chlorinated tap water, and well water intended for human consumption. For quality assurance of the R2A medium, the use of control strains of Bacillus subtilis subsp. spizizenii, Pseudomonas fluorescens and an additional strain of Sphingomonas paucimobilis was verified during the work. The R2A method is especially suitable for determination of micro-organisms forming colonies after a prolonged incubation time, enabling HPC enumeration from biostable waters with low nutrient levels and low temperature. Such waters usually produce zero counts when nutrient-rich formulations of culture media with short incubation times are employed. Further, the nutrient limited conditions of R2A minimize the colony size and overgrowth in all kinds of water providing improvement to the HPC determinations.

Prokaryotes, particularly those in extreme environments, are capable of diverse metabolic states resulting in altered cell envelope structure and function. However, these changes are difficult to assess as standard fluorescent probes are often incompatible with extreme conditions and/or extremophile cell physiology. Halophilic archaea present the challenge of near-saturated intra-/extra-cellular salts, high membrane potential, and extended survival in altered metabolic states including entrapped within salt crystal fluid inclusions. We evaluated the compatibility of six fluorescent markers of cell envelope stability and activity with two model species, Halobacterium salinarum and Haloferax volcanii. Redox activity markers alamarBlue and pure resazurin solutions, membrane potential probes MitoTracker Orange-CMTMRos and Rhodamine 123, and SYTO 9 and propidium iodide (LIVE/DEAD kit) to assess cell membrane integrity were evaluated for use in bulk (microplate reader) and cell-specific (microscopy) applications. Limitations of each probe were identified, clarifying the utilization of each based on cell physiology, growth phase, medium composition, and probe exposure time including extended timescales needed to simulate the environmental conditions of haloarchaea. Of particular note, propidium iodide behavior was unreliable leading to double-labeling of cells and false interpretation of cells as dead. These data provide important insights into the study of prokaryotes in non-standard conditions.

ImportanceMolecular confirmation of Listeria monocytogenes typically employs a multiplex PCR method that targets both genus-specific prs and species-specific hlyA genes. This study assessed the specificity of this assay within Nigeria, where local microbial diversity may influence performance outcomes. MethodsOut of eight phenotypically presumptive L. monocytogenes food isolates tested, six produced the expected prs and hlyA amplicons, with five classified as serogroup 1/2b. However, all six isolates tested negative for the crucial virulence regulator prfA, necessitating further investigation. ResultsDefinitive 16S rRNA gene sequencing revealed that only two of the six PCR-positive isolates were identified as L. monocytogenes, while the remaining four were identified as Enterococcus faecium. This results in a false-positive rate of 66.7% (4/6) for the assay in this particular context. Phylogenetic analysis corroborated the taxonomic distinction, exhibiting a robust clustering of the four E. faecium isolates with reference strains. In contrast, the two confirmed L. monocytogenes isolates formed a separate sub-clade, indicating regional divergence and further underscoring the assays inability to differentiate between L. monocytogenes and Enterococcus species. ConclusionThese findings highlight a significant lack of specificity, as the prs/hlyA primers exhibited cross-reactivity with non-target E. faecium. The anomalous negative result for prfA served as a critical diagnostic indicator. Consequently, the positive outcomes from this widely utilized confirmatory assay should be regarded as presumptive and necessitate additional verification.

BackgroundSurveys to monitor large-scale deworming programs against soil-transmitted helminthiases (STH) involve examination of stool samples from schoolchildren. These surveys are resource demanding and impact school activities. A potentially cost-saving alternative that does not involve children is to process fecal sludge samples from school pit latrines. To provide a proof-of-principle of latrine-based monitoring of STH programs, we optimized tools to collect fecal sludge and to quantify STH eggs in the samples. MethodsFirst, we designed, developed and field-tested three locally made fecal sludge sampling prototypes. Second, we developed a modified egg-counting method and conducted spiking experiments to explore its analytical performance. Third, we estimated the variation in egg counts in fecal sludge samples collected from six primary schools in Ethiopia at different pit latrine depths used by boys and girls and by repeatedly examining samples. Finally, field data were used to inform an egg count simulation model to quantify this variation in egg counts and to determine the sampling and analysis strategies that resulted in surveys as precise as a stool-based survey. ResultsThe modified fecal sludge sampling prototypes were generally successful, except for a few pit latrines with dried/solid sludge types and insufficient sludge volume. The egg-counting method had moderately high analytical sensitivity that varied across the consistency of the samples. The variation in egg counts was mainly explained by differences between squat holes followed by repeated fecal sludge sample processing. Latrine-based surveys were as precise as stool-based surveys only for Ascaris and when the intensity of infections was low. ConclusionsWe developed a sampling and diagnostic strategy that we will use in a follow-up study. This study will be conducted in Jimma Zone across 25 schools (52 children per school) and will compare the mean fecal sludge egg counts at school level with the STH prevalence in children. Author summaryProgress of large-scale deworming programs are currently monitored through screening individual stool samples of schoolchildren. A latrine-based program monitoring, without active participation of schoolchildren and interruption of routine school activities, is a potentially cost-effective alternative. As a proof-of-concept, we developed a method to quantify worm eggs in fecal sludge samples, conducted spiking experiments to determine its analytical performance and applied the method across schools in Ethiopia to explore variation in egg counts (e.g., squat holes, depth of sample collection, and repeated analyses). Based on our findings, we then determined the sample collection and analysis strategy that results a latrine-based survey as precise as a survey based on screening individual stool samples. The analytical performance was moderately high but varied across the consistency of the samples. The variation in egg counts was mainly driven by variation between squat holes on the same pit latrine, and thus it is better to sample more squat holes (at least 3) than repeatedly processing the same fecal sludge sample (one examination is sufficient). We will now apply this sample collection and analysis across 25 schools (52 children per school) in Ethiopia and compare it with a survey based on screening individual stool samples.

Validated inactivation procedures are required for the safe handling and downstream analysis of highly pathogenic organisms, particularly those categorized as biological select agents and toxins (BSATs). TRIzol-based extraction methods are widely used for nucleic acid and protein isolation, yet their reliability for bacterial inactivation has not been comprehensively evaluated. In this study, we assessed TRIzol-based extraction methods for sample quality and inactivation reliability across a series of mock failure scenarios using five attenuated bacterial isolates: Francisella tularensis holarctica LVS, Bacillus anthracis Sterne, Yersinia enterocolitica, Mycobacterium marinum, and Burkholderia cepacia. Dilution of TRIzol to induce incomplete cell lysis for the initial extraction step, including 0% TRIzol, consistently inactivated all surrogate organisms, suggesting that downstream precipitation and sample washing reagents, including isopropanol and 70% ethanol, were sufficient to inactivate organisms in the absence of TRIzol. Several protocol failure scenarios were then evaluated to simulate human error by omitting extraction, precipitation, and washing steps individually or in combination for the most resistant organism, B. anthracis Sterne strain. Failure-scenario testing demonstrated that reliable inactivation of B. anthracis required strict adherence to the complete protocol due to the spore-forming ability of B. anthracis. Collectively, this work provides a reference with experimental evidence supporting the use of TRIzol-based extraction as a bacterial inactivation strategy for a wide range of bacterial pathogens.

Bacteriophages are promising alternatives for antibiotics. One challenge of developing bacteriophage-based preparation for practical application is to understand expected host range of such products. We evaluated the host range and lytic activity of BAFASAL(R), a four-phage cocktail targeting Salmonella in poultry production. For that purpose, we developed a new composite metric, the Combined Lytic Score (CLS), integrating results from two in vitro assays: serial dilutions spot test on semisolid medium and spectrophotometric growth inhibition in liquid culture. Using this approach phage cocktail was tested against collection of 72 Salmonella strains, including 55 S. Enteritidis isolates representing diverse geographic origins and genomic backgrounds. Spot test patterns were transformed into a continuous scale using the Most Probable Number (MPN) approach to estimate the number of phages required for visible lysis. In parallel, growth inhibition was quantified as the area-under-curve-based inhibition score (ANS). Both metrics were normalized and combined into CLS as a projection onto the regression line describing their correlation (R{superscript 2} {approx} 0.82). More than 65% of S. Enteritidis strains, reached normalized CLS values higher 75%, indicating high susceptibility to BAFASAL(R) in vitro. Phage susceptibility did not correlate with either phenotypic antibiotic resistance or the number of resistance and virulence genes. CLS provides a quantitative method to integrate different experimental methods of determination of bacterial susceptibility to bacteriophages and to rank bacterial strains by phage susceptibility. This approach supports robust host range determination and may facilitate regulatory evaluation and rational design of phage-based interventions in food safety and animal production. IMPORTANCEAssessment of bacteriophage host range is an important step in characterization of bacteriophage strains both in basic and translational research, yet it is still commonly based on qualitative or poorly standardized assays. This lack of harmonization limits reproducibility and complicates comparisons across studies, laboratories, and application contexts. In this work, we propose the Combined Lytic Score (CLS) as a quantitative framework that integrates outcomes from two widely used experimental approaches: serial-dilution spot assays and microtiter-based growth inhibition kinetics. By converting spot-test results into a continuous, concentration-dependent metric and combining them with normalized kinetic inhibition data, CLS enables more consistent interpretation of phage-host interaction outcomes. Application of CLS to a diverse collection of Salmonella enterica strains demonstrates how this approach can support systematic, scalable host range analyses. The CLS framework provides a practical step toward improved standardization of phage susceptibility testing, facilitating clearer data interpretation and comparison in both environmental and applied microbiology research.

This paper evaluated and compared the relative microalgal biomass accumulation of rocking, floating, and stationary bag photobioreactors. Microalga Neochloris oleoabundans was grown in these photobioreactors in batch mode for 24 days under illumination. The 50 L plastic bags (cell suspension volume 25 L) were placed on the surface of a rocking platform, an artificial pond or a stationary platform. In the pond, waves were generated by electrical fans which shake and mix microalgal cells within the plastic bags. The bags were supplied with 5% CO2 in air under elevated pressure inside of the bags. The rocking bag method significantly increased biomass yields to approximately 3-4 g * L-1, as compared to 0.16 g * L-1 in the floating photobioreactor and only 0.03 g * L-1 in the stationary type photobioreactor.

Antimicrobial resistance remains a global existential threat. Given that antimicrobial therapy commonly starts before pathogen identification, rapid and scalable methods capable of determining effective antimicrobial compounds are needed. In this paper, we demonstrate a 2 x 2 array of parallelised microscopes that uses low numerical aperture (NA=0.25) detection optics and LED excitation to determine bacterial viability based on their fluorescence response to an electrical stimulus. Following a 2-hour incubation, the fluorescent viability readout requires less than one minute. We use K-means clustering to classify pixels in a time lapse sequence of widefield fluorescence images and extract changes seen within bacterial clusters. We demonstrate sufficient sensitivity to measure fluorescence changes after electrical stimulation in a bacterial monolayer. To capture these subtle fluorescence changes at high signal-to-background ratios, we place a limit on the minimum optical density of the bacterial sample. This novel approach is scalable to 96-well formats using a suitable consumable electrode array.

Yarrowia lipolytica is a yeast of industrial interest exhibiting remarkable lipolytic and proteolytic capacities, with a high potential for white biotechnology applications. This yeast can be isolated from a wide range of natural, polluted or anthropogenic environments, including various food products. The present study aims to increase the data on Y. lipolytica phenotypic diversity by evaluating the growth parameters and secreted enzymatic activities of 28 wild-type Y lipolytica (and Yarrowia sp.) strains isolated from various environments across 10 countries. These data could facilitate the selection of appropriate strains for specific research purposes, particularly when wild-type strains are prioritized over genetically engineered ones, like for food-related applications. Notably, strain SWJ-1b exhibited an outstanding combination of favourable characteristics, with optimum (or near) performances for both growth and enzymatic parameters.

Drug interaction outcomes-synergism, additivity, or antagonism-represent complex phenotypes. While drug repurposing aims to identify compounds that potentiate conventional antifungals, when given in combination, the modulators of these interactions in fungi remain largely unexplored. We hypothesize that the response to repurposed-conventional antifungal pairs is a complex trait modulated by genetic and environmental factors. To study the impact of genotype on the outcome, we screened six diverse Saccharomyces cerevisiae isolates, including clinical, wild, and fermentation strains, for their responses to combinations of ibuprofen with either clotrimazole or caspofungin. We evaluated the role of the environment using rich and minimal media and assessed the influence of assay type by comparing solid- and liquid-rich media assays. Our results reveal that ibuprofen-clotrimazole interactions are highly dynamic, predominantly antagonistic, with limited synergy observed. These outcomes are significantly modulated by genetic background, media composition, assay type, and, in specific genotypes, even by the drug dosage, reflecting a complex, multi-parametric phenotype. However, the ibuprofen-caspofungin combination is more predictable, exhibiting only synergy or additivity. Interaction outcomes correlate with baseline sensitivity to caspofungin: caspofungin-resistant isolates consistently demonstrate synergy, while sensitive strains exhibit additivity. These findings shift the paradigm of drug discovery by demonstrating that synergism and antagonism are not static properties of drug pairs but are dynamic, context-dependent outcomes. This study highlights the need to use clinically relevant models and patient-specific isolates before clinical application, as drug interactions cannot be generalized from a single dosage, strain, or environmental condition.