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The yeast Cryptococcus neoformans is an opportunistic human pathogen capable of surviving within various environmental conditions. The repertoire of antifungal agents effective in treating cryptococcal infection is limited, necessitating the identification of alternative treatment strategies. Nonsense-mediated decay (NMD) is an RNA decay mechanism that serves as a post-transcriptional regulator of gene expression. While the absence of NMD in C. neoformans sensitizes cells to the antifungal fluconazole, the mechanism underlying this sensitivity and role of NMD in C. neoformans biology remained unexplored. Using phenotypic analysis and RNA-sequencing analysis, we identify basal dysregulation of thermal- and nutrient-adaptive genes and demonstrate temperature- and/or nutrient-dependent phenotypic suppression of upf1{Delta} phenotypes, including fluconazole sensitivity and resistance to rapamycin. We determine rapamycin co-treatment also suppresses the upf1{Delta} fluconazole sensitivity, implicating dysregulation of Tor signaling in phenotypic outcomes when NMD is absent. We then investigate Tor-sensitive signaling in the upf1{Delta} mutant, finding inhibition of cell wall integrity (CWI) signaling and hyperactivation of the kinase Gcn2, both of which returned to wildtype-like levels by either rapamycin treatment, nutrient limitation, or constitutive thermal stress. These results indicated NMD is required for appropriate regulation of Tor signaling in unstressed conditions and suggested upf1{Delta} phenotypes are driven in part by Tor hyperactivation. A phenotypic screen of mutants lacking Tor regulators revealed that deletion of the Tor-suppressing IML1 gene recapitulates upf1{Delta} phenotypes and signaling defects, consistent with Tor hyperactivation. Taken together, our results suggest NMD participates in the regulation of Tor signaling in C. neoformans. Future work will investigate how specific targets of NMD impact Tor signaling and promote fluconazole sensitivity in C. neoformans. ImportancePulmonary and central nervous system infections cause by Cryptococcus neoformans are responsible for about 112,000 deaths annually. Ten-week mortality remains high at 25% with use of frontline antifungals which imposes major health risks due to inherent toxicity. Thus, a need arises to identify novel avenues of treatment, including ways of boosting the efficacy of widely available antifungals such as fluconazole against C. neoformans. The design of NMD inhibitors is an active pharmaceutical pipeline for use in treating human genetic diseases. Even though NMD is conserved across eukaryotes, underlying components and regulatory roles of NMD differ between humans and fungi. Therefore, understanding NMD within C. neoformans will inform the design and repurposing of NMD inhibitors to enhance the antifungal activity of fluconazole as a treatment for Cryptococcosis.

A fatal case of toxoplasmic encephalitis, and others like it, has caused microbiologists and clinicians to question whether different strains of T. gondii have more pathogenic potential than others. This raises significant concern, as T. gondii is a widely spread parasitic organism that is presumed to lie dormant in a third of the worlds general population. In this study, we expand on a previously published proteomicanalysis reactivated toxoplasmic encephalitis and have been able to identify T. gondii-specific peptides in the cerebrospinal fluid (CSF) of this patient and two additional cases of toxoplasmic reactivation. Multilocus PCR-restriction fragment-length polymorphism (PCR-RFLP) was used to genetically identify the T. gondii strain that resulted in this fatal case, which belonged to ToxoDB PCR-RFLP genotype #7. Using other T. gondii strains of the same genotype, we performed bioassays to compare the pathogenicity of this genotype with that of a clinically relevant strain, ME49. Both of the tested genotype #7 strains appear to have a greater pathogenic potential, although through likely different mechanisms. Of the most abundant T. gondii-specific CSF peptides across multiple patients, we identified a polymorphic region of the dense granule protein GRA5 that appears to have strain specificity. This approach could represent a "proteotype" that allows for T. gondii strain risk stratification within clinical samples. Ultimately spinal fluid could be a valuable tool in distinguishing between T. gondii exposed individuals with no cyst forms in the brain versus those exposed individuals that harbor "clinically silent" but viable brain cysts. SIGNIFICANCEA third of the worlds population is exposed to the parasite Toxoplasma gondii, residing in a dormant, encysted stage within neurons. T. gondii is a diverse microorganism with some strains having greater reactivation potential. There is no means of identifying which individuals are at risk of reactivation. Proteomic analysis of cerebrospinal fluid from patients with reactivated toxoplasmosis demonstrated a consistent pattern of T. gondii peptides, including GRA5, a secreted virulence factor. Postmortem analysis identified a ToxoDB PCR-RFLP genotype #7 strain associated with a fatality. Cerebrospinal fluid could provide clues to persistent brain infection and may identify strains able to reactivate.

Giardia duodenalis is an intestinal protozoan transmitted through contaminated food and water and is a major cause of diarrheal disease worldwide and has been linked to post-infectious sequelae and growth impairment in children. Quantifying parasite burden in vivo is essential for studying infection dynamics and host responses, yet commonly used methods (stool PCR, antigen detection, and terminal trophozoite counts from segments of the intestine) are limited by intermittent stool shedding, uneven parasite distribution, sampling error, and lack of longitudinal data. We developed a bioluminescent reporter system for the assemblage B strain GS to enable noninvasive tracking of infection in mice without antibiotic pretreatment. Using in vivo bioluminescent imaging (BLI), we observed peak signal in immunocompetent C57BL/6J and BALB/c mice at days 6-8 post-infection, followed by return to baseline by ~day 10, consistent with the self-limiting nature of giardiasis. In immunodeficient mice, radiance increased and persisted over time, demonstrating stable luciferase expression during chronic infection in vivo without continuous antibiotic selection. Whole-animal radiance strongly correlated with direct trophozoite counts from excised intestinal segments (r = 0.915, p < 0.001), validating BLI as a useful alternative for determining parasite burden. Together, these findings establish BLI as a robust platform for longitudinal studies of Giardia infection dynamics and for future applications that may examine host immunity, the microbiota, nutrient-dependent effects, and drug testing.

The mitochondrion is a versatile organelle involved in diverse processes, such as cell death, metal homeostasis, plasma membrane and cell wall integrity, stress response, oxygen concentration, temperature, and metabolic adaptation, in addition to its role in generating energy. Consequently, mitochondrial fitness is essential for the pathogenicity of various organisms, including fungi. Cryptococcus neoformans is a fungal pathogen responsible for over 180,000 HIV-related deaths each year. In this study, we analyzed C. neoformans metabolic plasticity when grown with non-fermentable carbon sources and their impact on virulence and mitochondrial homeostasis. Growth on non-fermentable carbon sources increased thermotolerance, glucuronoxylomannan (GMX) content in the capsule, melanization rate, urease activity, biofilm formation, and virulence. Moreover, cells grown on non-fermentable carbon sources manifested increased mitochondrial number and activity. Conversely, mutants of the master regulator of mitochondrial biogenesis, the Hap complex, the catalytic subunit 1 of protein kinase A, or media supplementation with antioxidants, decreased the use of alternative carbon sources, capsule formation, melanin synthesis, urease activity, mitochondrial number, and resistance to both fluconazole and macrophage killing. Our results implicate mitochondrial homeostasis in virulence regulation via the PKA pathway, suggesting that targeting fungal mitochondrial homeostasis could be a therapeutic approach for cryptococcosis.

BackgroundNon-invasive methods to colonize intact gut microbiota populations with specific bacterial species are useful for experimental studies that advance our understanding of this commensal microbial population in health and disease. Within the gut microbiota, the anaerobic muciniphile Akkermansia muciniphila has many established health benefits. We report the development of a new voluntary feeding protocol for non-invasive administration of bacteria into the intestine and use it to characterize the early life colonization of the intestinal tract by A. muciniphila. ResultsMice were voluntarily fed a human strain of A. muciniphila (MucT/BAA-835) in the week after weaning, whereupon they consistently and rapidly ingested the bacterium. At this developmental period, conventionally housed mice were rapidly colonized by human A. muciniphila that persisted until at least 8 weeks of age. In mice that contained a dysbiotic gut microbiota that lack endogenous A. muciniphila, voluntary feeding with human A. muciniphila similarly led to rapid and stable colonization. Colonization was similar in female and male mice. Also, in conventionally housed mice there was incomplete colonization of the intestinal tract with endogenous A. muciniphila between 3 - 4 weeks of age, which enabled its competitive exclusion by human A. muciniphila that was orally delivered. ConclusionsThese findings establish a new and non-invasive approach for colonizing the intestinal tract with commensal microbes that provides information on the early life colonization of the gut microbiota with A. muciniphila.

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 SummaryWe 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.

The invasive fungal pathogen Pseudogymnoascus destructans is responsible for the collapse of several North American bat species through an infectious fungal skin disease known as White-Nose Syndrome (WNS). Recent transcriptomic studies have suggested that trace copper ion acquisition is essential for P. destructans propagation on its animal hosts. However, little is known about the mechanistic details of P. destructans adaptation occurring at the protein level. In this study, we report the global proteomic adaptation of P. destructans under chronic Cu-stress growth conditions employing chemically defined media. We identify 4340 P. destructans proteins, or approximately 47.8% of the predicted proteome, spanning a dynamic intensity range of six orders of magnitude. Chronic Cu-withholding stress leads to substantial alterations in the proteome, with 1398 differentially abundant proteins (DAPs) exhibiting statistically significant (p < 0.05) changes in protein levels compared to control growth conditions. We find that Cu-withholding stress induces increased levels of proteins associated with high-affinity Cu-acquisition, changes in intracellular superoxide dismutase (SOD) levels, and alterations in mitochondrial proteins related to aerobic respiration. In contrast, chronic Cu-overload stress leads to 390 DAPs (p < 0.05), which are more widely distributed across the proteome, with several DAPs associated with genomic stability and basic metabolism. Additionally, in this report, we present assessment of antisera products against intracellular and cell-surface protein targets of P. destructans that are effective for indicating Cu-withholding stress by western blotting.

The human gut mycobiome, though less diverse than the bacterial microbiome, plays a significant role in health and disease. This study investigates the culturable fungal communities in fecal samples from hospitalized patients with diarrhea in Mexico City. We isolated and characterized 26 fungal strains using culture-dependent methods, including 20 yeasts and six filamentous fungi. The most prevalent organisms were Candida albicans, Rhodotorula mucilaginosa, Penicillium spp., and Paecilomyces spp. Fungal isolates were tested for their ability to withstand gut-like conditions, including temperature, pH, oxidative stress, and bile salts. Notably, Paecilomyces variotii demonstrated thermotolerance, surviving at 42{degrees}C, and exhibited competitive growth against other fungi. Co-occurrence analysis revealed associations between fungal isolates and bacterial pathogens such as Salmonella and Clostridioides difficile, suggesting potential interkingdom interactions. Cytotoxicity assays on Caco-2 cells showed that cell-free supernatants from Candida inospicua and filamentous fungi reduced cell viability by up to 40%. Finally, dereplication and untargeted metabolomic analyses of P. variotii, Penicillium crustosum, and Penicillium chrysogenum revealed the presence of several bioactive metabolites, including mycotoxins and antimicrobial compounds, highlighting their potential roles in gut ecology and disease. Overall, this study underscores the importance of the gut mycobiome in dysbiosis and its interactions with bacterial pathogens. The findings suggest that fungi, particularly thermotolerant species such as P. variotii, may contribute to gut dysbiosis and disease progression, particularly in immunocompromised patients. Further research is needed to elucidate the functional roles of these fungi and their metabolites in gut health and disease.

The human gut microbiome is a complex community that plays an important role in health, where perturbations can result in dysbiosis and disease. Bacteriophages (phages) can provide treatment for bacterial gastrointestinal disease, and commercial preparations such as the Intesti bacteriophage cocktail can be taken orally to target bacterial pathogens. However, interactions between these phages and the native gut microbiota are understudied. To investigate the impact of phage treatment, we used simulated gut models seeded with healthy donor microbiota from three individuals, sequenced the DNA, and analysed the bacterial and viral portion from samples obtained over time. Each donor had a unique bacterial composition which diverged with time. When comparing phage treated to control samples, we observed that Escherichia coli abundance accounted for the largest portion of bacterial community variance and was more associated with the controls. The lower abundance in phage treated samples may have resulted from the lytic action of phages from the cocktail. Additionally, our analyses of the viral portion revealed a phage bloom exclusive to phage treated samples. A highly abundant phage in this bloom was matched with the Intesti bacteriophage cocktail, showed similarity to Enterobacteria phage phi92, and provided evidence of productive infection within the model. While we did observe fluctuations in relative abundance of additional viral sequences in the presence of the phage cocktail, these changes were often transient. Furthermore, we detected only slight differences to typical members of the virome, and low numbers of active prophages. Our experiments suggest that the phage cocktail had minimal interruption to the native gut microbiota within the model. Impact statementBacteriophages are increasingly investigated and tested for their efficacy in treating infections and are a key component in fight against antimicrobial resistant bacterial infections. Because of their specificity, it has become almost a dogma to state that they do not alter the gut microbiome. We have now tested this in an in vitro study using a commercially available cocktail and real human faecal microbiota. We show minimal effects on the composition of the healthy microbiota with an individual-specific effect on Escherichia coli caused by productive infection of one phage in the cocktail.

Edhazardia aedis is an obligate microsporidian parasite of the arthropod vector Aedes aegypti, which is responsible for the spread of several vertebrate pathogens of global health importance. E. aedis can be highly virulent to Ae. aegypti and infection has severely detrimental effects on multiple life history traits that are relevant to the vectoral capacity of Ae. aegypti, including longevity, body size, propensity to host-seek and blood-feed, and reproductive capacity. Because E. aedis is also highly specific to Ae. aegypti and is incapable of completing its full life cycle in any other mosquito species, E. aedis merits investigation as a novel tool for biological vector control. In the present study, we queried the effect of E. aedis infection on the bacterial microbiota of adult female Ae. aegypti using high-throughput amplicon sequencing of the 16S rRNA gene. Analysis of sequencing data revealed that the bacterial microbiota community is strikingly robust to E. aedis infection, as we observed no significant effect on alpha or beta diversity, differential abundance of any taxa, predicted metabolic function profile, or overall bacterial load. The data show that E. aedis, despite dramatically impacting the health and fitness of the adult female mosquito, does not affect the microbiota. These results provide unique insight into tripartite relationships (or lack thereof) between hosts, pathogens, and the microbiota.

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.

BackgroundGut microbiome disruption is often characterized by loss of obligate anaerobic bacteria, which may lead to altered production of microbial metabolites that can be detected peripherally. The application of widely used sequencing-based microbiome analyses to clinical settings is limited by cost, turnaround time, and challenges with patients with very low stool output. Since some products of strictly bacterial metabolism detectable in blood, peripheral metabolites may provide a potentially rapid and scalable indicator of gut microbiome composition and function. We performed a systematic review and meta-analysis of studies reporting circulating microbial metabolites and gut microbiome composition to evaluate whether peripheral microbial metabolites could identify gut microbiome perturbation. ResultsCandidate metabolites were identified systematically across an independent set of studies reporting metabolite-microbiome associations, enabling assessment of reproducibility across disease states and cohorts. We performed a meta-analysis of 19 human cohorts comprising 3242 participants with paired blood metabolite and stool microbiome data. Anaerobe depletion (obligate anaerobe relative abundance <0.70 by sequencing) was associated with decreased products of anaerobic microbial metabolism. Combinations of metabolites distinguished individuals with anaerobe-depleted microbiomes from those without. Circulating metabolite levels distinguished between cases and controls with similar performance as gut microbiome composition across a range of health/disease states, and changed markedly within patients experiencing gut anaerobe depletion after antibiotic exposure. ConclusionsCirculating microbial metabolites are potentially informative indicators of gut microbiome disruption and may serve as a rapid and method for patient stratification in clinical trials or acute care settings. ImportanceCirculating microbial metabolites represent a practical and scalable approach to detecting significant gut microbiome disruption, particularly loss of obligate anaerobes. Unlike stool-based sequencing, which can be logistically challenging and slow, blood-based metabolite profiling could be actionably integrated into existing clinical workflows. Our findings suggest metabolites capture compositional consequences of microbiome collapse, with performance comparable to direct microbiome profiling in distinguishing disease states. Enabling diagnostic enrichment and real-time monitoring of microbiome injury (e.g., during antibiotic use or critical illness) has potential implications for both clinical care and research, including selection of patients for investigation of microbiome-targeted therapies. With further validation, circulating metabolites could provide an accessible surrogate for gut microbiome composition in settings where sequencing is impractical.

Corynebacterium bovis, the cause of Corynebacterium-associated hyperkeratosis (CAH), is an important pathogen in immunocompromised mice that is difficult to eliminate and can confound research outcomes. We recently observed that CAH severity varies among outbred athymic nude mouse stocks, but the relative contributions of host genetics and the microbiome remain unclear. We hypothesized that disease course and severity vary based on host genetic stock and/or microbiome composition. Three nude mouse stocks were rederived into the axenic state and either monoinfected with a pathogenic C. bovis isolate (104; CFU) or given sterile media (n=6/group). Axenic mice were also reassociated with their source microbiome or microbiomes from three other stocks with known differences in CAH severity, then inoculated with C. bovis (n=6) or sterile media (n=2). In a separate experiment, one axenic stock was used to assess the role of C. amycolatum via monoinfection, monoinfection followed by C. bovis challenge, or addition to a nonprotective microbiome followed by C. bovis challenge. Mice were monitored daily for 21 days and scored for skin lesions (0-5). C. bovis monoinfected mice developed disease comparable in severity and timing to conventionally raised controls. Notably, reassociation with Vendor A2s microbiome prevented clinical lesions and reduced histopathologic changes across all stocks. While C. amycolatum as a monoinfection did not cause disease nor reduce disease severity following C. bovis challenge, it delayed the onset and lowered peak scores when added to a non-protective microbiome. These findings demonstrate that C. bovis can cause CAH as a monoinfection, that both host genetics and microbiome composition influence disease progression, and, together with prior work, support its role as the etiologic agent consistent with Kochs postulates. Identifying protective microbiome constituents may inform strategies to reduce disease burden in susceptible mice.

The study of human enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) has been limited by the inability of these pathogens to effectively colonize murine models without prior antibiotic treatment. Because it mimics key features of human EPEC and EHEC infection, Citrobacter rodentium, a natural mouse pathogen that colonizes the lower intestine, has become the primary model for investigating these organisms. C57BL/6 mice are most commonly used for C. rodentium research, however, unless they carry specific genetic mutations, they typically develop only mild disease and clear the infection within weeks. As a result, models of severe disease in genetically unmodified hosts are lacking. Here, we describe the development of a non-genetically modified C57BL/6 mouse line with an undisturbed intestinal microbiota, highly susceptible to severe C. rodentium infection. Early infection in these mice was marked by significantly elevated cecal bacterial burdens and tissue pathology. Immune profiling revealed broad reductions in multiple lymphoid subsets, indicating impaired early mucosal activation. Although overall cytokine expression patterns were similar between groups, ceca of susceptible mice exhibited elevated baseline and early post-infection IL-18, as well as increased G-CSF at day 1. Microbiota analyses showed broadly comparable communities with wildtype controls, with some altered groups, such as Lachnospiraceae, Prevotellaceae, Desulfovibrionaceae, and Erysipelotrichaceae. Together, these findings characterize a robust C57BL/6 model that reproducibly develops severe C. rodentium-induced disease. This phenotype is driven by microbiota-associated alterations and impaired early cecal immunity, providing a valuable system for studying host-microbiota interactions in enteric infections.

Nakaseomyces glabratus is a globally distributed opportunistic fungal pathogen. An ongoing discussion in studies of N. glabratus population structure has been whether genetic clusters are best defined using multilocus sequence typing (MLST) or short-read whole-genome sequencing (WGS). To assess the concordance between MLST- and WGS-based phylogenies, we analyzed a dataset of 548 N. glabratus WGS sequences from 12 countries. Clusters identified from WGS largely recapitulated the MLST-defined sequence type (ST) groups: fourteen WGS clusters were composed of a single MLST ST, and the remaining contained STs with very closely related MLST profiles. We thus propose a pragmatic naming convention, consistent with the system used in other microbial species, which specifies WGS cluster labels based on the primary ST. From the large WGS isolate dataset, we determined the prevalence of admixture and genomic variants. Interestingly, seven of the nine singleton isolates were admixed, in addition to 58 isolates from six different clusters. Aneuploidy was detected in 4% of isolates, most commonly in chrE, which contains ERG11, the gene encoding the enzyme targeted by azole antifungals. Aneuploid chromosomes did not exhibit elevated heterozygosity relative to the sequencing error rate, consistent with instability of extra chromosome copies. Copy number variants were found in 3% of the isolates; some of the CNVs co-occurred with aneuploidies, and were primarily identified on chrD, chrE, chrI, and chrM. Our findings demonstrate that deep splits between clusters preserve the utility of MLST ST designations for clade-level designation, yet underscore the utility of WGS for high-resolution genomic analyses. Article SummaryThere is an ongoing debate in studies on Nakaseomyces glabratus about whether traditional MLST analysis is sufficient to determine population structure, or whether the precision of whole genome sequencing (WGS) is necessary. We analyzed WGS data from 548 isolates from around the world. We found a very strong agreement between the two methods. We propose a hybrid naming system, where cluster names are based on the dominant MLST group. We used the WGS data to show that admixed isolates, and those with extra chromosomes or CNVs are rare (<7% of isolates in each class) and are distributed throughout the phylogeny.

Characterizing the household bacterial microbiome allows for a stronger understanding of the various microbes that a person is exposed to everyday in their home. Exploring household microbiomes in different countries around the world increases - our understanding of the impact cultural differences might have on niche microbial communities in the house. The goal of this study was to use shotgun metagenomics to characterize the microbiome for ten locations around the home in ten different houses from three different countries (Mysuru, India; Dubai, United Arab Emirates (UAE); and Tucson, United States of America (USA)). There was a significant difference in alpha diversity between the three countries (ANOVA, p<0.05) with homes in Mysuru, India showing significantly higher bacterial diversity compared to Dubai, UAE and Tucson, AZ, USA. Beta diversity analysis of the homes found that bacterial communities significantly differed between cities (PERMANOVA, p<0.01) and within cities by household locations (PERMANOVA, p<0.001). Locations such as underneath the toilet rim, bathroom and kitchen sinks had the highest levels of bacterial diversity across the three cities compared to other sampling areas. A core microbiome of Actinomycetes and Gammaproteobacteria was found in all homes in all three cities. Within each city, a core microbiome was identified at the species level within specific household locations in each city. Over 90% of bacterial taxa found in the homes were a part of the human-associated phyla Actinomycetes (eg. genera Brevibacterium, Corynebacterium, and Microbacterium), Pseudomonadota (eg. genera Acinetobacter, Moraxella, Pantoea, Paracoccus, and Psuedomonas), and Bacillota (genus Streptococcus), which was comparable to previous studies. The household microbiome is variable in different locations in the house and on a global scale. Factors such as human activity, cultural practices, climate, and surface type and use may drive this diversity. Characterizing the household microbiome on a global scale allows for a better understanding of what drives microbial diversity, increasing our understanding of how microbial communities are shaped by the environment and how humans influence their dynamics, as well as any risks to human health that the built microbiome may potentially pose. Impact StatementThis research contributes to the understanding of the built microbiome, specifically how it varies within the house, within cities, and across the globe. This can aid in our understanding of microbial dynamics in environments with heavy human influence and help develop and improve hygiene habits and products which are mindful of the existing microbiome. Data SummaryDNA sequence data from this research is publicly available on the NCBIs Sequence Read Archive under BioProject PRJNA1416920. Data was analyzed using python and R code. Analysis protocols and information on software versions, packages, and more can be found within the text and in the following github repository: https://github.com/carolinescranton01/Global_Household_Microbiome. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

The gut-lung axis describes interactions between intestinal and respiratory mucosal systems through microbial, metabolic, and immune pathways, but the systemic impact of gut-targeted therapies on upper respiratory tract (URT) communities remains underexplored. We conducted a longitudinal study in adult patients undergoing fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (CDI) alongside healthy controls. Fecal, nasal, and oral samples were collected at baseline (Day 0) and on Days 14 and 56 following FMT. Shotgun metagenomic sequencing was performed to quantify microbial diversity, taxonomic composition, and the abundance of antimicrobial resistance genes (ARGs). FMT was associated with increased gut diversity and decreased levels of key intestinal taxa commonly considered pathobionts, including Klebsiella spp., Escherichia spp., Shigella spp., and Klebsiella pneumoniae. At the phylum level, fecal Bacteroidota increased, while Mucoromycota decreased following treatment. Post-FMT nasal microbiome changes included reduced richness and diversity, expansion of Moraxella, and decreases in taxa linked with respiratory colonization, including Staphylococcus aureus and Streptococcus pneumoniae. By Day 56, nasal communities partially recovered toward healthy profiles. Baseline nasal ARG abundance decreased following FMT, particularly among {beta}-lactam, aminoglycoside, and fluoroquinolone resistance genes, and remained comparable to healthy controls by Day 56. In contrast, the oral microbiome and oral resistome remained largely stable, with only minor fluctuations, and no consistent increases in respiratory pathobiont-associated taxa. In summary, FMT was associated with broader effects beyond the gut, including changes in the URT microbial ecology and antimicrobial resistance profiles. Together, these findings are consistent evidence of gut-lung microbial interactions, linking intestinal dynamics with respiratory microbial composition and antimicrobial resistance patterns.

Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma, is frequently accompanied by skin dysbiosis, with advanced lesions often dominated by multidrug-resistant Staphylococcus aureus. Increased S. aureus colonization is associated with clinical complications and accelerated disease progression, emphasizing the urgent need for effective antimicrobial strategies and a deeper understanding of bacterial adaptation to MF lesions. Here, we evaluated synergistic antibiotic combinations and performed integrated phenotypic, genomic, and metabolomic profiling of MF-associated S. aureus isolates derived from patch and plaque lesions to understand virulence and pathogenicity driving mechanisms in microbe-host interactions. Several antibiotic combinations, most notably carbenicillin with either gentamicin or levofloxacin, exhibited strong synergy and restored antimicrobial activity against highly resistant strains. Comparative genomic analyses revealed that plaque-derived isolates carried expanded resistomes and virulence repertoires, including increased enterotoxins, immune-evasion, and stress-response factors, whereas patch-derived isolates encoded more genes linked to interbacterial competition, such as accessory components of the T7SS. Metabolomic profiles further supported these findings: plaque isolates produced metabolites linked to host interaction, dysbiosis, and inflammation, whereas patch isolates showed profiles consistent with ecological competition. In summary, this work provides insight into the distinct adaptation strategies of S. aureus across MF disease stages. The differential virulence and resistance repertoires observed between patch- and plaque-derived isolates suggests progressive adaptation toward the host microenvironment, potentially influencing disease progression and patient outcomes. Additionally, our findings identify synergistic antibiotic combinations as promising therapeutic approaches for targeting multiresistant MF-associated S. aureus. Importance StatementThe role of multidrug-resistant Staphylococcus aureus in worsening clinical outcomes of mycosis fungoides remains poorly understood, despite its frequent dominance in advanced lesions. Bridging the gap between clinical observation and microbiological mechanisms is essential for clarifying how S. aureus (SA) persists within MF skin and for identifying therapeutic alternatives for SA-positive patients, where treatment options remain limited. This study sheds light on two major clinical needs: the lack of effective antibiotic strategies and the limited insight into bacterial factors that may accelerate MF progression. By integrating synergistic antibiotic testing with genomic, phenotypic, and metabolomic profiling, our work provides insight into stage-specific adaptation patterns of MF-associated S. aureus. These findings identify promising therapeutic directions and establish a framework for future studies to understand the role of S. aureus in MF pathogenesis and exploring how its effects may be therapeutically mitigated.

Sequencing of ribosomal marker genes remains a cornerstone for profiling complex microbial communities. In recent years, there has been a shift from Illumina to long-read technologies, including PacBio and Oxford Nanopore Technologies (ONT). ONT is attractive due to its low startup cost and portability; however, historically high error rates have prevented direct amplicon sequencing variant (ASV) generation from raw nanopore reads. This has forced most workflows to rely on mapping raw reads against reference databases constraining analyses to taxa covered by these. With recent improvements in ONT sequencing accuracy, we sought to challenge this view by sequencing samples of increasing complexity using primer sets targeting amplicons of different lengths, and by sequencing the exact same PCR libraries on both PacBio and ONT. We demonstrate that error-free ASVs can now be generated directly from raw nanopore reads using standard denoising algorithms originally developed for Illumina data. Current ONT read quality enables reliable reconstruction of amplicons spanning [~]250 bp to [~]4,200 bp and allows resolution of intragenomic rRNA gene variants. These results extend beyond simple mock communities to complex fecal, anaerobic digester, activated sludge, and soil samples. When sequencing depth is sufficient, ONT accurately recovers all or nearly all intra-genomic 16S rRNA gene copy variants, showing perfect sequence identity to curated reference sequences in mock communities and to ASVs inferred from PacBio data in complex communities. Across the primer sets, ONT required higher sequencing depth than PacBio to fully resolve the communities, with this requirement increasing with amplicon length. For complex samples, ONT required approximately 2-3x more reads for V4 ([~]250 bp) and V1-V3 ([~]500 bp), 4.1-5.6x more reads for V1-V8 ([~]1400 bp), and 25-42x more reads for rRNA operon (OPR) amplicons ([~]4200 bp). Consequently, sequencing complex communities with OPR primers on ONT is currently not feasible due to the unrealistically high read depth required. This study provides evidence that ONT amplicon sequencing has matured to the point where true ASV-resolved profiling is practically and economically feasible, moving ONT amplicon analysis beyond reliance on OTU clustering or reference alignment to enable application in environments lacking comprehensive reference databases. Key FindingsO_LIIt is now straightforward to generate ASVs on ONT platforms (250-4200 bp) C_LIO_LIONT can resolve intragenomic 16S rRNA gene variants C_LIO_LIASV recovery is successful in both simple and complex communities C_LI

The SWI/SNF complex comprising of the catalytic subunit, Snf2, is a key regulator of gene expression and DNA damage repair in eukaryotic cell. Candida albicans Snf2 is known to regulate hyphal formation. In this paper, we have investigated the role of this protein in DNA damage response. We show that CaSnf2 is required for cell division as deletion of both copies of SNF2 leads to increased duplication time. The mutant cells form clumps with increased chitin and {beta}-glucan deposition on the cell wall. The altered cell wall phenotype leads to reduced uptake of genotoxic stressors leading to increased resistance to both methyl methane sulfonate (MMS) and hydroxyurea (HU). In addition, resistance of Casnf2{Delta} cells to MMS also appears to be mediated by upregulation of CaRAD9 expression by CaFun30, an ATP-dependent chromatin remodeling protein, and CaRtt109, a fungal-specific histone acetyltransferase. The response of Casnf2{Delta} to genotoxic stressors is at variance with the response of Scsnf2{Delta} mutant, highlighting the differences in DNA damage response/repair pathway between the two organisms. Finally, we show that Casnf2{Delta} mutants are extremely sensitive to azoles due to downregulation of multi-drug resistance pumps leading to reduced efflux of the drug.