mSphere

Most gut microbiome studies have focused on bacteria, leaving a knowledge gap regarding gut associated fungi. We assessed fungal diversity in the gastrointestinal tract of the reptilian order Testudines (turtles and tortoises) using samples from 6 families, 19 genera, and 27 species. A highly diverse community affiliated with 17 phyla and 157 orders was encountered, with four phyla (Neocallimastigomycota, Chytridiomycota, Ascomycota, and Basidiomycota) representing 89.13% of the community. Neocallimastigomycota was identified in host families Testudinidae (land tortoises), Chelidae, Chelydridae, Emydidae, Geoemydidae, and Kinosternidae, with higher relative abundances in Testudinidae (40.18{+/-}37.97%) compared to all other families combined (2.71{+/-}4.04%). Neocallimastigomycota sequences were mostly affiliated with orders Testudinimycetales in the host family Testudinidae and Neocallimastigales in other host families. Chytridiomycota was identified in all host families, but was more ubiquitous and abundant in Kinosternidiae (45.17{+/-}34.12%), and exhibited a high level of variability across samples. Dikarya communities were highly diverse, with 108 orders identified, and, similar to Chytridiomoycota, exhibited a highly stochastic distribution pattern. Representatives of multiple yet-uncultured phyla (Candidatus "Algovoracomycota", "Sedimentomastigomycota", "Tartumycota" and "Cantoromastigomycota") were identified, as well as eight novel orders in Chytridiomycota and Rozellomycota. Deterministic selection shaped community assembly in the host family Testudinidae, while the process was more stochastic in other host families. Distinct community structure was driven by differences in abundance and identity of the Neocallimastigomycota when comparing Testudinidae to. Our results describe a diverse and dynamic fungal community, shaped by the co-occurrence of autochthonous (resident) and transient (allochthonous) members of the gut microbiome. ImportanceFungi are known to inhabit the gastrointestinal tract (GIT) of humans and mammals. However, information on the fungal community in the GIT of reptiles is relatively sparse. We investigated the diversity and community structure of fungi in the reptilian order Testudines. We conducted a culture-independent diversity survey on fecal samples obtained from 27 different host species. We identify representatives of 17 fungal phyla. As well, we demonstrate that the anaerobic gut fungi (phylum Neocallimastigomycota) are not restricted to the family Testudinidae (land tortoises) as previously suggested, but could successfully colonize and inhabit all other testudines families, including those exhibiting a predominantly omnivorous or carnivorous lifestyles. In addition, we expand on the known fungal diversity by identifying additional representatives of multiple recently described yet-uncultured phyla, and describe multiple novel orders and classes within existing phyla. Collectively, this effort adds to the growing body of knowledge of mycobiomes in underexplored animal hosts.

Clostridioides difficile is a healthcare-associated infection that arises when broad-spectrum antibiotic treatment disrupts the gut microbiota and is transmitted by highly resistant spores. Vancomycin-resistant Enterococcus faecium (VRE) is an opportunistic pathogen frequently co-isolated from C. difficile patients. We found that C. difficile sporulation is significantly reduced in VRE-C. difficile co-culture. Physical separation of C. difficile and VRE in transwell co-culture restored sporulation. Mixed macrocolony culture assays on solid agar confirmed physical contact is necessary for sporulation inhibition. We screened a panel of enterococci and found that most strains reduce sporulation, except Enterococcus saccharolyticus, which lacks predicted surface displayed virulence factors in its genome. We performed a candidate gene screen using an Enterococcus faecalis OG1RF transposon library and found that an insertion in the major pilin ebpC partially restored C. difficile sporulation in co-culture. These data were confirmed with in-frame deletions in the ebpABC pilus operon and a clinical isolate of E. feacalis lacking ebpABC. These findings suggest enterococci modulate C. difficile sporulation through a contact-dependent mechanism involving the Ebp pilus. ImportanceA characteristic of C. difficile infection is multiple episodes of acute disease. Spores are the transmission vector of C. difficile and are necessary for recurrence in models of disease. Our research demonstrates that C. difficile spore production is significantly reduced in the presence of enterococci, a common group of beneficial and pathogenic bacteria present in the gut microbiota. Physical contact with enterococci reduces C. difficile spore production. We attribute this effect to a protein structure on the surface of enterococci. This finding suggests a potential role for enterococci and the gut microbiota in general to uncover regulators of C. difficile spore formation. This may provide an avenue for innovative treatment strategies that reduce spore formation.

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.

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.

Spartina alterniflora, the dominant plant species in salt marshes along the Atlantic and Gulf of Mexico coastlines of the Americas, is affected by disease and sudden vegetation dieback. Despite the foundational role of S. alterniflora in low-elevation salt marshes, the response of the native leaf-associated microbiome (i.e., phyllosphere microbiome) to leaf damage resulting from disease and environmental stress has not been explored. We hypothesized that healthy and damaged plants would show differentiation in their phyllosphere microbiomes following primary infection or exposure to environmental stressors. Here, we analyzed changes in prokaryotic and fungal relative abundance, diversity, and community composition in the S. alterniflora phyllosphere microbiome. We compared natural marsh and greenhouse plants in Georgia and South Carolina, USA, and collected leaves from healthy and damaged natural plants across two contrasting Spartina phenotypes that differ in their exposure to environmental stress. Our results show that plant origin (i.e., greenhouse vs. natural marsh), plant health status (i.e., healthy vs. damaged), and plant phenotype (i.e., short vs. tall Spartina) affect microbial relative abundance, alpha diversity, and community composition in the S. alterniflora phyllosphere. Damaged leaves presented higher microbial abundance and alpha diversity than healthy leaves, suggesting microbial proliferation following leaf damage. Plants raised from seeds in the greenhouse presented the lowest microbial abundance and Shannon diversity for both prokaryotic and fungal communities, indicating that in natural ecosystems the phyllosphere microbiota is acquired predominantly through horizontal transmission from the environment. Overall, this study provides novel insights into the assembly of the S. alterniflora phyllosphere microbiome. ImportanceSalt marshes are tidally influenced coastal wetlands that provide a range of ecosystem services to global and local communities, including protection from storm surge, water purification, and carbon sequestration. Spartina alterniflora is the dominant plant species in Atlantic and Gulf of Mexico marshes within the Americas. Fungal disease and exposure to environmental stressors have previously been described in marsh ecosystems and linked to extensive and sudden vegetation dieback in the southeastern U.S. In this study, we show that microbial proliferation follows plant damage caused by either fungal disease or environmental stress, leading to a profound change in native leaf-associated microbiota abundance, diversity, and composition (i.e., leaf microbiome dysbiosis). Using greenhouse plants as a control, we also demonstrate that microbes colonizing marsh leaves are acquired predominantly from the environment. Overall, this study advances our understanding of the leaf-associated microbiome of S. alterniflora, with implications for ecosystem management and restoration practices.

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.

Soil microbial communities (SMCs) play an important role in various ecological processes, including plant growth, carbon cycling, and greenhouse gas production and consumption. There have been many prior studies of soil microbiome function and structure. However, soil is a complex environment in which to conduct biological studies. Therefore, simplified SMC models, often adapted to liquid culture, have been employed in the laboratory to study specific microbial interactions and individual microbial functions. Specific advantages of these laboratory liquid SMC models include the ability to modulate community membership, control environmental conditions, and employ high-throughput assay techniques. The disadvantages of current laboratory liquid SMC models include long cycles for growing bacteria in vitro, the obligatory use of strains that are culturable in isolation, intricate media requirements, and complex community assembly protocols. To address some limitations of current liquid SMC models, we sought to create a streamlined process for extracting and maintaining a liquid culture of an existing SMC. Soil-Extracted Solubilized Organic Matter (SESOM) was made from four different soil types, including rich organic potting soils and environmental samples, and filtered to maintain the SMC. These SESOM liquid SMC models were cultured for 28 days, and SMC composition was measured by 16S rDNA sequencing. The SESOM SMCs maintain high alpha and beta diversity over time, including strains that are not culturable in isolation, with the greatest stability correlated with higher soil organic carbon. Further, the SESOM SMCs maintain unique signatures of their starting solid soils, suggesting that drift in SMC composition over extended time in liquid culture does not eliminate the defining microbial relationships of a given soil type. Network analysis of SESOM SMCs relative to solid soils suggests the functional roles of bacterial taxa were maintained in the liquid models over time. We further demonstrate that the platform can be applied to monitor the survival and persistence of a model engineered microbe - the common synthetic biology chassis Pseudomonas putida - within a native SMC. We conclude that the SESOM model is a valuable tool for facilitating the study of SMCs in the laboratory.

BackgroundWhether the lung microbiome represents a stable microbial colonization or a transient ecosystem shaped by continuous microbial turnover and controlled by host immunity remains unresolved. The murine lung microbiome largely consists of species from the former Lactobacillus genus with Ligilactobacillus murinus as a dominant species, bacterial genera such as Streptococcus, Staphylococcus, Mammaliicoccus, Enterococcus and other less frequently detected bacteria. Here, we directly addressed the question of persistence and host interaction of a dominant murine lung commensal in vivo and focused on the host immune response towards lung commensal bacteria. ResultsWe developed a transformation strategy for stable genomic integration of a green fluorescent protein (GFP)-encoding gene to track the fate of a lung bacterium. Following intranasal administration of GFP-labeled L. murinus in mice, bacteria were readily detected in the lungs at early time points but declined rapidly and became undetectable after 72 hours, as determined by quantification of viable bacteria and qPCR. Flow cytometry and fluorescence imaging revealed efficient uptake of GFP-labeled bacteria by lung phagocytes. These findings indicate that even dominant members of the murine pulmonary microbiota normally detected at low abundances are transiently present in the lungs without causing infection. We further analyzed the effects of moderate and high bacterial concentrations. While moderate bacterial loads were efficiently controlled without clinical effects, high concentrations induced severe lethargy, indicating a threshold-dependent host response. Finally, we demonstrated that pulmonary commensals such as L. murinus, Staphylococcus xylosus, and Mammaliicoccus sciuri, as well as conidia of the opportunistic lung pathogen Aspergillus fumigatus, are phagocytosed at comparable rates in macrophage assays. ConclusionsOur data demonstrate that even lung-adapted bacterial species fail to establish stable colonization and are instead subject to rapid immune-mediated elimination contributing to the maintenance of a low microbial burden in the lungs. While this homeostatic balance supports health, elevated bacterial loads trigger immune activation and, at high levels, lead to health deterioration. Together, these results support a model of a highly dynamic and transient lung microbiome, maintained by continual microbial immigration rather than long-term colonization. Accounting for the lung microbiome dynamics is essential for understanding host-microbiota interactions and respiratory health.

We present a novel protocol for intranasal colonization of germ-free mice with human nasal microbiota, coupled with an optimized DNA extraction method for murine nasal samples compatible with 16S rRNA sequencing. This protocol may facilitate investigation of the functional roles and causal effects of nasal microbial communities in chronic rhinosinusitis and antimicrobial resistance.

Phyllosphere microbiomes are increasingly recognized as key regulators of plant health and stress responses, although they are also known to change considerably over both space and time. In the phyllosphere, members of the genus Methylobacterium are often abundant and ecologically important as plant growth promoting bacteria. However, knowledge about the temporal abundances and community dynamics of Methylobacterium in agricultural systems remains limited. To address this gap, we characterized seasonal shifts in Methylobacterium-specific and total phyllosphere bacterial loads and community structure on two common summer crops and one overwintering cover crop. Leaf samples of Zea mays (corn), Glycine max (soybean), and Thlaspi arvense L. (pennycress) plants were collected over one year in Minnesota, USA and analyzed with host-associated microbial PCR (hamPCR). Microbial loads and community composition varied strongly among hosts and across growing seasons. Corn supported the highest Methylobacterium and total bacterial loads, increasing towards senescence, while pennycress exhibited the lowest loads and the most distinct communities. While there were strong host-specific patterns, a group of most abundant genera were shared across all crops (Methylobacterium, Sphingomonas, Pseudomonas, and Massilia) and the most abundant Methylobacterium amplicon sequence variants were present on all three hosts. Our findings highlight how microbial loads and community composition change during phyllosphere assembly across diverse summer and overwintering crops, with a small core of versatile taxa dominating multiple agricultural hosts. Understanding these host and season-linked patterns provides a foundation of harnessing Methylobacterium strains to enhance crop productivity and resilience.

Within a single bacterial strain, DNA sequence variation is expected between individual clones. Whole genome sequencing (WGS) can be applied to clinical cultures to detect this polyclonal variation, enabling tracking of within-host evolution and transmission. Culture isolates from infected patients are often sequenced as individual colonies (c-seq). To increase the sensitivity of variant detection, cultures can also be sequenced to a high depth of coverage as a pool (p-seq), but the utility of this approach is not clear for most clinical specimens. To understand the performance of high-depth WGS in bacteremia, we applied p-seq to blood culture plates for 10 patients with persistent bacteremia due to methicillin-resistant Staphylococcus aureus. As a comparison, for six patients, we also applied c-seq to five colonies (c5-seq) from the same plates. p-seq was more sensitive than c5-seq for detecting low frequency variant alleles; however, the most important factor for new variant detection was the number of culture plates analyzed rather than the sequencing method used. We also used these data to construct Muller plots for three patients with especially diverse infecting populations, which enabled visualization of rapid evolutionary dynamics in response to antibiotic exposures. We identified 204 unique variant alleles, and our analysis provides additional evidence for parallel evolution of several different genes during S. aureus bacteremia. Overall, these data provide a detailed view of evolutionary dynamics during clinical cases of MRSA bacteremia and describe the merits and limitations of a c-seq versus p-seq strategy for analyzing blood culture plates using WGS. ImportanceAs bacterial whole genome sequencing (WGS) is increasingly used as a research tool for clinical samples, it is important to understand the pros and cons of different culture sampling methodologies. Here, we analyzed cases of persistent bacteremia due to methicillin-resistant Staphylococcus aureus by applying WGS to either each of five individual colonies isolated on blood culture plates (c5-seq) or the pooled bacterial population on each plate (p-seq). We found that c5-seq was a more practical and informative method to understand evolutionary dynamics.

Francisella tularensis is a highly virulent, Gram-negative bacterial pathogen that causes the zoonotic disease tularemia. F. tularensis infects a variety of host cells and replicates intracellularly while evading and interfering with host immune responses. The molecular mechanisms that facilitate the intracellular replication and virulence of F. tularensis are poorly understood. The Francisella genome contains a set of pil genes that code for the assembly of surface fibers termed type IV pili (T4P). T4P are major bacterial virulence determinants but the function of the pil system during F. tularensis infection and intracellular growth is unclear. T4P are closely related to the type II secretion pathway and the pil system of a related Francisella species, F. novicida, was shown to function in protein secretion as well as pilus assembly. To identify proteins secreted by F. tularensis, we analyzed the F. tularensis Live Vaccine Strain (LVS) using bio-orthogonal non-canonical amino acid tagging (BONCAT). Using BONCAT in conjunction with proteomics, we identified candidate proteins secreted by the wild-type LVS, as well as candidate proteins whose extracellular abundance decreased in the absence of the PilF ATPase or the PilE4 pilus subunit. Using epitope tagging of selected candidates, we validated T4P-mediated secretion of the ChiA and ChiD chitinases and the KatG catalase by the LVS. These results further our understanding of the pil system and protein secretion pathways in F. tularensis. IMPORTANCEFrancisella tularensis is a highly virulent Gram-negative bacterial pathogen and the causative agent of tularemia. F. tularensis lacks secretion systems utilized by other intracellular bacterial pathogens but contains pil genes that encode for type IV pili (T4P) and may also function in protein secretion. T4P are observed on the surface of all Francisella spp. but pil-mediated protein secretion has only been reported for F. novicida, which is not normally pathogenic in humans. In this study, we used bio-orthogonal non-canonical amino acid tagging to identify proteins secreted by F. tularensis, for which there is limited information. We demonstrate that the F. tularensis pil system is capable of protein secretion and validate T4P-medeated secretion of the ChiA and ChiD chitinases and the KatG catalase. These results will facilitate investigation of Francisella virulence mechanisms and may provide targets for therapeutic intervention.

Early childhood is a critical period for both nasal microbiome development and susceptibility to respiratory viral infections. While prior cross-sectional and limited longitudinal studies suggest that the nasal microbiome is shaped by both host and environmental factors, high-frequency longitudinal data linking microbial dynamics and respiratory viral disease in young children remain sparse. MethodsWe conducted the MINNE-LOVE prospective longitudinal cohort study of children under 5 years of age with weekly symptom surveillance and parent-collected anterior nasal swabs. Total nucleic acid was extracted and analyzed for nasal microbiome composition by 16S rRNA sequencing on short- and long-read platforms and viral pathogen detection by target-enriched metagenomic sequencing. Microbiome diversity, community structure, viral detection patterns, and correlations among dominant bacterial taxa were assessed over time. ResultsWithin individuals, microbiome composition was relatively stable over time, with acute shifts observed. We detected a broad array of respiratory viruses, including frequent viral co-detections and prolonged detection of select viruses across multiple weeks. ConclusionIn this longitudinal cohort of young children undergoing high-frequency sampling, we demonstrated the feasibility of multiomic assessment of nasal microbial communities. Key bacterial ecological relationships described in prior cross-sectional studies were recapitulated using dense temporal sampling. Target-enriched sequencing enhanced the range of viral pathogen detection, including co-infections and prolonged viral shedding. Full-length 16S long-read sequencing enabled clinically relevant species-level resolution not achievable with short-read approaches. These findings highlight the value of intensive longitudinal cohort designs for defining host-virus-microbiome interactions in early childhood and informing future mechanistic and interventional studies. Key pointsWe conducted weekly symptom assessment, nasal microbiome profiling, and respiratory virus detection by target-enrichment metagenomics in four Minnesota preschool children. We demonstrated high symptom and viral burden, intraindividual microbiome stability, and improved taxonomic resolution with long-read 16S gene sequencing.

Microbiome science is increasingly important in modern biology education because microbial communities influence human health, ecosystems, and environmental processes. However, undergraduate microbiome instruction is often limited by the high cost and technical complexity of sequencing-based workflows, restricting opportunities for authentic student-driven research. To address this challenge, we developed a low-cost, inquiry-based curriculum that enables undergraduate students to conduct complete microbiome studies using 16S rRNA gene sequencing. The module integrates project design, environmental sample collection, microbial cell processing, PCR amplification, sequencing, and bioinformatic analysis using open-source tools such as QIIME 2. Cost-reduction strategies included centrifugation-based cell collection and a surfactant-assisted direct PCR workflow that eliminated the need for commercial DNA extraction kits. Students designed independent research projects investigating microbial communities in local environments, including campus water sources and gym equipment surfaces. Assessment data from post-course surveys, knowledge checks, and student research products demonstrated strong learning gains in microbiome concepts, molecular biology techniques, scientific communication, and computational analysis. Students reported high confidence in PCR, experimental design, and microbiome interpretation, while also identifying bioinformatics as the most challenging yet rewarding component of the curriculum. All participants expressed increased interest in future research in microbiology or bioinformatics. Overall, this curriculum provides an accessible, scalable framework for integrating next-generation sequencing into undergraduate education while promoting inquiry-driven learning, student ownership, and engagement in authentic scientific research.

BackgroundIntestinal helminth infections trigger complex host responses, determining parasite survival and tissue homeostasis. Primary Echinostoma caproni infection disrupts epithelial metabolism, differentiation, and repair in an IL-25-deficient environment, as shown in a previous study by our research group; however, the adaptive mechanisms during homologous superimposed infections remain unclear. Methodology/Principal findingsMale ICR mice were assigned to control, primary infection, and homologous superimposed infection groups, and ileal epithelial cells were isolated for proteomic profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with data-dependent acquisition (DDA) and sequential window acquisition of all theoretical mass spectra (SWATH). Differential protein expression was analyzed with Elastic Net regression, partial least squares discriminant analysis, and fold-change ranking, while functional enrichment and protein-protein interaction networks were explored using gene set enrichment analysis (GSEA) and STRING. Notably, homologous superimposed infection revealed proteomic signatures associated with lysosomal and peroxisomal lipid metabolism, PPAR pathway activation, cytoskeletal reorganization, epithelial barrier reinforcement, a specialized antimicrobial peptide repertoire, and interactions between IgE receptor-associated proteins, consistent with a restoration of intestinal homeostasis influenced by IL-25. ConclusionsHost adaptation to repeated E. caproni exposure involves coordinated metabolic, signaling, and tissue repair responses that partially restore intestinal homeostasis, with IL-25 emerging as a central regulator linking metabolic reprogramming, epithelial integrity, and anti-helminth immunity, thereby providing a proteomic framework for understanding how repeated helminth exposure drives partial resistance through integrated epithelial and immunometabolic adaptations. Author summaryWe investigated how repeated intestinal worm infections affect the cells lining the small intestine in mice. Infections with intestinal trematodes can disrupt the normal balance of the gut, leading to tissue damage and altered metabolism. Using a proteomics approach, we measured changes in thousands of proteins in intestinal epithelial cells during a first infection and after a second, repeated infection. We found that the first infection caused stress in the cells, impaired oxygen use, and reduced the activity of pathways that normally help repair tissue. In contrast, the repeated infection triggered a coordinated response that restored many cellular functions. Cells increased protein activity related to fat metabolism, tissue structure, barrier integrity, and antimicrobial defense. We also observed evidence that the immune signaling molecule interleukin-25 plays a central role in coordinating these protective and repair processes. These results suggest that the gut epithelium can adapt to repeated infections by reorganizing its metabolic and structural functions, which may help limit tissue damage and promote partial resistance to parasites. Our study provides a detailed map of the molecular changes that underlie this adaptation, improving our understanding of how the intestinal lining responds to repeated worm infections.

Trichophyton mentagrophytes genotype VII (TmVII) is an emerging sexually transmitted dermatophyte that causes skin infections characterized by inflammatory, erythematous-squamous, painful, and persistent lesions. This genotype is part of the T. interdigitale/T. mentagrophytes Species Complex (TiTmSC), which comprises 28 genotypes. To enable rapid and specific differentiation of TmVII from other genotypes, a real-time polymerase chain reaction (rt-PCR) assay was developed targeting three unique single-nucleotide polymorphisms in the ITS1 region of TmVII. Assay specificity was further improved by introducing an additional mismatch at the 3 ends of both forward and reverse primers. The rt-PCR assay demonstrated high sensitivity, with a detection limit of 0.0002 ng of TmVII genomic DNA. The assay was highly specific, with no cross-reactivity observed with either closely or distantly related fungal pathogens when a cycle threshold (Ct) cutoff of 37 was applied. Among 497 mold isolates tested, 47 were confirmed as TmVII by rt-PCR, and the results were fully concordant with conventional ITS-PCR/Sanger sequencing. The rt-PCR assay demonstrated high sensitivity, specificity, reproducibility, and speed, with a turnaround time of one day after DNA extraction, compared with seven to ten days for Sanger sequencing. The first rapid molecular assay developed using TaqMan chemistry for TmVII identification is expected to enhance patient care and support infection control measures.