mSphere

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.

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.

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.

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.

Non-typeable Haemophilus influenzae (NTHi) is a prominent opportunistic pathogen relevant to chronic respiratory diseases. NTHis metabolic diversity enables its survival in a wide range of environmental conditions within the host. As such, deeper research into the metabolic pathways of NTHi may open an avenue for novel therapies aimed at combating NTHi-associated respiratory diseases. Draft genome sequences from nine NTHi clinical strains from three isolation sites - ear (ear sample, ES), pharynx (pharynx sample, PS), and lower respiratory tract (Lungs) - were analyzed and annotated using RAST, PROKKA, KEGG KAAS, and antiSMASH. Pathway module coverage per-strain was computed and summarized by per-group for significant annotated metabolites. Metabolites were analyzed by LC/HRMS, identified by Metaboscape, and statistically compared using MetaboAnalyst and R software. Gene content across the tested NTHi strains was largely conserved, with limited core-genome SNP variation. Gene annotation for metabolite-related pathways revealed that all nine strains possessed largely similar sets of metabolic pathway genes, despite minor nucleotide-level differences, indicating broadly comparable metabolic capacities. In contrast, metabolomics data revealed differential metabolic profiles among the body-site groups. In a principal component analysis (PCA), the ES group was significantly separated from both the PS and Lung groups, which overlapped considerably. Detailed metabolite analyses showed that inosine, hypoxanthine, and uracil were highly significant in the ES group compared to the PS and Lung groups. For the first time, our study sheds light on the extent of metabolic differences associated with NTHi inhabiting diverse host niches. The observed metabolic differences suggest that NTHi may modulate its metabolism in a site-specific manner that is affected by environmental factors. These findings add to our understanding of how NTHi metabolism contributes to site-specific colonization. Author summaryHaemophilus influenzae is widely recognized as a causative agent of meningitis and pneumonia. In particular, H. influenzae strains with a polysaccharide capsule--known as H. influenzae type b (Hib)--were historically a major cause of invasive disease. However, Hib has been largely eradicated following implementation of the Hib vaccine. Nonetheless, there are H. influenzae strains that lack this capsule and are therefore not targeted by the vaccine. These are known as non-typeable H. influenzae (NTHi). Following the decline of Hib, NTHi has rapidly occupied the ecological niche in the lower respiratory tract, becoming the most prominent pathogen in patients with chronic respiratory infections--particularly in those with chronic obstructive pulmonary disease (COPD), where it frequently triggers exacerbations. Importantly, NTHi is also a common component of the normal microbiome in healthy individuals, typically residing in the upper respiratory tract without causing disease. In our study, we investigated the metabolic characteristics of NTHi isolates obtained from different body sites in patients to better understand what distinguishes strains capable of colonizing specific anatomical niches. We successfully identified several distinct metabolic features associated with NTHi strains from the ear, pharynx, and lung. These findings may serve as a foundation for future research into patient-tailored biomarkers and targeted therapies, ultimately aiming to eradicate NTHi in chronic lung infections.

Powassan virus (POWV) is an emerging tick-borne flavivirus that can cause severe encephalitis in humans. Currently no vaccines or therapeutics are approved to treat POWV. POWV is spread by the deer tick, Ixodes scapularis, which is ubiquitous across the Northeastern United States. To better understand POWV prevalence in high-risk populations, we examined POWV seroprevalence in Cadets at United States Military Academy (USMA) in West Point, New York. Cadets at USMA, located in a heavily wooded area, are at high risk for tick exposure during outdoor military training. 1,051 serum samples from the Cadet class of 2017 were screened for POWV seropositivity using a POWV Envelope (E) DIII ELISA. A seropositivity rate of 1.3% was determined. Several ELISA-positive samples were also able to neutralize both reporter virus particles bearing the POWV E protein and authentic POWV. This study demonstrates populations at risk for tick exposure may have significant seroprevalence of POWV.

Shigella sonnei is a leading cause of bacterial dysentery and a high priority WHO pathogen because of the spread of multidrug resistant strains. Understanding microbiome-Shigella-host interactions during colonization of the gastrointestinal tract, and the development of vaccines have been hampered by the lack of small animal models of shigellosis. Here, we developed a murine model of intestinal colonization with S. sonnei. Pre-treatment of mice with antibiotics disturbed the intestinal microbiome and rendered mice susceptible to high level, gastrointestinal colonization with S. sonnei for over one week. Infection with S. sonnei CS14 harbouring a stable virulence plasmid induced an initial inflammatory response in wild type mice, with weight loss and elevated levels of fecal lipocalin 2; the S. sonnei Type III Secretion System was responsible for this inflammatory response. Expression of O-antigen and Group IV capsule by S. sonnei promoted sustained intestinal colonization, with infected mice developing mucosal and systemic antibody responses predominantly directed at these glycans. Finally, infection with S. sonnei induced a degree of protection against subsequent re-challenge. Overall, this murine model successfully mimics aspects of S. sonnei colonization and should be helpful in understanding how S. sonnei successfully survives within the gastrointestinal tract and competes with the microbiota as well as the evaluation of vaccine candidates.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that regulates diverse cellular processes, including colony morphology, motility, biofilm formation, and virulence. It is synthesized by diguanylate cyclases (DGCs) containing the GGDEF domain and degraded by phosphodiesterases (PDEs) containing the EAL domain. However, studies on the genetic and physiological characteristics of c-di-GMP metabolism in Pantoea ananatis are lacking. In this study, we identified 26 predicted c-di-GMP metabolism-related genes in the P. ananatis PA13 genome: 9 encode GGDEF-only domain proteins, 5 encode dual GGDEF/EAL domain proteins, and 12 encode EAL-only domain proteins. We constructed overexpression strains and mutants of 26 DGC- and PDE-encoding genes, and then assessed their Congo Red binding, mucoid and rugose phenotypes, pellicle formation, and swimming motility. We identified 14 of 26 DGC and PDE proteins that affect phenotype changes. Among the 26 DGC- and PDE-overexpressing strains, 13 exhibited the phenotypic changes described above, with some showing alterations in multiple phenotypes simultaneously. Notably, overexpression of dgcM induced changes across all phenotypes. Among the 26 DGC and PDE mutants, the pdeC mutant increased pellicle formation and Congo red binding, the pdeM mutant reduced the mucoid phenotype, and the pdeS mutant, which shows high similarity to ydiV, an anti-FlhD factor, increased swimming motility. Overexpression strains and mutants of 14 DGC and PDE proteins that exhibited phenotypic changes had higher intracellular c-di-GMP levels than the wild type. This study provides important insight into the role of the c-di-GMP network in the plant pathogen P. ananatis. IMPORTANCEPantoea ananatis is a versatile bacterium that causes significant diseases in various economically important plants. To survive and infect hosts, bacteria use a key signaling molecule called c-di-GMP to switch between swimming freely and forming protective communities known as biofilms. Despite its importance, the specific genes governing this signaling network in P. ananatis remained unknown. In this study, we systematically identified and characterized 26 genes responsible for regulating c-di-GMP levels in P. ananatis PA13. By analyzing mutants and overexpressing these genes, we pinpointed 14 critical factors that control essential behaviors such as motility, pellicle formation, and colony appearance. Notably, we discovered specific genes, such as dgcM and pdeS, that act as master regulators of these traits. This comprehensive functional map of the c-di-GMP network provides essential insights into how this pathogen adapts to its environment, offering potential targets to control plant infections.

Microbiomes can influence the outcomes of virus infection, while viral infections can disrupt microbiome composition. These reciprocal interactions may contribute to patterns of host susceptibility, but it remains unclear whether microbiome-virus interactions are consistent across host species, or whether host evolutionary relationships influence microbiome variation and its association with viral susceptibility. Here, we investigate interactions between bacterial microbiomes and Drosophila C virus (DCV) infection across 32 Drosophilidae host species, using experimental infections and 16S rRNA amplicon sequencing. Using phylogenetic mixed models, we tested for phylogenetic structure in microbiome diversity and composition, and whether specific bacterial taxa were associated with among-species variation in viral load. Drosophilidae microbiomes were consistently dominated by a small number of bacterial genera and showed limited phylogenetic structuring. DCV infection was associated with a small reduction in bacterial richness, but microbiome composition was only weakly affected by wounding or infection and largely dominated by within-species variation. However, specific taxa were associated with large differences in viral replication, including reduced DCV load in flies harbouring Streptococcus before infection or Bacillus during infection. These results suggest that, at least in the conditions used in this study, microbiome composition may not generate strong, generalisable host phylogenetic patterns in virus susceptibility.

Understanding how host-microbiome interactions influence tree disease is critical for understanding forest resilience. Here, we present foliar microbiome ITS2 metabarcoding transcriptomic datasets from Pinus sylvestris to investigate susceptibility to Dothistroma needle blight (DNB), a globally important foliar disease caused by Dothistroma septosporum. We hypothesised that host genotype shapes foliar microbial communities and their interactions, thereby influencing disease outcomes. Samples were collected from a progeny-provenance field trial in the south of Scotland representing a broad spectrum of disease susceptibilities. The dataset comprises ITS2 metabarcoding samples from 200 genotypes across three timepoints and RNAseq samples from 48 genotypes across two timepoints. Sampling captured key stages of pathogen exposure and disease progression. Both standardised and bespoke protocols were used for nucleotide extraction, sequencing, and quality control, including multiple negative and positive controls. These datasets, available in the European Nucleotide Archive (project accession PRJEB88228), enable analysis of temporal dynamics in foliar fungal communities, host-microbiome transcriptional responses, and genotype-dependent variation in disease susceptibility.

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium that causes scrub typhus, a potentially life-threatening disease. To systematically identify host factors regulating early stages of infection, we performed a microscopy-based genome-wide siRNA screen in HeLa cells. This approach identified 2,989 genes grouped into 55 functional networks that modulate bacterial entry and intracellular translocation. In addition to confirming previously described pathways, including endocytosis and microtubule-dependent trafficking, the screen revealed an association between Ot infection and host cell cycle regulation. We found that Ot preferentially infects and/or replicates in host cells in the S and G2 phases, where intracellular bacterial accumulation is increased relative to G1. Early infection was associated with a shift in host cell cycle distribution, consistent with a delay in progression through S and G2 phases. Longitudinal analysis further showed that these cell cycle states support enhanced bacterial expansion. In parallel, infected cells exhibited reduced proliferation compared to uninfected cells, suggesting that Ot infection alters host cell division dynamics. Together, these findings support a model in which host cell cycle state influences susceptibility to Ot infection and intracellular growth. This work provides a systems-level map of host pathways involved in early infection and identifies cell cycle regulation as an important component of host-pathogen interactions in scrub typhus. Author SummaryScrub typhus is a potentially life-threatening disease caused by the bacterium Orientia tsutsugamushi, which can only survive and replicate inside human cells. Although some host factors involved in infection have been identified, many remain unknown. In this study, we used a large-scale screening approach to systematically identify human genes that influence the bacteriums ability to enter and move within host cells. Our analysis uncovered multiple pathways required for infection, including a role for the host cell cycle. We found that O. tsutsugamushi preferentially accumulates in cells during specific stages of the cell cycle, particularly when cells are preparing to divide. At the same time, infection slows host cell division, suggesting that the bacterium alters the cellular environment to support its own growth. These findings provide new insight into how O. tsutsugamushi interacts with human cells and identify potential host processes that could be targeted to limit infection.

Campylobacter jejuni is a major cause of food-borne gastroenteritis and is responsible for substantial mortality and economic losses in meat and dairy production. Detecting C. jejuni in contaminated food samples remains difficult because current assays are culture-based, slow, and can yield false positives. As a result, contamination may not be identified for several days, limiting detection at the point of production. Developing improved assays has also been challenging because Campylobacter genetics and the biology of clinical isolates remain poorly understood. Here, we expand the C. jejuni genetic toolbox by sequencing two strains, HC1 and RM1164, derived from patient and food samples. We identified two cryptic plasmids in HC1, one potentially capable of conjugation and another conferring tetracycline resistance. We also engineered a mobilizable plasmid carrying an OriT sequence that can be transferred from Escherichia coli donor strains to C. jejuni RM1164 by conjugation. Together, these clinical isolates and the plasmid system expand the genetic tools available for C. jejuni.

Anaerobic gut fungi (AGF) are key members of the herbivorous gut microbiome. While AGF communities have been well-studied in foregut and hindgut fermenters, they remain poorly characterized in pseudoruminants such as camels. Here, we present a comprehensive culture-independent diversity survey of 142 fecal samples from all three extant camel species (Camelus dromedarius, Camelus bactrianus, and Camelus ferus). The AGF community in Camelus was highly diverse, with representatives of 42 AGF genera identified. However, this diversity was unevenly distributed, with three genera (Neocallimastix, Caecomyces, and Orpinomyces) accounting for 70.7% of sequences encountered, and only 12 genera exceeding 1% relative abundance in the entire dataset. While several of the genera identified as major components of the AGF community in camels are highly ubiquitous in all herbivores, others, such as Oontomyces, Aestipascuomyces, Liebetanzomyces, and the yet uncultured genera NY09, NY03, and JV-2025d are extremely rare in ruminants and hindgut fermenters, hinting at their preference and potential co-evolution with the Camelidae. Ordination approaches identified host species and biogeography as key determinants driving AGF community structure differences between various camel species. Comparative community structure analysis between AGF community in camels versus reference foregut and hindgut fermenters identified the relative enrichment of the genera Oontomyces and Aestipascuomyces in pseudoruminants datasets. Our results demonstrate a distinct AGF community composition in Camelidae, elucidate factors impacting AGF diversity and community structure variations in Camelus, and identify key distinct taxa differentially enriched in psuedoruminants compared to ruminants and hindgut fermenters. The ecological and evolutionary drivers of such patterns are discussed.

Tick-borne pathogens can reshape vector microbiomes in ways that influence pathogen colonisation and transmission, yet the interplay between Ehrlichia ruminantium and the microbiota of its tick vectors remains uncharacterised. We profiled bacterial communities in haemolymph, midgut, and salivary glands of infected (n = 11) and uninfected (n = 12) Am. gemma ticks, a vector of E. ruminantium in East Africa, collected from cattle in Kajiado County, Kenya, using near-full-length 16S rRNA gene amplicon sequencing on the Oxford Nanopore platform. Community composition, alpha and beta diversity, co-occurrence networks, keystone taxa, and PICRUSt2-inferred functional profiles were compared across tissue-infection status groups. We identified 226 bacterial genera dominated by Coxiella, Pseudomonas, Acinetobacter, Proteus, and Rickettsia. Infection was associated with tissue-specific shifts in community composition (PERMANOVA R{superscript 2} = 0.14, p < 0.001) and co-occurrence network structure, with midgut networks showing complete hub taxon turnover (Jaccard = 0.000, p = 0.043). Haemolymph communities converged around Luteimonas as a keystone taxon, while opportunistic Proteobacteria, including Acinetobacter and Serratia, emerged as keystones in infected midgut. Endosymbiotic Rickettsia was near-absent in infected tissues (0.3% vs 9.3% mean relative abundance in midgut), consistent with competitive exclusion. Functional inference identified FDR-significant enrichment of predicted aerobactin siderophore biosynthesis, antimicrobial efflux, and oxidative stress response gene families in infected microbiota. These findings show tissue-specific restructuring of the Am. gemma microbiome associated with E. ruminantium infection and point to candidate targets for microbiome-based interventions against heartwater. ImportanceHeartwater, caused by the bacterium Ehrlichia ruminantium and transmitted by Amblyomma ticks, kills up to 90% of susceptible ruminants and is one of the most devastating tick-borne diseases in sub-Saharan Africa. Controlling heartwater requires understanding how the pathogen interacts with the microbial communities living inside its tick vector. In this exploratory study, we show that E. ruminantium infection is associated with tissue-specific shifts in the Amblyomma tick microbiome, including reduced abundance of beneficial symbionts, elevated representation of opportunistic bacteria among community hubs, and enrichment of iron acquisition and antimicrobial resistance functions. The midgut, the first tissue colonised during infection, showed the most marked structural reorganisation. These tissue-resolved microbiome signatures point to potential targets for novel control strategies, such as anti-microbiota vaccines or approaches that reinforce natural colonisation resistance, offering new strategies to reduce heartwater transmission and protect livestock livelihoods across Africa.

Human metapneumovirus (HMPV) causes acute lower respiratory infections, primarily affecting young children and older adults, with seasonal outbreaks peaking annually in March or April in the United States and other temperate regions in the Northern hemisphere. However, the factors driving HMPV seasonality in the United States remain poorly understood. We analyzed laboratory-confirmed HMPV cases and age-specific emergency department visits across 10 US regions, fitting an age-stratified dynamic transmission model to assess spatiotemporal patterns and investigate the influence of environmental variables and viral interference from RSV on HMPV transmission rates. We found that models incorporating climate variables into the transmission rate, including vapor pressure, precipitation, potential evapotranspiration, and minimum temperature, could not capture the timing of HMPV activity across all regions. Instead, HMPV timing was associated with RSV activity, with the HMPV transmission rate reduced in the presence of RSV. We showed that, unlike RSV, only models incorporating viral interference could reproduce the biennial pattern of HMPV observed in some regions, characterized by alternating late-small and early-large epidemics. Furthermore, our model successfully reproduced post-COVID-19 HMPV and RSV epidemics and predicted that RSV interventions are not likely to lead to a substantial increase in HMPV activity despite decreasing competition from RSV. Our work unravels the spatiotemporal dynamics of HMPV and its interaction with RSV, informing future seasonal forecasting and intervention strategies for HMPV.

The Burkholderia cepacia complex (BCC) is comprised of 24 species of Gram-negative bacteria that cause opportunistic infections. While antimicrobial susceptibility testing (AST) has historically been used to guide treatment for BCC infections, recent work highlighting problems with AST for these organisms led the Clinical and Laboratory Sciences Institute (CLSI) to remove disk diffusion (DD) and minimal inhibitory concentration (MIC) breakpoints for BCC from its M100 standards document. Epidemiological cut-off values (ECVs) may be helpful to clinicians in the absence of breakpoints, as they may be used to determine whether an isolate has a wild-type or non-wild-type phenotype. Here we present an analysis of BCC ECVs for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX). ECVs were calculated using MIC data from 3 previous studies and 3 independent laboratories for 1,896 BCC isolates. ECVs were 16 g/ml for CAZ, 8 g/ml for LVX, 16 g/ml for MEM, and 8 g/ml for MIN. The ECV for TMP-SMX varied depending on the analysis from 2 g/ml, 8 g/ml, and 16 g/ml and therefore could not be reliably established. Challenges with establishing ECVs for BCC include limitations with the pooled MIC dataset, broad MIC distributions, and high ECVs that are above the obsolete susceptible MIC breakpoints. These challenges limit the clinical utility of ECVs for these organisms and supported removal of ECVs from the CLSI M100 standards document. IMPORTANCEThe Burkholderia cepacia complex is a group of bacterial species that cause difficult-to-treat opportunistic infections. Recently, clinical breakpoints, which are used to determine whether organisms are susceptible to certain antimicrobials, were removed from Clinical and Laboratory Standards Institute (CLSI) standards for these organisms due to problems with antimicrobial susceptibility testing performance. Clinicians are now faced with the challenge of how to treat these complex infections without clinical breakpoints. Here we determine epidemiological cut-off values (ECVs) for relevant antimicrobials for the B. cepacia complex. While we established ECVs for four antimicrobials, we encountered significant challenges in our analyses, including limitations with data for these organisms and high ECVs that are not clinically useful. These challenges limit the practical use of these ECVs in helping guide clinicians on treatment and supported the eventual removal of ECVs from the CLSI M100 standards document.

SummarySpaceflight introduces environmental stressors that can alter human microbiomes and immune responses. We analyzed 259 biospecimens from six astronauts across two commercial ISS missions: Axiom 2 (10-day mission) and Axiom 3 (21-day mission). Samples included saliva, stool, urine, and body swabs from 10 anatomical sites, profiled via 16S ribosomal RNA (rRNA) gene sequencing. Gut and oral microbiomes remained stable, while skin-associated communities exhibited transient diversity shifts post-flight. Taxonomic analysis revealed individual and site-specific patterns as well as a possible microbial acquisition from the ISS/space-flight environment and microbiome exclusivity. Cytokine profiling from single cell data indicated immune activation, with IL-32 and IL-16 elevated in Axiom 2 and Axiom 3, coinciding with microbial changes. These findings provide an integrated view of microbiome individuality, exclusivity and immune dynamics during two short-duration commercial spaceflights of three weeks, informing strategies for crew health on future long-duration missions.

Montane fens are rare and microbiologically poorly characterised wetland ecosystems in south-eastern Australia, and their microbial communities remain virtually unexplored. Here, we profile the microbiomes of Victorian montane fens using 16S rRNA metabarcoding of 12 soil cores collected along a 12-m transect and sampled across four depth horizons. Surface soils exhibited slightly higher alpha diversity than deeper layers, but the most pronounced differences occurred in community composition, with surface microbiomes significantly distinct from all subsurface depths. To contextualise these communities within global environmental diversity, we compared them with 9662 Earth Microbiome Project samples spanning 24 environmental materials processed using comparable methods. Montane fen microbiomes were one of the most diverse environmental materials analysed and compositionally distinct from all comparator biomes. Overrepresentation analysis identified signature microbial taxa, including archaeal lineages from the Crenarchaeota and Methanomicrobia and bacterial phyla such as Acidobacteria, highlighting taxa involved in ecological processes associated with acidic, saturated, and organic-rich soils. Notably, the majority of sOTUs detected in montane fens were unique to this environment - the highest proportion of source-specific taxa among all biomes analysed. Together, these findings demonstrate that southeastern Australian montane fens harbour a highly distinctive and largely uncharacterised microbial community, underscoring their ecological uniqueness and the importance of conserving these rare alpine wetlands. Data SummarySequencing data is available in SRA BioProject PRJNA1398590; accessions SRR36684598 through SRR36684645. Metadata and accessions for collected montane fen samples are included in Table S1 and metadata for Earth Microbiome Project samples included in this study are listed in Table S4. Impact StatementWetland ecosystems are increasingly recognised as important reservoirs of microbial diversity, yet many remain poorly characterised in global microbiome surveys. In this study, we provide the first characterisation of microbial communities inhabiting montane fens in southeastern Australia and place them in a global context using publicly available environmental microbiome data. We show that these fens harbour exceptionally diverse microbial communities that are compositionally distinct from other environmental sources processed using comparable methods, with a high proportion of taxa that are not present in any other sample in an existing reference dataset. By extending global comparisons to an under-sampled wetland type, this work adds to the growing body of evidence that significant microbial diversity remains undocumented in geographically and ecologically restricted environments. The findings are relevant to researchers working in microbial ecology, environmental genomics, and biogeography, as well as those interested in wetland function and conservation. While largely descriptive, this study represents an important step in expanding environmental genome catalogues and provides a baseline framework for future genomic, functional, and mechanistic investigations of montane wetland microbiomes.

Bacterial pathogens must adapt to dynamic host tissue environments to proliferate. Accordingly, elegant regulatory systems evolved to overcome challenges presented by the host and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcripts generated in a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur-starvation response in chemically defined growth conditions. Results demonstrate that the sulfur starvation and sulfur replete CymR regulons exhibit considerable overlap, including previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. The link between iron acquisition, oxidative stress, and sulfur metabolism is validated further by the finding that sulfur-containing glutathione (GSH) mitigates heme and peroxide toxicity. In addition to GSH, Cys and thiosulfate fulfill the S. aureus sulfur requirement. Transcriptional responses to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic thiosulfate were quantified, revealing sulfur source-specific expression patterns. Thiosulfate induced the largest number of differentially expressed genes. Consequently, the thiosulfate transporter (SAUSA300_RS10985) has been confirmed as essential for S. aureus growth when thiosulfate is the sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth on thiosulfate. Collectively, a resourceful transcriptomics framework is provided which underscores the dynamic nature of S. aureus sulfur metabolism.