Animal Microbiome

Sarbecoviruses, a subgenus of Betacoronavirus, display both respiratory and gastrointestinal tropism, suggesting potential interactions with host gut microbial communities. However, ecological signatures of infection in wild bats remain poorly understood. We investigated associations between Sarbecovirus infection status, gut microbiome structure, and diet composition in Rhinolophus shameli roosting in northeastern Cambodia. Fecal samples collected across dry and wet seasons (2023-2024) were subject to full-length 16S rRNA gene sequencing and arthropod DNA metabarcoding. Sarbecovirus-positive bats exhibited stable alpha diversity but consistent shifts in gut community composition and increased interindividual variability consistent with the Anna Karenina Principle, suggesting infection-associated destabilization of community assembly rather than diversity erosion. Infection status was associated with enrichment of Shigella and Escherichia species, taxa linked to inflammatory or epithelial stress states in bats. In contrast, dietary composition showed no strong global structuring by infection status and weak coupling with bacterial community structure, suggesting that trophic ecology is unlikely to be the main driver of the infection-associated microbiome signal. Although causal directionality cannot be inferred, our results reveal measurable and consistent microbiome restructuring associated with Sarbecovirus detection in a natural reservoir host and highlight the potential of microbiome profiling for monitoring wildlife disease processes.

The female reproductive tract harbors complex microbial communities that may influence reproductive success. In previous work using 16S rRNA gene sequencing, we identified bacterial taxa in the vagina and uterus of beef cattle associated with pregnancy outcomes, but taxonomic resolution and functional inference was limited. Here we used shotgun metagenomic sequencing to characterize the taxonomic composition, functional potential, and antimicrobial resistome of vaginal and uterine microbiomes at the time of artificial insemination (AI) in cows that subsequently became pregnant or remained open. Vaginal (pregnant n = 54; open n = 7) and uterine (pregnant, n = 41; open, n = 9) samples were collected prior to AI. Microbial community structure did not differ between pregnancy outcome groups in either anatomical site (PERMANOVA; P > 0.05). However, cows that remained open showed significantly greater species-level richness and diversity in the vaginal microbiome (P < 0.05). No diversity differences were observed in the uterine microbiome. In contrast, significant differences were detected between anatomical sites, with distinct dominant taxa and functional profiles. Vaginal microbiomes were enriched in pathways related to genetic information processing, whereas uterine microbiomes exhibited greater representation of metabolic pathways. A total of 105 ARGs spanning 11 antimicrobial classes were identified, with tetracycline resistance genes [tet(Q), tet(W), and tet(M)] predominating, and blaTEM-116 more abundant in the uterine microbiome. Overall, while vaginal and uterine microbiomes were compositionally and functionally distinct, no robust pregnancy-associated taxonomic or functional signatures were detected, likely reflecting limited statistical power and challenges inherent to low-biomass metagenomic datasets. IMPORTANCEUnderstanding the role of the reproductive tract microbiome in fertility could improve reproductive efficiency in cattle. We used shotgun metagenomic sequencing to characterize the taxonomic composition, functional potential, and antimicrobial resistome of vaginal and uterine microbiomes at the time of artificial insemination in cows that subsequently became pregnant or remained open. Using paired samples from the same animals, we directly compared microbial communities between the upper and lower reproductive tract to identify shared and site-specific features. Although no distinct microbial signatures associated with pregnancy outcomes were detected, this may reflect limited statistical power and low microbial biomass inherent to these samples. Despite these challenges, our study provides high-resolution insights into the composition, functional potential, and resistome of bovine reproductive microbiomes and highlights important technical considerations for studying low-biomass microbial ecosystems.

Spotty Liver Disease (SLD) is an acute bacterial infection of layer chickens in production, caused by Campylobacter hepaticus, and occurs most frequently in barn-housed and free-range systems. The disease is characterized by a sharp decline in egg production and increased mortality. The hallmark pathological feature is 1-2 mm white to grey necrotic foci distributed across the liver surface. Despite its growing economic impact on commercial poultry, the molecular mechanisms underlying host responses to C. hepaticus infection remain poorly understood. To address this gap, we performed a comprehensive transcriptome analysis of liver tissue from chickens naturally infected with SLD compared to uninfected controls. High-throughput transcriptome sequencing, yielding 9,277 differentially expressed genes (DEG), of which 3,063 were upregulated and 6,214 were downregulated. Functional pathway enrichment analysis revealed significant alterations in immune and metabolic processes associated with SLD pathophysiology. Infected chickens exhibited significant activation of immune response pathways, particularly cytokine-cytokine receptor interactions involving interleukins IL-22, IL-21, and IL-6, along with enhanced cell signaling, and cell adhesion. Among the individual genes, C1QTNF1 and the adhesion molecule gene ADGRD1 were notably overexpressed, indicating enhanced inflammatory activity. In contrast, core hepatic metabolic functions were profoundly reduced, as evidenced by downregulation of oxidative phosphorylation, fatty acid metabolism, iron ion binding, and heme binding pathways. A marked increase in serum amyloid A gene (SAA) expression further confirmed robust acute-phase responses and compromised liver function during infection. Together, these findings demonstrate a complex interplay between inflammatory activation and metabolic dysregulation during SLD. The strong upregulation of acute-phase proteins and pro-inflammatory cytokines demonstrates the hosts vigorous attempt to combat bacterial infection, whereas the concurrent suppression of essential metabolic pathways reflects the pathological consequences of SLD. This study provides a transcriptomic characterization of host responses to C. hepaticus infection, offering insights into SLD pathogenesis and potential avenues for targeted intervention.

Animal-associated bacteria (microbiomes) can have important effects on host phenotypes and fitness. Microbiomes can also vary across individuals in ways that depend on host genotype and environment. Temperature is an especially important environmental factor that can affect the microbiome in a way that depends on host genotype and affects organismal fitness. Thermal stress, in particular, can have dramatic effects on animal microbiomes, including dysbiosis and immune dysregulation. However, most previous work on extreme temperature effects has focused on heat stress. To investigate how low temperatures affect the microbiome of a warm-adapted animal, we characterized the bacterial communities associated with house fly (Musca domestica) males raised at cool (18{degrees}C) and warm (29{degrees}C) temperatures. We sampled two distinct genotypes in these experimental flies, each of which is associated with a particular thermal environment (warm or cool). We contrasted our experimental results with the microbiomes we characterized in wild house flies from two collection sites with different large animals present. We found that temperature has a much stronger effect on the house fly microbiome than the host genotype in our experimental flies. Consistent with the strong environmental effects in our experiment, we found that wild house fly microbiomes differed between the two collection sites. Despite these environmental effects on the house fly microbiome, we did not detect evidence for dysbiosis associated with either cool or warm temperatures. We therefore conclude that the environment has more of an effect on the house fly microbiome than host genotype, but dysbiosis does not occur within the temperature range we considered.

Centrohelid heliozoans are a monophyletic group of free-living, ubiquitous, predatory protists widely distributed in aquatic and soil ecosystems. Centrohelids are known as cytotrophic protists that feed on bacteria, algae, and small unicellular eukaryotes. While algal and chloroplast symbioses have been documented in this group, their bacterial associations remain largely unexplored. In this study, we characterize the bacterial communities associated with centrohelids isolated from freshwater habitats using full-length 16S rRNA PacBio sequencing. Amplicon sequencing revealed 5 phyla, 6 classes, and 58 genera in the bacterial communities associated with seven centrohelid isolates. Alphaproteobacteria, Bacteroidia, and Gammaproteobacteria were the most abundant classes, while Arcicella, Sphingobium, Pseudomonas, Sphingomonas, Azospirillum, Shinella, Flavobacterium, Variovorax, and Rhodococcus were the most abundant genera. Notably, Arcicella, Variovorax, Sphingobium, and Pseudomonas constituted the core microbiome. Unexpectedly, we detected bacteria known as opportunistic pathogens, providing the first evidence that centrohelids may serve as environmental reservoirs for bacteria with pathogenic potential (e.g., Acidovorax, Acinetobacter, Anaerococcus, Bosea, Corynebacterium, Escherichia, Moraxella, Mycobacterium, Prevotella, Pseudomonas, Ralstonia, and Sphingomonas). In addition, this study provides the first evidence of Rickettsiaceae associations with centrohelids. IMPORTANCEThis study reveals that centrohelid heliozoans, ubiquitous microbial predators, harbor diverse and host-specific bacterial communities. Critically, we show they can serve as environmental reservoirs for bacteria with pathogenic potential, a role previously overlooked outside of model protist groups. These findings expand our understanding of pathogen ecology, suggesting that a wider range of protists may contribute to the persistence and dispersal of opportunistic pathogens in aquatic ecosystems.

Bilophila wadsworthia is a gut pathobiont implicated in dysbiosis-driven inflammation, yet its pathogenic mechanisms remain poorly investigated. Here, we evaluated the suitability of Galleria mellonella larvae as an in vivo model to study B. wadsworthia infection. Two infection routes were compared: oral inoculation to mimic gastrointestinal colonization and hemolymph injection to model systemic infection. Oral challenge had minimal impact on larval health, whereas hemolymph injection caused marked morbidity, including reduced mobility, impaired cocoon formation, and progressive melanization, indicating that access to the circulatory system is required for overt disease. Infection required live bacteria, with B. wadsworthia capable of intracellular replication within hemocytes, leading to transient depletion of circulating immune cells followed by compensatory hemocyte proliferation. These findings reveal tight coupling between bacterial proliferation and host immune dynamics. Comparison with other sulfidogenic bacteria suggests that Bilophila pathogenicity is likely to involve host-specific interactions. Overall, our results establish G. mellonella as a practical and ethically favorable model to investigate B. wadsworthia virulence, host-pathogen interactions, and mechanisms relevant to gut-associated infection.

Foodborne non-typhoidal Salmonella remains a major public health concern, yet routine surveillance recovers large numbers of isolates from food that are not associated with human illness. Studies have shown foodborne isolates can be genetically linked to clinical cases, highlighting a critical challenge for risk assessment and outbreak prioritisation. This study aimed to determine whether genomic markers can distinguish foodborne Salmonella strains with an increased likelihood of causing infection. Whole-genome sequencing data from over 900 Salmonella isolates recovered from food and the environment through UK Health Security Agency surveillance were analysed using hierarchical clustering to define genetically related groups. These clusters were expanded using the global EnteroBase database to provide broader epidemiological context. Genome-wide association analyses identified genetic markers associated with clusters containing clinical isolates, including phage-associated regions. A highly conserved 7 kb marker identified in S. Agona demonstrated strong predictive performance at a global scale, with high sensitivity and specificity for infection-associated lineages and strict serovar restriction. Comparative genomic analysis revealed that all markers localised to a shared chromosomal hotspot corresponding to a prophage integration site. The 7 kb risk-associated marker formed part of a larger prophage closely related to the well-characterised S. Typhimurium Fels-2 phage, which encodes a DNA invertase linked to phase variation, a mechanism known to promote phenotypic heterogeneity and host adaptation. As these S. Agona isolates are monophasic, our findings indicate that our genome-wide association approach has rediscovered this DNA invertase known to contribute to infection risk but in a different serovar via an alternative regulatory mechanism. Overall, this work demonstrates the potential to move beyond treating all foodborne Salmonella isolates as equivalent hazards, towards a genomics-informed framework for risk stratification. This approach provides a foundation for improved risk-based decision-making, enhance outbreak investigations and enable earlier prioritisation of public health responses during Salmonella surveillance and control. Author summaryFoodborne Salmonella infections remain a major public health concern, but not all strains pose the same risk to human health. Here we investigated whether genetic differences could explain why some foodborne strains are more likely to cause human infection. We analysed over 900 genomes from food and environmental sources, grouping closely related strains before placing them in a global context using EnteroBase. By combining pangenome and genome-wide association analyses, we identified distinct lineages within several serovars that differed in their association with human cases. In Salmonella Agona, all clinical isolates belonged to a single lineage carrying a highly conserved 7 kb marker that was absent from low-risk strains. This marker demonstrated strong sensitivity and specificity across global datasets and was located within a prophage closely related to the well-characterised Fels-2 phage. This region encodes a DNA invertase previously linked to phase variation, a mechanism that promotes bacterial adaptability. Our findings indicate that infection risk can be structured at the lineage level and influenced by mobile genomic elements, particularly prophages, that enhance environmental persistence and host adaptation. This work advances genomic surveillance from retrospective linkage towards mechanistic and predictive risk assessment, with direct relevance for supporting risk-based decision-making during outbreak investigations.

Enteric methane emissions from ruminant livestock contribute to global warming, creating an urgent need for effective mitigation strategies that do not compromise animal productivity and welfare. Methanogenic archaea within the rumen microbiome drive enteric methane emissions. However, large-scale rumen-fluid sampling in commercial production systems is impractical, due to its invasive nature and the associated logistical challenges. This study hypothesised that rumination enables the capture of rumen microbial signals within the oral cavity and using oral microbiome profiles to provide a practical, non-invasive alternative method for proxy methane phenotyping in commercial production systems. To test the hypothesis, we estimated the oral microbiability, defined as the proportion of phenotypic variance in methane emissions explained by oral microbiome variation. Samples were collected from 209 animals across two trials in Queensland, Australia. Oral microbiome samples were obtained from all animals, with paired rumen samples in one trial, and methane emissions were measured using either the sulphur hexafluoride (SF6) tracer technique or the GreenFeed system. Microbial features were characterised using taxonomic and functional annotations, and microbiability was estimated using mixed linear models incorporating microbiome-based relationship matrices. Although the small sample size limited strong conclusions, the oral microbiability estimates reported in this study were comparable to those derived from rumen samples. Functional microbial profiles generally explained a greater proportion of methane variation than taxonomic profiles, suggesting that microbial function is more closely linked to methane production than community composition alone. However, these differences were not statistically significant due to large standard errors. These findings suggest that oral microbiome sampling potentially provides a practical, minimally invasive, scalable proxy method for methane emissions of individual cattle in grazing systems, where direct methane gas measurements are labour-intensive and difficult to implement. Integrating oral microbiome profiles in the existing breeding model with the host genetics, weight and environmental factors could provide a promising pathway for enabling selection for low emissions and advancing reduced emissions livestock farming under real-world production conditions. Lay summaryCattle produce methane as part of their normal digestion and this contributes to climate change. Reducing methane emission in grazing livestock systems is therefore important. However, measuring methane from individual grazing animals is difficult, costly, and often impractical under commercial conditions. The rumen microbiome has been used as a proxy for estimating methane emissions, but collecting rumen samples is invasive and impractical for large-scale use. Because rumination transfers material from the rumen to the mouth, we investigated whether microbes found in cattle mouths could also be used to estimate how much methane an individual animal produced. We suggest that mouth-swab sampling method can be an alternative to rumen fluid sampling because it was less invasive, relatively quick and practically applicable in commercial conditions. Importantly, the microbiome explained a meaningful proportion of the between-animal variation for methane emission. This suggests that collection of mouth swabs is a potentially scalable alternative proxy method to identify cattle that naturally produce less methane. Overall, our findings support the potential use of oral ruminant microbial information to improve breeding and management strategies aimed at reducing methane emissions while maintaining productive livestock systems. Teaser TextThis study demonstrates that collecting oral swabs from the mouths of grazing beef cattle could provide a scalable method to estimate individual methane emissions in commercial production systems, offering a practical alternative to invasive rumen sampling and complex gas measurement systems. These findings support the development of scalable breeding and management strategies for methane mitigation in large-scale livestock production systems.

AimsSponge microbiomes have been extensively studied, in part due to their potential as sources of novel antimicrobials and other biologics, with most research focusing on Demosponges. Here, we investigate the Hexactinellid sponge Pheronema carpenteri, previously identified as a promising source of antibiotic-producing bacteria. MethodsUsing next-generation sequencing of bacterial 16S rRNA genes and a single sponge metagenome, we examined the composition of bacterial communities of P. carpenteri sponges recovered from the Porcupine Seabight, along with local water and sediment samples. ResultsOur results show that P. carpenteri harbours a microbiome abundant in Proteobacteria (47.1-59.4%) and Actinobacteria (11.5-27.5%), with consistent intra-aggregation similarities and structured intra-sponge communities. A metagenomic analysis revealed the presence of several nitrogen cycling genes (nirK, nosZ, nirS homologues of proteobacterial origin), supporting a suggestion that these sponges may play a role in nitrogen cycling, while biosynthetic gene clusters (BGCs) were limited (4 complete clusters). Notably, bacterial community structures within P. carpenteri aggregations resemble those observed in both low and high microbial abundance (LMA/HMA) sponges. ConclusionsHexactinellids are traditionally considered LMA sponges, so identifying species that deviate from this dichotomy provides new insights into sponge microbiome ecology. Integrating Hexactinellids into both culture-dependent and culture-independent studies will advance our broader understanding of sponge-associated microbial diversity and could inform biodiscovery programmes in marine environments. Impact StatementOur findings support the suggestion that a combination of culture-based and molecular analyses is required to generate a comprehensive picture of the biosynthetic potential of P. carpenteri sponges. We also reveal insights into the ecosystem services that sponge microbiomes may contribute towards. These observations could facilitate a deeper understanding of the biotechnological and environmental value of key marine resources.

Myrmecocystus honeypot ants rely on specialized workers, repletes, to store dissolved carbohydrates in their crops long term. The repletes store this liquid, which does not spoil in their crops, for many months at a time. When resources are scarce, repletes redistribute the stored nutrients to their colony members via trophallaxis. While we suspect that the gut microbiome of honeypot ants may aid in spoilage prevention, before we can investigate this, we must first characterize it. Here, we used 16S rRNA gene sequencing to determine the microbial community composition across six Myrmecocystus honeypot ant species, sampling multiple colonies, castes, and organs. We found that microbiome community composition was strongly shaped by species, with variation between colonies in M. arenarius, M. depilis, and M. mexicanus. Organ level differences were observed in the crop and midgut in M. mexicanus. Caste differences were observed in M. flaviceps and M. mexicanus. Replete crops of M. mexicanus and M. depilis were enriched in Fructilactobacillus, other lactic acid bacteria, and acetic acid bacteria, whereas halophiles were more prominent in the gut of species such as M. flaviceps and M. wheeleri. In this study we demonstrate that Myrmecocystus ants host species-specific gut microbiomes and identify an association between lactic acid bacteria, acetic acid bacteria, and halophiles within replete crops. While much work remains in understanding the roles of the microbes in the symbiosis with their host ants, the dominance of these particular taxonomic groups suggests an association with a high sugar environment and a potential microbial role in preventing spoilage of the crop contents.

BackgroundTransduction is a form of horizontal gene transfer in which bacterial DNA is packaged and transferred by virus-like particles (VLPs). Transductomics is a sequencing-based method used to detect DNA carried by VLPs. During transductomics analysis, reads from a samples ultra-purified VLPs are mapped to metagenomic contigs assembled from the same samples whole-community. The read mapping produces coverage patterns that require a time-consuming manual inspection and classification process which makes the methods use unfeasible for datasets with many samples. ResultsWe developed a novel algorithm, TrIdent (Transduction Identification), that uses pattern-matching to automate the transductomics data analysis and that is available as an R package (https://jlmaier12.github.io/TrIdent/). There is no software equivalent to TrIdent so we compared TrIdents classifications of transductomics datasets to classifications made by human classifiers. TrIdents classifications were generally comparable to the manual classifications on a previously generated, manually classified transductomics dataset. When applied to newly generated transductomics data from the murine microbiota, TrIdent agreed with two independent human classifiers as much as the two independent human classifications agreed with each other. TrIdent classified transductomics datasets in a fraction of the time needed by human classifiers, and the classifications produced by TrIdent are fully reproducible. We used TrIdent to explore three murine gut transductomes and found that bacterial DNA associated with the Oscillospiraceae and Turicibacteraceae families was highly enriched in the DNA packaged by VLPs as compared to the whole community metagenomes. ConclusionsThe TrIdent software is a more accessible, more efficient, and more reproducible alternative to the manual inspection of read coverage patterns previously required for transductomics data analysis. To demonstrate the application of TrIdent, we analyzed transductomics datasets from murine fecal pellets and showed that specific low abundance bacterial families appear to be heavily involved in transduction.

Avian influenza virus (AIV) epidemiology is well-documented in temperate regions but remains poorly understood in isolated ecosystems like tropical oceanic islands. On these islands, seabirds nest in dense interspecific colonies where the role of different species as reservoirs and dispersers of AIV may vary greatly. Here, we examine the role of noddies (Anous spp.) as potential reservoirs for low pathogenic AIV and evaluate their potential as sentinel species for highly pathogenic AIV introduction on tropical oceanic islands. We analyzed blood samples from 11 seabird species across eight islands in the southwestern Indian Ocean (2015-2020). Noddies exhibited high, stable seroprevalence (30-45%), comparable to reservoir host species in temperate regions. The detection of two N7-positive noddies, sampled the same year on two distinct islands, provided direct molecular evidence that AIV actively circulates on these island colonies. While most other species showed low exposure, Bridled Terns (Onychoprion anaethetus) had exceptionally high seroprevalence (80%), though their reservoir status requires further investigation due to limited sampling. Given noddies consistent exposure and regional distribution, we recommend prioritizing islands with large noddy populations for AIV surveillance. Continued investigation of viral dynamics within and among islands is now called for to elucidate the ecological drivers of AIV maintenance and transmission.

BackgroundStunting, defined as height-for-age below -2 standard deviations of the WHO child growth standards median, is influenced by nutritional and environmental factors. It remains a public health challenge in Tanzania, particularly in Iringa (prevalence 57%, exceeding the national average of 30%), despite abundant food production. This study explored the gut bacteriome as a potential biomarker for child growth and its association with water, sanitation, and hygiene (WaSH) practices in food-secure settings. MethodsA community-based cross-sectional study (September-October 2024) enrolled children aged 5-23 months in Iringa, collecting fecal samples and household data on growth metrics, WaSH, feeding practices, and illness. The V3-V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq and analysed with QIIME2 and R for alpha and beta diversity, differential abundance (ANCOM-BC), and random forest (RF) modelling. ResultsOverall, 60.5% of 297 children were stunted. Stunting was associated with older age, male gender, discontinued breastfeeding, poor feeding diversity, toilet sharing, and residence location (p < 0.001, p = 0.049, p = 0.001, p = 0.001, p = 0.001, and p = 0.005, respectively). Significant differences in bacterial community composition were observed between stunted and normally growing children (Shannon p = 0.0053; Bray-Curtis p = 0.001). A shared core bacteriome was identified in both groups, influenced by environmental and dietary factors. Normally growing children were enriched with Bifidobacterium, Rothia, Olsenella, Slackia, Lactobacillus, Gemella, and Oscillibacter, while stunted children showed enrichment of Prevotella, Akkermansia, Fusobacterium, Acinetobacter, Alistipes, Odoribacter, Fournierella, and the Ruminococcus torques group. ConclusionAge was the most consistent predictor of gut microbial diversity. Stunting does not appear to be caused by a completely different gut microbiome; instead, shared environmental and dietary factors shape both gut bacteria and child growth. Promoting diverse complementary feeding, continued breastfeeding, and improved hygiene could mitigate risks and inform targeted interventions in food-secure regions.

The microbiota and microbiome associated with zooplankton remains rather understudied compared to other animal groups and/or taxa. The present study aimed at investigating the whole-body bacterial microbiota of Daphnia spp. in two contrasting Greek lakes, the shallow and hypertrophic Lake Koronia vs. the deep and mesotrophic Lake Vegoritida, including both egg-bearing and non-egg-bearing individuals. In both lakes, 2,060 bacterial operational taxonomic units (OTUs) were found, with 223 of them being conditionally rare (crOTUs) with low contribution even for the dominant phyla, with L. Vegoritida having more crOTUs than L. Koronia. The individuals microbiota had inconsiderable overlap with the surrounding water microbiota in both lakes. The two lakes showed significant differences in their Daphnia -associated microbiota. L. Koronia had richer OTUs and rather homogeneous bacterial communities, with higher occupancy. Overall, no significant differences in between the microbiota of egg-bearing and non-egg-bearing Daphnia individuals in both lakes. However, regarding the most important OTUs (miOTUs), the L. Koronia miOTUs were highly overlapped between the individuals with and without eggs, with only one missing from the individuals without eggs. In L. Vegoritida the individuals without eggs had only six miOTUs and while egg-bearing individuals had nine different ones; the two lakes had no shared miOTUs., considerable differences occurred.. A total of 27 miOTUs, was found and belonged to the Pseudomonadota, unclassified Bacteria, Cyanobacteria, Bacteroidota, Bacillota and Actinomycetota. Those miOTUs, where assignment to the genus level was possible, they were related to Cyanobium, Mucilaginibacter, Flavobacterium and Staphylococcus. This study showed that lake morphotype and ecological status can exert some impact on Daphnia-associated bacterial microbiota, with more pronounced effects on egg-bearing and non-egg-bearing individuals.

Severe acute respiratory infections (SARI) are a leading cause of hospitalisation and mortality globally. Many SARI cases remain undiagnosed because kit-based PCR diagnostic panels are typically limited to one or a small number of known pathogens and may fail to identify low-abundance infections or novel, poorly characterised organisms. Here, we used metatranscriptomic sequencing to profile the total infectome of 300 PCR-negative SARI nasopharyngeal samples collected through sentinel hospital-based surveillance in New Zealand between 2014-2021. Our analysis revealed actively transcribing potential pathogens in 43% of SARI cases, comprising 10 RNA viruses, three DNA viruses, nine bacterial species and four fungal species. Notably, co-infections occurred in 26% of cases, revealing polymicrobial infections missed by routine diagnostics. Human rhinoviruses were the most frequently identified, despite not being detected by PCR, and multiple common-cold coronaviruses, human parechovirus A1 and parainfluenza virus type 4, were identified, although these were not included in the PCR screening panel. We also detected a range of bacterial and fungal species and uncovered highly expressed virulence and antimicrobial resistance genes. Infectome composition and diversity were shaped by key demographic and epidemiological factors, with strongest effects observed for age and year of sample collection, indicating that host characteristics and temporal dynamics influence both microbial richness and community structure. These findings highlight the limitations of current diagnostic strategies and the value of metatranscriptomics for comprehensive microbial identification. Integrating such genomic approaches into both clinical and public health frameworks could improve diagnostic accuracy, enabling more sensitive detection and characterisation of potential pathogens while also strengthening surveillance and outbreak response.

BackgroundThe virulence-persistence trade-off is considered a fundamental organizing principle of Listeria monocytogenes population biology: hypervirulent clonal complexes dominate clinical cases but are rarely found in processing environments, while hypovirulent lineages dominate industrial niches but are underrepresented in severe disease. However, whether this dichotomy operates as an absolute paradigm has not been quantitatively evaluated at the population scale. Here we develop a multi-dimensional genomic scoring approach that simultaneously quantifies virulence potential (V), environmental persistence capacity (P), clonal epidemiological context (C), and antimicrobial resistance (R) across 903 genomes from four independent datasets spanning five countries, and apply it to test the universality of the trade-off and to characterize the ecological strategies of L. monocytogenes at the population level. MethodsThe scoring approach integrates four components into a composite 0-100 score through empirically calibrated weights (V: 30%, P: 40%, C: 20%, R: 10%). Validation employed 903 L. monocytogenes genomes from four public BioProjects: longitudinal industrial surveillance in Norway (Fagerlund et al. 2022, n = 513, PRJNA689484), retail environments in the United States (Stasiewicz et al. 2015, n = 191, PRJNA245909), clinical-environmental context in China (Wang et al. 2021, n = 151, PRJNA759341), and meat processing in Poland (Kurpas et al. 2020, n = 48, PRJNA629756). ResultsThe composite score achieved excellent discriminatory performance for identifying persistent clones (AUC = 0.933; 95% CI: 0.910-0.954) with perfect specificity (1.000; zero false positives). The inverse V-P correlation was statistically significant across all four datasets (Spearman {rho} from -0.144 to -0.713; p < 0.01), providing the first cross-dataset quantitative confirmation of the trade-off. However, simultaneous evaluation of V-P profiles at the population scale revealed that the species does not conform to a binary dichotomy but rather exhibits three quantitatively distinguishable ecological strategies, for which we propose a functional trophic taxonomy: nosotrophic lineages (22.7%; V > 65, P < 35), specialized in the pathogenic niche; saprotrophic lineages (5.8%; V < 30, P > 45), with irreversible virulence attenuation and industrial specialization; and, as the central finding, amphitrophic lineages (39.1%; V [&ge;] 35, P [&ge;] 40), which simultaneously retain functional inlA and stress tolerance determinants (SSI-1) without detectable genomic sacrifice. The three strategies differed significantly (Kruskal-Wallis H = 138.7; p = 7.6 x 10-3{superscript 1}). The correspondence between trophic strategy and CC was predominant but not absolute, demonstrating that this phenotypic classification captures intra-CC functional heterogeneity inaccessible through conventional typing. Furthermore, comparison between genome-based and surveillance-informed classifications revealed that 60 hypervirulent isolates (CC1/CC14), genetically classified as nosotrophic, persisted for up to 8 years in industrial facilities despite lacking any recognized persistence markers -- indicating that their prolonged survival reflects environmental opportunity rather than intrinsic genomic adaptation. ConclusionsMulti-dimensional genomic profiling reveals that the virulence-persistence trade-off, while statistically robust, does not operate as an absolute paradigm. The amphitrophic strategy -- documented here for the first time as a quantitatively distinguishable category encompassing 39.1% of the analyzed population -- challenges the prevailing dichotomous model and identifies a previously unrecognized combined ecological niche. The ability to discriminate between genome-encoded persistence capacity and environmentally facilitated persistence provides a biological framework for understanding the ecological determinants of L. monocytogenes population dynamics in anthropogenic environments.

BackgroundPeriodontitis (Perio) is a progressive oral disease characterized by inflammation and degradation of the periodontal apparatus and is associated with local and systemic morbidity including loss of teeth, cardiovascular disease, and diabetes mellitus, among others. Perio is highly prevalent in domestic canines and exhibits certain parallels in pathogenesis and pathophysiology to Perio in humans, although standard treatments are less effective. In both species, a complex interplay between oral microbiota and host immune response is implicated in the etiology of Perio but is not fully understood. ResultsUsing shotgun metagenomics and RNA-seq on oral samples from companion dogs, we identify features of the oral microbiome and host transcriptional profile that are associated with Perio and its progression. We observe differences in microbiota composition between Perio and non-Perio animals that are largely consistent with what has been described in humans but also identify several species that are distinctly associated with canine Perio. We observe an abrupt shift in host gene expression related to immune response and tissue structure that is associated with disease severity, specifically the progression from mild periodontal disease (PD) to more severe Perio and the initiation of clinical attachment loss. The gingival plaque microbiota exhibits a parallel dynamic, with distinct compositional profiles in mild, moderate, and severe PD. We then examine several of the known mechanistic components of the keystone pathogen hypothesis of PD, identifying specific commonalities between canine and human pathologies, including the involvement of Porphyromonas species and related virulence factors. Additionally, we show infiltration of gingival tissue by Porphyromonas and Tannerella spp. via fluorescence microscopy. Finally, we assess correlations between host gene expression and microbial metabolic pathways which suggest additional potential virulence factors. ConclusionsThis work elucidates the metagenomic and transcriptomic signatures of Perio in companion dogs with the goals of informing veterinary medicine, evaluating the potential of canines as a model organism for the study of Perio, and clarifying the relationship between Perio development and progression, the oral microbiota, and the localized host response. Our findings provide insight into the etiopathogenesis of canine Perio and its relationship to human Perio and suggest novel targets of potential translational interest.

BackgroundCholera outbreaks remain a major public-health challenge in sub-Saharan Africa, where diagnostic capacity is limited and clinical case definitions are non-specific and reply heavily on syndromic diagnosis. Rapid identification of Vibrio cholerae is critical, yet cholera-suspected diarrhoea can have multiple infectious causes not captured by targeted diagnostics. MethodsWe evaluated a mobile, culture-independent metagenomic sequencing workflow for on-site detection of gastrointestinal pathogens directly from faecal samples in Burundi. The offline workflow combined long-read ONT sequencing with rapid, laptop-based taxonomic and antimicrobial resistance (AMR) screening and was deployed across a health centre, a district hospital, and a refugee transit camp. The frontline and real-time results were verified using both conventional culturing and in-depth bioinformatic analyses. ResultsV. cholerae signals were only detected in a subset of suspected cholera cases, while many samples were dominated by alternative bacterial taxa, most frequently Escherichia coli. V. cholerae abundance correlated strongly with detection of the cholera toxin phage CTX{varphi}, supporting differentiation between toxigenic signal and background exposure. AMR genes were detected across samples, providing early situational insight into resistance determinants among gastrointestinal bacteria. ConclusionsMobile, offline metagenomic sequencing enables rapid frontline characterization of gastrointestinal disease, especially cholera-suspected, in resource-limited settings and complements existing diagnostics by improving etiological resolution and outbreak response. Author SummaryCholera remains a major cause of severe diarrhoeal disease in many low-resource settings, where diagnosis often relies on symptoms and limited laboratory testing. However, patients suspected of cholera can be infected by a wide range of other pathogens that are not detected by standard diagnostics. In this study, we evaluated a portable, sequencing-based approach that allows direct identification of pathogens from stool samples at the point of care, without the need for laboratory infrastructure, internet access, or culture. Using this approach in multiple settings in Burundi, including a health centre, hospital, and refugee camp, we found a subset of suspected cholera cases were associated with Vibrio cholerae. Other cases were also dominated by other bacteria, particularly Escherichia coli. We also detected antimicrobial resistance genes across samples, providing additional information relevant for treatment and surveillance. Our findings demonstrate that mobile metagenomic sequencing can improve the identification of disease causes directly in outbreak settings and help distinguish true cholera cases from other gastrointestinal infections. This approach has the potential to strengthen outbreak response, improve patient management, and support more accurate disease surveillance in resource-limited environments.

Sourdough starters contain simple microbial communities typically consisting of a few bacterial species and one or two yeast species. The yeast Maudiozyma humilis and the lactic acid bacterium Fructilactobacillus sanfranciscensis often co-occur in sourdough starters, and have been presumed to exist in a trophic relationship supported by glucose cross-feeding. However, previous research has highlighted a lack of evidence showing that yeast strains consume the glucose that F. sanfranciscensis produces. We have investigated the interaction between sourdough isolates of M. humilis and F. sanfranciscensis in a synthetic wheat sourdough medium, allowing us to control substrate composition and use flow cytometry to enumerate living and dead cells. M. humilis fitness was found to be lower in co-culture with F. sanfranciscensis than when grown alone. Analysis of spent medium composition highlighted the reliance of M. humilis on glucose rather than maltose for growth. Comparisons of predicted and measured co-culture metabolite content also revealed that F. sanfranciscensis consumed less maltose in co-culture than when grown alone. For the first time, we examined potential amino acid cross-feeding between M. humilis and F. sanfranciscensis, and found that within the pairing, F. sanfranciscensis was the main producer of amino acids. Our findings suggest that the M. humilis-F. sanfranciscensis interaction is likely to be neutral, or even competitive, with the strain identity of F. sanfranciscensis playing a defining role in the observed dominance of the bacteria and spent medium metabolite composition. ImportanceThe association of the yeast Maudiozyma humilis and the bacterium Fructilactobacillus sanfranciscensis in sourdough starters is well-documented, and together this pairing makes key functional and organoleptic contributions to the final bread product. Their relationship has historically been thought to be stabilised by cross-feeding of glucose to M. humilis. However, this theory has been drawn into question by recent research which found no evidence that M. humilis consumes the glucose produced by F. sanfranciscensis. Our understanding of cooperation, coexistence, and competition in microbial consortia affects approaches to ecosystem management in a broad variety of applied fields. The significance of our research is in demonstrating that this pairing does not interact mutualistically within a specified setting, providing support for neutral or competitive interactions as drivers of ecological stability. Research areas:

Despite their importance for individual fitness and population processes, the microbiota of many ecologically significant insects remains poorly explored. Even less is known about the interactions between microbial communities inhabiting insects and their surrounding environment. Ant infrabuccal pockets (IBPs), representing the interface between the digestive tract and the external environment, provide an opportunity to study these interactions. Here, we aimed to characterize ant-microbial interaction networks in the forest floor by profiling fungal and bacterial communities associated with the IBP of Formica polyctena ants, known as ecosystem engineers in temperate forests. We used direct microscopy, culturing, and sequencing amplicons of ITS1, ITS2, 18S rRNA marker regions to describe fungal communities, and 16S rRNA metabarcoding to characterize bacterial communities. Classical methods combined with a multi-marker amplicon sequencing allowed for a comprehensive description of the IBP microbiota. Fungal communities consistently contained representatives of 15 ecologically diverse genera, including insect-associated yeasts and primarily saprotrophic or endophytic fungi. Bacterial communities were dominated by genera previously reported from ant guts, mainly Bacilli and Alphaproteobacteria, and showed greater stability among ant colonies than fungal communities. Further studies on red wood ants IBP microbiota would enhance our understanding of their role in shaping ecological networks in forest ecosystems.