Frontiers in Microbiology

Methanogens, a diverse group of archaea that produce methane during energy metabolism, are prevalent in anoxic habitats where they are exposed to viral infections. The head-tailed virus {Psi}M2 is one of few virus isolates that infect a methanogen host and was described as a lytic virus that lyses its host, Methanothermobacter marburgensis. Here, we present evidence that {Psi}M2 does not exclusively follow the classic lytic lifecycle but can also enter an alternative, chronic infection-like state. Growth experiments showed that infected cultures in minimal medium, optimized for the methanogen host (MS medium) did not exhibit complete lysis but continuously released infective virions. In contrast, in a suboptimal complex medium (SAB medium), the {Psi}M2-infected culture released virions in the exponential growth phase and eventually lysed after 14 hours post infection. Although the genome of {Psi}M2 encodes a putative integrase, a prerequisite for lysogeny, whole genome sequencing of {Psi}M2-infected M. marburgensis cultures revealed that the viral DNA remained extrachromosomal rather than integrating into the host genome, likely due to a mutation in the integrase gene. These findings suggest that {Psi}M2 enters a non-lytic lifecycle alternative to lysogeny under suboptimal host growth conditions. Sequencing further identified a heterogeneous viral population including previously undescribed variants. A high density of non-synonymous mutations and frameshift-inducing insertions was observed within ORFs predicted to encode a capsid protein and a major spike protein, suggesting these viral variants to differ in host recognition and infectivity. Overall, our results show more complex viral behavior and greater genetic variability than previously described.

Marine microbial communities vary seasonally and spatially, shaped by physiochemical gradients and biotic interactions. Spatiotemporal variability of seawater microbiomes in tropical coastal regions is comparatively understudied, especially in Southeast Asia with high pressures from development and anthropogenic activities. Here, we examined the microspatial and temporal variability of bacterioplankton in the Singapore Strait, which is a tropical system in Southeast Asia influenced by monsoons. By applying total community amplicon sequencing to three particle size fractions reflecting microspatial habitats of seawater (0.22-1 {micro}m ''free-living'', 1-10 {micro}m ''large cells and small aggregates'' and 10-150 {micro}m ''particle-associated''), we show that the seawater microbial community was structured by their microspatial habitats, monsoon seasons and physiochemical variables. Monsoon-driven alternation of water masses originating from the South China Sea to the East and from off the Sumatran coast to the West resulted in a distinct community during the Southwest monsoon across all three size fractions. Total DNA (biomass) was lowest in the 10-150 {micro}m fraction, but these particle-associated sequences had the highest diversity and were surprisingly enriched with picocyanobacterial and some pathogenic taxa, potentially reflecting aggregate formation and/or host-derived particle association. This fraction also exhibited greater temporal variability than the two smaller fractions, which could be related to a more heterogenous environment in particle microhabitats. Overall, this study demonstrated compositional variability in the bacterioplankton across space and time in an equatorial region influenced by monsoons.

Stratified benthic deposits offer crucial modern analogs for Precambrian biogeochemical dynamics, yet whether the vertical organization of deep sedimentary columns reflects depositional history, present-day geochemical conditions, or both remains poorly understood, particularly under extreme oligotrophy. Here, we present a depth-resolved exploratory survey of a continuous 34-cm sediment core partitioned into 11 discrete horizons (L1 to L11) from Poza Roja 2, an alkaline, sub-hypersaline pond in the Cuatro Cienegas Basin, Mexico. Integrating physical stratigraphy, organic carbon and nutrient stoichiometry, and 16S rRNA gene metabarcoding, we evaluated down-core microbial community structure alongside acute phosphorus scarcity. Molar C:Po and N:Po ratios exceeded canonical Redfield proportions (106:16:1) in every layer, by up to 48.5-fold and 87.8-fold, respectively, both peaking in layer L4. Unconstrained ordination (PCoA) and hierarchical clustering grouped the layers into three compositional zones: upper (L2-L4), intermediate (L5-L6), and basal (L8-L11), with L1 and L7 occupying transitional positions. Archaea accounted for 31.0% of prokaryotic reads, ranging from 8.3% (L5) to 43.7% (L9), and were dominated by SM1-50 (Thermoplasmatota) and Halobacteriota. The phototrophic heliobacterium Heliorestis peaked in the intermediate zone (47.6% of bacterial reads in L6). Constrained Redundancy Analysis models lacked statistical significance (P >= 0.12), highlighting the exploratory nature of a single core (n = 11). This pilot characterization provides a baseline for understanding deep biogenic archives under severe nutrient limitation.

Accelerated retreat of the Greenland Ice Sheets is increasing freshwater discharge to coastal fjords and promoting transitions from marine-terminating glacier (MTG) regimes to land-terminating (LTG) ones. These changes strongly affect the physical and chemical features of the water column, which in turn impact the structure and functioning of fjord ecosystems. We used high-throughput imaging and DNA metabarcoding approaches to investigate seasonal (spring vs summer) differences in the structure of pelagic microbial communities in two contrasting fjord systems (MTG vs LTG) in southwest Greenland, with focus on the bacterial and heterotrophic protist components, which to date remain understudied in Greenland fjords. Our analyses revealed a marked seasonal restructuring in the microbial communities, especially in the surface layers. In spring, heterotrophic community composition in both fjords manifested a strong link with the diatom spring bloom, being dominated by typical phytoplankton-associated and copiotroph bacterial groups that can utilize algal-derived organic matter, and protists grazing on phytoplankton. In summer, heterotroph communities strongly diverged between the two fjords, most likely caused by the differential impact of MTG vs LTG glaciers on water column structure and chemistry. There was a marked increase in pico- and nano-sized heterotrophs, parasitic protists (dinoflagellate Syndiniales), and bacterial groups indicative of low-nutrient environments. In addition, bacterial, phytoplankton and heterotrophic protist communities become more strongly coupled, either through stronger interactions and/or stronger environmental filtering acting on all groups simultaneously. These observations are indicative of a shift from resource-replete systems in spring to more resourced-depleted systems in summer. However, this shift was less pronounced in the MTG fjord, where subglacial upwelling enabled the persistence of diatom bloom related communities into summer. Finally, we uncovered the presence of a unique, ice melange associated inner fjord microbial community, which shows similarities with both supraglacial and sea-ice associated communities, in the inner part of the MTG impacted fjord.

Anaerobic gut fungi (AGF) are an integral part of the herbivore gut microbiome, primary decomposers of lignocellulose, and play a crucial role in establishing foundational links of syntrophic associations. The syntrophy with methanogens appears beneficial for mitigating feedback inhibition and results in the augmentation of AGF activity. However, the thermodynamic advantages of the presence of methanogens to AGF remain poorly understood. This study explores the preferred pathways adopted by Cyllamyces sp. and Methanobrevibacter olleyae, using 13C-glucose as a substrate and spiked with malate and pyruvate in different experimental set-ups. The results indicated reduced biomass formation by Cyllamyces sp. in monoxenic culture with Methanobrevibacter olleyae compared to axenic AGF culture supplemented with glucose. Additionally, metabolic analyses revealed the pyruvate:ferredoxin oxidoreductase (PFO) as a key contributor to pyruvate metabolism. Malate supplementation notably enhanced the growth of Cyllamyces sp. and M. olleyae, whereas pyruvate supplementation enhanced the growth of Cyllamyces sp. only in axenic culture. This investigation provides insights into the preferred metabolic pathways operating in the hydrogenosomes of axenic anaerobic gut fungi (AGF) cultures and monoxenic AGF-methanogen co-cultures.

Acinetobacter sp. strain Tol 5 is an attractive microorganism because of its ability to utilize various aromatic compounds, including toluene. However, because Tol 5 is a non-model microorganism, the utilization of carbon sources in central carbon metabolism remains unclear. In this study, the activity of the central carbon metabolism was characterized using 13C-metabolic flux analysis (13C-MFA). Tol 5 was cultured in synthetic medium using ethanol or acetate as the sole carbon source. In addition, because toluene is degraded into pyruvate and acetyl-CoA via the toluene dioxygenase pathway, this situation was mimicked by culturing with ethanol and lactate as co-substrates. The 13C-MFA confirmed flux through the glyoxylate shunt, which bypasses part of the TCA cycle when either ethanol or acetate was used as the sole carbon source. In ethanol, the flux ratio of isocitrate dehydrogenase at the isocitrate branch point decreased markedly. Unlike acetate, the conversion of ethanol to acetyl-CoA is accompanied by NADH production. Therefore, the observed change in this flux ratio was influenced by the excess NADH generated. Under ethanol-lactate conditions, Tol 5 simultaneously utilized ethanol and lactate. The ethanol consumption rate decreased to less than half of that observed when ethanol was the sole carbon source. Most of the lactate uptake was converted to pyruvate and metabolized via the TCA cycle. The glyoxylate shunt flux was lower than that in ethanol. These metabolic characteristics provide valuable insights into the use of Tol 5 as a next-generation microbial cell factory.

Microbial biological control agents (mBCAs) offer a promising alternative to conventional disease management, but identifying effective strains requires screening large microbial collections against many pathogens. Here, we developed a scalable screening approach to characterize the antagonistic activity of potato-associated bacteria against multiple pathogens and investigated how bacterial origin and phylogeny relate to antagonistic phenotypes. A collection of 600 bacterial strains isolated from two potato cultivars, three plant compartments using four cultivation media was screened against six major potato pathogens: the bacteria Dickeya solani and Pectobacterium carotovorum, the fungi Alternaria solani and Rhizoctonia solani, and the oomycetes Phytophthora infestans and Pythium ultimum. A scalable confrontation method was developed for non-filamentous bacteria, while complementary lower-throughput assays were used for filamentous strains. This approach enabled successful assessment of 92% of non-filamentous and 97% of filamentous strain-pathogen combinations. Isolation cultivar, plant compartment and cultivation medium had only limited effects on the proportion of antagonistic strains. In contrast, pathogen sensitivity varied markedly: P. infestans was the most susceptible pathogen, whereas D. solani and P. carotovorum were generally resistant to bacterial antagonism. Antagonistic activity clustered phylogenetically, with Bacillus and Streptomyces containing many broad-spectrum inhibitors. In addition, several taxa showed preferential activity against the oomycetes, including P. infestans-specific antagonists from the Pseudomonas genus and P. ultimum-specific antagonists from the Frigoribacterium, Curtobacterium, Pedobacter and Phyllobacterium genera. Overall, multi-pathogen screening revealed distinct generalist and specialist antagonistic profiles and identified candidate strains and taxa for further evaluation as mBCAs.

The human gut is home to more than 100 trillion bacteria, where various environmental factors, such as nutrients and gases, exist and influence them. The levels of fermentation gases in the gut, including CO2, fluctuate depending on the host's diet. However, how CO2, an environmental factor, affects the physiological states of gut anaerobes remains poorly understood, but CO2 is often utilized in their cultivation. Akkermansia muciniphila (A. muciniphila) is widely studied as a beneficial human gut microbe. However, the effects of CO2 on the physiological state of A. muciniphila are not fully understood. In this study, we focused on CO2 as an environmental factor and aimed to clarify how CO2 affects the physiological state of A. muciniphila. Culture experiments showed that compared with the absence of CO2, the presence of CO2 significantly increased the growth of A. muciniphila. The results of the metabolomic analysis revealed distinct metabolic profiles in the presence or absence of CO2. The tracing analysis using stable 13C-labelled CO2 demonstrated that CO2 was incorporated into various metabolites in the TCA cycle, the de novo purine/pyrimidine synthesis pathways, and other pathways. In addition, the activity of pyruvate carboxylase, which catalyses an anaplerotic reaction involving CO2 fixation to supply oxaloacetate (OAA) to the TCA cycle, was significantly higher in A. muciniphila cells grown in the presence of CO2. Moreover, supplementation with OAA increased the growth of A. muciniphila in a dose-dependent manner, mirroring the effects of CO2 supplementation. These findings suggest that A. muciniphila utilizes CO2 for an anaplerotic reaction in the TCA cycle and other pathways involving CO2 incorporation, facilitating metabolic flux to promote its robust growth.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

Spontaneous vegetable fermentation is driven by the indigenous microbiota associated with the raw ingredients. The process often involves an initial heterofermentative phase followed by a predominantly homofermentative phase, both ensured by lactic acid bacteria (LAB). While microbial dynamics during fermentation are well characterized, phage communities remain poorly understood, despite evidence linking phage activity to LAB succession. We monitored viral and bacterial succession during sauerkraut and carrot fermentations using a metagenomics approach to characterize community dynamics. To further assess the impact of phage PLE001 on bacterial community dynamics, we developed a simplified carrot fermentation model comprising representative strains of Rahnella aquatilis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum. Two consortia were assembled: one containing the PLE001-susceptible strain L. mesenteroides CIM3004, and the other containing also its phage-resistant derivative, CIM3004 BIM. We observed major shifts in the viral community during the first week of fermentation, coinciding with changes in bacterial structure during hetero- and homofermentative phases transition. In the simplified model, phage PLE001 reduced the population of the host during the heterofermentative phase but did not affect overall fermentation dynamics nor metabolite production. These results suggest that bacteriophages contribute to bacterial succession during fermentation, while communities remain resilient to phage infection, preserving fermentation stability.

Mangrove reforestation is expanding across the Caribbean, but restoration success is still judged almost exclusively by tree survival and canopy cover. Whether the sediment microbiome, which governs carbon burial and nutrient cycling, follows the recovery of the vegetation remains largely unknown. We combined 16S rRNA gene metabarcoding (V4, Illumina MiSeq) with CHNS elemental analysis, pH and salinity measurements to compare sediment prokaryotic communities along 0-100 cm profiles in a reference, a reforested (Rhizophora mangle plantings since 2019) and an altered mangrove within the Las Dunas de Las Calderas Natural Monument, Dominican Republic (39 cores pooled into 28 depth-resolved composite samples). Reference sediments contained 2.5-5.5-fold more organic carbon (median 7.5% vs 3.0% in altered and 1.3% in reforested sediments), had higher C/N ratios and were less alkaline (pH 8.2 vs 8.8). Reference communities were more diverse and even, and differed in composition from both disturbed stands (pairwise PERMANOVA, R^2 ~ 0.09, adjusted P = 0.0015), whereas reforested and altered communities were statistically indistinguishable (R^2 = 0.04, P = 0.25). Disturbed sediments were dominated by Cyanobacteriota (~41-45% of reads), which tracked high pH, whereas reference sediments were enriched in Chloroflexota, Thermodesulfobacteriota and Thermoproteota, lineages linked to anaerobic degradation of complex organic matter and sulfate reduction. Depth structured communities by separating surface from subsurface layers, and sediment organic carbon and nitrogen were the only significant constraints in distance-based redundancy analysis. These results indicate that, within a few years of the first plantings (2019), reforestation had not restored the carbon-rich, anaerobic sediment habitat of the reference forest. We propose sediment organic carbon and microbial community structure as complementary indicators for mangrove restoration monitoring in the Caribbean.

Carbohydrate inducible promoters on expression plasmids are widely used for biotechnology and basic science research. However, using carbohydrates for inducers has drawbacks including influencing bacterial metabolism, the inducer being used as a carbon source by the bacteria, high background expression, and only a subset of cells experiencing promoter activation. Furthermore, some bacteria including the pathogen Acinetobacter baumannii currently lack good carbohydrate-inducible promoters. To address this issue, a tetracycline-inducible promoter, PtetB, isolated from A. baumannii was cloned and evaluated in a number of important bacteria: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Serratia marcescens. A highly sensitive luxCDABE reporter was used to evaluate promoter activity. S. marcescens strains with elevated and reduced cyclic-AMP levels were used to evaluate the impact of cAMP on PtetB function. The PtetB promoter was induced over 800-fold in A. baumannii and 10-750-fold inducible in the other tested species. Background expression was lower from PtetB in the pseudomonads than previously described carbohydrate inducible promoters. Data indicated that the PtetB promoter system may be a useful tool for future basic science research with the tested bacteria.

Due to their highly polyphagous capacities, black soldier fly (Hermetia illucens) larvae (BSFL) are increasingly valued for their ability to convert organic waste into valuable biomass that can be used for a variety of purposes. These remarkable digestive capabilities are highly dependent on an extremely plastic gut microbiota. However, the distribution and functioning of bacterial communities in the various gut compartments - particularly in the hindgut - remain little understood. In this study, we used a metabarcoding approach based on 16S gene sequencing to investigate the effect of three carbohydrate-rich diets with distinct molecular compositions on the functional diversity of the BSFL gut microbiota. Our results showed that the midgut harbors a highly substrate-sensitive microbiota, with a high abundance of Actinomyces spp., regardless of the substrate. A bacterial diversity oriented toward fatty acid biosynthesis pathways is promoted by starch-rich environment, whereas a lignocellulosic substrate fosters a midgut microbiota dominated by Paenibacillus spp. In contrast, the hindgut exhibits a distinctly stable and homogeneous bacterial composition dominated by Dysgonomonas spp. Overall, our results provide clear evidence of a two-compartment microbial system, in which the midgut primarily serves as a substrate-adaptive primary degradation chamber, while the hindgut functions as a stable terminal compartment for the final processing of residual substrates and the recycling of nutrients. These findings contribute to our understanding of the functional diversity of the bacterial microbiota along the BSFL digestive tract, which is a key factor in explaining this insect's remarkable polyphagous behavior and optimizing its use for industrial purposes.

Little is currently known about the regulation of PL fimbriae produced by some pathogenic E. coli strains. We identified PlfB, a transcriptional regulator of the PapB family, as a negative regulator of PL fimbriae (Plf). Deletion of plfB significantly increased plfA transcription and Plf production. PlfB bound specifically to the plfA promoter region. Using a PplfA-lux reporter system, the deletion of plfB caused sustained and increased expression across serial passages. Global regulatory genes such as lrp, argR, and hns were also implicated in regulation of plf expression. Loss of lrp or argR decreased plf expression, suggesting these regulators positively regulate PL fimbriae. By contrast, loss of hns increased levels of plf expression, supporting its role as a negative regulator. Interestingly, loss of plfB significantly increased bacterial colonization in the lung of 6-day-old turkey poults. Further, complementation of plfB restored plf fimbrial expression and colonization of turkey lungs to wild-type levels. This further supports an important role for PL fimbriae for colonization of turkey lungs, since loss of plfB which resulted in increased plf gene expression in turkey lungs also correlated with higher bacterial numbers in the lungs. Taken together, these results indicate that PlfB acts as a negative regulator to reduce levels of PL fimbrial expression, under some conditions, but that it is not required for plf expression. This contrasts with the role the PapB regulator, which has been shown to play an essential role in expression of P fimbriae in uropathogenic E. coli strains.

Abstract The Bacillus cereus group is a complex of closely related species, which vary in terms of their geographic spread, industrial utility, and pathogenic potential. In addition to encompassing well-known species represented by thousands of genomes (e.g., bioterrorism agent B. anthracis, foodborne pathogen B. cereus, biopesticide B. thuringiensis), the B. cereus group houses "rare" species discovered/first sequenced over a decade ago. Until recently, four rare species (B. manliponensis, B. bingmayongensis, B. gaemokensis, "B. clarus") were each represented by a single unique genome; as such, virtually nothing is known about these species beyond their type strains. We hypothesized that additional representatives of rare B. cereus group species may already be present in public databases as yet-unassembled metagenomes. To this end, we used species-specific markers to identify 7 metagenomic samples in NCBI's Sequence Read Archive, which harbored B. manliponensis, B. bingmayongensis, or B. gaemokensis. Metagenomic assembly and binning yielded an isolate-quality B. manliponensis metagenome-assembled genome (MAG) from avocado orchard soil in Israel, plus a partial B. bingmayongensis MAG from commercial spinach spiked with Shiga toxin-producing Escherichia coli. For rare species represented by >1 unique genome, we compared our novel MAGs to all available genomes and identified strain- and lineage-specific genomic elements (e.g., sphingomyelinase C and hemolysin BL homologs, variably present in B. manliponensis and B. bingmayongensis, respectively). Altogether, our study indicates that some rare B. cereus group species may be more common and geographically widespread than previously thought, and highlights the utility of culture-independent methods for recovering rare/under-isolated taxa. Impact Statement Individual B. cereus group strains vary in terms of their pathogenic potential and industrial utility. While some strains cause illness in humans and/or animals (e.g., anthrax, emetic intoxication, diarrheal toxicoinfection, non-gastrointestinal illnesses), other strains have important biotechnological applications (e.g., biocontrol, plant growth promotion, production of antimicrobials and other natural products). Because many virulence- and biotechnological-related phenotypes are strain-dependent (rather than species-dependent), phenotypes associated with an individual strain (e.g., a species type strain) cannot be extrapolated to an entire B. cereus group species. Within this context, rare B. cereus group species represent a major gap in our understanding of B. cereus group evolution, ecology, and pathogenicity, as until 2021, each rare species was represented solely by its type strain genome. Here, we aggregate all publicly available genomes/MAGs of rare B. cereus group species and supplement them with their first novel representatives since 2024. Our efforts bring the total number of unique B. manliponensis and B. bingmayongensis genomes/MAGs to >2, allowing us to conduct the first-ever phylogeographic survey and strain-level assessment of functional potential for each species. Overall, our study represents a critical first step toward understanding rare B. cereus group species beyond their type strains.

Gut microbiome plays an important role in human health. Urbanization has been suggested to alter the gut microbiome. We comprehensively evaluated differences in gut microbiome profiles of 76 urban and 81 rural female residents of Vietnam using shotgun metagenomic data. We evaluated rural-urban differences in gut microbial diversity, individual microbial taxa, and metabolic pathways. Rural residents had higher alpha and beta diversities in age-adjusted models (p<0.05). Additionally, significant rural-urban differences were observed for 35 microbial taxa in age-adjusted models (FDR<0.1). Rural residents had higher relative abundance of classes, orders, and families of FGB2982 and FGB38642 and strain SGB15332 of the species Faecalibacterium prausnitzii, whereas urban residents had higher relative abundances of species Bacteroides thetaiotaomicron, Coprobacter fastidiosus, Bilophila wadsworthia, Enterobacter cloacae, Enterobacter hormaechei, Klebsiella aerogenes, Dielma fastidiosa, and Holdemania massiliensis. Notably, these associations were attenuated after further adjustment for socioeconomic characteristics, lifestyle, dietary factors, and comorbidity (FDR>0.1). In the fully adjusted model, significant rural-urban differences were observed in the abundance of 17 microbial taxa, including Ruminococcus sp AF13 28 and Prevotella pectinovora, as well as in three metabolic pathways (FDR<0.1). This study suggests that urbanization-related factors may contribute to differences in the gut microbiome between urban and rural Vietnamese women.

Skin ulcers, such as those observed in cases of the non-venereal disease yaws and the venereal disease syphilis, are traditionally diagnosed based on their anatomical location and visible appearance. Yet various evidence suggests that the pathogens that cause these ulcers may not be restricted to genital or non-genital body sites. We explored this by profiling the bacterial communities of genital and non-genital skin ulcers collected in Ghana using 16S rRNA gene amplicon sequencing. Swab samples were collected from 156 clinically diagnosed skin lesions (110 non-genital and 46 genital). The V1-V2 region of the 16S rRNA gene was sequenced to characterize bacterial communities. Microbial diversity, community composition, differential abundance, and associations between lesion diversity and common skin bacteria were analysed using bioinformatics and statistical methods. Following quality filtering, 135 samples were retained, revealing 23 bacterial phyla, 143 families, and 406 genera. Alpha diversity indicated similar bacterial richness and diversity in genital and non-genital skin lesions. Beta diversity analyses indicated differences between genital and non-genital lesions, accompanied by significant differences in within group dispersion. A log2 fold change analysis evidenced genital lesions were enriched in Gardnerella, whereas non-genital lesions showed more abundance of Staphylococcus, Streptococcus, Corynebacterium, Bradyrhizobium, and Pelomonas. Clinically important genera, including Treponema, Haemophilus, and Mycobacterium, were also detected, even in samples with low sequencing depths. Statistical modelling indicated that greater microbial diversity was associated with intermediate abundances of common skin taxa, particularly Corynebacteriaceae, Micrococcaceae, Cutibacterium, and Staphylococcaceae, thereby attesting the presence of polymicrobial communities in skin ulcers. Overall, genital and non-genital skin lesions showed similar microbial diversity but differed in composition. Our findings support the view of skin lesions as ecological niches containing diverse bacterial communities rather than being consistently dominated by a single bacterial taxon. They provide a basis for investigating possible polymicrobial interactions in skin ulcers.

Histomonosis, caused by Histomonas meleagridis, is a severe disease in poultry with main pathological lesions in ceca and liver, leading to significant animal suffering and economic losses, with no licensed treatments available. While cecal pathology and microbiota disruption have been documented, the metabolic consequences of infection and vaccination remain poorly understood. Here, we used untargeted LC-MS metabolomics to profile cecal tissue and luminal digesta in a controlled vaccination-challenge chicken trial (n=100, 25 birds/group), including uninfected and unvaccinated but challenged controls, together with birds vaccinated either cloacally, or orally via an edible gel following challenge, across five post-challenge timepoints. Results showed that H. meleagridis infection caused extensive, time-dependent disruption of the cecal metabolome, especially at 7-14 days post-infection (DPI). At DPI 14, 325 tissue and 359 digesta metabolites were significantly altered in unvaccinated challenged birds. Infection was associated with depletion of microbiota-related metabolites, including dicarboxylic acids, tryptophan indoles, phytochemical derivatives, bilirubin catabolites, phenolics, vitamins, and secondary bile acids, alongside accumulation of microbial substrates and fermentation products. Changes in the host-associated metabolites indicated mucosal inflammation, epithelial injury, mitochondrial and amino acid metabolic disturbance, oxidative stress, and membrane lipid remodeling. Cloacal vaccination provided greater metabolic protection than oral vaccination, with 78.3% of disrupted metabolites preserved at DPI 14, compared with 60.7% vaccination by oral gel delivery, pointing towards the importance of vaccine take. By DPI 21, cloacally vaccinated birds showed nearly complete metabolic recovery across most pathway groups, whereas gel-vaccinated birds retained broader metabolic disturbance. These findings reveal histomonosis as a disruption of the cecal host-microbiota metabolic interface and provide a metabolite-level framework for future studies of pathogenesis, vaccine development, and prevention strategies.

The molecular epidemiology of Acinetobacter, which is almost exclusively culture-dependent, has centered mainly on clinical isolates of A. baumannii. Yet, this overlooks sources not amenable to microbial culture and many non-A. baumannii species. Here, by analysing metagenomic projects from public transport systems worldwide, we show that metagenome-assembled genomes (MAGs) are highly relevant for assessing the unrecognized diversity of Acinetobacter. We recovered 839 Acinetobacter MAGs, with 269 being high-quality MAGs. The high-quality MAGs were across more than 20 well-defined Acinetobacter species; however, five species (A. nosocomilalis, A. ursingii, A. radioresistens, A. schindleri, and A. variabilis) had the most MAGs. Pangenome and phylogenetic diversity analyses of 685 genomes plus the 269 high-quality MAGs from those 5 species showed that MAGs are an important source of both lineage and gene diversity. Also, FST analyses and genome phylogenies show that for some species there is no genetic differentiation between MAGs and clinical isolates, implying transmission between hospitals and public transport settings. In addition to finding the antibiotic resistance genes (ARGs) in MAGs, we implemented an ad hoc strategy (leveraging the use of genomes) to recover considerably more ARGs. PCoA and PERMANOVA analyses show that, whereas in A. nosocomialis and A. ursingii, the ARG profiles of MAGs and clinical genomes are different, in A. radioresistens and A. schindleri, the ARG profiles are not different. Finally, some clinically important ARGs, such as sul1 and sul2, aadA, and blaOXA23, were found in all or some of the 5 species. Our study shows how combining MAGs and regular genomes is a powerful strategy for better appraising the genomic diversity (including the resistome) of Acinetobacter. On a broader level, our study showcases the importance of MAGs for genomic surveillance of man-made environments with clear public health implications.

Human rhinovirus (HRV), a major cause of upper respiratory infections, has increasingly been associated with lower respiratory tract disease. Although tools to investigate HRV pathogenesis are needed, they remain limited. Reporter viruses expressing fluorescent or luminescent proteins are powerful tools for analyzing viral distribution and replication; however, insertion of a reporter gene often impairs viral replication, and the inserted gene is readily lost during serial passage. To address these problems, we established a versatile and stable strategy for generating reporter HRV and other enteroviruses. The gene encoding mScarlet3, a red fluorescent protein, was inserted between the 5'UTR and VP4 gene, a site previously used in other enteroviruses but not in HRV. Optimizing the 2A cleavage sequence between mScarlet3 and VP4 yielded a reporter HRV with growth kinetics comparable to those of the wild-type virus. To improve the genetic stability of the reporter insert, we evaluated GC-content optimization of the reporter gene and relocation of the cis-acting replication element (CRE). GC-content optimization substantially improved reporter stability in both HRV-B14 and EV-A71, whereas CRE relocation provided additional stabilization in EV-A71 but not in HRV-B14, demonstrating the potential applicability of this strategy to other enteroviruses. This strategy for constructing stable reporter HRV and other enteroviruses will facilitate studies of enterovirus pathogenesis.