npj Biofilms and Microbiomes

Bacteria exhibit two lifestyles: planktonic free-floating individual cells or sessile multicellular aggregates known as biofilms. The biofilm lifecycle is characterised by three distinct stages: attachment, maturation and dispersal. Distinct adaptations occur in each stage, determining cellular behaviours such as surface attachment or synthesis and degradation of extracellular matrix components. Characterising stage-specific bacterial profiles therefore represents a valuable strategy for the development of novel antibiofilm therapies. Here, we used the model biofilm-forming bacterium Pseudomonas aeruginosa PAO1 to characterise the transcriptional profiles of each stage of the biofilm life cycle: attachment, biofilm maturation and spontaneous dispersal in closed cultures. We report that surface attachment was accompanied by the upregulation of genes comprising the Pil-Chp mechanosensory system, whereas biofilm maturation was characterised by the upregulation of genes involved in Pel polysaccharide synthesis, siaD and PA4396 diguanylate cyclases as well as pipA, fimX and PA5442. In contrast, dispersing cells upregulated genes responsible for the biosynthesis of alginate, rhamnolipid, and extracellular nucleases (eddA, eddB), as well as the transcriptional regulator of dispersal amrZ. Additionally, genes encoding the spontaneous dispersal molecule cis-2-decenoic acid (dspS and dspI), canonical phosphodiesterases (nbdA and rbdA), four non-canonical HD-GYP phosphodiesterases and seven other c-di-GMP-related enzymes were also upregulated during dispersal. Our comprehensive analysis of transcriptional changes across biofilm stages therefore provides benchmarking stage-specific transcriptional profiles for P. aeruginosa biofilms in closed culture systems. Furthermore, it allowed the identification of a subset of fourteen genes as transcriptional biomarkers of dispersal, which were used to build reporter plasmids as tools to determine the onset of dispersal. ImportanceBiofilm infections by P. aeruginosa are a major medical challenge due to the increased tolerance to antimicrobials displayed by bacteria living in sessile communities, which is reduced during spontaneous biofilm dispersal. Attachment, biofilm maturation and dispersal represent the main stages of a dynamic process known as the biofilm lifecycle. However, the global regulatory responses governing transitions between these stages remain understudied. Here, we combine live microscopy and biomass quantification to track the progression of P. aeruginosa cultures through the three main stages of the biofilm lifecycle. We show that cells from each stage recapitulate canonical, stage-specific transcriptional responses and identify a set of biomarkers associated with the onset of dispersal. These biomarkers may offer a practical tool for rapidly screening dispersal-inducing compounds, aiding in the discovery of the next generation of antibiofilm therapeutics.

1.Fermentable fibres such as inulin can support metabolic health but may exacerbate gastrointestinal symptoms in individuals with irritable bowel syndrome (IBS) due to rapid fermentation and gas production. The gel-forming fibre psyllium improves IBS symptoms, although the underlying mechanisms remain unclear. We hypothesised that fibre gelation alters fermentation by modulating microbial access to substrates. To test this, we compared psyllium with methylcellulose, a chemically modified, gel-forming fibre, to determine the effects of gelation on inulin fermentation. Inulin alone or combined with psyllium or methylcellulose was fermented for 48 hrs in a colonic fermentation model inoculated with healthy human faeces. Gas production, metabolite profiles, microbial community composition and microbial localisation within fibre gels were assessed. Bioactivity of fermentation products was evaluated in STC-1 cells. Psyllium co-fermentation significantly accelerated fermentation and enhanced production of metabolites, while methylcellulose had minimal effects. Psyllium maintained higher diversity and enriched polysaccharide-degrading taxa including Bacteroides and Phoecaeicola species, which were strongly associated with metabolic activity. Bacterial penetration into the psyllium matrix was observed but not into methylcellulose. Fermentation products from psyllium but not methylcellulose stimulated GLP-1 and 5-HT secretion in STC-1 cells. These findings demonstrate that delayed-onset fermentable gel-forming fibres enhance microbial access to entrapped substrates, driving metabolic and hormonal responses.

The cervicovaginal microbiome is pivotal to reproductive health, yet its dynamics in HPV-negative women with gynaecological disorders remain underexplored. We investigated microbial diversity and taxonomic shifts in HPV-negative women from Bangladesh using 16S rRNA gene sequencing and shotgun metagenomics. Of 224 women screened, 136 were HPV-negative; 29 underwent 16S profiling, and three infertility-associated cases were further analyzed by shotgun metagenomics. Healthy controls exhibited low alpha diversity and a Lactobacillus-dominated profile (98.2%), reflecting ecological stability. In contrast, pathological cases displayed significantly elevated richness and evenness, reduced Lactobacillus (28.0%), and enrichment of anaerobic and opportunistic taxa, including Bifidobacterium (23.4%), Achromobacter (12.9%) and Sneathia (7.5%). Distinct microbial signatures emerged across clinical subgroups: pelvic inflammatory disease was enriched in Bifidobacterium, intra-menstrual bleeding retained moderate Lactobacillus, while infertility exhibited prominent dominance of Achromobacter (45.5%). Shotgun metagenomics confirmed Achromobacter spp. (A. ruhlandii, A. dolens, A. xylosoxidans) as the predominant taxa (84.9%) in infertility cases, accompanied by depletion of protective Lactobacillus. Functional inference revealed conserved metabolic backbones but disease-specific enrichment of stress-response and biosynthetic pathways, particularly in infertility and PID. Co-occurrence network analysis identified condition-specific microbial consortia, with Achromobacter forming infertility-associated clusters. This study represents the first integrated application of amplicon and shotgun metagenomic approaches to profile the cervicovaginal microbiota in HPV-negative women. It identifies Achromobacter as a potential microbial biomarker of infertility and highlights the urgent need for microbiome-informed diagnostics and targeted interventions to restore cervicovaginal homeostasis.

Staphylococcus aureus is a leading cause of biofilm-associated infections, in which communities of bacterial cells are encased in an extracellular matrix composed of polysaccharides, proteins, and extracellular DNA (eDNA) that protect bacteria from host immune defense and antibiotics. Despite their importance, the mechanisms by which matrix components are released from bacterial cells and incorporated into the biofilm matrix remain poorly understood. Using a drip-flow biofilm system, we showed that MVs were associated with the biofilm matrix formed by S. aureus clinical isolate MN8. Proteomic analysis of biofilm matrix proteins and purified MVs showed that biofilm-derived MVs carried cytoplasmic, membrane, and extracellular proteins that closely resembled the protein composition of the biofilm matrix but differed significantly from MVs produced by planktonic cultures. Biofilm-derived MVs carried significantly higher levels of DNA than MVs from planktonic cultures, and MV-associated DNA was resistant to DNase treatment. Although strain MN8 is known to form polysaccharide-dependent biofilms, exogenously added DNase or proteinase K significantly impaired biofilm formation and integrity. Notably, these inhibitory effects were reversed by the addition of biofilm-derived MVs, which significantly restored biofilm formation in enzyme-treated cultures. Together, these findings provide evidence that S. aureus MVs are generated within biofilms, and that these MVs serve as an important resource of matrix components and contribute to biofilm formation. ImportanceExtracellular membrane vesicles (MVs) are important mediators of intercellular communication and have been implicated in the physiology and pathogenesis of bacterial infections. While MV production in S. aureus planktonic cultures has been recognized for over one decade, their presence and function in S. aureus biofilm formation have remained unexplored. Here, we report for the first time the purification and characterization of MVs derived from S. aureus biofilms. Our studies demonstrate that S. aureus MVs are important components of the biofilm matrix that contribute to biofilm formation by serving as key carriers of matrix proteins and eDNA. This work advances our limited understanding of MVs in Gram-positive bacteria and reveal a previously unrecognized mechanism underlying S. aureus biofilm formation.

Mucosal barriers serve as a multifunctional interface and nutrient-rich habitat for diverse microbes, including bacteria and bacteriophages. Some phages can bind to mucin glycoproteins via carbohydrate-interacting modules and provide an additional layer of mucosal immunity by shielding the underlying epithelium from invading bacteria. However, the role of mucins in shaping phage-bacterium interactions remains poorly understood. We investigated the dynamics between highly pathogenic Yersinia enterocolitica serotype O:8 and its mucus-adherent phage fMtkYen801 under in vitro mucosal environment. We assessed how mucin supplementation, varying phage doses, nutrient and temperature conditions influence phage-bacterium dynamics and biofilm formation. We found that pre-exposure to mucins led to enhanced phage replication in the bacterial host, with a 2-log increase in phage titers, and high abundance of surviving bacteria. Interestingly, mucin glycoproteins also provided Y. enterocolitica a nutrient source and a chemical cue to modulate its growth and biofilm biogenesis. Genomic analysis of phage-resistant bacterial variants revealed mutations in virulence, quorum sensing, and antibiotic resistance genes in both mucin enrichment and control groups, suggesting potential fitness tradeoffs during resistance evolution. Collectively, these findings highlight the importance of mucosal surfaces as an important ecological driver of phage-host interactions in Y. enterocolitica, a significant enteric pathogen, and emphasize the need for investigating these dynamics under complex, physiologically relevant systems to inform better phage therapy strategies against mucosal bacterial infections.

Unicellular microorganisms can make a transition to multicellular states that enhance survival under environmental fluctuations. In bacteria, one of these states is the biofilm, defined by the production of an extracellular matrix. Although biofilm maturation and dispersion have been extensively studied, where and how initial matrix production is induced within a growing population remains largely unknown. Here we show that production of colanic acid, an important matrix component, is initiated around topological defects, where cell orientation mismatches and growth-induced pressure builds up, in bacterial monolayers. Using Escherichia coli reporting mechanically induced production of colanic acid in response to cell contact and deformation, we found matrix production accompanied by out-of-plane growth under agar-pad confinement. Controlling confinement geometry using microfluidic devices dictated the positions of topological defects and thereby localized regions of high matrix production. These findings reveal that the cell orientation patterning spatially organizes mechanical cues to induce matrix production for biofilm initiation of bacteria.

In a healthy host, the residential microbes help regulate the growth of pathobionts, which are common members of the human gut microbiome, preventing them from causing diseases, including infections, under certain conditions. In cases of dysbiosis, this protection may be compromised. Targeted microbiome modulation offers a promising approach to restore healthy conditions in a disrupted community and consequently prevent infections using the natural colonization resistance of the microbiome. Elucidating the interaction mechanisms between microbial species within a microbiome is crucial for understanding how a microbiome can be modulated precisely and effectively to benefit the hosts well-being. Here, we investigated the interactions between the pathobiont C. perfringens and human gut commensals on physiological and molecular levels. We found that commensal strains affect C. perfringens growth by competing for substrates such as amino acids or a carbon source other than glucose. We further observed that Bacteroidaceae strains altered the levels of C. perfringens proteins, among others, the host-directed {theta}-toxin. Our findings reinforce the notion that modulating the composition of the gut microbiome is an effective strategy to prevent infections.

The incidence of colorectal cancer (CRC) has been increasing in Taiwan and is associated with multiple risk factors, including aging, obesity, and dietary habits. Increasing evidence suggests that gut microbiota dysbiosis contributes to CRC development. This study aimed to characterize microbial and metabolic alterations across premalignant and malignant colorectal lesions and to identify potential microbiome-associated biomarkers. Individuals undergoing colonoscopy for screening or surveillance at Taipei Veterans General Hospital were enrolled. Gut microbial composition was analyzed using full-length 16S rRNA gene sequencing to achieve high-resolution taxonomic profiling. Predicted functional pathways were inferred from microbial communities, and targeted metabolomic profiling was performed to evaluate microbial metabolic outputs. A total of 122 individuals were included, comprising 62 healthy controls, 15 adenoma cases, and 45 CRC cases. Progressive shifts in microbial composition and predicted functional pathways were observed along the adenoma-carcinoma sequence. Several bacterial taxa, including Phocaeicola dorei, Anaerotignum faecicola, Negativibacillus massiliensis, and Dysosmobacter segnis, were enriched in CRC. At the functional level, CRC samples showed enrichment of pathways associated with energy metabolism and bacterial stress responses. Metabolomic analysis further revealed increased levels of tauro-ursocholanic acid in CRC samples, whereas short-chain fatty acids (SCFAs) were reduced compared with controls. Integrative analysis combining full-length 16S sequencing, functional pathway prediction, and metabolomic profiling revealed coordinated microbial and metabolic alterations across the adenoma-carcinoma sequence. These findings provide insight into microbiome-associated processes in colorectal tumorigenesis and suggest potential microbial and metabolic biomarkers for CRC. ImportanceColorectal cancer (CRC) develops through a adenoma-carcinoma sequence, yet the microbial and metabolic alterations accompanying this progression remain incompletely understood. In this study, we integrated full-length 16S rRNA gene sequencing with metabolomic profiling to characterize taxonomic, functional, and metabolic changes across healthy controls, adenoma, and CRC. Our results reveal synchronized shifts in specific microbial taxa, predicted metabolic pathways, and fecal metabolites along the adenoma-carcinoma sequence. Several bacterial species, including Phocaeicola dorei, Anaerotignum faecicola, and Dysosmobacter segnis, increased in CRC, whereas short-chain fatty acids decreased progressively from controls to adenoma and CRC. Functional pathway analysis further indicated alterations in microbial fermentation, amino acid metabolism, and energy-related pathways. Together, these findings highlight the potential role of microbiome-associated metabolic changes in colorectal tumorigenesis and suggest candidate microbial and metabolic markers that may aid in understanding disease development and improving risk stratification.

Trichomonas vaginalis is an extracellular parasite that inhabits the human genital tract, yet little is known about how it senses and responds to the complex vaginal microbial ecosystem. Here, we show that T. vaginalis exhibits chemotactic behavior on semisolid surfaces, forming multicellular assemblies that coordinate collective migration. Parasite colonies display both positive and negative chemotactic responses, indicating the ability to detect and react to diffusible signals. Different parasite strains display marked mutual avoidance between neighboring colonies, highlighting specific recognition mechanisms. Furthermore, we show that T. vaginalis is strongly attracted to acidic environments, revealing a niche-adapted pH taxis. Given that vaginal bacteria critically shape local pH, we examined parasite responses to representative members of the vaginal microbiota. T. vaginalis exhibited preferential chemotactic migration toward Lactobacillus gasseri, a hallmark species of eubiotic community state types (CSTs), over Gardnerella vaginalis, which is associated with dysbiotic CST-IV communities, while showing no detectable attraction to Escherichia coli. This selective migration correlated with a robust chemotactic response to lactic acid, a major metabolite produced by lactobacilli. Additionally, when the parasite is co-cultured with the equal number of L. gasseri and G. vaginalis, T. vaginalis exhibits a clear preferential binding to L. gasseri, as demonstrated by flow cytometry and fluorescent microscopy. We show that co-culture of T. vaginalis with either L. gasseri or G. vaginalis results in enhanced parasite growth only in the presence of L. gasseri. Collectively, these findings reveal pH taxis; bacteria-directed migration and preferential association with Lactobacillus as previously underappreciated behavioral traits of T. vaginalis. Such behaviors may destabilize protective microbial communities and drive the transition toward a CST-IV-type dysbiotic state which is frequently associated with trichomoniasis.

Polyethylene (PE) is the most produced synthetic polymer and as a result, a major source of microplastic waste accumulating globally. Exposure to photo- and thermo-oxidative conditions in the environment can cause PE to degrade into carbonyl-containing compounds, hydrocarbons, and low molecular weight PE (LMWPE). In both marine and freshwater ecosystems, fish, including Atlantic salmon, can ingest PE and its derivatives, creating opportunities for interactions with their gut microbes. Here, we investigated the ability of a bacterial isolate from the gut of salmon, Rhodococcus sp002259485 strain ASF-10, to grow on an LMWPE model substrate for partially depolymerized and oxidized PE. Comparative genomic analyses showed that ASF-10 has a smaller genome than other Rhodococcus species yet retaining conserved functions including those related to utilization of medium- and long-chain hydrocarbons. In-depth characterization of the substrate following growth with ASF-10 confirmed depletion of alkanes and 2-ketones deriving from LMWPE, while the polymeric component remained unchanged. Proteomic analysis identified multiple enzymes that were likely to be involved in the degradation of LMWPE-derivatives, including an alkane 1-monooxygenase, cytochrome P450 hydroxylases and Baeyer-Villiger monooxygenases, as well as proteins for production of biofilm and a surfactant that may enhance accessibility to the substrate. Collectively, our findings advance the understanding of the ecology and enzymatic mechanisms underlying utilization of medium- to long-chain alkanes and oxidized variants thereof, that resemble molecules that can occur from abiotic PE degradation, by a fish gut-associated microbe. This metabolic capacity could be harnessed to develop sustainable strategies for bioremediation of oxidized, LMWPE-derivatives. ImportanceThe widespread presence of plastics in marine and freshwater environments has raised concerns due to their toxicity when ingested by fish. Microbial mechanisms driving breakdown of microplastic components, such as LMWPE and derivatives, in gut systems remain poorly understood. This study reveals how a bacterium isolated from the gut of salmon, Rhodococcus sp002259485 strain ASF-10, metabolizes alkanes and oxidized variants thereof, that can result from abiotic PE decomposition. We identified key enzymes that are potentially involved in this process as well as in the production of biofilm and surfactants that may facilitate access to the substrate. Besides extending the knowledge of the enzymatic basis for degradation of PE-derivatives in gut-associated microbes from aquatic organisms, our results provide a framework that couples advanced compositional characterization of the substrate with omics techniques, offering valuable insight to support future studies aimed at unequivocally identifying microbes and their enzymes implicated in transformation of PE-derivatives.

The high prevalence of aerotolerant human Campylobacter jejuni isolates suggests a correlation between the ability to survive in aerobic conditions, virulence and resistance to harsh stress conditions. However, the mechanisms are still unclear. Here, we investigated the role of bacterial extracellular vesicles (bEVs) in the adaptation of the clinical aerotolerant C. jejuni Bf strain to thermal and oxidative stress. We show that C. jejuni Bf survives and actively multiplies under this combined stress. Stress exposure induced cell rounding and loss of motility, remodeling of membrane composition, decreased membrane fluidity, and metabolic reprogramming with increased intracellular ATP levels. Lipidomic analyses further revealed that bEVs composition is markedly different from that of the parent membranes indicating that vesicle formation is selective and regulated. Although bEVs were produced in similar amounts under both microaerophilic and stress conditions, stress exposure generated significantly larger vesicles with greater diameter and dry mass, and altered their protein and lipid profiles. bEVs derived from stressed cells showed increased toxicity toward the epithelial barrier of Caco-2 cells. Taken together, these results indicate that C. jejuni bEV secretion is part of a survival strategy that connects environmental adaptation with pathogenicity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/714464v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@17aa2aforg.highwire.dtl.DTLVardef@4eab9dorg.highwire.dtl.DTLVardef@e4fba8org.highwire.dtl.DTLVardef@146109a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Plastic biodegradation in natural environments is increasingly recognized as a multi-organism process, yet the mechanisms enabling coordinated depolymerization and metabolism of polyethylene terephthalate (PET) remain poorly understood. Previously, we demonstrated that a full consortium containing three Pseudomonas and two Bacillus strains isolated from hydrocarbon-rich coastal soils of Galveston Bay, Texas, can synergistically depolymerize PET plastic and utilize it as a sole carbon source, a capacity not observed in individual isolates. In this report, using integrated comparative genomics, proteomics, and chemical analyses, we show that PET degradation in this system reflects exaptation of hydrocarbon metabolism reinforced by metabolic division of labor. Within this naturally occurring consortium, Bacillus strains persist under environmental stress, establish biofilms, and perform essential secondary hydrolysis, while Pseudomonas strains catabolize aromatic monomers and buffer oxidative stress. Genes supporting these functions are enriched within the accessory genomes of the consortium strains, indicating consortium-enriched horizontal gene transfer (HGT). In addition to the canonical two-step hydrolytic pathway well documented in PET biodegradation, we identify a secondary methylation-and redox-associated process, mechanisms where the full consortium acts on the oligomer mono(2-hydroxyethyl) terephthalate (MHET), yielding nearly complete conversion to terephthalic acid (TPA) and methylated MHET (MMHET). Together, these findings demonstrate how cooperation and competition within consortia facilitate targeted gene exchange, enabling emergent plastic biodegradation in natural microbial communities. IMPORTANCEEnvironmental plastic degradation is rarely accomplished by a single organism, yet the microbial mechanisms enabling community-level PET plastic breakdown remain poorly understood. This study shows that a bacterial consortium isolated from crude petroleum-contaminated beaches biodegrades PET through exaptation of ancestral hydrocarbon pathways, metabolic division of labor, and targeted gene exchange rather than specialized PET-specific metabolic pathways. Pseudomonas strains initiate PET cleavage, while stress-tolerant Bacillus strains persist long enough to clear inhibitory intermediates and enable downstream aromatic and diol metabolism. PET degradation is observed to be an emergent property of ecological interactions and distant evolutionary history. These findings provide a community-level model for understanding how natural microbial communities may adapt to novel anthropogenic substrates such as synthetic polymers, sustaining prolonged biodegradation.

The stability of gut bacterial communities is determined by complex inter-cellular interactions such as competition, cooperation and host dynamics. A mechanism proposed to mediate these interactions is bacterial secretion systems: specialized protein complexes that secrete effector molecules into neighbouring cells or the surrounding environment to influence community stability. However, the forces driving secretion system distribution and evolution in host-associated microbiomes remain unclear. Here, we show that secretion systems in the bee gut microbiome are predominantly vertically inherited and evolve primarily through recurrent gene loss rather than horizontal acquisition. Using comparative genomic analysis, we found that bee gut symbionts mostly encode type I, V, and VI secretion systems. In contrast, the pathogen-associated type II and III systems are missing, but they retain evolutionarily related pili and flagella. We found weak association between the presence of specific secretion systems and the bee hosts, suggesting that these systems are maintained for interbacterial interactions rather than host-specific adaptation. Co-phylogenetic analyses show congruence between bacterial strain phylogenies and most secretion system phylogenies, indicating a vertical mode of transmission. Only a subtype of the type VI system in the Orbaceae and the type IV system in Snodgrassella spp. show evidence of horizontal transmission. The lack of horizontal transfers means that losses of secretion systems is a permanent evolutionary event in almost all lineages of the bee gut microbiota. Our study provides a uniquely comprehensive analysis of secretion systems across an entire gut bacterial community, giving insight into how microbiomes evolve and maintain functional interactions within host-associated environments.

The intimate cohabitation between humans and their pets facilitates bidirectional microbial exchange, yet the extent and functional consequences of this transfer within the oral niche remain underexplored. Here, we employed metagenomic sequencing to characterize the oral microbiome of dogs and their owners across distinct geographic regions in China, integrating taxonomic, gene-centric, and functional analyses using public databases (BacMet, CARD, eggNOG, KEGG) to assess microbe-host associations. We found that dog-owner pairs exhibited significantly higher gene-level similarity compared to unrelated individuals, indicating a strong cohabitation-driven microbial linkage. While no major taxonomic shifts were observed in the human oral microbiome associated with pet ownership, we identified a marked enrichment of antibiotic resistance genes (ARGs)--particularly those conferring resistance to peptides, fluoroquinolones, antiseptics, diaminopyrimidines, cephalosporins, and carbapenems--in cohabiting pairs. This enrichment, together with the identification of exclusively shared ARGs (e.g., mdtF, macB, RanA), suggests the potential for horizontal gene transfer (HGT) between pet- and human-associated microbiomes. Functional profiling further revealed greater similarity in microbial metabolic pathways between cohabiting pairs than between unrelated individuals, reinforcing the likelihood of HGT as a mechanism underlying functional convergence. Collectively, these findings reveal that cohabitation with dogs reshapes the human oral microbiome at the genetic and functional levels, with potential implications for antimicrobial resistance transmission. This study provides a foundational framework for assessing the health risks associated with pet-human microbial exchange in shared living environments.

Short-chain fatty acids (SCFAs) like butyrate and propionate are abundant microbiota-derived metabolites that influence bacterial physiology in host-associated niches such as the gastrointestinal tract. However, their effects on Staphylococcus aureus under varying nutritional conditions remain incompletely understood. Here we investigated how SCFAs interact with glucose or galactose to regulate anaerobic growth, biofilm formation, and global transcription in S. aureus. Both SCFAs inhibit growth in a dose-dependent manner. Biofilm formation was differentially affected, with butyrate promoting and propionate suppressing biofilm formation. Glucose and galactose alleviated SCFA-mediated growth inhibition, with glucose exerting the strongest effect. Notably, glucose enhanced butyrate-associated growth and biofilm formation beyond glucose alone, whereas galactose produced more modest effects. Enzymatic and genetic analyses indicated that SCFA-sugar biofilms contain proteins and extracellular DNA and involve VraSR-dependent regulation. Transcriptomic profiling revealed broad metabolic reprogramming, including induction of urease genes, amino acid biosynthesis, and stress response pathways. Synergistic effects between butyrate and glucose were partially dependent on anaplerotic metabolism via pyruvate carboxylase, linking the TCA cycle to SCFA adaptation. Together these findings demonstrate that the nutritional environment dictates whether SCFAs impair S. aureus growth or reprogram its physiology, promoting metabolic adaptation and biofilm formation under sugar-replete conditions.

BackgroundPelagic Sargassum has undergone significant range expansion and dramatic blooms in the Atlantic over the past 15 years. This algaes microbiome provides symbiotic functions that are believed to contribute to its ecological success. Recent research shows that Sargassum-associated bacteria are enriched in integrated prophages compared to the surrounding seawater and that these prophages are inducible by chemical and ultraviolet treatment. ResultsHere, we investigated a Sargassum-derived in vitro multispecies biofilm encompassing the dominant heterotrophic microbial members associated with Sargassum to probe the impacts of prophage induction on the composition of Sargassum microbiomes. Induction was quantified by coverage-based virus-to-host ratios in chemically induced treatments with Mitomycin C and non-induced controls, and the community composition and metabolic profiles were analyzed after a period of recovery post-induction. Chemical induction led to a significant increase in abundance and virus-to-host ratio of viral genomes linked to Vibrio metagenome-assembled genomes. This was accompanied by altered biofilm community composition, with a reduction in Vibrio bacterial abundance that opened niche space for other biofilm members in the genera Pseudoalteromonas, Alteromonas, and Cobetia. The induced Vibrio-associated phages encoded genes involved in quorum sensing, biofilm formation, virulence, and host metabolism. Induction led to a relative loss of 17 metabolic modules, including functions related to energy metabolism and nitrogen utilization. ConclusionDue to the high frequency of lysogeny in the Sargassum microbiome and the susceptibility of prophages to chemical and ultraviolet light induction, these results suggest that prophage integration and induction are mechanisms that significantly contribute to structuring the Sargassum microbiome and its functional profiles, potentially aiding in microbiome flexibility in changing environmental contexts.

Biofilm formation is a major cause of chronic implant-related bone infections and is associated with impaired immune responses. In a previous study, we identified the cGAS-STING pathway as a potential therapeutic target, as its activation--observed in response to planktonic Staphylococcus aureus (SA)--was absent in the corresponding biofilm setting. The present study aimed to identify potential mechanisms underlying the lack of cGAS activation in the biofilm environment. As biofilm-derived nucleases might degrade cGAS ligands, we assessed presence and activity of micrococcal nuclease in conditioned media from planktonic and biofilm-grown SA and evaluated the impact of extracellular DNases on cGAS pathway activation in macrophages. In addition, we examined altered cGAS expression, the requirement for continuous biofilm exposure and potential downstream inhibition resulting from degradation of the cGAS product. Biofilm formation was associated with dynamic nuclease expression, and exposure to the biofilm environment led to reduced cGAS levels in macrophages, accompanied by a lack of interferon response. Exogenous cGAS activation by G3-YSD failed to restore signaling, independent of nuclease activity or continuous biofilm exposure. In contrast, supplementation with the cGAS product and STING ligand 2'3'-cGAMP fully restored interferon responses and enhanced macrophage activation, indicating that increased degradation of the second messenger in the biofilm environment is not responsible for impaired pathway activation. Similar effects observed with Staphylococcus epidermidis and primary macrophages suggest a broader mechanism that is not SA- or cell line-specific. In conclusion, our data provide novel mechanistic insight into biofilm-mediated impairment of cGAS-STING signaling, revealing a previously unrecognized mechanism of immune evasion in staphylococcal biofilms. These findings extend our previous work and support the therapeutic potential of targeting STING as promising strategy to restore immune responses in chronic implant-related bone infections. HighlightsO_LIBiofilm-derived factors impair cGAS-STING pathway activation and suppress interferon responses in macrophages. C_LIO_LIImpaired signaling is not primarily explained by extracellular micrococcal nuclease-mediated degradation of potential cGAS ligands. C_LIO_LIBiofilm exposure reduces cGAS expression levels and inhibits exogenous cGAS activation independently of continuous presence. C_LIO_LIExogenous 2'3'-cGAMP fully restores interferon responses, indicating that impaired signaling is not due to degradation of the cGAS product. C_LIO_LIDirect activation of STING broadly enhances macrophage activation and by this could amplify overall immune responses. C_LIO_LIBypassing cGAS via direct STING targeting represents a potential therapeutic strategy to overcome immune evasion in chronic implant-related bone infections. C_LI

Listeria monocytogenes relies on a tightly controlled set of surface-associated and secreted proteins to mediate host interaction and infection. The correct localization and exposure of these proteins at the bacterial surface are critical for virulence, yet the role of cell wall components in organizing this process remains incompletely understood. In particular, wall teichoic acid (WTA) glycosylation has been implicated in anchoring and function of selected surface proteins, but its global impact on protein distribution across the bacterial cell envelope is unclear. Here, we performed a comprehensive proteomic analysis to investigate how WTA glycosylation influences protein distribution in L. monocytogenes. Using isogenic mutants lacking rhamnose ({Delta}rmlT) or GlcNAc ({Delta}lmo1079) WTA glycosylation, we compared the exoproteome, the surface-accessible proteome and the surface-exposed proteome. Loss of WTA glycosylation did not result in a global disruption of the surface proteome but instead induced a redistribution of proteins across extracellular and surface-associated fractions. This effect was dependent on protein anchoring mechanisms, with limited changes observed for LPXTG-anchored proteins, moderate effects on non-covalently associated proteins, and a marked enrichment of lipoproteins in the surface-exposed proteome, particularly in the {Delta}lmo1079 mutant. In parallel, virulence-associated proteins displayed altered accessibility and exposure, with a progressive shift towards increased surface localization and a combination of shared and mutant-specific responses. This global effect was supported by functional annotation, which revealed that the affected proteins were associated with similar biological processes across fractions, highlighting a broad rather than pathway-specific impact of WTA glycosylation loss Together, these findings indicate that WTA glycosylation plays a key role in organizing the bacterial surface by modulating protein retention, exposure and release. Rather than affecting specific proteins, WTA glycosylation broadly shapes the spatial distribution of proteins across the cell envelope, with potential consequences for host- pathogen interactions.

The human skin is repeatedly exposed to mechanical and environmental stress, particularly in common skin diseases such as eczema, and yet the determinants of recovery remain poorly understood. Using longitudinal, multimodal profiling of skin physiology, structure (Raman spectroscopy), and microbial communities (shotgun metagenomics), we investigated in a human cohort (n=36 subjects, x2 sites, x6 timepoints) how host-microbe interactions could jointly shape recovery. Despite baseline variability in physiological parameters, we established that our protocol enables a defined disruption of the stratum corneum. While recovery trajectories for host attributes were notably consistent across age groups and body sites, individual-specific differences in recovery timelines were observed. To assess the role of the skin microbiome, several key time-dependent changes in microbial species were identified including enrichment of select Cutibacterium and Staphylococcus species and depletion of Corynebacterium and Malassezia species. Clustering of microbiome stability profiles across subjects and sites identified 6 distinct groups which associate with varying host-recovery patterns and microbial functions. Finally, joint hazards modelling of recovery timing revealed significant contributions from microbial taxa, functions and stability groups, highlighting the under-appreciated role of host-microbial interactions in response to skin stress and in the recovery process.

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.