npj Biofilms and Microbiomes

The human duodenum harbours a complex, dynamic microbial community that is challenging to study due to inaccessibility, particularly postprandially when nutrient-rich chyme and fluctuating metabolites create unique microbial niches. We used naso-duodenal intubation to longitudinally sample duodenal luminal contents following pea-based meals of differing food structure, alongside parallel blood collection. Shotgun metagenomic sequencing, comprehensive metabolomic profiling and gut hormone measurements were combined to explore microbe-metabolite-hormone interactions. Food structure significantly affected postprandial bacterial composition, with saccharolytic oral taxa increasing after meals with intact structure. Alpha diversity was influenced by structure type (P = 0.025), with whole pea seeds promoting greater diversity than pea flour. Network analysis revealed complex interactions between the duodenal microbiome, luminal metabolites and gut hormones, with most microbial associations linked to glucose-dependent insulinotropic polypeptide (GIP) rather than glucagon-like peptide-1 (GLP-1). Metabolic profiling showed meal-dependent changes in amino acid metabolism, including shifts in D/L amino acid ratios over time consistent with microbial metabolism. The duodenal microbiome showed close phylogenetic relationships with the oral microbiome, with composition influenced by food structuring and swallowing. These findings reveal dynamic microbe-metabolite interplay in the human duodenum during digestion and its relationship to gut hormone responses.

The vaginal microbiome is a critical determinant of womens health. We investigated the genetic basis of common vaginal microbiome species and their biofilm formation. Genomic analysis of Gardnerella vaginalis (Gv) revealed a fundamental phylogenetic split correlating with high- versus low-biofilm phenotypes, driven by clade-specific genomic islands and allelic variants. In a dual-species coculture model of five key vaginal bacteria, Gv achieved numerical dominance, triggering extensive, asymmetric proteomic reprogramming in partner species while showing limited shifts itself. Proteins from biofilm-associated modules showed functional divergence, supported by AI-predicted structural variations in a type II secretion/Tad pilus system, which is first discovered from Gv strains. Integrated metabolomics identified a methyl-{beta}-carboline compound that is elevated in cocultures containing Prevotella bivia (Pb). This compound acts as a potent and selective inhibitor of Gv and Pb biofilms, sparing Lactobacillus crispatus. This work establishes a direct genomic basis for Gv virulence and demonstrates how interspecies interactions govern community dynamics and antimicrobial metabolite production. HighlightsO_LIComprehensive genomic resource comparing with high-quality long-read whole genomes and reference Gardnerella vaginalis and Lactobacillus iners strains. C_LIO_LIIntegrated multi-omics and functional analysis on the most common vaginal microbiome species using 16S rRNA gene sequencing, proteomics, metabolomics, and in vitro assays. C_LIO_LIKey phenotypes quantified, including biofilm formation and polymicrobial interactions. C_LIO_LIConserved Type II Secretion/Tad Pilus System identified across all Gardnerella vaginalis strains, with AI-predicted structural modeling. C_LIO_LIEvaluation of growth inhibition using metabolites against a panel of relevant microbes, including vaginal microbes and opportunistic pathogens. C_LI

BackgroundThe female lower reproductive tract harbors a complex microbiome that plays a critical role in reproductive health. A vaginal microbiome dominated by Lactobacillus crispatus (LC; Community State Type (CST) I) supports vaginal health, whereas a microbiome enriched with anaerobic species, such as Gardnerella vaginalis (GV) and Mobiluncus mulieris (MM) (CST IV) is linked to bacterial vaginosis (BV) and adverse outcomes, including sexually transmitted infections, infertility, and preterm birth. Although antibiotics such as metronidazole and clindamycin are commonly prescribed to treat BV, recurrence rates remain high, and the impact of these treatments on bacterial extracellular vesicles (bEVs), critical mediators of host-microbe interactions, is poorly understood. ResultWe investigated how antibiotic treatment at a dose below minimum inhibitory concentration alters the production and immunomodulatory function of bEVs derived from GV, MM, and LC. Using nanoparticle tracking analysis, cytokine profiling, and TLR pathway analyses, we found that antibiotic treatment significantly enhanced the inflammatory properties of bEVs in a species- and antibiotic-specific manner. Notably, bEVs from antibiotic-exposed GV and MM cultures induced elevated cytokine responses in epithelial and immune cells, primarily through TLR2 activation for GV bEVs, and through both TLR2 and TLR5 activation for MM bEVs. While LC bEVs are typically non-inflammatory, exposure to metronidazole, even at a lower dose than what is used clinically, rendered them immunostimulatory, suggesting a potential unintended proinflammatory consequence of treatment on beneficial microbes. We also detected bEVs in human vaginal swabs, including vaginolysin-positive bEVs, even in CST I microbiomes, indicating that low-abundance microbes, including pathogens, remain transcriptionally active. ConclusionsThese findings suggest that antibiotics not only reduce microbial load but also reshape bacterial communication via bEVs, potentially contributing to inflammation, epithelial barrier disruption, persistent dysbiosis, and recurrent infections. This work underscores the need for precision antimicrobial strategies that eliminate pathogens while preserving beneficial bacteria and their functional bEVs. Future therapies may benefit from considering the ecosystem-wide effects of antibiotics on the vaginal microbiome and its bEV-mediated signaling network.

Urinary tract infections (UTIs) are the most prevalent bacterial infections globally, and their management increasingly challenged by antimicrobial resistance (AMR). Probiotics offer a promising approach to mitigate AMR by competitively excluding uropathogens and enhancing host immunity by producing immune modulators. Despite being potential, key gaps persist between the discovery of uroprotective probiotic strains and optimization of formulations for urinary tract delivery. Here, we analyzed the urinary microbiome of UTI patients and healthy individuals to identify potential probiotic candidates for the prevention and management of UTIs. Publicly available 16S rRNA amplicon sequencing data of the urinary tract were processed using a standardized pipeline for sequence quality assessment, taxonomic assignment, and microbial function prediction. Comparative analysis showed a significant shift in microbial composition between UTI patients and healthy controls. The dominated phyla identified included Acidobacteriota, Actinobacteriota, Bacteroidota, Campylobacterota, Cyanobacteria, Firmicutes, Fusobacteriota, Patescibacteria, Proteobacteria, and Synergistota. Overall differential abundance analysis revealed Escherichia coli as the predominant UTI-associated species, while Lactobacillus crispatus was enriched in healthy samples. Additionally, predictive functional analysis indicated that metabolic pathways associated with beneficial microbes were enriched in the healthy group. Overall, the study highlights the association of distinct urinary microbiome signatures with infection status, which supports L. crispatus as the most promising probiotic for UTI prevention and control.

Industrialization has been identified as the single biggest factor driving global microbiome diversity. While many studies examining gut microbiomes attribute these shifts to dietary increases in fat and reductions in protein, oral microbiome responses to industrialization remains debated. The oral microbiome is more resilient due to long-standing coevolution with host tissues and biofilm stability. However, limited geographic and historical representation has constrained our understanding of how these transitions unfolded globally in the oral microbiomes. Here, we investigate oral microbiome variation in Batwa rainforest hunter-gatherers and neighboring Bakiga subsistence farmers from southwestern Uganda, comparing them with publicly available data from Tanzanian, Venezuelan, and industrialized populations from North America, Europe, and Australia. Using 16S rRNA gene sequencing, we characterized salivary microbiota and evaluated differences in local and global diversity, composition, and differential abundance. Ugandan populations contained significant compositional differences but similar levels of diversity, suggesting that shared environments and dietary overlap may shape microbial assemblages despite distinct cultural histories. Globally, strong continental and industrialization effects were observed in the oral microbiome, with all industrial populations clustering separately from people living in other locations. African populations also clustered separately from non-African groups. Oral microbiome diversity was highest in Ugandan individuals and lowest in industrialized populations, mirroring patterns previously observed in the gut microbiome. Together, these findings demonstrate that both geography and subsistence strategy structure global oral microbiome variation. They also clarify the position that oral microbial communities record biocultural transitions and highlight the need to better understand the industrial mechanisms that shape microbial diversity in the oral cavity.

Enterococcus faecalis is a Gram-positive intestinal commensal and opportunistic pathogen capable of causing serious infections, including urinary tract infections, endocarditis, and wound infections. A major contributor to its persistence during infection is the ability to form biofilms on host tissues and medical devices. Biofilm cells have higher phenotypic tolerance to antimicrobial treatment than planktonic bacteria. While mechanisms governing biofilm assembly in E. faecalis have been widely studied, the processes that regulate biofilm dispersion, the final stage of the biofilm life cycle, remain poorly understood. In this study, we found that dispersion is triggered by a tenfold step-change increase in nutrient availability and by cell free supernatant (CFS) of E. faecalis OG1RF cultures. Cells released from biofilms regain sensitivity to antibiotics similar to planktonic cells but maintain a high potential for adherence. We characterized the glycosyltransferase epaOX, which contributes to the structure of the enterococcal polysaccharide antigen as necessary for nutrient step-change induced dispersion, CFS induced dispersion, and adhesion of dispersed cells. Supplementation of epaOX mutant CFS with galactose and N-acetylgalactosamine was sufficient to restore CFS induced dispersion. Together these data suggest that dispersion in OG1RF occurs with fast kinetics, affects antibiotic sensitivity and is regulated in part by known virulence factors. ImportanceE. faecalis causes difficult to treat infections at numerous body sites in human patients. E. faecalis biofilms are adherent populations that require high levels of antibiotics for treatment. Biofilms undergo a disassembly process named dispersion that allows individual cells to leave the biofilm and colonize new locations. Dispersed cells in other species are killed by lower amounts of antibiotics than biofilm cells. Here we showed that dispersion occurs in E. faecalis and lowers the level of antibiotics needed to kill dispersed cells. Dispersion triggers could be used in the future to design treatments that increase the effectiveness of antibiotics.

AimsPseudomonas aeruginosa biofilm-associated infections pose a significant clinical challenge due to their inherent antibiotic tolerance. This study aimed to evaluate the antibacterial and antibiofilm activity of Placentrex, a standardised aqueous placental extract, against P. aeruginosa and to elucidate its molecular mechanism of action using RNA sequencing (RNA-seq). Methods and ResultsPlacentrex exhibited potent bactericidal activity against P. aeruginosa at 50 mg/mL. Biofilm formation was significantly inhibited by [~]87% at 50mg/mL after 72 hours. Preformed biofilms were eradicated by [~]93% and [~]89% at 50 and 25 mg/mL, respectively. Interestingly, biofilm viability was reduced by [~]93% and [~]87% upon treatment with 50 mg/mL and 25 mg/mL of Placentrex, respectively. EPS characterisation revealed that the EPS contain a single large polysaccharide, and chromatography data suggested that it is made up of glucose as a monomer. RNA-seq identified coordinated downregulation of seven key genes, namely, flp major pilin (surface attachment), extracellular solute binding protein (ABC transporter-mediated nutrient sensing and biofilm maintenance), gntP permease (carbon metabolism), AraC family transcriptional regulator (quorum sensing and polysaccharide biosynthesis), ureE (urease nickel metallochaperone), aromatic amino acid permease (pyoverdine and PQS biosynthesis), and MFS transporter (efflux and autoinducer export). ConclusionsPlacentrex exerts comprehensive antibiofilm and antibacterial activity through simultaneous disruption of surface attachment, nutrient-sensing-driven biofilm maintenance, quorum sensing, carbon metabolism, urease virulence maturation, and efflux-mediated persistence. This polypharmacological mechanism supports Placentrex as a promising multi-target antibacterial agent against P. aeruginosa biofilm-associated infections. Impact statementPlacentrex is a potential anti-biofilm agent against Pseudomonas aeruginosa.

Host-associated microbiomes are typically maintained in stable configurations that support host fitness, yet the mechanisms by which metabolic perturbations destabilize these communities remain poorly understood. Using the freshwater cnidarian Hydra vulgaris AEP, we systematically assessed microbiome responses to 326 single-metabolite perturbations. Only 17 metabolites, mostly amino acid-related compounds, induced significant compositional shifts in the microbial community. Most shifts are accompanied by transitions from Curvibacter- to Pseudomonas-dominated or Legionella-dominated states, indicating the existence of three alternative community states which can be induced by metabolic triggers. Integrating 16S sequences with functional genomic information, we found that {beta}-diversity strongly predicted functional shifts, whereas reduced -diversity was associated with loss of metabolic functions. The metabolite perturbations also altered host-microbe interactions, affecting pathogenicity-, glycocalyx-, and nitrogen-related functions. In particular, nitrogen metabolism shifted from ammonia oxidation in Curvibacter-dominated communities to ammonia reduction in Pseudomonas-dominated states. Experimental validation confirmed that Pseudomonas metabolizes L-arginine and drives environmental ammonia accumulation to levels that could impair Hydras fitness and induce disease phenotypes. Conversely, Limnobacter was found to scavenge the environmental ammonia, potentially mitigating the adverse effects. These results demonstrate that metabolite-driven niche reconfiguration can destabilize host-associated microbiomes by coupling compositional shifts to functional change and host pathology, identifying metabolite-driven niche restructuring as a mechanism linking microbial community instability to host dysfunction.

The evolution of antimicrobial resistance (AMR) in chronic biofilms is often viewed as a unidirectional path toward higher fitness, yet the metabolic constraints governing these trajectories remain poorly understood. We performed a four-passage evolution experiment using a murine lung biofilm model to assess the impact of prolonged ciprofloxacin (CIP) exposure on resistance and host response. This approach integrated population-level adaptive dynamics, whole-genome sequencing (WGS), and NMR-based metabolomics, alongside histopathology and cytokine analysis. Prolonged CIP treatment accelerated resistance, with isolates reaching MICs of 8-12 mg/L (a 32- to 48-fold increase) by the fourth passage. WGS revealed distinct evolutionary trajectories: control isolates accumulated metabolic and regulatory mutations without susceptibility changes, while CIP-treated isolates exhibited a stepwise progression from metabolic adaptation to high-level resistance, marked by early nfxB and late gyrA mutations. Metabolomic profiling revealed progressive divergence, with PCA identifying the nfxB genotype as the primary driver of variation (49.1% of variance). This resistant metabolic state was characterized by the depletion of central carbon metabolites, including glucose and tyrosine, alongside the accumulation of essential amino acids. Importantly, these changes were accompanied by a distinct trade-off; high-level CIP resistance triggered collateral sensitivity to tobramycin and aztreonam. While CIP treatment ultimately reduced neutrophilic inflammation (p = 0.011) and mucin production (p = 0.0496), early-passage lungs exhibited transient elevations in pro-inflammatory cytokines (CXCL2, MMP2, TNF-). In conclusion, the adaptive trajectory to CIP resistance involves metabolic rewiring and collateral sensitivity, offering a framework to exploit the evolutionary costs of resistance in chronic biofilm infections.

The fish skin microbiome serves as a protective barrier, influencing host health and facilitating interactions between the host and its environment. While several studies have characterised the composition and roles of the fish skin microbiome, there remains a paucity of data on how environmental variation influences these microbes in natural populations. Here, we used 16S rRNA gene sequencing to characterise the skin microbiome of wild three-spined stickleback populations and examine how environmental factors influence microbial diversity and community composition across 17 freshwater lochs on the island of North Uist, Scotland. Analysis of 239 samples revealed a set of dominant bacterial genera commonly associated with other fish species, including Janthinobacterium, Pseudomonas, Acinetobacter, and Psychrobacter, that constituted a core skin microbiota across lochs. Microbiome composition was primarily shaped by environmental variables, particularly habitat, water pH, conductivity, and metal concentrations, with pH emerging as a key driver of community structure. Host sex also influenced microbiome variation, with several taxa differing in relative abundance between males and females. Alpha-diversity was higher among stickleback fish from lochs with a neutral pH compared with those from alkaline and acidic environments. Differential abundance analyses identified 27 and 24 amplicon sequence variants (ASVs), respectfully, associated with variations in pH and host sex, including members of Psychrobacter, Sphingobacterium, Carnobacterium, Chryseobacterium, and Arthrobacter, highlighting the combined influence of environmental and host factors on microbiome composition in wild fish populations in freshwater environments.

Micro- and nanoplastics (MNPs) are now recognized as ubiquitous dietary and environmental contaminants, yet practical strategies to reduce gastrointestinal exposure remain limited. This study evaluated whether Qi601, a heat-inactivated Limosilactobacillus fermentum biofilm-derived postbiotic, could bind plastic particles and reduce intestinal epithelial plastic burden. Prior probiotic studies have demonstrated live bacterial adsorption of MNPs and mitigation of MNP-associated toxicity in vivo; here, we evaluate whether a nonviable postbiotic preparation can produce analogous MNP-binding and epithelial-protective effects. Qi601 durably bound polystyrene nanoplastics under in vitro simulated digestion conditions. In Caco-2 intestinal epithelial monolayers, Qi601 reduced surface-associated and intracellular nanoplastic burden in both protection and rescue models, indicating decreased epithelial particle interaction both before and after established nanoplastic exposure. Multimodal imaging, including confocal microscopy, atomic force microscopy, and scanning electron microscopy, confirmed close physical association between Qi601 and nanoplastics. Finally, a first-in-human proof-of-concept chewing-gum study showed Qi601 binding in the human mouth to heterogeneous gum-derived microplastic fragments released during mastication. Together, these findings support the concept of postbiotic intervention for gastrointestinal epithelial protection against ingested MNPs.

Background: Surgical site infection (SSI) is the leading cause of perioperative morbidity following oral cancer surgery, yet the role of the oral microbiota in SSI pathogenesis remains poorly defined. This study prospectively investigated microbiota dynamics in relation to SSI occurrence in patients undergoing resection for oral squamous cell carcinoma (OSCC). Methods: A total of 172 oral swab samples were collected from 45 OSCC patients across four longitudinal time points: baseline (~29 days pre-surgery), immediately pre-surgery (hospital admission), early post-surgery (within 5 days), and late post-surgery (6 to15 days). Bacterial composition was profiled by 16S-rDNA V3-V4 sequencing (172 successfully sequenced samples), and bacterial/human DNA ratios were quantified by qRT-PCR (170 samples evaluated). SSI was assessed within 30 days post-surgery using adapted CDC criteria. Results: Fourteen of 45 patients (31.1%) developed SSI. Younger age was significantly associated with SSI occurrence (median age 53.2 years in SSI group vs. 67.4 years in non-SSI group; p=0.011), with each one-year decrease in age conferring a 7% increased risk. Notably, younger patients presented with larger and more advanced tumors (T3/T4: median age 57.2 vs. 72.9 years for T1/T2; p=0.033), leading to more extensive surgical procedures. Across all 172 samples, surgery induced a marked post-operative reduction in bacterial load and diversity. However, at the late post-surgery time point (collection IV), patients with SSI exhibited significantly higher alpha-diversity compared to non-infected patients (p<0.05 for Observed, Shannon, and Simpson indices). Beta-diversity also differed significantly between groups at this time point (weighted UniFrac, p=0.043). Prevotella and Porphyromonas dominated SSI patients at infection, together accounting for ~40% of reads versus 9.5% in non-infected patients. Among the 172 samples analyzed longitudinally, Aggregatibacter abundance at the early post-surgery time point (collection III) emerged as a significant predictor of subsequent SSI (OR per 1% increase: 1.10; p=0.012), with frequencies >0.044% conferring a 5.7-fold higher risk. Conclusions: Our longitudinal analysis demonstrate that while OSCC surgery profoundly disrupts the oral microbiota, non-SSI patients restore their preoperative profile within 12 days. In contrast, SSI is characterized by persistent dysbiosis dominated by Prevotella and Porphyromonas. Younger patients with advanced tumors are at particular risk. Early post-surgical Aggregatibacter abundance may serve as a novel risk indicator for SSI, potentially enabling timely preventive interventions in high-risk patients.

Polydimethylsiloxane (PDMS) is an excellent material for the construction of biomimetic leaf replicas which reproduce leaf surfaces with high fidelity. This allows for the study of leaf surface-colonizing bacteria and the impact of the leaf topology on bacterial distributions and behavior. However, their application is limited to short-term experiments, as long term survival of microorganisms on their surface is not possible due to a lack of nutrient replenishment. On living leaves, nutrients diffuse across the cuticle via leaching, a process not yet replicated in biomimetic systems. Here, we explore whether water and fructose can be supplied to microbial colonizers on PDMS membranes by mimicking leaching. We created hybrid membranes by incorporating polymers (Carbopol, Pemulen, cellulose microfibers, cellulose nanocrystals, and polyvinylpyrrolidone) to enhance nutrient transport. We determined that bulk diffusion of water correlated negatively with membrane thickness and positively with polymer concentration. Further, fructose diffusion across hybrid membranes reached similar rates compared to isolated Populus x canescens leaf cuticles. Under high relative humidity, these membranes supported long-term bacterial survival. Our findings represent important steps towards the development of topomimetic leaf surfaces that sustain microbial life, enabling further investigation into the microbe-microbe interactions that take place on leaves.

Recent evidence suggests that the gut microbiome plays a role in the development and treatment response of pancreatic ductal adenocarcinoma (PDAC). However, the functional impact of tumor location and preoperative biliary stenting (PBS) on microbial composition and metabolism remains poorly understood. In this prospective study, preoperative stool specimens were collected from patients undergoing surgery for PDAC at Heidelberg University Hospital, Germany, between March 2020 and July 2021. Whole-genome shotgun metagenomic sequencing was performed to characterize microbial composition and functional pathways. A total of 63 preoperative stool samples were analyzed, including 40 patients with pancreatic head tumors (63.5%) and 23 with body/tail tumors (36.5%). Microbial community composition differed significantly according to tumor location (Bray-Curtis, p=0.005), with enrichment of Ruminococcus bromii in body/tail tumors. Among patients with pancreatic head tumors, PBS was associated with reduced alpha diversity (Shannon index, p=0.04), depletion of taxa including members of the Eubacteriales and Clostridiales orders as well as the genera Raoultella and Prevotella, and reduced abundance of selected genes involved in secondary bile acid metabolism. PBS was also associated with a higher rate of major postoperative complications according to Clavien-Dindo >3a (28.6% vs 3.8%; p=0.04). These findings suggest that biliary intervention may induce functional dysbiosis characterized by reduced microbial diversity and impaired bile acid metabolism, potentially disrupting host- microbiome crosstalk and contributing to adverse postoperative outcomes in pancreatic cancer.

Periodontal disease is an inflammatory disorder that arises from dysbiosis of the subgingival microbiota, with Porphyromonas gingivalis acting as a keystone pathogen in the shift from health to disease. P. gingivalis employs multiple strategies to subvert host immune defenses, and its capsular K-antigen serves as a key virulence determinant. Here, a pre-adsorbed antiserum (pAds106) was generated by removing nonspecific antibodies using cells from a K-antigen-null mutant (W83{Delta}PG0106), resulting in exceptional specificity for the P. gingivalis K1-antigen. Immunofluorescence analysis revealed that the K-antigen preferentially coats outer membrane vesicles (OMVs), rather than attaching to the bacterial cell surface. This localization was further confirmed by ELISAs of density gradient ultracentrifuge-purified OMVs, with background signal detected in OMVs derived from K-antigen-deficient strains, non-K1-strains, and other oral Bacteroidetes. K-antigen-coated OMVs exhibited higher hydrophilicity and elicited weaker inflammatory responses compared to K-antigen-deficient OMVs, consistent with previously reported properties of encapsulated strains. Importantly, the antiserum detected K-antigen-coated OMVs in subgingival plaque from periodontal patients, suggesting that K-antigen is actively produced at diseased sites. These findings revise the prevailing view that K-antigen solely encapsulates the bacterial cell body and suggest that K-antigen-coated OMVs produced by P. gingivalis play distinct roles in immune evasion during periodontal disease.

Antibiotic use in agricultural systems can unintentionally disrupt beneficial rhizosphere microorganisms, yet the consequences of this dysbiosis for plant fitness remain insufficiently understood. Building on previous findings that application of streptomycin to the roots decreases cyanobacteria and increases tomato plant susceptibility to foliar Xanthomonas infection, this study aimed to determine whether this relationship reflects causation or correlation. We evaluated whether targeted inoculation with the filamentous nitrogen-fixing cyanobacterium Cylindrospermum sp. (CI) or a complex rhizosphere microbiome transplant (RMT) could mitigate antibiotic-induced dysbiosis. As expected, streptomycin treatment significantly increased bacterial spot disease severity and reduced microbial richness in the rhizosphere, marked by a pronounced decline in cyanobacterial and Cylindrospermum operational taxonomic units. Co-occurrence network analysis revealed that this dysbiotic state was defined by reduced community connectivity and increased negative associations, indicating a breakdown in cooperative microbial relationships. Notably, both CI and RMT reduced plant disease severity, though they caused distinct rhizosphere community reassembly outcomes. While RMT relied on microbial functional redundancy, the targeted CI approach achieved more robust colonization and effectively "patched" the functional gap left by dysbiosis. Microbiome restoration directly influenced host physiology, significantly reducing the overactivation of ethylene-mediated defense genes, such as ERF1, and partially reinstating auxin-responsive signaling pathways (IAA21) that were disrupted under dysbiosis. These findings suggest that targeted microbial inoculation could reverse dysbiosis and enhance plant resilience under pathogen pressure as effectively as complex microbial transplants. This work highlights a shift in microbiome management: from the complex rebuilding of communities to the strategic repair of specific functional gaps.

Biofilms formed by soil microbes hold immense potential for bioremediation, carbon dioxide sequestration, and the development of sustainable cementitious materials. However, quantifying the complex temporal coupling among bacterial growth, extracellular matrix (ECM) production, and mineralization dynamics remains a significant challenge due to the inherent nonlinearity of these processes and signal noise in high-throughput assays. To address this, we utilized an automated real-time kinetic analysis framework integrating connectivity-based segmentation, automated baseline alignment, and robust sliding-window algorithms to quantify the biomineralization competence of Bacillus subtilis under varying calcium regimes. Crucially, our results demonstrate that calcium carbonate promotes microbial growth as effectively as the highly soluble calcium acetate, providing strong evidence that B. subtilis actively solubilizes this crystalline powder to facilitate its metabolic requirements. Despite this growth efficacy, we found that calcium carbonate is an inadequate source for macro-calcite production compared to organic salts. By quantifying the expression efficiency of the sinI reporter gene, we determined that calcium-acetate-driven ECM expression significantly enhances the structural compatibility required for robust biomineralization. Furthermore, kinetic modeling suggests that ECM overproduction can partially compensate for defects in crystal growth-when provided crystalline calcium carbonate powder. These findings, enabled by high-resolution automated signal processing, underscore the critical role of self-mediated carbonate supply and present new engineering pathways for upcycling mineral-rich construction waste.

Vaginal dysbiosis (VD), characterized by low abundance of vaginal lactobacilli and increased bacterial community diversity, is implicated in multiple adverse reproductive outcomes and is an emerging target for preventive interventions, including live biotherapeutic products (LBPs). The most common clinical presentation of VD is bacterial vaginosis (BV), but at least half of people are asymptomatic. We investigated how two commonly used diagnostic criteria for BV, namely Amsel and Nugent, align with 16s rRNA gene sequencing-defined community state types (CSTs) demonstrating VD. We analyzed screening specimens from a Phase 1b randomized trial of LBP conducted at two sites (CAPRISA, South Africa; MGH, USA), as well as a single follow-up visit from enrolled participants. Using sequencing-based CST as the reference and multinomial mixed-effects logistic models, we evaluated the association of Amsel BV and Nugent BV with CST IV (including subtypes IV-A and IV-B) and tested for site-specific effects. Amsel BV was significantly associated with CST IV-A, and IV-B; however, the strength of association was significantly diminished at CAPRISA compared to MGH, pointing to site-specific assessment differences or underlying biological variation. Nugent BV yielded stronger associations with CST IV-A, and IV-B and showed no evidence of a site-specific interaction, indicating consistent performance across sites. These findings indicate that diagnostic performance for VD varies by framework: Amsel criteria are susceptible to geographical site effects, whereas Nugent score demonstrates stronger and more site-agnostic associations. For clinical studies targeting VD, Nugent scoring and/or sequencing-based approaches should be prioritized for VD endpoint definition and stratification.

Patescibacteria are an elusive linage of "microbial dark matter" bacteria predicted to represent [~]25% of total bacterial diversity. Despite this abundance and ubiquity, these organisms are challenging to cultivate, resulting from their specialized episymbiotic lifestyle. All cultivated representatives to date, predominantly composed of Saccharibacteria from the oral microbiome, depend on cognate prokaryotic hosts for growth and reproduction. Studying the growth dynamics of episymbiotic bacteria and their hosts in batch cultures has suggested that many episymbionts initially reduce host populations, and that hosts eventually adapt to episymbiont stress after serial passaging. However, discontinuous batch cultures do not reflect natural interactions between these organisms due to their drastically different growth rates. An episymbiont requires several ([~]2-4) serial passages alongside its host to reach the high cell densities needed to impact host growth, which complicates investigation of host inhibition and adaptation to episymbiont stress. To describe these dynamics accurately, we utilized continuous culture via small-scale Raspberry Pi powered bioreactors, called Pioreactors. Within a bioreactor, host bacteria can be cultivated at a consistent growth rate indefinitely, providing the perfect substrate for cultivation of model Saccharibacteria. Quantification of time until host crash, crash severity, time until recovery, and stable co-culture density provides mechanistic ways to describe episymbiont-host interactions. First, we used these techniques to compare episymbiont infection by three different episymbionts, revealing distinct infection patterns ranging from mild inhibition with rapid host adaptation, to rapid host collapse followed by "arms race" oscillation dynamics. Then, bioreactors were used to quantify the episymbiotic role played by a known host-binding type 4 pili (T4P-2), demonstrating that loss of long-distance host binding significantly delayed the host crash without altering general crash dynamics. These experiments reveal that episymbionts can have drastically different effects on bacterial communities and provide the tools necessary to describe strain/species differences and molecular interactions. ImportanceEpisymbiotic Patescibacteria represent one of the largest branches of life on Earth, as well as one of the least understood. Furthermore, because Patescibacteria can manipulate their hosts growth and morphology they have immense ecological potential to be shaping the communities they occupy, both environmental and microbiome-associated. Our study highlights for the first time the potential of small-scale continuous cultivation for studying episymbiotic interactions that cannot be captured in discontinuous cultures. Herein we used these techniques to interrogate inter-species variation in host inhibition potential and to determine how loss of a long-distance episymbiosis factor mechanistically alters the cycle of episymbiont infection; however, this cultivation platform will enable researchers to answer many new questions about these ubiquitous host-episymbiont interactions.

PurposeBacterial vaginosis (BV) is associated with disruption of the vaginal microbiome and extracellular matrix (ECM) remodeling, yet the contribution of host proteases to this process remains unclear. This study investigated whether expression and activity of cathepsins K, L, S, and V differ by BV diagnosis and community state type (CST). We hypothesized that BV and BV associated CSTs would exhibit increased expression and activity of collagen and elastin-degrading cathepsins. MethodsVaginal fluid samples were collected and classified by BV diagnosis and CST. Cathepsin expression was evaluated by Western blotting to distinguish inactive and active enzyme forms. Proteolytic activity was assessed using multiplex cathepsin zymography. Statistical analyses compared cathepsin expression and activity across diagnoses and CSTs. Principal component analysis and linear regression were performed to assess associations between cathepsin activity, microbial diversity, and CST. ResultsProcathepsin K expression was significantly increased in BV-positive and CST IV samples, while total cathepsin L expression was significantly elevated in samples with Nugent-intermediate scores. Cathepsins S and V showed variation in inactive and active forms in Nugent-intermediate and CST III samples. In contrast, total cathepsin activity, including cathepsins K and V, did not significantly differ across BV diagnoses or CSTs. Overall, cathepsin activity varied between individuals rather than by clinical classification. ConclusionsCathepsin expression and maturation state differ by microbiome composition, suggesting that the vaginal microbiome may regulate post-translational processing of cathepsins. As a result, cathepsin activity appears to be regulated at the individual level rather than strictly by BV diagnosis or CST. These findings link vaginal microbiome composition to ECM remodeling and potential adverse reproductive outcomes.