npj Biofilms and Microbiomes

Streptococcus mutans is the major microbial etiological agent of dental caries and can adhere to surfaces such as type-I collagen, present in dentin and periodontal tissues. Recent studies have characterized planktonic S. mutans bacterial extracellular vesicles (bEVs) and demonstrated environmental-induced changes due to sugar presence or pH alterations. However, to date there are no studies exploring if surface-derived changes - such as tissue glycation - can modulate bEV production in the context of oral biofilm formation in the elderly. Therefore, the aim of this work was to determine the role of biofilm formation and collagen glycation on the morphology and composition of S. mutans bEVs. For this, bEVs from S. mutans biofilms on native and glycated collagen surfaces were isolated, characterized, and compared to bEVs from planktonic cells. Nanoparticle tracking analysis and microscopy confirmed bEV production and showed that bEVs from biofilms are smaller in size and less abundant than those from planktonic cells. Furthermore, proteome analysis revealed that S. mutans biofilm formation on native and glycated collagen led to the enrichment of several key virulence proteins such as Eno, LuxS, Tpx, and ScrB. Also, a shift towards proteins involved in metabolic processes was found in bEVs following biofilm formation on collagen surfaces, whereas glucan metabolism proteins were overexpressed in vesicles from the planktonic state. These results demonstrate that biofilm formation, as well as the glycation of collagen associated with aging and hyperglycemia, can modulate bEV characteristics and cargo and could play a central role in S. mutans virulence and the development of diseases such as dental caries and periodontal disease.

Biofilm-associated diseases like peri-implant mucositis (PIM) and peri-implantitis (PI) are significant clinical challenges affecting millions of dental implant patients globally. Although studies have described the role of microbial, host, or environmental factors in disease development, their complex interplay, particularly during dysbiosis remains poorly understood. This cross-sectional study characterized the microbiome composition and metatranscriptomes of 125 peri-implant biofilms from 48 individuals uncovering molecular signatures linked to peri-implant health (PIH), PIM, and PI. Distinct variations were observed in biofilm amount, composition, activity, phage populations and host response. Biofilms were categorized into four community types (CTs) based on the bacterial transcriptional activity: one linked to PIH, one to PI, and two to PIM. PIH and PIM were primarily characterized by aerotolerant taxa with increased anabolic processes, while PI was dominated by obligate anaerobes with complex biofilm morphology, and heightened catabolic activity and virulence. PIM samples, relative to PIH were characterized by biofilm expansion with minimal functional changes, except for the Neisseria-rich PIM subtype showing higher pyruvate and lipoic acid metabolism. The phagome mirrored the bacterial compositional variations across disease states. Furthermore, human transcriptome responses varied indicating increased keratinization in PIH, enhanced expression of ribosome components in PIM, and inflammatory signaling and hypoxia in PI. Additionally, we identified complex species-enzyme, phage-bacteria, and host-microbe associations within the peri-implant ecosystem. Our integrative multi-omics approach provides a comprehensive view of microbial, biochemical, host, and ecological factors associated with dysbiosis, offering novel insights into peri-implant disease dynamics. ImportancePeri-implant mucositis and peri-implantitis are highly prevalent inflammatory conditions that compromise the long-term survival and success of dental implants, yet their underlying biological mechanisms are largely unresolved. While next-generation sequencing has advanced our understanding of microbial composition across health and peri-implant diseases, it falls short of capturing microbial activity and the broader molecular context of peri-implant dysbiosis. Metatranscriptomics overcomes this limitation by profiling actively transcribed genes within the biofilm, offering direct insights into microbial community functions. In this study, we integrated full-length 16S rRNA gene amplicon sequencing with metatranscriptomic profiling to simultaneously assess microbial taxonomy, functional activity, phage dynamics, and host gene expression in peri-implant biofilms. Importantly, we provide a systems-level view and report previously undescribed associations between different molecular signatures in peri-implant ecosystem.

Treating chronic bacterial infections is challenging due to the formation of biofilms, making bacteria less susceptible to antimicrobials. In vitro models have limitations in replicating biofilm physiology. To address this problem, we have created a hydrogel substrate that combines crosslinked agarose and gelatine presented as beads, providing stability and resistance to autoclaving. Bacterial pathogens rapidly colonise these biogel beads when submerged in liquid culture. The substrate was tested with Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showing more robust biofilm growth than its glass bead counterpart. Additionally, this led to increased virulence factor production and served as a reservoir for biofilm quorum sensing molecules. These features closely resemble clinical situations, suggesting a more accurate representation of biofilm-associated infections than current approaches. This new substrate offers a practical and convenient model for studying biofilms of bacterial pathogens, providing an efficient solution to the research community and holding promise for future breakthroughs.

Clostridioides difficile is a major cause of hospital-associated diarrhoea worldwide. The intricate interactions between C. difficile and the resident gut microbiota play a crucial role in determining the outcome of C. difficile infection (CDI), although the molecular mechanisms underlying many C. difficile-commensal interactions are not understood. Here we show that selected Bacteroides species can inhibit C. difficile growth within mixed biofilms. A transcriptomic analysis of C. difficile-Bacteroides biofilms showed significant metabolic shifts, with distinct changes in carbohydrate and amino acid metabolism and, interestingly, a downregulation of C. difficile toxin gene expression. A significant reduction in C. difficile toxin production was evident in C. difficile-Bacteroides cocultures, irrespective of the extent of C. difficile growth inhibition. Notably, Stickland fermentation of proline, which is known to repress toxin synthesis, was upregulated in C. difficile, while proline synthesis was induced in the cocultured species B. vulgatus and B. dorei. Furthermore, upregulation of proline reductase pathways and consequent toxin repression were evident within a synthetic 9-species gut commensal biofilm community containing multiple Bacteroides spp. Thus, leveraging multiomics approaches, we demonstrate a potential cross-feeding mechanism where proline produced by B. dorei and B. vulgatus is utilised by C. difficile through Stickland fermentation to drive toxin repression. Our study reveals a new mechanism of microbiota-mediated control of a key virulence factor involved in C. difficile pathogenesis while enabling pathogen co-existence within a polymicrobial commensal community. ImportanceC. difficile infection, characterised by severe diarrhoea and colitis, has a significant impact on healthcare settings globally due to the high rates of recurrence. CDI is closely associated with the gut microbiota status and the use of antibiotics, yet the mechanistic basis of interactions between the causative bacterium C. difficile and individual gut commensal species remains poorly defined. Here, we demonstrate inhibitory effects of Bacteroides species on C. difficile through nutrient competition and a cross-feeding mechanism between these abundant gut commensals and this pathogen which blocks expression of key C. difficile virulence factors. Our findings offer insights into the effective design of microbiota consortia to prevent and treat CDI.

Several mechanisms have been described to explain how bacterial species colonize and persist in the mammalian gut. However, biofilm formation remains underexplored as a mechanism for gut microbiota symbiont persistence. While evidence of biofilm formation by individual gut symbionts is beginning to emerge, its occurrence and relevance in multispecies gut microbiota communities remain poorly studied. Here, we established an in vitro biofilm model for the Oligo-Mouse Microbiota 12 (OMM12) consortium, a defined community of murine gut isolates, and used it to investigate community biofilm formation and responses to bile acids, host-derived detergent-like molecules released into the gut that can perturb bacterial growth and community structure. We identified distinct contributions of two OMM12 members: removal of Enterococcus faecalis strongly reduced community biofilm biomass, whereas removal of Bacteroides caecimuris had limited effect on biomass but strongly altered species associations. These results, together with monoculture assays, show that individual biofilm capacity does not directly predict community-level contribution. Although planktonic and biofilm communities had broadly similar compositions, their response to bile acid stress were markedly distinct. Planktonic cultures, while more susceptible to bile, impaired in biomass and species associations, showed resilience by recovering biomass within 24 hours upon bile stress removal. Community biofilms, in contrast, showed greater tolerance to bile acid stress and preserved or recovered more species associations. Overall, our findings support biofilms as a community-level lifestyle that can buffer defined gut microbiota communities against host-associated chemical perturbations. ImportanceDespite decades of research, how the gut microbiota maintains diversity and persistence remains to be completely understood. Gut bacterial species must withstand harsh host-derived stresses while navigating complex interspecies interactions, many of which being highly competitive. In host-associated contexts, biofilms have largely been viewed as a detrimental trait because of their role in pathogen persistence and protection from clearance, leaving the potential contribution of commensal gut biofilms to microbiota stability underexplored. Our work establishes a simple and adaptable experimental framework to study biofilm formation in a defined multispecies gut bacterial community. We show that biofilms alter how this community responds to bile acids, host-derived molecules that can disrupt bacterial growth and community structure. Our findings support biofilm formation as a protective lifestyle that can help gut symbionts withstand bile acid stress, raising the possibility that community biofilms contribute to microbiota persistence under chemical stress encountered in the host.

Bacillus subtilis has been extensively used to study the molecular mechanisms behind the development and dispersal of surface bacterial multicellular communities. Well-structured spatially organised communities (colony, pellicle, and submerged biofilm) share some similarities, but also display considerable differences at the structural, chemical and biological levels. To unveil the spatial transcriptional heterogeneity between the different communities, we analysed by RNA-seq nine spatio-physiological populations selected from planktonic and spatially organised communities. This led to a global landscape characterisation of gene expression profiles uncovering genes specifically expressed in each compartmental population. From this mesoscale analysis and using fluorescent transcriptional reporter fusions, 17 genes were selected and their patterns of expression reported at single cell scale with time-lapse confocal laser scanning microscopy (CLSM). Derived kymographs allowed to emphasise spectacular mosaic gene expression patterns within a biofilm. A special emphasis on oppositely regulated carbon metabolism genes (gapA and gapB) permitted to pinpoint the coexistence of spatially segregated bacteria under either glycolytic or gluconeogenic regime in a same biofilm population. Altogether, this study gives novel insights on the development and dispersal of B. subtilis surface-associated communities.

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.

In Bacillus subtilis colony biofilms, phenotypic diversification confers tissue-like properties and enhanced competitive fitness within a structural framework that allows both colony expansion and long-term survival via endospore formation. Cannibalism is thought to delay sporulation by enabling one subpopulation to produce the sporulation delay protein SDP, the sporulation killing factor SKF and the epipeptide EPE. These toxins are thought to lyse susceptible nonproducers, thereby releasing nutrients to prevent premature sporulation. However, the molecular mechanisms orchestrating this bacterial programmed cell death during biofilm development are poorly understood. Here, we comprehensively characterized mutants defective in either toxin production or the corresponding autoimmunity by a multiscale approach, combining luminescence reporters, colony biopsy, multi-parameter flow cytometry and MALDI-mass spectrometry imaging to resolve cannibalism function and distribution. The toxins are produced in distinct, only partially overlapping areas of the colony and interdepend in their spatial distribution. Both EPE and SDP, but not SKF, are crucial for delaying sporulation. Loss of EPE or SDP autoimmunity resulted in severe morphological changes and stress-induced occurrence of suppressor mutants. The absence of all three toxins led to small, hyper-sporulating colonies with excessive wrinkle formation, indicating that cannibalism is essential for maintaining biofilm structure and lateral expansion. Our results provide the first evidence for the complex interactions between the cannibalism toxins that shape biofilm architecture through bacterial programmed cell death. Localized toxin production and their spatial distribution affect the spatiotemporal organization, morphology and subpopulation dynamics within B. subtilis biofilms. ImportanceProgrammed cell death (PCD) is a ubiquitous and crucial mechanism to structure eukaryotic multicellular tissues. PCD-like processes have also been described in bacteria, but their contribution to the multicellular development is poorly understood. Cannibalism in Bacillus subtilis has been described as a sporulation delay strategy, in which one subpopulation produces antimicrobial peptides that kill susceptible nonproducing siblings. Their lysis is thought to release nutrients that delay the sporulation in the producing subpopulation. This study comprehensively analyses the role of the three cannibalism toxins in shaping colony biofilms. By combining MALDI-mass spectrometry imaging, colony biopsy, flow cytometry, and luminescence reporters, we demonstrate that cannibalism toxins are crucial for biofilm structure. They show a discrete and interdependent localization within the biofilm. While cannibalism inhibits sporulation and causes severe envelope stress within colonies, our data challenges the established role of cannibalism-dependent killing as the mechanism behind this sporulation delay.

The human protozoan parasite Entamoeba histolytica is responsible for amebiasis, a disease endemic to developing countries. E. histolytica trophozoites are released from the cysts to colonize the large intestine, where they primarily feed on bacterial cells. In these scenarios, bacterial cells form aggregates or structured communities too large for phagocytosis. Our results show that E. histolytica can degrade pre-established biofilms of Bacillus subtilis and Escherichia coli in a dose- and time-dependent manner. Surprisingly, trophozoites incubated with B. subtilis biofilm exhibit a unique transcriptome signature compared to those incubated with planktonic cells or without bacteria. Biofilm-induced genes include cysteine proteases (CPs), and the general inhibition of CPs by E64D or by the use of specific small-RNA (sRNA)-based RNA interference impairs the degradation of biofilms by E. histolytica. The degradation of B. subtilis extracellular matrix (ECM) protein TasA by CPs is associated with partial biofilm digestion and activation of the stress response in the interacting B. subtilis cells. The interaction with B. subtilis biofilms was also associated with lower levels of oxidoreductases. Oxidoreductase downregulation can be a readout of the embedding of E. histolytica trophozoites within the biofilm-produced extracellular matrix, reducing their exposure to oxidative stress (OS). Our results indicate that parasites may digest biofilms by a controlled mechanism of digestive exophagy as secretion of digestive enzymes as a conserved mechanism for biofilm degradation allows phagocytic digestion of biofilm cells. Furthermore, the partially digested biofilms can serve as an unexpected shield protecting parasites from oxidative environments and thereby may regulate the persistence and virulence of the parasite.

Biofilm represents a complex aggregation of bacteria embedded within a self-produced extracellular polymeric substance (EPS). We investigated the characteristics of mycobacterial biofilm using Mycobacterium smegmatis (Msm) as model organism. By combining transcriptomic (RNA-seq) and proteomic (LC-MS) analyses, the research captures dynamic changes during the establishment and maturation of the biofilm. Transcriptomics analysis showed a distinct gene expression profile as compared to its planktonic form. Interestingly, clear differences were seen between initial (~2-day old) and mature (~5-day old) biofilm stages, highlighting phasic gene expression throughout biofilm development. Marked alteration in oxidative stress-related genes and energy metabolism from ATP to NADH was observed. Furthermore, quantitative mass spectrometry-based proteome examination of EPS showed an abundance of cytoplasmic proteins present differentially between initial and mature biofilm stages. Pathway enrichment revealed enhanced oxidative stress responses and metabolic shifts in mature biofilms, including upregulation of NADH dehydrogenase and downregulation of ATP synthase, indicating altered energy metabolism. Our findings thus provide insights into the molecular adaptations, including production of mycofactocin, occurring during mycobacterial biofilm establishment and maturation, and advance our understanding of mycobacterial biofilm physiology.

Diet is an important ecological modulator of the oral microbiome, yet population-level evidence on a broader spectrum of food components remains limited. This cross-sectional study investigated associations among dietary intake, oral rinse microbiome, and oral disease conditions in a nationally representative sample of United States adults from the National Health and Nutrition Examination Survey. A total of 3,254 participants with oral rinse microbiome sequencing data were included, with oral conditions classified as oral health, caries-only, periodontitis-only, or co-existing disease. Dietary intake was assessed using 24-hour dietary recalls and summarized as dietary indices and energy-adjusted food components. Associations between diet and the oral microbiome were evaluated using community-level analyses, regression models, mediation analyses, and unsupervised clustering, while accounting for oral conditions. This study found that dietary intake, as a combined variable set, explained 3.6% of the variance in oral rinse microbial community structure; this was comparable to oral disease status or smoking and larger than sociodemographic factors. Healthier dietary profiles, including higher health-associated dietary index scores and greater vegetable and fruit intake, were associated with taxa commonly linked to oral health (e.g., Neisseria, Cardiobacterium and Lautropia). In contrast, added sugars, alcoholic drinks, cured meat, potatoes, dairy products, and higher dietary inflammatory index scores showed opposite association patterns. Mediation analyses suggested that coordinated microbial groups may partly link dietary exposures with oral disease outcomes, particularly for vegetables and added sugars. Additionally, three population-level dietary patterns were identified, among which the plant-rich pattern was associated with more favorable oral health and microbial profiles enriched in nitrate-reducing commensals, including Neisseria and Haemophilus. Overall, dietary intake was associated with oral microbiota composition and oral health conditions, supporting ecological influences of dietary components beyond sugar on oral bacteria and dental diseases. Longitudinal studies are needed to clarify the direction and causality of these relationships.

Non-toxic, biocompatible materials that inhibit bacterial biofilm formation on implanted medical devices and so prevent infection are urgently required. Weakly amphiphilic acrylate polymers with rigid hydrocarbon pendant groups resist bacterial biofilm formation in vitro and in vivo but the biological mechanism involved is not known. By comparing biofilm formation on polymers with the same acrylate backbone but with different pendant groups, we show that poly(ethylene glycol dicyclopentenyl ether acrylate; pEGdPEA) but not neopentyl glycol propoxylate diacrylate (pNGPDA) inhibited the transition from reversible to irreversible attachment. By using single-cell tracking algorithms and controlled flow microscopy we observed that fewer Pseudomonas aeruginosa PAO1 cells accumulated on pEGdPEA compared with pNGPDA. Bacteria reaching the pEGdPEA surface exhibited shorter residence times and greater asymmetric division with more cells departing from the surface post-cell division, characteristic of reversible attachment. Migrating cells on pEGdPEA deposited fewer exopolysaccharide trails and were unable top adhere strongly. Discrimination between the polymers required type IV pili and flagella. On pEGdPEA, the lack of accumulation of cyclic diguanylate or expression of sadB were consistent with the failure to transit from reversible to irreversible attachment. Constitutive expression of sadB increased surface adhesion sufficient to enable P. aeruginosa to form biofilms in a Mot flagellar stator dependent manner. These findings were extendable to other biofilm resistant acrylates highlighting their unique ability to inhibit reversible to irreversible attachment as a mechanism for preventing biofilm-associated infections. SignificanceBacteria readily attach to surfaces forming biofilms. These are commonly associated with medical device-associated infections and highly refractory to antibiotics. Biocompatible, weakly amphiphilic acrylate polymers with large hydrophobic pendant groups that inhibit biofilm formation and can prevent such infections have been described. However, the biological mechanism involved is not understood. By comparing a biofilm-inhibiting with a biofilm-supporting acrylate, we showed that Pseudomonas aeruginosa PAO1 cells responded differentially to the two polymers and were unable to accumulate and adhere strongly, activate cyclic diguanylate signalling or transit from reversible to irreversible attachment on the inhibitory polymer. Constitutive expression of sadB increased surface adhesion sufficient to enable P. aeruginosa to form biofilms in a flagellar stator dependent manner overcoming the biofilm inhibitory properties of the polymer.

The distribution of microorganisms in built environments with high human traffic, such as food centres, can potentially have a significant impact on public health, particularly in the context of increasing worldwide incidence of food and fomite-related outbreaks. In several major Asian cities, public food centres are the main venue for food consumption and yet we lack a baseline understanding of their environmental microbiomes. We conducted city-wide metagenomic surveillance of food-centre microbiomes in Singapore (16 centres, n=240 samples) to provide a detailed map of microbial (bacteria, archaea, fungi, viruses) as well as non-microbial DNA abundances across two timepoints. Food-centre microbiomes were found to be enriched in food-related DNA signatures compared to other environments such as hospitals and offices, with specific food-microbe associations (e.g. Enterobacteriaceae and fish) and food DNA providing a partial explanation for the microbial profiles observed (44% of variation explained). Machine learning analysis identified a small set of microbial species (n=22) that serve as highly accurate (>80%) location-specific signatures for various food centres, some of which persist even after 3 years. Profiling of antibiotic resistance genes (ARGs) and pathogens identified a surprising enrichment of ARGs in food centres relative to other non-healthcare environments (>2.5{xi}), and an order of magnitude enrichment of key pathogenic species (e.g. Klebsiella pneumoniae, Enterobacter spp) even compared to hospital environments. These results highlight the contribution of diverse biotic and abiotic factors in shaping the unique microbiome profiles of different food-centre environments, and the potential for using metagenomic surveillance to understand the risk for infections and antibiotic resistance gene transmission.

Entamoeba histolytica (Eh) is a formidable intestinal pathogen, yet the ecological principles governing its invasion of the gut microbiome remain elusive. Upon colonization, Eh encounters resident bacteria typically sequestered within aggregates and biofilms. While Eh utilizes cysteine proteinases to degrade biofilm matrices and access bacterial prey, the strategies bacteria employ to sense and evade this predation are largely unknown. Here, we identify a metabolic signalling axis that allows the probiotic bacterium Bacillus subtilis to perceive and respond to predatory Eh. In the absence of mitochondria, Eh relies on fermentative glycolysis, using alcohol dehydrogenase to produce distinct metabolic byproducts. Using quantitative proteomics and single-cell imaging, we demonstrate that B. subtilis detects the Eh-derived metabolite acetaldehyde as a proxy for predator presence. By mapping the acetaldehyde-responsive regulatory network, we show how this metabolic input is transduced to control the motility machinery, while our data suggest that the broader predatory secretome acts as a multi-modal signal influencing multiple bacterial physiological programs. This chemical cue triggers a rapid phenotypic switch in B. subtilis, driving a transition towards a motile, planktonic "flight" response. Our findings reveal that commensal bacteria exploit the unique metabolic signature of anaerobic parasites to coordinate defensive behaviours, highlighting how inter-kingdom signalling shapes microbiome architecture during infection.

Mucosal biofilms play an important role in intestinal health; however, the mucosal bacterial community has been implicated in persistent infections. Clostridioides difficile is an important nosocomial pathogen, with an unacceptable high rate of recurrence following antibiotic treatment. As C. difficile is a known biofilm producer, a property which may contribute to this suboptimal therapeutic response, we have investigated the transcriptional changes and regulatory pathways during the transition from planktonic to biofilm mode of growth. Widespread metabolic reprogramming during biofilm formation was detected, characterised by an increased usage of glycine metabolic pathways to yield key metabolites, which are used for energy production and synthesis of short chain fatty acids. We detected the expression of 107 small non-coding RNAs that appear to, in some part, regulate these pathways; however, 25 of these small RNAs were specifically expressed during biofilm formation, indicating they may play a role in regulating biofilm-specific genes. Similar to Bacillus subtilis, biofilm formation is a multi-regulatory process and SinR negatively regulates biofilm formation independently of other known mechanisms. This comprehensive analysis furthers our understanding of biofilm formation in C. difficile, identifies potential targets for anti-virulence factors, and provides evidence of the link between metabolism and virulence traits.

Previous transcriptional profiling of the enteropathogen Yersinia pseudotuberculosis during persistent stages of colonisation of mouse cecal lymphoid follicles indicated the possible involvement of biofilm in infection maintenance. Not much is known about the mechanisms responsible for biofilm formation by this pathogen, and most current knowledge is based on results of experiments conducted using the related Y. pestis pathogen that forms biofilm in the flea gut. In this study, we performed transcriptional profiling of Y. pseudotuberculosis in biofilms from different biofilm-inducing conditions, bile exposure, amino acid deprivation and in vivo mimicking conditions with and without oxygen. The comparison of differential expression of genes in biofilm versus planktonic bacteria showed a set of 54 core genes that were similarly regulated, independent of inducing condition. This set included many genes that were previously shown to be associated with biofilms, such as hutG, hsmF, hmsT and cpxP that were upreg-ulated and other genes such as hmsP and rfaH that were downregulated. There were also novel biofilm-associated genes, including genes encoding hypothetical proteins. To identify the genes involved in inducing biofilm formation, the gene expression of bacteria during an early initial phase when biofilm starts to form after induction by bile or amino acid depletion was determined. Comparisons of the resulting gene expression profiles with the profiles of non-induced bacteria incubated for the same period of time showed a set of core genes associated with early biofilm formation. This set included genes involved in quorum sensing, pili biogenesis and genes indicative of a potential metabolic shift involving nitrogen utilisation. Genes encoding components of sugar phosphotransferase systems were also up-regulated during biofilm induction. Assays of biofilm formation by bacteria deleted of some of these core genes showed that strains lacking hpr and luxS, which are known to be important for functional sugar phosphotransferase systems and quorum sensing, as well as glnL encoding a sensory histidine kinase were most negatively affected. Most of the deletion mutant strains tested were affected, but the effect was less severe, suggesting high levels of redundancy in the pathways involved in biofilm formation by this pathogen.

The gut microbiome composition varies across human populations, but its characteristics and determinants in a multi-ethnic setting remain incompletely understood. Singapore, a multicultural city-state, provides a context in which culturally diverse groups share a broadly similar built environment. Using the Health for Life in Singapore (HELIOS) cohort, we profiled the gut microbiome of ethnic Chinese, Indian, and Malay participants (n=861) who resided in the country. Despite substantial overlap in the overall microbial compositions, each group displayed distinct microbial signatures that paralleled culturally rooted dietary habits. Specifically, ethnic Indian participants showed enrichment of multiple Bifidobacterium species associated with greater intake of traditional grain-based staples such as idli and thosai; ethnic Malay participants exhibited higher abundance of Ruminococcaceae associated with coconut-and rice-based dishes; and ethnic Chinese participants had greater levels of Bacteroides associated with seafood- and meat-rich diets. These ethnicity-diet-microbiome relationships were further corroborated by additional data from two independent Malaysian cohorts (n=544), a cohort from the United States (n=210), and an in vitro microbial culture model showing selective Bifidobacterium expansion by a fermented rice-based batter. Analysis of fecal microbiome-based risk scores revealed ethnic gradients in colorectal neoplasia scores that mirrored population-level cancer incidence patterns. Together, these findings characterize gut microbiome variations across major Asian ethnicities residing in a shared urban environment, providing a reference for precision health strategies relevant to over two billion ethnically relevant people in the Asia-Pacific region and beyond.

Bacterial cells detached from Staphylococcus epidermidis biofilms are found to release predominantly as small oblate clusters ([~]1.9 {micro}m) in both untreated biofilms and biofilms treated with matrix-targeted disruptors. Quantitative image analysis common to colloidal science was applied to quantitatively evaluate the physical properties of 9,147 bacterial clusters detached from S. epidermidis biofilms with and without targeted disruption of individual matrix components (polysaccharides, proteins, extracellular DNA) or solubilization of the extracellular polymeric substances (EPS). Concentrations of S. epidermidis biofilm-detached cells are highest after matrix-targeted disruption of polysaccharides. K-means clustering, an unsupervised machine learning technique, was used to reveal that S. epidermidis biofilm-detached cells are released in five distinct phenotypes: small oblate, mid-sized oblate, large oblate, small spherical, and mid-sized prolate clusters. S. epidermidis biofilm detached cell clusters are predominantly oblate across three size groups (79.5%), with the small oblate phenotype representing 60.1% of cell clusters that have 3.1 {+/-} 1.2 cells per cluster, Euclidean diameters of 1.9 {+/-} 0.4 {micro}m, anisotropy indices of 0.98 {+/-} 0.05, and asphericities of -1.75 {+/-} 0.31 on average. The proportion of S. epidermidis cell clusters within each biofilm-detached cell phenotype differs between matrix-targeted disruptors. There are also variations in the abundance of S. epidermidis biofilm detached cells after matrix-targeted disruption between growth conditions and strains. Evaluating the physical properties of biofilm-detached cells after matrix-targeted disruption is critical to understanding their translocation in fluid flow and susceptibility to the host immune response as well as in evaluating matrix-targeted disruption for biofilm control.

Dietary fiber has been consistently associated with a decreased risk of colorectal cancer (CRC) development. While the apoptotic effect of dietary fiber microbial fermentation products on tumor colonocytes is well established, the role of these products on other components of the tumor microenvironment remains unexplored. Tumor associated macrophages play a critical role in tumor development in the colon; however, the effect of dietary fiber fermentation by microbiota on macrophage-colonocyte interaction in colorectal cancer has been difficult to dissect due to a lack of complex in vitro models of CRC containing both immune cells and microbiota. Recently, we developed a microfluidic model that facilitates the coculture of CRC spheroids with complex microbial communities. Here, we expand our model to include macrophages and employ it to study the impact of dietary fiber on macrophage-colonocyte interaction. We optimized monocyte differentiation parameters in vitro and demonstrated the capacity of our model to recapitulate changes in microbiota composition and metabolic output associated with dietary fiber administration in vivo. Combinatorial coculture of colonocytes with microbiota and macrophages revealed that alterations in microbial production of SCFA derived from dietary fiber fermentation correlated with enhanced colonocyte death, possibly mediated by an increase in transcription of tumor pro-apoptotic signals by macrophages. Our work highlights the capacity of complex in vitro systems to study the role of microbial metabolism of dietary molecules on CRC colonocyte viability and macrophage activity.

Antibiotic-resistant biofilm infections have emerged as public health concerns because of their enhanced tolerance of high-dose antibiotic treatments. The biofilm life cycle involves multiple developmental stages, which are tightly regulated by active cell-cell communication via specific extracellular signal messengers such as exosomes. This study was aimed at exploring the roles of Pseudomonas aeruginosa exosomes secreted at different developmental stages in controlling biofilm growth. Our results show that exosomes secreted by P. aeruginosa biofilms during their exponential growth phase (G-Exo) enhance biofilm growth. In contrast, exosomes secreted by P. aeruginosa biofilms during their death/survival phase (D-Exo) can effectively inhibit/eliminate P. aeruginosa PAO1 biofilms up to 4.8-log10 CFU/cm2. The inhibition effectiveness of D-Exo against P. aeruginosa biofilms grown for 96 hours improved further in the presence of 10-50 M Fe3+ ions. Proteomic analysis suggests the inhibition involves an iron-dependent ferroptosis mechanism. This study is the first to report the functional role of bacterial exosomes in bacterial growth, which depends on the developmental stage of the parent bacteria. The finding of D-Exo-activated ferroptosis-based bacterial death may have significant implications for preventing antibiotic resistance in biofilms. Significance statementAntibiotic-resistant bacterial infections caused 1.27 million deaths in 2019 [1], and this number is projected to increase to 10 million deaths annually worldwide by 2050 [2]. Of these infections, up to 80% are caused by biofilm-associated infections [3, 4], which pose a significant challenge to human health. The treatment of biofilm infections remains a formidable problem because of the limited effectiveness of the currently available antibiotics against drug-resistant biofilms [5, 6]. The development of new therapeutic approaches that can effectively combat biofilm infections is required. This study represents a promising solution to antibiotic-resistant biofilm infections. The successful use of exosomes against biofilms opens new possibilities for combating challenging antibiotic-resistant biofilm infections.