Biofilm

1Opportunistic, biofilm-forming pathogens such as Pseudomonas aeruginosa can employ an array of strategies to reduce the impact of antibiotics on their survival. The biofilm matrix can prevent antibiotics from reaching bacteria embedded within it; general changes in metabolic activity alter susceptibility to specific drugs dependent on the target; changes in the membrane and the expression of channel or pump proteins embedded within it affect drug uptake and efflux; and production of antibiotic-degrading enzymes can remove the threat. In this study, we report that biofilm-deficient mutants of two well-studied lab strains of P. aeruginosa (PA14 and PAO1) have wild-type (WT) levels of tolerance to colistin and meropenem when allowed to establish mature populations in an ex vivo pig lung model of cystic fibrosis lung infection. The biofilm defects in the mutants were confirmed using SEM, and cryoSEM was used to visualise the hydrated biofilm matrix in the WT. Using RNA sequencing of the PA14 WT and an isogenic mutant lacking the pel polysaccharide, we were able to identify a small number of differences in the responses of the two genotypes to the lung environment and to exposure to sub-bactericidal colistin in the lung model. Notably, there was differential upregulation of the MexXY-OprM and MexEF-OprN multidrug efflux pumps. However, the relative roles of biofilm matrix versus cellular changes in physiology in conferring antibiotic tolerance in this environment remain to be fully elucidated.

2.Quantitative image analysis is central to understanding microbial growth, morphology, and spatial organisation. However, conventional metrics such as mean intensity or object count often do not capture the complex structural heterogeneity and patterning characteristic of microbial colonies and biofilms. To address this limitation, Analysis of Biofilm Complexity in 3D (ABC3D), an open-source Python framework for automated extraction of fractal, textural, and statistical descriptors from volumetric microscopy images, is reported. ABC3D computes a set of parameters including fractal dimension, lacunarity, entropy, grey-level co-occurrence matrix features, and wavelet sub-band energies from three-dimensional (3D) image datasets. ABC3D is demonstrated in macrocolony biofilms formed by cell shape mutants of Escherichia coli, where it is shown that nutrient availability accounts for the majority of structural variance, while cell shape produces additional structural variation that differs between nutrient conditions. ABC3D provides researchers with an accessible, quantitative approach to assessing biofilm morphology in microscopy datasets. SummaryAn open-source, quantitative analysis pipeline is presented that integrates fractal, lacunarity, entropy, texture and wavelet descriptors to characterise colony biofilm architecture in three dimensions. Application to Escherichia coli cell shape mutants demonstrates that macrocolony biofilm architecture is best understood as a coordinated, multiscale phenotype rather than as an aggregate of independent structural metrics. 3. Impact statementBiofilm architecture is pivotal for microbial survival, antimicrobial tolerance, and ecological function but tools to quantify structural organisation in these cell communities remain limited. The commonest metrics describe bulk properties such as width, thickness, or cell number, but they do not capture multiscale spatial heterogeneity. Here, an open-source framework for Analysis of Biofilm Complexity in 3 Dimensions (ABC3D) is reported. This software integrates measurements of fractal geometry, lacunarity, entropy, texture statistics, and wavelet energy. ABC3D is demonstrated in Escherichia coli macrocolony biofilms, where it is shown that nutrient environment has a leading role in determining colony architecture in E. coli biofilms, while cell shape has a lesser but still significant influence on structural variation. The ABC3D pipeline can be applied to any microbial communities imaged by confocal microscopy and other volumetric imaging methods and has the potential to give a deeper understanding of how cells organise in biofilms. 4. Data summaryFull code for ABC3D and data analysis is available at https://github.com/gailmcconnell/ABC3D. Image data are available upon request. The author confirms all supporting data, code and protocols have been provided within the article or through supplementary data files.

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.

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.

ObjectivesBiofilms are the dominant mode of bacterial life. The gut microbiota itself has characteristics of a biofilm that grows on the intestinal mucosa. C. difficile and VRE are commonly co-isolated from patients but biofilm formation has not been studied in a multi-species context. Here we study the interactions between C. difficile and VRE in surface adherent community. ResultsWe found that VRE inhibits C. difficile biofilm formation in dual-species culture in the presence of excess glucose. Robust dual-species biofilms were produced when the carbon source was changed to a non-fermentable sugar such as fucose and xylose. We observed a high level of vancomycin tolerance in C. difficile biofilms that was not affected by the presence of VRE. Finally we also found that a nutrient step-change is sufficient to induce dispersion of single and dual-species biofilms. ConclusionsVRE can inhibit the development of C. difficile biofilms in the presence of a fermentable carbon source. VRE does not appear to affect vancomycin tolerance or nutrient-induced dispersion of C. difficile biofilms. Highlights- VRE inhibits C. difficile biofilm formation in the presence of fermentable glucose. - Stable VRE - C. difficile biofilms are formed by managing the available carbon source. - VRE does not affect C. difficile vancomycin tolerance in this model. - A 10-fold increase in available nutrients is sufficient to induce biofilm dispersion in C. difficile and VRE.

1.1Antimicrobial resistant infections present a concerning and expanding healthcare problem that is compounded by the reduction of antimicrobial research by the pharmaceutical industry. Additionally, the currently used antimicrobials consistently present less than ideal clinical treatment outcomes. This contention is supported by in vitro analysis in appropriate models. Here, in mature methicillin resistant Staphylococcus aureus (MRSA) in vitro cultures, we tested multiple antimicrobials and showed that for a biofilm grown for 72 hours, no antimicrobial tested was able to completely eradicate the biofilm even after 24 hours of exposure. However, the addition of an enzymatic biofilm-dispersal agent (DNase I or Proteinase K), greatly improved the performance of vancomycin and tigecycline in this in vitro model. Despite the improved performance in the presence of the dispersal agent, a high concentration of antimicrobial, 2000 {micro}g/mL, was needed to completely eradicate the infection as demonstrated by analyses using both a traditional XTT assay as well as a subculture assay to account for persister cells. It was shown that the addition of DNase I improved the diffusion of vancomycin through the biofilm. This suggests vancomycin efficacy is limited by the biofilm. The presented work provides a potential avenue for future treatments of MRSA lung infections by utilizing a traditional antibiotic combined with a passive dispersive agent.

ObjectivesBacteriophage (phage) therapy is being explored as a strategy for clinical control of periprosthetic joint infections, addressing the challenges of antibiotic-resistant bacteria and limitations around the complete eradication of methicillin-resistant Staphylococcus aureus biofilms by conventional antibiotic treatment. Additionally, phage-antibiotic combinations may enhance treatment success through synergistic interactions. This study aimed to compare the efficacy of phage Silviavirus remus (Remus) and vancomycin, independently and together, against S. aureus biofilms on Kirschner-wire (K-wire) implants using Galleria mellonella as an in vivo model, and to assess the biocompatibility of K-wire implantation. ResultsSurvival of G. mellonella larvae implanted with S. aureus biofilms and treated with Remus ([~]3 x 104 PFU/worm) and/or vancomycin (5 {micro}g/worm) after 72 hours was assessed using Kaplan-Meier curves. Statistical analyses among treatment groups were performed using log-rank tests at p-value < 0.10. Implantation of uncolonized K-wires with treatment administration did not affect survival (100%). Compared with untreated biofilm-infected larvae (77%), vancomycin alone (97%; p = 0.023) or in combination with Remus (93%; p = 0.087) improved survival, whereas Remus alone did not increase survival (67%, p = 0.33). Scanning electron microscopy confirmed the presence of biofilm-associated attachment of S. aureus on K-wires, although adherence was non-uniform.

AbstractBeneficial associations between bobtail squids (Cephalopoda: Sepiolidae) and Vibrio bacteria encompass a unique association where symbionts are obtained environmentally from the surrounding environment. Vibrio symbionts are susceptible to a number of ecological pressures such as protozoan grazing whilst in their free-living state. Impacts of grazing have several consequences for symbiosis characteristics such as biofilm formation, a trait crucial for survival both in and outside the squid. Therefore, in order to ascertain how biotic factors such as grazing in the environment effect symbiotic success, two V. fischeri strains, ES114 and ETBB1-C were experimentally evolved in separate biofilm grazing experiments with the amoeba, Acanthamoeba castellanii and ciliate Tetrahymena pyriformis. Both ES114 and ETBB1-C biofilms were evolved up to 50 generations through serial passaging. At 50 generations, ES114 biofilms displayed variability in response to predation by both predators, whereas ETBB1-C biofilms were more stable across generations of grazing. A. castellanii decreased in population numbers when co-inoculated with ETBB1-C, whereas T. pyriformis increased in numbers with biofilm growth. Growth of V. fischeri biofilms in the presence of grazers such as T. pyriformis has an important role in inducing biofilm growth by acting as a chaperone for recycling nutrients back into the environment. Additionally, V. fischeri colonization fitness in the host was dependent on which grazer was used to evolve the biofilms. Such variation in response by V. fischeri to different types of predation demonstrates the versatility of this symbiont in its free living state and has subsequent impacts on the eventual association with squids. ImportanceThis manuscript demonstrates the importance of biotic factors (such as protozoan grazing) in the environment that effect host colonization in a beneficial symbiosis. Using an experimental evolution approach, this work demonstrates how symbiotic biofilms can adapt to pressures such as grazing that subsequently influences the ability to colonize its invertebrate host.

Air-liquid interfaces (ALIs) at the upper layer of oceans, lakes and rivers cover the majority of the earths surface. Microbes are known to accumulate at these resource-rich boundaries, but the mechanisms of ALI colonization are often assumed to mirror the formation of pellicle biofilms by non-aquatic organisms. Here, we analyzed ALI colonization by natural aquatic bacteria. We used samples from a freshwater lake to enrich for microbes that colonize the ALI in liquid growth medium. Mixed-species pellicles formed rapidly in these enrichments, were structurally stable for weeks and displayed a pronounced ecological succession. We isolated 31 members of the genus Pararheinheimera from early stages of mixed-species pellicle maturation. Five phylogenetically distinct Pararheinheimera clades were identified, each with a shared colony morphology. We used representative isolates to show that only one Pararheinheimera clade formed thin, adherent films at the ALI resembling classical pellicles. Isolates from the four remaining clades formed floating structures that could be categorized either as non-adhesive films or large viscous masses. Viscous mass (VM) pellicle formation was a polyphyletic trait that correlated with a highly mucoid appearance on agar plates, suggesting that the process is driven by copious secretion of extracellular matrix. Matrices from VM biofilms were largely non-adhesive, contained a mixture of acidic polysaccharides and proteins and formed thermally stable, shear-thinning hydrogels. Our results demonstrate that ALI colonization strategies vary widely even among closely related aquatic bacteria and identify VM pellicles as a distinct biofilm architecture with unique mechanical properties. ImportanceLakes, rivers and oceans contain a boundary between the air and the waters surface known as the air-liquid interface (ALI). Microbial communities that populate the ALI play crucial roles in nutrient cycling, but how aquatic microbes partition to these sites remains poorly characterized. Our study investigated how bacteria from a freshwater lake accumulate at the ALI. Lake water samples incubated in nutrient medium formed a layer of cells known as a pellicle biofilm at the ALI, and we isolated 31 different bacteria from a genus (Pararheinheimera) that was abundant during the early stages of pellicle formation. Only a subset of Pararheinheimera isolates formed traditional pellicle biofilms. Most formed either thin, non-adhesive films or large, gelatinous aggregates that appeared to persist at the ALI due to buoyancy. These findings expand our understanding of biofilm diversity in aquatic systems and suggest that the production of buoyant hydrogels may play an important role in structuring microbial communities at air-water boundaries.

The study presented here shows Biofilm quantification in microtiter plates in strains of Candida auris and Candida albicans evaluated by means of Crystal violet, MTT, ATP-Luminescence and NBTZ/BCIP assays. The results showed significant differences in biofilm formation between Candida auris and Candida albicans but also within Candida auris outbreak strains in contrast to Candida auris DSM 21092 reference strain.

Rock-inhabiting fungi thrive in subaerial oligotrophic environments such as desert rocks, solar panels and marble monuments where organic carbon and nitrogen are scarce. We tested whether the rock-inhabiting fungus Knufia petricola showed a preference regarding nitrogen ([Formula] or [Formula]) and carbon (glucose or sucrose) sources and whether it was sensitive towards carbon and nitrogen limitation. As this fungus produces the carbon-rich, nitrogen-free 1,8-dihydroxynaphthalene (DHN) melanin, we tested whether a melanin-deficient mutant would be less sensitive to carbon limitation. The carbon and nitrogen concentrations were the primary predictors of growth, with a broad optimum partially explained by an optimal fungal C:N ratio. Limiting carbon or nitrogen supply decreased biomass formation, CO2 production and biofilm thickness but promoted substratum penetration through filamentous growth. The nitrogen content of the biomass was flexible within limits, increasing upon increasing nitrogen supply or decreasing carbon supply. The carbon use efficiency was fairly constant, whereas melanization correlated with a higher nitrogen content of the biomass despite melanin being nitrogen-free. In conclusion, in vitro, K. petricola switches to explorative growth under nutrient limitations, like fast-growing fungi, revealing universal fungal resource-acquisition patterns. Graphical abstract text and imageCarbon and nitrogen availability affect biofilm growth and morphology of the extremotolerant fungus Knufia petricola Abolfazl Dehkohneh, Julia Schumacher, Bastiaan J. R. Cockx, Karin Keil, Tessa Camenzind, Jan-Ulrich Kreft, Anna A. Gorbushina, Ruben Gerrits Growth of the rock-inhabiting fungus Knufia petricola was studied by varying carbon and nitrogen sources and concentrations. Overall, growth was best predicted by the carbon and nitrogen concentrations. Carbon and nitrogen limitation promoted substratum penetration through filamentous growth. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/712823v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@6d98bdorg.highwire.dtl.DTLVardef@146aac5org.highwire.dtl.DTLVardef@757fa8org.highwire.dtl.DTLVardef@ff709_HPS_FORMAT_FIGEXP M_FIG C_FIG

Staphylococcus aureus is the leading cause of soft tissue infections that can be treated with antibiotics. However, it can also cause significant mortality and morbidity due to systemic infections and infections of surgical implants. Implant infections typically require invasive surgery, and treatment often necessitates removal of the implant because S. aureus biofilms are extremely difficult to eradicate with antibiotic treatment alone. Therefore, there is a significant need for improved diagnostic tools for rapid, non-invasive confirmation of S. aureus infections. We recently developed an activity-based probe containing an oxadiazolone electrophile that selectively labels the S. aureus-specific serine hydrolase, FphE, by covalent binding to its active site serine residue. Here we describe a Cy5-labeled version of the probe, JJ-OX-012, and its characterization as an imaging agent for detecting biofilms both in vitro and in vivo. The probe labeled S. aureus biofilms in vitro, with virtually no background labeling of bacteria that lack FphE expression. Furthermore, we demonstrate that JJ-OX-012 can be used for non-invasive fluorescent imaging as a way to detect S. aureus biofilms in vivo. Overall, these findings support the potential for using covalent probes targeting FphE as imaging agents for rapid detection and diagnosis of staphylococcal infections in vivo.

Biofilms in the built environment (BE) can harbor pathogens and have been linked with negative health outcomes, particularly in hospital environments. The formation of biofilms requires bacterial cell attachment on surfaces, such as hospital plumbing, which can have varying properties, including roughness, wettability, chemistry, and charge. Despite the importance of bacterial attachment to surfaces, the role of multiple surface properties has been minimally investigated. Using seven materials with differing surface characteristics, this work considers the initial attachment of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus to investigate the impact of several surface characteristics. Initial attachment was evaluated using column experiments and compared to batch experiments in which bacterial growth on coupons was monitored. The attachment of all bacterial species was not influenced by material surface properties, with similar attachment seen across materials tested. Bacterial cell envelope morphology affected attachment, with gram-negative species displaying greater attachment than gram-positive species. Attachment efficiency () was found to be a good predictor of bacterial attachment, with greater sensitivity than batch tests. Establishment of commensal communities should be the focus for limiting pathogens in the BE, as engineering surfaces to reduce microbial attachment appears to offer limited benefit.

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

The gut microbiota influence many aspects of their hosts health and physiology including the digestion of food, and food intake in turn influences the composition of the gut microbiome. However, the ways in which food can alter the behavior of intestinal bacteria remain largely unknown, due in large part to the difficulty of assessing behavior in situ. Larval zebrafish provide a model for addressing this gap because of their optical transparency and their ability to be prepared germ-free and then associated with specific microbial species. Using light sheet fluorescence microscopy to visualize bacteria inside the intestines of live zebrafish larvae, we examine the properties of two commensal strains with markedly different physical characteristics. One is a zebrafish-commensal Enterobacter species that forms large aggregates in unfed larvae, and the other is a pathobiont Vibrio species that is motile and planktonic. Following host consumption of rotifers, a common food, Enterobacter clusters disintegrate into motile individuals. Vibrio remains planktonic in fed larvae but decreases the activity of its Type VI Secretion System, leading to a strong decrease in damage to host tissue. Our results reveal that feeding can have major impacts on bacterial behavior that should be considered in models of normal gut microbiome dynamics as well as pathogenesis.

BackgroundThe nasal microbiome is a collection of diverse microbial populations that inhabit the nose. Staphylococcus aureus is the most common opportunistic pathogen that colonizes the nasal mucosa, increasing the risk of invasive infections in immunocompromised and hospitalized patients. Clinicians usually prescribe antibiotics to decolonize the nasal cavities of at-risk patients from S. aureus. However, their broad antimicrobial activity can damage the resident nasal microbiome. Instead, naturally occurring compounds or resident bacteria in nasal microbiomes can effectively and safely exclude S. aureus from the nose. Cell culture and animal models have been used for nasal microbiome studies. However, their unstable microbiomes reduce the accuracy and reliability of the results. Recently, continuous bioreactors have been proposed as alternatives to these models. ResultsWe designed and operated a continuous bioreactor system to maintain stable nasal microbiomes. Next, we inoculated the bioreactor with nasal-swab specimens that we had collected from healthy volunteers. We operated the bioreactors under varying conditions (i.e., operating mode, dilution rate, temperature, pH, and medium composition), and determined the optimal conditions (continuous mode, 1 d-1, 30xlink:href=" pH 6.5, and synthetic nasal medium 3), resulting in stable microbiomes consisting of the main nasal bacterial species. The nasal microbiomes in the optimized bioreactors showed high reproducibility and resilience during a pH perturbation. Moreover, all microbiomes in the bioreactor, which were inoculated with six different nasal-swab specimens, maintained stable bacterial and metabolite compositions. In addition, we applied a synthetic microbial community (SynCom), which was derived from one of the volunteers, to demonstrate a S. aureus decolonization strategy. The bioreactor, inoculated with this SynCom, maintained a stable nasal microbiome for more than one month. Finally, different S. aureus strains that we inoculated in the SynCom showed distinct growth patterns within the otherwise stable community. ConclusionsThe continuous bioreactor enables the cultivation of stable nasal microbiomes for longer than one month by mimicking the environmental conditions of the human nose. The bioreactor is a valuable model for understanding the functions of the nasal microbiome and devising new decolonization strategies against S. aureus.

Spatial organization is a defining feature of multispecies biofilms and critically influences microbial interactions and emergent community properties. However, understanding and manipulating how microbes assemble into spatially structured biofilms remains challenging because most experimental frameworks emphasize species composition and pairwise interactions, while often overlooking the spatial constraints on biofilms imposed by the environment. In this study, we focus on how carbon substrate type, distinguishing between diffusible sugars and polymeric substrates, affects biofilm self-organization in a four-member synthetic bacterial community (SynCom). Across all tested conditions, the SynCom consistently formed more biofilm biomass than any of its subsets, indicating a robust synergistic phenotype. Using chemically defined, 3D-printed hydrogel substrates with consistent physical properties, we varied carbon source composition to identify its impact on biofilm assembly. Microscopic imaging showed that carbon substrate type strongly influenced biofilm self-organization with diffusible simple carbon substrates yielding relatively intermixed communities, whereas polymer-rich carbon substrates promoted a highly structured biofilm organization characterized by the dominance and peripheral localization of polymer-degrading species. Bioinformatic analyses of carbohydrate-active enzymes (CAZymes) annotation and genome-scale metabolic modeling suggested that metabolite exchange networks in the SynCom may drive more complex metabolic interactions beyond the commonly observed degrader-exploiter-scavenger relationship within planktonic microbial communities. Together, our findings demonstrate carbon substrate type as an important ecological determinant of biofilm self-organization, highlighting the need to integrate environmental factors alongside species composition and metabolic potential to fully understand and manipulate natural and engineered multispecies biofilms.

Pseudomonas aeruginosa infections in adults with cystic fibrosis (CF) are comprised of heterogeneous populations, most often tracing ancestry back to a single recent common ancestor. What is not clear is the physical spatial structure within the lung infection population, its stability over time and whether this physical structure leads to different evolutionary trajectories in different adaptive environments. To compare the P. aeruginosa populations across a single lung, we performed whole genome sequence analyses of 450 isolates recovered from lavage samples of the three different lobes of the right lung from a person with mild-to-moderate CF lung disease at three time points over the course of [~]1.5 years. We found that isolates fell into five distinct phylogenetic lineages with evidence for repeated translocation of isolates from different lineages across lobes and loss-of-function mutations in lasR and mucA were present in all 450 isolates. The well-resolved phylogenetic analyses revealed a structured population in which we find the coexistence of a slowly evolving lineage and more rapidly evolving lineages. There is also support for numerous migration events. Further, strong evidence for parallel adaptive mutations in multiple genes revealed distinct evolutionary paths affecting mucoid phenotypes and genetic variation in antibiotic resistance-associated pathways across coexisting populations within a single individual over time. These results provide an example of within-host evolution leading to microheterogeneity that may be useful to consider in future study of infection metapopulations dynamics over the course of chronic infection. IMPORTANCEIndividuals with cystic fibrosis (CF) commonly have chronic lung infections that contain clonally derived Pseudomonas aeruginosa populations with genotypic and phenotypic diversity. This study describes a substantial dataset containing 450 isolates from different lobes of the right lung across three timepoints from an individual with mild-to-moderate CF lung disease. Some regional enrichment for specific lineages with parallel mutations among individual lobes of the lung was observed, but longitudinal analysis also demonstrated that compartmentalization is not strictly maintained and that isolates migrate between lobes of the lung over time. Perspectives on within lung evolution will be important for understanding the pathogen populations in chronic respiratory infections in CF and other diseases.

ObjectivesTo quantify how urine sample type and polymicrobial context impact antimicrobial resistance (AMR) in urinary tract infections (UTIs), using routine diagnostics at scale. MethodsIn this retrospective, single-centre study, we analysed 188,687 urine cultures from the Institute of Medical Microbiology, University of Zurich, Switzerland (January 2015 to May 2023). We compared midstream urine (MU), indwelling catheter (IDC), and intermittent catheter (IMC) samples. Samples were classified as negative, bacteriuria, or UTI, by meeting a microbiological UTI threshold ([&ge;]105 CFU/mL). We compared sample types using covariate-adjusted regression and constrained ordination for community composition. In bimicrobial cultures, we assessed co-occurrence using adjusted pairwise odds ratios and degree-preserving permutation null models, supported by partner-choice analyses. AMR was modelled as acquired resistance (AR) and total resistance (TR: acquired + intrinsic) probabilities, with predictor contributions quantified using mutual information. ResultsAmong 186,819 MU, IMC, IDC samples, 56,867 met the UTI threshold. Catheter-associated UTIs (IDC and IMC) were ~60% more likely to be polymicrobial than MU samples. Community composition differed by sample type (p<0{middle dot}001). In IDC, Escherichia coli was less prevalent than in MU, but device-associated pathogens like Pseudomonas aeruginosa and Candida albicans were enriched. Most species-pairs showed no increased co-occurrence after adjusting for covariates, but a subset showed reproducible enrichment across methods (e.g., C. albicans-C. glabrata). Organism identity was the dominant determinant of AMR, with the highest mutual information across AR and TR. AR was higher in IDC for common uropathogens (e.g., E. coli). Co-isolation with hospital-associated partners (e.g., Enterococcus faecium) was associated with further AR increase. From 2015 to 2023, AR increased from ~48% to ~60%, with rising {beta}-lactam (+{beta}-lactamase inhibitor) resistance and declining fluoroquinolone resistance in Enterobacterales. ConclusionsSample type and co-isolated partners provide clinically actionable information beyond pathogen identity and could support more context-aware reporting and empiric prescribing.

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.