Fluvial biofilms develop through the establishment of microbial communities on submerged mineral surfaces. However, they are routinely studied after detachment from their colonized substrate by scraping or brushing, a standard procedure that homogenizes biofilm architecture and disrupts the biofilm–rock interface. Using an in situ microscopy approach that examines biofilms without separating them from their colonized substrate, we studied undisturbed biofilm-covered rocks collected from Antarctic meltwater streams. Our observations revealed: (i) a highly organized three-dimensional architecture characterized by spatially structured microbial assemblages embedded in a matrix of extracellular polymeric substances; (ii) a close association among microbial cells, the colonized rock surface and fine-grained sedimentary components; (iii) the development of an endolithic biofilm fraction through the colonization of internal rock fissures and cavities; and (iv) a frequent laminated structure with alternating layers rich in microbial cells and layers composed mainly of sediment particles, which may preserve a physical signatures of hydrological variability and sediment deposition. Together, these observations challenge the widespread view of fluvial biofilms as uniform microbial layers developing on inert mineral surfaces. Integrating in situ microscopy approaches with conventional destructive approaches offers new opportunities to link microbial diversity with its spatial organization, providing a more complete understanding of fluvial biofilm ecology and microbial–mineral interactions.
Acinetobacter baumannii, a notorious nosocomial pathogen, exhibits enhanced antibiotic resistance through biofilm formation. However, a comprehensive understanding of the heterogeneity and regulatory dynamics underlying biofilm development at single-cell resolution is lacking. This study outlines the transcriptional landscape of A. baumannii biofilm formation at single-cell resolution and explores potential therapeutic targets. We monitored the dynamic formation process of biofilms using single-cell RNA sequencing (scRNA-seq) technology. Subsequently, we conducted characteristic genes, gene ontology (GO) enrichment, and pseudotemporal analysis on each identified cluster. In this study, scRNA-seq and pseudotemporal trajectory results showed the transition from planktonic to biofilm states in A. baumannii. Increased cellular heterogeneity was observed during biofilm maturation: planktonic subpopulations (AB_0 h) displayed a metabolic divergence between phenylacetate catabolism (paa genes) and the tricarboxylic acid cycle (acnD, atp genes), whereas the 12 h mixed population (M_12 h) exhibited co-upregulation of ribosomal (rpl, rps) and stress response genes (recA, uvrA), facilitating protein synthesis and environmental adaptation. Mature biofilm subpopulations (BF_48 h) activated iron acquisition (bauA, basD) and sulfur/nitrogen metabolism pathways (ssuC, purine degradation genes) under nutrient limitation, alongside DNA repair (uvrB, uvrC) and proteostasis mechanisms (clpB, clpX). Pseudotemporal analysis identified a critical branchpoint (Node 2) that marked the transition from the high-metabolism planktonic to the low-metabolism biofilm state, characterized by the downregulation of ribosomal (rpl, rps) and transporter (putP) genes. These findings characterize transcriptional programs associated with biofilm maturation and reveal subpopulation-specific metabolic features that may represent potential vulnerabilities warranting further investigation through targeted mutagenesis and functional assays.
Mudflats play an important role in coastal carbon cycling. The colonization of diatom-dominated microphytobenthic biofilms and associated heterotrophic microbial communities, affects the biogeochemical cycling in mudflats. Yet, the influence of biofilm colonization on the microbial communities and organic matter profiles in mudflats remains poorly understood. Here, 84 samples were collected in different seasons, locations and depths with/without biofilms in tidal flats of Zhejiang, China, and a multiparametric analysis was performed on environmental factors, microbial communities and dissolved organic matter (DOM) profiles to reveal the multidimensional influence of biofilm colonization on mudflats. The results showed that, compared with surface adjacent bare sediment (surface ABS), biofilms exhibited higher carbon stocks, lower bacterial diversity, and a shift in the dominant community assembly process from dispersal limitation to homogeneous dispersal. The microbial communities in biofilms and related sediments showed a higher abundance of functional genes involved in carbon, nitrogen and sulfur cycling, including carbon fixation and metabolism, leading to high proportion of recalcitrant DOM in the biofilm-related sediments, in which, Pseudomonadota, Bacteroidota, Chloroflexota and Bacillota acted as key functional groups. This study highlights the association between biofilm occurrence, microbial community composition, functional potential, and organic matter characteristics in mudflat sediments, highlighting the potential contribution of biofilms to carbon retention and transformation processes in mudflat ecosystems.
Microorganisms are pivotal agents in the process of sandstone weathering; nevertheless, the ecological mechanisms that govern their transition from mere colonization to sustained weathering activity remain ambiguous. This study systematically elucidated microbe-mediated weathering mechanisms through amplicon and metagenomic sequencing of bacteria, fungi, and archaea across a sandstone weathering sequence—from original unweathered sandstone (OS), biofilm-covered sandstone (BS), to weathered sandstone (WS). The findings indicate that microbial communities undergo associations across a weathering gradient, with biofilms constituting a unique transitional state. Community assembly mechanisms undergo a transition from stochastic processes in original sandstone to deterministic processes during the processes of biofilm formation and weathering. Biofilm communities formed modular, tightly interconnected putative association networks enriched with keystone taxa. Metagenomic analysis revealed significant enrichment of functional pathways related to iron acquisition, organic acid metabolism, and sulfur cycling during weathering, with functional annotation directly linking these traits to pivotal microbial groups. The findings of this study, as suggested by partial least squares path modeling (PLS-PM), indicate that environmental changes are associated with deterministic processes and with increased microbial richness. These factors are further linked to the composition of putative keystone taxa along the weathering gradient. These pivotal groups subsequently influence the abundance of weathering-related functional genes, directly accelerating weathering processes. This finding unveils a distinct ecological cascade pathway, commencing with environmental selection and culminating in the enrichment of functional gene potentials. The present study proposes a universal framework demonstrating that sandstone weathering is associated with deterministic processes, putative keystone taxa, and synergistic gene networks. This mechanism is not only applicable to sandstone systems, but also offers novel insights into the understanding of microbially mediated mineral weathering in terrestrial environments. This process is fundamental in influencing global biogeochemical cycles, soil formation, and the preservation of geological and cultural heritage.
Staphylococcus aureus is a major human pathogen that causes persistent infections characterised by the formation of aggregates such as surface-attached biofilm and staphylococcal abscess communities (SACs). Both consist of dense bacterial populations associated with self-produced matrices that impair immune cell and antibiotic access. Surface-attached biofilms have mostly protein- or polysaccharide-rich matrices, whereas SACs are spherical structures within tissue encased in a fibrin pseudocapsule and microcolony-associated meshwork. It remains unclear whether SAC are simply biofilms within tissue or if they display distinct features with unique genetic regulation. Here, we investigated genetic determinants of SAC and biofilm formation using four S. aureus deletion mutants targeting staphylokinase (Δsak), coagulase (Δcoa), the alternative sigma factor SigB (ΔrpoF), and sortase A (ΔsrtA). SACs were grown in collagen gels, whilst biofilms were grown on titanium discs, with bacterial viability, fibrin deposition, and biofilm biomass assessed by microscopy, image analysis, and crystal violet staining. Although deletion of coa or sak did not significantly impact either SAC or biofilm formation, biofilm surface coverage was markedly increased in ΔsrtA and biomass decreased in ΔrpoF; however, these mutations had no effect on SAC. These findings suggest that certain mutations can have different effects in the two experimental systems, and that the tested gene deletions were more important for surface-associated biofilm development than SAC formation.
Biofilms represent the predominant mode of bacterial life at solid-liquid interfaces, and understanding their composition, structure, and dynamics is critical to addressing key challenges across medical, environmental, and engineering applications. This study presents a deep learning-based framework for rapid morphological characterisation of biofilms using optical coherence tomography (OCT) imaging and an automated image processing pipeline. Images were used to train two state-of-the-art segmentation models: YOLOv8 and SegFormer. Both models delivered impressive results in delineating biofilm structures; YOLOv8 achieved 0.99 for accuracy and an intersection over union (IoU) of 0.9, while SegFormer scored 0.97 and 0.87, respectively. Model robustness was assessed across eight challenging biofilm conditions, with YOLOv8 showing superior performance in discriminating thin and non-growing biofilms, and SegFormer's superiority with stable morphologies. Additionally, we developed a framework to extract key morphological characteristics from the segmented images, including thickness, roughness and density distribution. The model-derived measurements showed strong agreement with manually generated ground truth data, confirming the reliability of the automated pipeline. Furthermore, an experiment involving four taxonomically distinct multi-species biofilms demonstrated the utility of the approach for discriminating biofilms based on their morphology. The software, containing both segmentation models, is openly available to the community and provides a foundation for future high-throughput studies examining biofilm responses to taxonomic or environmental variation.
Biofilms are widely recognised as a dominant mode of microbial life, yet their visual representation in the scientific literature still often relies on simplified diagrams derived from laboratory systems. While these schemes have strong pedagogical value, they do not adequately reflect the heterogeneity, structural complexity, and environmental dependence of biofilms encountered in real settings. Because visual representations shape both scientific communication and conceptualisation, the way biofilms are illustrated matters for how they are understood.Here, we present the outcome of a collaboration between a biofilm microbiologist and a scientific illustrator to develop a conceptual visual framework for representing the classical biofilm life cycle across scales and environments. Rather than replacing the classical sequence, the framework embeds it within complex microbial scenes and uses graphical guidance, including colour coding, to make its most explicit trajectory readable within heterogeneous biofilm organisation. In this way, adhesion, growth, matrix production, remodelling, and dispersal are represented not as rigidly separated stages, but as processes that may coexist locally within structured communities.To connect microscopic mechanisms with real-world situations, these processes were integrated into domestic environments such as bathrooms and kitchens. The illustrations include both undesirable and beneficial biofilms, and selected domestic biofilm structures were informed by real microscopy observations to preserve biological plausibility while maintaining graphical coherence. Particular attention was given to microbial and structural diversity across domestic habitats.The visual framework relies on isometric projection and multiscale transitions that connect experimental systems, colonised surfaces, biofilm architectures, individual cells, and extracellular matrix components within a coherent visual continuum. This approach is introduced through a progressive view extending from a laboratory flow-cell device to the molecular network surrounding cells within the biofilm. The framework was implemented across complementary media, including static figures, animated slides, a simplified movie, and a large-format poster. Together, these materials form a multiformat visual framework intended to support reasoning, teaching, and interdisciplinary communication about biofilm organisation.
Listeria monocytogenes persistence in food processing environments is closely associated with biofilm formation on food-contact surfaces and subsequent transfer to fresh produce. However, the role of native environmental microbiota in shaping pathogen behaviour within biofilms remains poorly understood. This study evaluated biofilm formation, transfer dynamics, cell surface hydrophobicity, and biofilm organization of L. monocytogenes in mono- and multispecies biofilms on stainless-steel (SS), silicone rubber (SR), and polycarbonate (PC) surfaces under low-shear conditions. Multispecies biofilms enhanced L. monocytogenes persistence and modulated cross-contamination dynamics in a surface-dependent manner. On SS, multispecies biofilms showed reduced initial transfer but sustained cumulative transfer across sequential contacts compared to monospecies biofilms, indicating greater structural stability. In contrast, PC exhibited high initial transfer followed by rapid transfer plateauing, whereas SR supported limited accumulation after the first contact event. Higher cell surface hydrophobicity in multispecies biofilms, particularly on SS, was associated with increased biofilm cohesion. Together with fluorescence microscopy observations, these results suggest that biofilm organization, rather than total cell numbers alone, plays a key role in determining persistence and transfer behaviour. Overall, both surface properties and microbial community composition influence L. monocytogenes biofilm stability and cross-contamination risk. Incorporating surface-dependent and ecological factors into hygienic design and sanitation strategies may improve contamination control in fresh produce processing environments.
Background Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and is strongly associated with polymicrobial biofilms, which contribute to antimicrobial tolerance and high recurrence rates. New strategies targeting BV-associated biofilms are therefore needed. This study evaluated the antimicrobial and antibiofilm activity of octenidine dihydrochloride/phenoxyethanol (Octiset®) compared with octenidine dihydrochloride (octenidine) alone in in vitro and ex vivo BV biofilm models. Methods Planktonic cultures and biofilms of BV-associated species (Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia, Peptostreptococcus anaerobius and Mobiluncus curtisii) were established under anaerobic conditions. Planktonic suspensions were exposed for 24 h to octiset® (50%, 25%) or octenidine (0.1%, 0.05%). Single-species and polymicrobial biofilms were formed for 24 h and treated with octiset® or octenidine up to 12 h. Additionally, polymicrobial biofilms were established on reconstructed human vaginal epithelium (SkinEthic™ HVE) and treated for 8 h. Antimicrobial efficacy was assessed by CFU quantification. Recovery experiments were performed to determine whether residual culturable cells persisted after treatment, by incubating samples in fresh medium for up to 72 h and subsequently quantifying CFUs. Antimicrobial activity was also tested against species associated with healthy vaginal microbiota, by assessing vaginal lactobacilli susceptibility to both compounds. Results Both compounds showed time- and concentration-dependent activity. Octiset® consistently demonstrated superior killing compared to octenidine, including eradication of several single-species biofilms. Polymicrobial biofilms showed increased tolerance; however, extended exposure (≥8 h) reduced CFUs below detection for both agents, with no regrowth observed after 72 h. In the ex vivo model, both treatments reduced biofilm growth below the limit of detection. All tested lactobacilli also showed susceptibility to both compounds. Conclusion Octiset® and octenidine exhibited strong antibiofilm activity against BV-associated polymicrobial biofilms in vitro and ex vivo, supporting their potential as biofilm-targeted BV treatment approaches.
Phage-derived depolymerases represent a promising antibiotic alternative for treating Klebsiella pneumoniae infections. Depolymerases can increase the sensitivity of bacteria to the host immune system and complement-mediated killing by specifically degrading capsular polysaccharides (CPS). However, the impact of depolymerases on bacterial biofilm formation remains unclear. This study found that depolymerase treatment significantly enhances the biofilm formation capability of hypervirulent K. pneumoniae (hvKp) strains, which inherently exhibit relatively weak biofilm formation due to their thick capsular polysaccharide (CPS) layers. Further investigation revealed that depolymerase-mediated CPS degradation relieved its repression on the type 3 fimbriae gene cluster mrkABCDF, thereby promoting biofilm formation. In vivo experiments in mice also showed that CPS can inhibit virulence functions associated with type 3 fimbriae. Furthermore, we found that CPS-mediated biofilm inhibition appears to be a common phenomenon among hvKp strains. In summary, by elucidating the dual role of depolymerase in modulating both virulence and biofilm in hvKp, our work reveals a potential interaction between CPS and type 3 fimbriae, providing deeper insights into the pathogenicity of this clinically important bacterium.
Biofilm extracellular matrix (ECM) varies with environmental conditions and substrate properties. Understanding the surface-biofilm relationship helps to perfect antibacterial strategies and to design new engineered living materials (ELMs). In this work, we studied how cationic and anionic polyelectrolyte coatings affect macroscopic features of Escherichia coli curli-producing biofilms, as well as the properties of their curli amyloid fibers. Cationic coatings limited biofilm spreading, increased their surface density and water absorption, which correlated with a higher yield of curli amyloid fibers with looser structure. In contrast, anionic surfaces allowed for standard biofilm spreading, with a lower fiber yield but a more compact and chemically stable fiber structure. Higher biofilm rigidity and adhesion were measured on both types of charged surfaces. Thus, we propose that the differences in biofilm macroscopic properties result from a trade-off between curli quantity and quality in the ECM, namely fiber density and molecular packing, as well as their interaction with water. Our findings provide insights on how the biophysical properties of the ECM can be controlled by tuning the substrate physico-chemical characteristics with charged coatings. This work opens up new avenues for developing antimicrobial strategies, as well as tailoring the properties of amyloid-based ELMs.
Cupriavidus necator is a metabolically versatile β-proteobacterium of growing interest for auto- and heterotrophic bioprocesses, yet the genetic determinants governing its biofilm formation remain largely uncharacterized, particularly under process-relevant heterotrophic conditions. Here, we applied a forward-genetics transposon-enrichment approach to identify loci which promote surface-associated growth. A high-density mini-Tn5 mutant library (26,185 insertion clones, exceeding the >17,000 required for genome-wide coverage) was cultivated as a biofilm in a microfluidic flow-cell system on fructose for 168 h, and the surface-associated community was characterized by deep sequencing. Twelve genes showed significantly elevated insertion frequencies, several with documented links to biofilm formation in other bacteria, including the ferrous-iron uptake system (feoA/feoB), galU, and a GSDEF/EAL dual-domain protein. The gene B2043 (E6A55_RS29530), encoding this c-di-GMP-metabolizing protein, was selected for validation by markerless deletion. Under static conditions, the ΔB2043 mutant showed a 1.69 ± 0.06-fold increase in biofilm-associated biomass (p = 5.16 × 10-15). Under flow-through conditions, the mutant attached faster, entered exponential growth ∼10 h earlier, reached its biovolume plateau ∼16 h earlier than the wild-type, and formed distinct tower-like structures. These results identify B2043 as a negative regulator of biofilm formation acting predominantly during attachment, provide the first experimental evidence for c-di-GMP-dependent biofilm regulation in C. necator H16, and establish a functional-genomics framework — together with eleven further candidate loci — for engineering productive biofilms in this organism.
Staphylococcus aureus adapts to hostile host-associated niches by dynamically switching between planktonic growth and biofilm lifestyles. Acidic environments, such as the skin surface and intracellular compartments, impose substantial stress on bacterial survival; however, the contribution of the type VII secretion system (T7SS) to biofilm adaptation under acidic conditions remains poorly understood. Here, we investigated the role of the T7SS ATPase EssC in regulating S. aureus biofilm formation under acidic stress. Using an essC deletion mutant in the USA300 background, we demonstrate that loss of EssC markedly enhances biofilm biomass and thickness at pH 5.0, despite reducing bacterial viability within mature biofilms. Mechanistically, essC deletion reprograms multiple stages of biofilm development, including enhanced initial adhesion mediated by upregulated fibronectin-binding proteins (FnBPA and FnBPB), increased intercellular aggregation driven by elevated polysaccharide intercellular adhesin (PIA) production, and biofilm stabilization through augmented autolysisdependent extracellular DNA release. These phenotypic changes are accompanied by coordinated transcriptional remodeling, characterized by downregulation of the biofilm repressor agr and activation of the arlS-icaA and sigB-icaA regulatory axis. Collectively, our findings uncover an unrecognized link between the T7SS core component EssC and biofilm regulation under acidic stress, highlighting EssC as a potential modulator of S. aureus survival strategies in hostile host microenvironments.
Pathogenic multispecies biofilms are major drivers in the persistence and virulence of bacterial infections, complicating treatment due to their pronounced antibiotic resistance. To better understand the temporal and spatial dynamics within these complex communities, we established dual-species biofilm models focusing on the pathogen Stenotrophomonas maltophilia in combination with Pseudomonas aeruginosa and Staphylococcus aureus. Using Lattice Light Sheet Microscopy (LLSM) and automated cell quantification, we demonstrated the dynamic growth and complex spatial organization of these dual-species biofilms. The study identified both shared and species-specific strategies of biofilm formation, ultimately underscoring the dynamic and adaptive nature of the S. maltophilia K279a biofilm architecture. A vertical zonation was a general and pronounced trait of S. maltophilia biofilms. Marker gene expression in S. maltophilia was generally heterogeneous across the biofilm layers but followed a clear time-dependent on/off pattern. Iron transport and cytochrome biosynthesis appear to be key traits involved in niche competition. Furthermore, S. maltophilia attenuated P. aeruginosa N-acyl-homoserine lactone (AHL) quorum sensing (QS)-regulated gene expression involved in surface colonization, outer membrane biogenesis, and cyclic di-GMP signaling. Thereby we identified key drivers involved in the S. maltophilia dualspecies biofilm lifestyle, representing potential drug targets to combat multi-species and heterogeneous biofilm infections.
Achromobacter xylosoxidans is an opportunistic pathogen in both cystic fibrosis (CF) and non-CF patients, in whom biofilm formation contributes to bacterial persistence and antibiotic tolerance. This study aimed to characterize early and mature biofilm formation in 57 clinical A. xylosoxidans isolates using complementary and physiologically relevant approaches and to compare biofilm phenotypes according to isolate origin (CF/non-CF). Early adhesion was assessed using the Biofilm Ring Test®, mature biofilm viable biomass was quantified under static conditions by colony-forming units counts, and biofilm dynamics were analyzed in a continuous-flow microfluidic system. The effects of five clinically relevant antibiotics (trimethoprim-sulfamethoxazole, piperacillin-tazobactam, meropenem, imipenem, and cefiderocol) were evaluated under dynamic conditions at sub-inhibitory concentrations (0.5 × Minimum Inhibitory Concentration (MIC)) and on preformed biofilm at inhibitory concentrations (10 × MIC). Non-CF isolates displayed faster early adhesion than CF isolates, whereas mature biofilm biomass was comparable between groups. If early adhesion did not predict mature biofilm biomass, dynamic biofilm coverage under flow conditions correlated with static mature biofilm levels. Sub-inhibitory antibiotic concentrations failed to prevent initial adhesion and elicited three distinct responses: biofilm formation enhancement (piperacillin-tazobactam, meropenem, imipenem), no effect (trimethoprim-sulfamethoxazole), or biofilm reduction (cefiderocol). Exposing mature biofilm to 10 × MIC identified trimethoprim-sulfamethoxazole and cefiderocol as the most effective agents in biofilm biomass reduction, whereas carbapenems and piperacillin-tazobactam were less effective. These findings provide new insights into A. xylosoxidans biofilm biology and may help guide therapeutic strategies for infections caused by this emerging, increasingly drug-resistant pathogen.
Antimicrobial resistance (AMR), particularly among carbapenemase-producing organisms, poses a major global health threat. Although hospital wastewater is considered an AMR hotspot, its functional contribution to resistance dynamics remains poorly defined. We developed in situ biofilms in hospital wastewater and applied integrated metagenomic, metatranscriptomic, and culture-based analyses to characterize community structure and gene expression. Biofilms exhibited greater biomass and higher contamination with extended-spectrum β-lactamase-producing Escherichia coli than planktonic wastewater. Biofilms were enriched in surface-adapted Flavobacteriaceae species and a broader array of carbapenemase genes, whereas wastewater showed higher abundance of gut-associated Bacteroidaceae species and virulence factors. Mobile genetic elements linked multiple AMR genes and showed increased expression in biofilms, including blaIMP family carbapenemases. Culture confirmed blaIMP-1 in four biofilm isolates and one wastewater isolate. These findings indicate that hospital-wastewater biofilms can serve as important reservoirs that promote the persistence and potential dissemination of clinically relevant carbapenem resistance.
Background Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells hamper the eradication and contribute to the recurrence of biofilm-related infections, especially in cystic fibrosis (CF) patients, often experiencing difficult-to-treat lung infections. The siderophore-cephalosporin cefiderocol, which hijacks bacterial iron-uptake systems, has emerged as a last-resort antibiotic against antibiotic-resistant Gram-negative bacteria and represents a desirable therapeutic option; however, there is still limited information about its impact on bacterial persisters. Methods P. aeruginosa biofilms were exposed to either tobramycin, ceftazidime, or cefiderocol at their minimum biofilm eradication concentrations under iron depletion in a rich or minimal medium. The bacterial survivors were quantified by combining cultural enumerations, quantitative PCR and confocal microscopy, to detect both culturable and VBNC cells. To extend these observations in vivo, a murine model of P. aeruginosa lung infection was employed, and bacterial burden and VBNC frequency were assessed following antibiotic treatment. Results A higher amount of culturable P. aeruginosa cells was recovered after cefiderocol challenge (∼105 CFU/ml) compared to tobramycin and ceftazidime (103-104 CFU/ml). Notably, it induced a significantly lower proportion of VBNC cells (∼85.2%) than the aminoglycoside (98.95%) or ceftazidime (95.80%).Consistently, cefiderocol exposure resulted in a higher bacterial survival in vivo, but in a lower frequency of VBNC subpopulation (71.89%) compared to tobramycin (81,58%). Conclusions Overall, these findings emphasize the capacity of P. aeruginosa to withstand treatment with cefiderocol and highlight the need to account for the roles of bacterial persisters and VBNC cells in the recurrence of chronic infections, particularly among CF patients.
Numerous lab-scale bioreactor systems exist to cultivate biofilms under desired growth conditions. However, existing systems have not been able to simultaneously deliver low shear stress, high gas transfer, and intermittent wetting. The industrial surfaces biofilm reactor (ISBR) was specifically developed to overcome these limitations by simultaneously providing these conditions. This study presents the first standard operating procedure (SOP) for the growth of a model biofilm-forming organism, Pseudomonas aeruginosa, within the ISBR, providing a reproducible framework for investigating biofilm development under conditions that closely mimic industrial environments. Statistical analyses confirmed the SOP's repeatability, ruggedness, and reproducibility using biofilm density and biovolume data, supported by an analytical shear stress model for ISBR coupons. Biofilm densities were rugged to recycle and rotation rate deviations but increased with influent feed rate. Conversely, biovolumes were rugged to influent feed and rotation rate changes but declined with higher recycle rates. Both metrics demonstrated excellent repeatability and reproducibility, underscoring the need to assess these characteristics for consistent biofilm formation. Additionally, as a proof-of-concept, two multidomain biofilms (a Legionella pneumophila-Vermamoeba vermiformis model and a Rhodotorula mucilaginosa yeast-P. aeruginosa bacterial coculture) were cultivated and analyzed. Both exhibited high repeatability, and the coculture remained stable over a month-long growth period.
Urinary tract infections are common healthcare associated infections, a large subset of which are caused by indwelling catheters. Long term catheterization causes persistent, asymptomatic, polymicrobial colonization despite catheters changes and antibiotic usage. In these polymicrobial populations, P. mirabilis, E. faecalis, and E. coli were found as the most common co-colonizing species. We investigated how interactions between P. mirabilis, E. coli, and E. faecalis contribute to biofilm formation and colonization of urinary catheters. Our results show that the interaction between these three species leads to enhanced biofilm biomass driven by an increase in total protein content of the biofilm. Biofilm enhancement required all three species and was also media-dependent, especially for dual-species combinations. Importantly, triple species biofilms also demonstrate biofilm enhancement when established under flow conditions in a biofilm reactor model using silicone urinary catheters. Additionally, triple species biofilm enhancement occurred in co-colonizing isolates from catheterized patients and was found to be specific to interactions between these three species. Triple species biofilms also demonstrated a species-dependent resistance to two commonly used antibiotics, ciprofloxacin and nitrofurantoin. By examining priority effects, E. coli was found to be the main facilitator of biofilm enhancement in a flow model. Finally, proteomics revealed that an L-fucose utilization pathway in E. coli was a key contributor to triple species biofilm enhancement. Overall, our results demonstrate the significant impact of polymicrobial interactions on biofilm formation in the catheterized environment and highlight ways in which complex microbial interplay and priority effects can shape the establishment of persistent colonization.
Acetogenic bacteria are attractive biocatalysts for the conversion of CO2 with H2 into acetate, as in gas fermentation. Gas fermentation reactors may benefit from biofilm formation, but attachment by acetogens is often limited. This study indeed found that the acetogen Sporomusa ovata 2663 was mainly planktonic and aimed to increase its biofilm formation through adaptive laboratory evolution. The adaptation strategy consisted of growing S. ovata on plastic (HDPE) carriers in bottles with a H2:CO2 headspace and transferring two carriers to a bottle with fresh carriers over eight serial transfers. This procedure resulted in the evolved S. ovata 2663-BF, which had a consistent increased propensity to attach. In heterotrophic growth conditions, four times more cells attached to the bottom of well plates in comparison to the wild type, while the adapted S. ovata produced 1.8 times more extracellular polysaccharides. Moreover, twice as many cells adhered to carriers when grown on H2:CO2. This improved attachment, however, did not lead to higher acetate production rates in simple trickle bed reactors, as the experimental setup rather stimulated planktonic growth. Only medium replacement sometimes favored the rate of the adapted strain. Interestingly, the evolved S. ovata had a point mutation in the gene galU, which likely increased the activity of the encoding UDP-glucose pyrophosphorylase, i.e. an enzyme involved in the synthesis of extracellular polysaccharides. Overall, this study demonstrates that cell attachment by S. ovata was increased through adaptive laboratory evolution, offering the prospect of investigating the importance of biofilm formation in biofilm-based gas fermentation reactors.
