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Abstract Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire’s Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.

Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire's Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems. [ABSTRACT FROM AUTHOR]

Agata Lange,1 Arkadiusz Matuszewski,2 Marta Kutwin,1 Agnieszka Ostrowska,1 Sławomir Jaworski1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 2Department of Animal Environment Biology, Institute of Animal Sciences, Warsaw University of Life Sciences, Warsaw, PolandCorrespondence: Sławomir Jaworski, Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Ciszewskiego 8, Warsaw, 02-786, Poland, Tel +48 225936675, Email slawomir_jaworski@sggw.edu.plPurpose: Biofilms, which are created by most microorganisms, are known for their widely developed drug resistance, even more than planktonic forms of microorganisms. The aim of the study was to assess the effectiveness of agents composed of farnesol and nanoparticles (silver, gold, copper, and zinc oxide) in the degradation of biofilms produced by pathogenic microorganisms.Methods: Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were used to create the biofilm structure. Colloidal suspensions of silver, gold, copper, and zinc oxide (Ag, Au, Cu, ZnO) with the addition of farnesol (F) were used as the treatment factor. The size distribution of those composites was analyzed, their zeta potential was measured, and their structure was visualized by transmission electron microscopy. The viability of the microorganism strains was assessed by an XTT assay, the ability to form biofilms was analyzed by confocal microscopy, and the changes in biofilm structure were evaluated by scanning electron microscopy. The general toxicity toward the HFFF2 cell line was determined by a neutral red assay and a human inflammation antibody array.Results: The link between the two components (farnesol and nanoparticles) caused mutual stability of both components. Planktonic forms of the microorganisms were the most sensitive when exposed to AgF and CuF; however, the biofilm structure of all microorganism strains was the most disrupted (both inhibition of formation and changes within the structure) after AgF treatment. Composites were not toxic toward the HFFF2 cell line, although the expression of several cytokines was higher than in the not-treated group.Conclusion: The in vitro studies demonstrated antibiofilm properties of composites based on farnesol and nanoparticles. The greatest changes in biofilm structure were triggered by AgF, causing an alteration in the biofilm formation process as well as in the biofilm structure.Keywords: farnesol, nanocomposites, nanoparticles, biofilm structure, toxicity

Staphylococcus aureus forms biofilms consisting of cells embedded in a matrix made of proteins, polysaccharides, lipids, and extracellular DNA (eDNA). Biofilmassociated infections are difficult to treat and can promote antibiotic resistance, resulting in negative healthcare outcomes. eDNA within the matrix contributes to the stability, growth, and immune-evasive properties of S. aureus biofilms. eDNA is released by autolysis, which is mediated by murein hydrolases that access the cell wall via membrane pores formed by holin-like proteins. The eDNA content of S. aureus biofilms varies among individual strains and is influenced by environmental conditions, including the presence of antibiotics. eDNA plays an important role in biofilm development and structure by acting as an electrostatic net that facilitates protein-cell and cell-cell interactions. Because of eDNA's structural importance in biofilms and its ubiquitous presence among S. aureus isolates, it is a potential target for therapeutics. Treatment of biofilms with DNase can eradicate or drastically reduce them in size. Additionally, antibodies that target DNABII proteins, which bind to and stabilize eDNA, can also disperse biofilms. This review discusses the recent literature on the release, structure, and function of eDNA in S. aureus biofilms, in addition to a discussion of potential avenues for targeting eDNA for biofilm eradication. [ABSTRACT FROM AUTHOR]

Introduction: Periodontal diseases are known to be associated with polymicrobial biofilms and inflammasome activation. A deeper understanding of the subgingival cytological (micro) landscape, the role of extracellular DNA (eDNA) during periodontitis, and contribution of the host immune eDNA to inflammasome persistence, may improve our understanding of the mechanisms underlaying severe forms of periodontitis. Methods: In this work, subgingival biolfilms developing on biologically neutral polyethylene terephthalate films placed in gingival cavities of patients with chronic periodontitis were investigated by confocal laser scanning microscopy (CLSM). This allowed examination of realistic cytological landscapes and visualization of extracellular polymeric substances (EPS) including amyloids, total proteins, carbohydrates and eDNA, as well as comparison with several single-strain in vitro model biofilms produced by oral pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus gordonii, S. sanguinis and S. mitis. Fluorescence in situ hybridization (FISH) analysis was also used to identify eDNA derived from eubacteria, streptococci and members of the Bacteroides-Porphyromonas-Prevotella (BPP) group associated with periodontitis. Results: Analysis of subgingival biofilm EPS revealed low levels of amyloids and high levels of eDNA which appears to be the main matrix component. However, bacterial eDNA contributed less than a third of the total eDNA observed, suggesting that host-derived eDNA released in neutrophil extracellular traps may be of more importance in the development of biofilms causing periodontitis. Discussion: eDNA derived from host immunocompetent cells activated at the onset of periodontitis may therefore be a major driver of bacterial persistence and pathogenesis. [ABSTRACT FROM AUTHOR]

Industrial biocides aim to keep water systems microbiologically controlled and to minimize biofouling. However, the resulting dead cells are usually not removed from the water streams and can influence the growth of the remaining live cells in planktonic and sessile states. This study aims to understand the effect of dead Pseudomonas fluorescens cells killed by industrial biocides—benzalkonium chloride (BAC) and 2,2-dibromo-3-nitrilopropionamide (DBNPA)—on biofilm formation. Additionally, the effect of different dead/live cell ratios (50.00% and 99.99%) was studied. The inoculum was recirculated in a Parallel Plate Flow Cell (PPFC). The overall results indicate that dead cells greatly affect biofilm properties. Inoculum with DBNPA–dead cells led to more active (higher ATP content and metabolic activity) and thicker biofilm layers in comparison to BAC–dead cells, which seems to be linked to the mechanism of action by which the cells were killed. Furthermore, higher dead cell ratios (99.99%) in the inoculum led to more active (higher culturability, metabolic activity and ATP content) and cohesive/compact and uniformly distributed biofilms in comparison with the 50.00% dead cell ratio. The design of future disinfection strategies must consider the contribution of dead cells to the biofilm build-up, as they might negatively affect water system operations. [ABSTRACT FROM AUTHOR]

IntroductionPeriodontal diseases are known to be associated with polymicrobial biofilms and inflammasome activation. A deeper understanding of the subgingival cytological (micro) landscape, the role of extracellular DNA (eDNA) during periodontitis, and contribution of the host immune eDNA to inflammasome persistence, may improve our understanding of the mechanisms underlaying severe forms of periodontitis.MethodsIn this work, subgingival biolfilms developing on biologically neutral polyethylene terephthalate films placed in gingival cavities of patients with chronic periodontitis were investigated by confocal laser scanning microscopy (CLSM). This allowed examination of realistic cytological landscapes and visualization of extracellular polymeric substances (EPS) including amyloids, total proteins, carbohydrates and eDNA, as well as comparison with several single-strain in vitro model biofilms produced by oral pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus gordonii, S. sanguinis and S. mitis. Fluorescence in situ hybridization (FISH) analysis was also used to identify eDNA derived from eubacteria, streptococci and members of the Bacteroides–Porphyromonas–Prevotella (BPP) group associated with periodontitis.ResultsAnalysis of subgingival biofilm EPS revealed low levels of amyloids and high levels of eDNA which appears to be the main matrix component. However, bacterial eDNA contributed less than a third of the total eDNA observed, suggesting that host-derived eDNA released in neutrophil extracellular traps may be of more importance in the development of biofilms causing periodontitis.DiscussioneDNA derived from host immunocompetent cells activated at the onset of periodontitis may therefore be a major driver of bacterial persistence and pathogenesis.

Bacteria usually dwell in microbial communities of biofilms surrounded by a self-secreted extracellular matrix. This work assessed effects of different media and temperature on the development of Bacillus subtilis 168 biofilms. We applied Scanning Electron Microscopy to analyse structural differences of the pellicle biofilms grown on LB, NB, and DMS media. Significant differences were observed in biofilm morphology, and unit cell length, highlighting the significance of intricate aspects such as nutrients in bacterial/ biofilm growth. Our findings reveal the adaptation tendency of bacterial through spatial modification of its biofilm structure in response to surrounding environmental conditions such as nutrients, temperature among others, perhaps explaining variations in biofilm morphologies observed. The results present an information on biofilm diversity which could be essential for biofilm applications. [ABSTRACT FROM AUTHOR]

Extracellular DNA (eDNA) is a major component of bacterial biofilms. In this study, we performed a three-dimensional analysis of Leptospira biofilm using advanced imaging by confocal laser scanning microscopy (CLSM) and multi-parameter analysis by COMSTAT 2 software, with quantification of Leptospira and eDNA fluorescence. To investigate the role of eDNA in Leptospira biofilm, we treated Leptospira biflexa biofilms with DNase I enzyme (DNase), which digested eDNA, and compared DNase treated biofilms and controls. There was a significant reduction of the biomass of biofilms treated with DNase, by spectrophotometry and COMSTAT analysis. The multiparameter analysis evidenced for DNase-treated biofilms a significant decrease in the surface area and the average thickness; opposing to a significant augmentation of the surface/biovolume ratio and the roughness coefficient (Ra*), when compared to controls. We analyzed the parameters of DNase-treated biofilms by Pearson's correlation coefficient and found significant positive correlations between biomass and average thickness; biomass and surface area; surface area and average thickness. On the other hand, there were significant negative correlations between Ra* and biomass; Ra* and average thickness; Ra* and surface area. These findings suggest that eDNA digestion results in biofilm instability and alteration of the three-dimensional architecture, justifying the negative correlation between Ra* and the above-mentioned parameters. In conclusion, our study showed that eDNA digestion produced a massive structural loss, instability, and dramatic changes in the three-dimensional architecture of Leptospira biflexa biofilm. These findings contribute to a better understanding of the role of eDNA and highlight the importance of eDNA as a key component in Leptospira biofilms. [ABSTRACT FROM AUTHOR]

The interest in microalgae biofilm-based systems has been increasing lately due to their high potential for biomass production. However, more studies focusing on the first stages of this bioprocess, such as support selection and inoculum properties, which may finally affect biomass productivity, are required. The aim of this study was therefore to assess the impact of support nature and inoculum properties on microalgae biofilm productivity and physiology. Results suggest that physico-chemical properties of the support (micro-texture, hydrophobicity and chemical functional groups) affect the attachment of Chlorella vulgaris. Significant differences in cell-distribution pattern and biofilm structure on polyamide-based (Terrazzo) and cotton-based fabrics were observed. Compared to Cotton, cells grown on Terrazzo showed higher biomass productivity (3.20-fold), photosynthetic capacity (1.32-fold) and carbohydrate pool (1.36-fold), which may be explained by differences in light availability due to support micro-texture. A high inoculum density resulted in a lower biofilm growth, likely due to a lower light/nutrient availability for the cells. Furthermore, when immobilized on fabrics, cells pre-acclimated to 350 μmol photons m−2 s−1 grew faster than those pre-acclimated to low light (50 μmol photons m−2 s−1), demonstrating the influence of light-history of the inoculum cells on biofilm productivity. Therefore, this work confirmed the importance of support and inoculum properties for biofilm-based systems. [ABSTRACT FROM AUTHOR]

As a typical sulfur-containing volatile organic compound, dimethyl disulfide (DMDS) is known for its high toxicity and resistance to degradation, necessitating efficient control in environmental media. To address the limitations of biological treatment in degradation capacity, this study employs electro-stimulation to promote DMDS elimination by a porous polyaniline@carbon nanotube bioanode developed on graphite sheet (PANI@CNT/GS). Compared with the unmodified GS bioanode, the PANI@CNT/GS bioanode demonstrates significant advantages in biofilm activity, redox property, and DMDS degradation efficiency. Kinetics analysis shows that the maximum degradation rate of the PANI@CNT/GS bioanode was 0.60 mM h−1, which is 1.36 times higher than that of the control. Characterization results reveal that the highly active biofilms in PANI@CNT/GS bioanode possess 1.40 times the amount of living cells and a 12.5% increase in thickness, contributing to the notable enhancement in DMDS degradation capacity. Additionally, functional gene annotation indicates that the PANI@CNT/GS electrode facilitates the motility and activity of microbial cells and enriches the genes encoding key enzymes involved in DMDS metabolism. This work validates the feasibility of electro-stimulation for enhancing DMDS degradation and further provides in-depth insights into the process intensification mechanism from the perspectives of biofilm spatial structure and key functional genes. [Display omitted] • Highly active biofilms with a 40% increase in living cells were formed in PANI@CNT/GS electrode. • DMDS up to 63.75 mg L−1 was degraded completely in 8 h under electro-stimulation. • PANI@CNT/GS bioanode had a maximum degradation rate 1.36 times higher than the control. • Both the EggNOG and KEGG metabolic pathway were annotated. • The genes encoding key enzymes took part in sulfur metabolism were enriched. [ABSTRACT FROM AUTHOR]

Bivalent cations are known to affect the structural and mechanical properties of biofilms. In order to reveal the impact of Fe2+ ions within the cultivation medium on biofilm development, structure and stability, Bacillus subtilis biofilms were cultivated in mini-fluidic flow cells. Two different Fe2+ inflow concentrations (0.25 and 2.5 mg/L, respectively) and wall shear stress levels (0.05 and 0.27 Pa, respectively) were tested. Mesoscopic biofilm structure was determined daily in situ and non-invasively by means of optical coherence tomography. A set of ten structural parameters was used to quantify biofilm structure, its development and change. The study focused on characterizing biofilm structure and development at the mesoscale (mm-range). Therefore, biofilm replicates (n = 10) were cultivated and analyzed. Three hypotheses were defined in order to estimate the effect of Fe2+ inflow concentration and/or wall shear stress on biofilm development and structure, respectively. It was not the intention to investigate and describe the underlying mechanisms of iron incorporation as this would require a different set of tools applied at microscopic levels as well as the use of, i.e., omic approaches. Fe2+ addition influenced biofilm development (e.g., biofilm accumulation) and structure markedly. Experiments revealed the accumulation of FeO(OH) within the biofilm matrix and a positive correlation of Fe2+ inflow concentration and biofilm accumulation. In more detail, independent of the wall shear stress applied during cultivation, biofilms grew approximately four times thicker at 2.5 mg Fe2+/L (44.8 µmol/L; high inflow concentration) compared to the low Fe2+ inflow concentration of 0.25 mg Fe2+/L (4.48 µmol/L). This finding was statistically verified (Scheirer–Ray–Hare test, ANOVA) and hints at a higher stability of Bacillus subtilis biofilms (e.g., elevated cohesive and adhesive strength) when grown at elevated Fe2+ inflow concentrations. [ABSTRACT FROM AUTHOR]

Localization and structural organization of microbial biofilms developing in anthropogenic ecological niches of meat-processing plants using different raw materials (poultry, pork, or mixed materials) were investigated. Mature biofilms were revealed both at the sites subject to routine sanitary control (equipment, sewage traps, and walls) and at other locations (ceilings, reverse (lower) side of bridges, vehicle wheels, and tunnel walls at subzero temperatures), indicating high adaptability of microbial communities. This finding indicates the urgency of modification (extension) of the list of cleansing sites and of the relevant protocols. The information on the composition and diversity of microbial biofilms in this ecotope, as well as on the physiological state of component microorganisms (e.g., active, dormant, and lysing cells) was obtained using transmission electron microscopy. The morphotypes facilitating bacterial survival in these biofilms were described (old cells and several types of specialized dormant forms). [ABSTRACT FROM AUTHOR]

Experimental observations reveal the existence of anoxic microzones within oxygen‐rich aquatic sediments. The microzones are hotspots of enhanced removal of nitrate and production of greenhouse gas nitrous oxide. Here we established a new microscale flow, reactive transport and biofilm growth model and explore how anoxic microzones form and evolve within hyporheic zones under dynamic interactions among pore‐scale flow, reactive transport and biofilm growth. The microzones can form and evolve by two patterns as streambeds are progressively clogged due to biofilm growth: (a) physical and biogeochemical conditions‐dependent perennial microzones for permeable biofilms, and (b) physical and biogeochemical conditions‐independent ephemeral microzones for low permeability biofilms. During biofilm growth, persistent transport‐dominated systems sustain for the former, whereas the transformation of transport‐dominated to reaction‐dominated systems occurs for the latter. These findings are significantly different from previous single pattern of ephemeral microzones. Plain Language Summary: Stream water is typically rich in oxygen and nutrients such as organic carbon, nitrate. Some proportion of stream water enters streambed at an upstream location of the water‐sediment interface and then exits at a downstream location. Along the subsurface flow path, oxygen concentration gradually decreases due to microbial consumption, and thus the subsurface flow zone can often be divided as generally oxic and anoxic zones. Recent studies have indicated that anoxic microzones can form within the generally oxic zone, and the microzones are the hotspots of enhanced removal of nitrate and production of greenhouse gas nitrous oxide. Here, from the microscopic view, we apply computer model to investigate how the microzones develop and change over time and space. As microbial biomass grows and streambeds are progressively clogged, the microzones can form and change following two distinct patterns depending on the streambed biofilms in which the microbes reside. Specifically, the microzones can be perennial for well permeable biofilms, whereas the microzones are ephemeral for low permeability biofilms because the microzones would disappear and an anoxic macrozone forms across the streambed. Key Points: Both the patterns of perennial and ephemeral anoxic microzones likely occur as streambeds are gradually clogged due to biofilm growthPermeable biofilms can support perennial microzones, whereas low permeability biofilms‐induced severe bioclogging lead to ephemeral onesPersistent transport‐dominated hyporheic system during biofilm growth is an essential prerequisite for presence of perennial microzones [ABSTRACT FROM AUTHOR]

Corynebacterium matruchotii is a reported calcifying bacterium that can usually be isolated from dental calculus and induce mineralization in vitro. In recent years, based on in situ hybridization probe and sequencing technology, researchers have discovered the central "pillar" role of C. matruchotii in supragingival plaque, and many studies focused on bacterial interactions in the biofilm structure dominated by C. matruchotii have been conducted. Besides, C. matruchotii seems to be an indicator of "caries-free" oral status according to imaging and sequencing studies. Therefore, in this review, we summarize C. matruchotii 's role in supragingival plaque based on the structure, interactions, and potential connections with oral diseases. [ABSTRACT FROM AUTHOR]

The anaerobic membrane biofilm reactor (An-MBfR) using dead-end hollow fiber membranes (HFMs) inevitably suffers the limited supply of gaseous electron donors to biofilms, as a result of the back-diffusion of inert gases. The microbial mechanisms, underlying the biofilm formation and decontamination performance of the An-MBfR disadvantaged by limited active gas supply, are still obscure in the literature. Herein, we investigated the evolution laws of biofilm ecology and function in a denitrifying H 2 -based An-MBfR, from a multidimensional perspective. Results showed that despite the operating parameters of the reactor were set at the optimal values, the ununiform biofilms were developed on the HFMs, exhibiting a variation trend that with increasing distance from the near-gas end, the thickness and biomass of biofilms were decreased accompanied by their morphological change from the compacted to loosened. As hydrogenotrophic denitrifying bacteria (DNB) suffered limited H 2 supply to the biofilm, they could not produce abundant extracellular polymeric substances (EPS) and result in a high ratio of protein/polysaccharide (PN/PS) ratio in the EPS to facilitate the biofilm growth; their proliferation slowed down, especially in the outer layer of the biofilm at the far-gas end. The propagation of heterotrophic DNB was more active in the outer layer rather than inner layer of biofilms, ascribed to the abundant presence of PN and PS as well as increased NO 3 − availability. The variation trends in abundance of functional genes pertinent to the biofilm formation and NO 3 − reduction coincided well with the evolution laws of biofilm characteristics and DNB distribution. The findings provided mechanistic insights into the biofilm structure and microbial interaction in the denitrifying An-MBfR. [Display omitted] • Biofilm at far-gas end showed less thickness, biomass and loosened morphology. • Higher EPS content and protein/polysaccharide ratio promoted biofilm development. • Hydrogenotrophic and heterotrophic denitrifying bacteria enriched at near-gas end. • More abundant heterotrophic denitrifying bacteria located in outer layer of biofilms. • Metabolism-related pathways of biofilm formation and NO 3 − reduction were revealed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fresh-cut lettuce production chain is vulnerable to B. cereus group contamination. • Biofilm-forming B. cereus group was highly prevalent at the harvesting stage. • The average maximum sporulation rate of B. cereus group isolates was 24.6%. • B. cereus group isolates can form dense biofilms at 10 °C. • Sanitization practices and temperature control are required to ensure the safety of vegetables. Bacillus cereus and Bacillus thuringiensis , which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124 B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus , whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to β-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain. [ABSTRACT FROM AUTHOR]

Microbes interact in natural communities in a spatially structured manner, particularly in biofilms and polymicrobial infections. While next generation sequencing approaches provide powerful insights into diversity, metabolic capacity, and mutational profiles of these communities, they generally fail to recover in situ spatial proximity between distinct genotypes in the interactome. Hi‐C is a promising method that has assisted in analysing complex microbiomes, by creating chromatin cross‐links in cells, that aid in identifying adjacent DNA, to improve de novo assembly. This study explored a modified Hi‐C approach involving an initial lysis phase prior to DNA cross‐linking, to test whether adjacent cell chromatin can be cross‐linked, anticipating that this could provide a new avenue for study of spatial‐mutational dynamics in structured microbial communities. An artificial polymicrobial mixture of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli was lysed for 1–18 h, then prepared for Hi‐C. A murine biofilm infection model was treated with sonication, mechanical lysis, or chemical lysis before Hi‐C. Bioinformatic analyses of resulting Hi‐C interspecies chromatin links showed that while microbial species differed from one another, generally lysis significantly increased links between species and increased the distance of Hi‐C links within species, while also increasing novel plasmid‐chromosome links. The success of this modified lysis‐Hi‐C protocol in creating extracellular DNA links is a promising first step toward a new lysis‐Hi‐C based method to recover genotypic microgeography in polymicrobial communities, with potential future applications in diseases with localized resistance, such as cystic fibrosis lung infections and chronic diabetic ulcers. [ABSTRACT FROM AUTHOR]

External resistance is important for the anode and cell performance. However, little attentions were paid on the effect of external resistance on the variation of biofilm structure. Here, we used external resistance ranged from 4000 to 500 Ω for anodic acclimation to investigate the correlation between anode performance and biofilm structure. With the reduce of external resistance, the maximum current density of anode increased from 1.0 to 3.4 A/m2, which was resulted from a comprehensive effect of reduced charge transfer resistance and increased diffusion resistance. Biological analysis showed that with the reduce of external resistance, biomass and extracellular polymeric substances content increased by 109 and 286%, cell viability increased by 22.7%, which contributed to the reduced charge transfer resistance. But the porosity of anodic biofilm decreased by 27.8%, which led to an increased diffusion resistance of H+. This work provided a clear correlation between the electrochemical performance and biofilm structure. [ABSTRACT FROM AUTHOR]

Approximately four million people contract fungal infections every year in Brazil, primarily caused by Aspergillus spp. The ability of these fungi to form biofilms in tissues and medical devices complicates treatment and contributes to high rates of morbidity and mortality in immunocompromised patients. Psd2 is a pea defensin of 5.4 kDa that possesses good antifungal activity against planktonic cells of representative pathogenic fungi. Its function depends on interactions with membrane and cell wall lipid components such as glucosylceramide and ergosterol. In the present study, we characterized Aspergillus nidulans biofilm formation and determined the effect of Psd2 on A. nidulans biofilms. After 4 hours, A. nidulans conidia adhered to polystyrene surfaces and formed a robust extracellular matrix-producing biofilm at 24 h, increasing thickness until 48 h Psd2 inhibited A. nidulans biofilm formation in a dose-dependent manner. Most notably, at 10 μM Psd2 inhibited 50% of biofilm viability and biomass and 40% of extracellular matrix production. Psd2 significantly decreased the colonized surface area by the biofilm and changed its level of organization, causing a shortening of length and diameter of hyphae and inhibition of conidiophore formation. This activity against A. nidulans biofilm suggests a potential use of Psd2 as a prototype to design new antifungal agents to prevent biofilm formation by A. nidulans and related species.

Significant progress has been made previously in the research and development of graphene oxide (GO) membranes for water purification, but their biofouling behavior remains poorly understood. In this study, we investigated the biofilm formation and biofouling of GO membranes with different surface microstructures in the context of filtering natural surface water and for an extended operation period (110 days). The results showed that the relatively hydrophilic and smooth Fe(OH) 3 /GO membrane shaped a thin and spatially heterogeneous biofilm with high stable flux. However, the ability to simultaneously mitigate biofilm formation and reduce biofouling was not observed in the weakly hydrophilic and wrinkled Fe/GO and H-Fe(OH) 3 /GO membranes. Microbial analyses revealed that the hydrophilicity and roughness distinguished the bacterial communities and metabolic functions. The organic matter-degrading and predatory bacteria were more adapted to hydrophilic and smooth GO surfaces. These functional taxa were involved in the degradation of extracellular polymeric substances (EPS), and improved biofilm heterogeneity. In contrast, the weakly hydrophilic and wrinkled GO surfaces had reduced biodiversity, while unexpectedly boosting the proliferation of EPS-secreting bacteria, resulting in increased biofilm formation and aggravated biofouling. Moreover, all GO membranes achieved sustainable water purification during the entire operating period.

Bivalent cations are known to affect the structural and mechanical properties of biofilms. In order to reveal the impact of Fe2+ ions within the cultivation medium on biofilm development, structure and stability, Bacillus subtilis biofilms were cultivated in mini-fluidic flow cells. Two different Fe2+ inflow concentrations (0.25 and 2.5 mg/L, respectively) and wall shear stress levels (0.05 and 0.27 Pa, respectively) were tested. Mesoscopic biofilm structure was determined daily in situ and non-invasively by means of optical coherence tomography. A set of ten structural parameters was used to quantify biofilm structure, its development and change. The study focused on characterizing biofilm structure and development at the mesoscale (mm-range). Therefore, biofilm replicates (n = 10) were cultivated and analyzed. Three hypotheses were defined in order to estimate the effect of Fe2+ inflow concentration and/or wall shear stress on biofilm development and structure, respectively. It was not the intention to investigate and describe the underlying mechanisms of iron incorporation as this would require a different set of tools applied at microscopic levels as well as the use of, i.e., omic approaches. Fe2+ addition influenced biofilm development (e.g., biofilm accumulation) and structure markedly. Experiments revealed the accumulation of FeO(OH) within the biofilm matrix and a positive correlation of Fe2+ inflow concentration and biofilm accumulation. In more detail, independent of the wall shear stress applied during cultivation, biofilms grew approximately four times thicker at 2.5 mg Fe2+/L (44.8 µmol/L; high inflow concentration) compared to the low Fe2+ inflow concentration of 0.25 mg Fe2+/L (4.48 µmol/L). This finding was statistically verified (Scheirer–Ray–Hare test, ANOVA) and hints at a higher stability of Bacillus subtilis biofilms (e.g., elevated cohesive and adhesive strength) when grown at elevated Fe2+ inflow concentrations.

In nature, Listeria may interact competitively and cooperatively with other organisms, resulting in unique spatial organization and functions for cells within the community. This study was undertaken to characterize the biofilm architecture of binary biofilms of Listeria monocytogenes and Lactobacillus species and to assess their effect on the survival of Listeria during exposure to hypochlorite. Three L. monocytogenes strains, ATCC 19115 (Lm5), ATCC 19117 (Lm7), and Coleslaw (LmC), were selected and combined individually with three Lactobacillus strains: L. fermentum (Lf), L. bavaricus (Lb), and L. plantarum (Lp). In binary Lm-Lp biofilms, the Lm cell counts were similar to single-species biofilms (8.5 log CFU/well), and the Lp cell numbers declined by 1.0 log CFU/well. In the presence of Lb, the Lm cell counts were reduced by 1.5 log CFU/well (p < 0.05), whereas the Lf cell counts increased at least by 3.5 log CFU/well. Confocal laser scanning microscopy (CLSM) determined that interspecies interactions significantly affected the spatial organization of three binary biofilms. Biofilm surface-to-volume ratio increased from 0.8 μm2/μm3 for Lm5 in the monoculture to 2.1 μm2/μm3 for Lm5-Lp in the dual-species model (p < 0.05), and was characterized by a thicker structure with a largely increased surface area. Biofilm roughness increased from 0.2 for Lm7 to 1.0 for Lm7-Lb biofilms (p < 0.05), which appeared as interspecific segregation. Biofilm thickness increased from 34.2 μm for LmC to 46.3 μm for LmC–Lf (p < 0.05), which produced flat and compact structures that covered the entire surface available. The biomass of the extracellular matrix was higher in the case of some binary biofilms (p < 0.05); however, this effect was dependent upon the species pair. When treated with hypochlorite, Lm5 in binary biofilms had an approximately 1.5 log CFU/well greater survival than individually. The unique spatial organization and greater protein production may explain the protective effect of Lp after hypochlorite exposure.

Agglutinin-like sequence protein 3 (Als3) is a cell surface glycoprotein of Candida albicans that plays essential roles in the processes of adherence and biofilm formation in vitro. In this study, we focused on the contribution of Als3 to the structure and drug susceptibility of biofilms. The C. albicans wild-type (WT) strain DAY185, the als3 Δ/Δ null strain and the als3 Δ/Δ + p ALS3 complemented strain were used. Colony-forming unit enumeration, crystal violet and cell surface hydrophobicity assays, scanning electron microscopy and confocal laser scanning microscopy coupled with analyses using COMSTAT software were performed to evaluate the biomass and architecture of the biofilms. The detailed architectural analysis showed a significant variation in the biofilm parameters of the als3 Δ/Δ biofilms compared with those of the WT biofilms. Fluconazole, miconazole and amphotericin B were selected as the antifungal agents for the antimycotic susceptibility test, and increased susceptibility was found with the ALS3 deletion biofilms. A quantitative real-time polymerase chain reaction analysis showed downregulation of biofilm formation-related genes (ALS1 , EFG1 , HWP1 and CSH1) and drug resistance-related genes (ERG11 , CDR1 , CDR2 and MDR1) in the als3 Δ/Δ biofilms. We concluded that Als3 contributes to biofilm formation by changing the biofilm architecture and is involved in the antifungal resistance of C. albicans biofilms. [ABSTRACT FROM AUTHOR]

In nature, Listeria may interact competitively and cooperatively with other organisms, resulting in unique spatial organization and functions for cells within the community. This study was undertaken to characterize the biofilm architecture of binary biofilms of Listeria monocytogenes and Lactobacillus species and to assess their effect on the survival of Listeria during exposure to hypochlorite. Three L. monocytogenes strains, ATCC 19115 (Lm5), ATCC 19117 (Lm7), and Coleslaw (LmC), were selected and combined individually with three Lactobacillus strains: L. fermentum (Lf), L. bavaricus (Lb), and L. plantarum (Lp). In binary Lm-Lp biofilms, the Lm cell counts were similar to single-species biofilms (8.5 log CFU/well), and the Lp cell numbers declined by 1.0 log CFU/well. In the presence of Lb, the Lm cell counts were reduced by 1.5 log CFU/well (p < 0.05), whereas the Lf cell counts increased at least by 3.5 log CFU/well. Confocal laser scanning microscopy (CLSM) determined that interspecies interactions significantly affected the spatial organization of three binary biofilms. Biofilm surface-to-volume ratio increased from 0.8 μm2/μm3 for Lm5 in the monoculture to 2.1 μm2/μm3 for Lm5-Lp in the dual-species model (p < 0.05), and was characterized by a thicker structure with a largely increased surface area. Biofilm roughness increased from 0.2 for Lm7 to 1.0 for Lm7-Lb biofilms (p < 0.05), which appeared as interspecific segregation. Biofilm thickness increased from 34.2 μm for LmC to 46.3 μm for LmC–Lf (p < 0.05), which produced flat and compact structures that covered the entire surface available. The biomass of the extracellular matrix was higher in the case of some binary biofilms (p < 0.05); however, this effect was dependent upon the species pair. When treated with hypochlorite, Lm5 in binary biofilms had an approximately 1.5 log CFU/well greater survival than individually. The unique spatial organization and greater protein production may explain the protective effect of Lp after hypochlorite exposure. [ABSTRACT FROM AUTHOR]

Mansour Amin,1,2 Tahereh Navidifar,2,3 Farkhondeh Saleh Shooshtari,2,4 Mohammad Rashno,3,5 Mohammad Savari,1,2 Fatemeh Jahangirmehr,6 Mania Arshadi1 1Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 2Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 3Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 4Infertility Research and Treatment Center of Jahad Daneshgahi, Ahvaz, Khuzestan, Iran; 5Department of Immunology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 6Pain Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IranCorrespondence: Tahereh NavidifarCellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Golestan Street, Ahvaz, IranTel +989307292597Email roya_67@ymail.comBackground: The ability of biofilm formation is an effective way for Acinetobacter baumannii survival from stressed conditions. This present study was aimed to evaluate the association between biofilm formation, structure, the expression levels of genes related to biofilm formation and biofilm-specific resistance of A. baumannii strains isolated from burn infections in Ahvaz, Iran.Methods: In this study, we assessed the antibiotic susceptibilities, ERIC-PCR typing, capacity of biofilm formation and biofilm structure of 64 A. baumannii isolates collected from burn infections. The distribution and the expression levels of genes involved in the biofilm formation including bap, ompA, abaI, pgaA and csuE were assessed by PCR and real-time PCR, respectively.Results: We classified A. baumannii isolates in 14 clonal types of ERIC-PCR. Most A. baumannii isolates were resistant to all antibiotics tested except to tigecycline and colistin and had the biofilm formation capability but with different capacities. There was a significant inverse relationship between resistance to antibiotic agents and biofilm formation. The biofilm matrix of 50 strains consisted of polysaccharides together with DNA or proteins. The genes involved in the biofilm formation were detected in both biofilm-forming and non-biofilm forming; however, the expression levels of these genes were higher in biofilm producers compared with non-producers.Conclusion: The biofilm cells exhibited dramatically decreased susceptibility to antibiotic agents; hence, they have great significance for public health. Therefore, the determination of antibiotic susceptibilities in biofilm and planktonic mode, molecular typing, and capacity of biofilm formation in clinical setting is essential.Keywords: Acinetobacter baumannii, biofilm formation, gene expression, biofilm structure

Biofilm formation has been shown to be critical to the success of uropathogens. Although Staphylococcus saprophyticus is a common cause of urinary tract infections, its biofilm production capacity, composition, genetic basis, and origin are poorly understood. We investigated biofilm formation in a large and diverse collection of S. saprophyticus (n = 422). Biofilm matrix composition was assessed in representative strains (n = 63) belonging to two main S. saprophyticus lineages (G and S) recovered from human infection, colonization, and food-related environment using biofilm detachment approach. To identify factors that could be associated with biofilm formation and structure variation, we used a pangenome-wide association study approach. Almost all the isolates (91%; n = 384/422) produced biofilm. Among the 63 representative strains, we identified eight biofilm matrix phenotypes, but the most common were composed of protein or protein–extracellular DNA (eDNA)–polysaccharides (38%, 24/63 each). Biofilms containing protein–eDNA–polysaccharides were linked to lineage G and environmental isolates, whereas protein-based biofilms were produced by lineage S and infection isolates (p < 0.05). Putative biofilm-associated genes, namely, aas, atl, ebpS, uafA, sasF, sasD, sdrH, splE, sdrE, sdrC, sraP, and ica genes, were found with different frequencies (3–100%), but there was no correlation between their presence and biofilm production or matrix types. Notably, icaC_1 was ubiquitous in the collection, while icaR was lineage G-associated, and only four strains carried a complete ica gene cluster (icaADBCR) except one that was without icaR. We provided evidence, using a comparative genomic approach, that the complete icaADBCR cluster was acquired multiple times by S. saprophyticus and originated from other coagulase-negative staphylococci. Overall, the composition of S. saprophyticus biofilms was distinct in environmental and clinical isolates, suggesting that modulation of biofilm structure could be a key step in the pathogenicity of these bacteria. Moreover, biofilm production in S. saprophyticus is ica-independent, and the complete icaADBCR was acquired from other staphylococci.

Biofilm formation has been shown to be critical to the success of uropathogens. Although Staphylococcus saprophyticus is a common cause of urinary tract infections, its biofilm production capacity, composition, genetic basis, and origin are poorly understood. We investigated biofilm formation in a large and diverse collection of S. saprophyticus (n = 422). Biofilm matrix composition was assessed in representative strains (n = 63) belonging to two main S. saprophyticus lineages (G and S) recovered from human infection, colonization, and food-related environment using biofilm detachment approach. To identify factors that could be associated with biofilm formation and structure variation, we used a pangenome-wide association study approach. Almost all the isolates (91%; n = 384/422) produced biofilm. Among the 63 representative strains, we identified eight biofilm matrix phenotypes, but the most common were composed of protein or protein–extracellular DNA (eDNA)–polysaccharides (38%, 24/63 each). Biofilms containing protein–eDNA–polysaccharides were linked to lineage G and environmental isolates, whereas protein-based biofilms were produced by lineage S and infection isolates (p < 0.05). Putative biofilm-associated genes, namely, aas , atl , ebpS , uafA , sasF , sasD , sdrH , splE , sdrE , sdrC , sraP , and ica genes, were found with different frequencies (3–100%), but there was no correlation between their presence and biofilm production or matrix types. Notably, icaC_1 was ubiquitous in the collection, while icaR was lineage G-associated, and only four strains carried a complete ica gene cluster (icaADBCR) except one that was without icaR. We provided evidence, using a comparative genomic approach, that the complete icaADBCR cluster was acquired multiple times by S. saprophyticus and originated from other coagulase-negative staphylococci. Overall, the composition of S. saprophyticus biofilms was distinct in environmental and clinical isolates, suggesting that modulation of biofilm structure could be a key step in the pathogenicity of these bacteria. Moreover, biofilm production in S. saprophyticus is ica -independent, and the complete icaADBCR was acquired from other staphylococci. [ABSTRACT FROM AUTHOR]

Biofilms are structured communities formed by a single or multiple microbial species. Within biofilms, bacteria are embedded into extracellular matrix, allowing them to build macroscopic objects. Biofilm structure can respond to environmental changes such as the presence of antibiotics or predators. By adjusting expression levels of surface and extracellular matrix components, bacteria tune cell-to-cell interactions. One major challenge in the field is the fact that these components are very diverse among different species. Deciphering how physical interactions within biofilms are affected by changes in gene expression is a promising approach to obtaining a more unified picture of how bacteria modulate biofilms. This review focuses on recent advances in characterizing attractive and repulsive forces between bacteria in correlation with biofilm structure, dynamics, and spreading. How bacteria control physical interactions to maximize their fitness is an emerging theme. [ABSTRACT FROM AUTHOR]

• Microalgal biofilm structures formed under different light conditions are different. • Biofilm formed under white light is heterogeneous with many voids. • Biofilms formed under red and blue lights are homogeneous with low porosity. • Biofilms formed under different photoperiods are different. • Biofilm structures under different lights is interpreted by cell–cell interaction. Biofilm structure plays an important role in microalgae biofilm-based culture. This work aims to understand microalgal biofilm structures formed under different light conditions. Here, Scenedesmus obliquus was biofilm cultured under the light spectra of white, blue, green, and red, and the photoperiods of 5:5 s, 30:30 min, and 12:12 h (light : dark period). Biofilms were observed with confocal laser scanning microscopes and profilometry, then the porosity and roughness of biofilm were determined. We found that cells under white light formed a heterogeneous biofilm with many voids, high porosity, and roughness. While under red and blue lights, cells formed homogeneous biofilms with low porosity. Biofilm structures formed under different photoperiods were different. The mechanism of forming different biofilm structures under different light conditions was interpreted from the aspect of cell–cell interactions. Moreover, the results revealed that biomass accumulation increased with the increasing biofilm porosity due to the high effective diffusion coefficient. [ABSTRACT FROM AUTHOR]

The biofilm phenotype offers bacterial communities protection from environmental factors, evidenced by its role in the viability, persistence, and virulence of cells under conditions in which flow is present, such as in riverbeds, industrial piping networks, and the human circulatory system. Here we examine the hypothesis that temperature - an environmental factor that affects the growth of the Gram-positive bacteria Staphylococcus epidermidis - controls, through a dual mechanism, persistence of this bacterial strain in a shear environment characteristic of the human circulatory system. We demonstrate that temperature and antibiotics impact the surface-adhered biofilm and material disseminated downstream in different ways. Specifically, by means of 3D confocal and scanning electron microscopy, an increase in surface adhered biofilm heterogeneity is observed with increasing temperature. Additionally, we find a four-log decrease in cell viability at the biofilm surface as the perfusate temperature is increased from 37° to 50°C. Finally, the viability of cell containing fragments that are disseminated from the substrate is assessed by downstream sampling, culture, and optical density measurement. We find that although temperature decreases the viability of the surface adhered biofilm, the downstream material remains viable. Yet, in the presence of antibiotics, the growth of disseminated material is nearly completely inhibited, even though the addition of antibiotics has no significant impact on the viability of the surface adhered biofilm. The mechanism involves both biofilm structural damage, as quantified by morphology of debrided material, and reduced cell viability, as quantified by assay of bacterial cells present in the surface adherent biofilm and in the downstream effluent. The results potentially identify parameter ranges in which elevated temperature could augment current antibiotic treatment regimens. thermal stress is a foundational step toward the development of an in-situ treatment/remediation method for biofilm growth in medical devices; such an application could use oscillatory flow of heated fluid in a catheter as an adjuvant to antibiotic treatment. The work furthermore provides new insight into the viability of disseminated biofilm material. [ABSTRACT FROM AUTHOR]

In some regions, climate change is increasing the variability of rainfall and the frequency of extreme events such as drought. Consequently, non‐flow periods have grown in length and frequency, both in temporary and in formerly permanent streams. Water abstraction for human use may further prolong these dry periods.We analysed the resistance and resilience of biofilms from permanent and temporary streams to non‐flow conditions. This was achieved by exposing cobbles (collected from permanent and temporary streams) with intact biofilm to 31 days of non‐flow, followed by 20 days of stream flow in artificial stream channels. Biofilm resistance and resilience were assessed at a structural (algal biomass, pigment composition, and algae and cyanobacteria composition) and functional level (photosynthetic efficiency and community metabolism).Algal taxa in biofilms from permanent and temporary streams differed throughout the experiment. Biofilms from permanent streams were less resistant to non‐flow than those from temporary streams at structural level. Permanent stream biofilms also presented lower resilience at a structural level, but responded similarly to temporary stream biofilms at a functional level.Our investigation shows how the non‐flow period disturbed permanent stream biofilms, and suggests that temporary stream biofilms will have greater adaptive capacity as hydroperiod becomes shorter due to climate change. [ABSTRACT FROM AUTHOR]

The impact of feed water quality on biofilm formation during membrane distillation (MD) was investigated in this study, particularly emphasizing the interrelationship between organics, salts, and microbes. Two types of typical natural surface waters in Nanjing, China, were chosen as feed solutions for long-term MD operation, including the Qinhuai River and Xuanwu Lake. The biofilms that developed under different feed water qualities exhibited distinct Foulant compositions and structures, causing different flux decline trends for the MD system. Accordingly, two typical patterns of biofilm formation were suggested for the MD operation of the two different kinds of surface waters in this study. Organics from a primal feed solution and dead bacteria were the key to the establishment of a biofilm on the membrane, and this needs to be effectively removed from the MD system through pre-treatment and process control strategies. Finally, a feasible strategy for MD biofouling control was suggested. [ABSTRACT FROM AUTHOR]

• A new mechanistic 1D biofilm model introduces a distinction between PN and PS. • A volume detachment process that depends on the physical properties is considered. • The stratification of physical properties and biofilm composition is captured. • The influence of COD/N, shear stress and SOLR on EPS composition is considered. • The model captures qualitatively the influence of these growth conditions. In industry, treatments against biofilms need to be optimized and, in the wastewater treatment field, biofilm composition needs to be controlled. Therefore, describing the biochemical and physical structures of biofilms is now required to better understand the influence of operating parameters and treatment on biofilms. The present study aims to investigate how growth conditions influence EPS composition, biofilm physical properties and volume detachment using a 1D biofilm model. Two types of EPS are considered in the present model, proteins and polysaccharides. The main hypotheses are that: (i) the production of polysaccharides occurs mainly under strong nutrient limitation(s) while the production of proteins is coupled to both the substrate uptake rate and the lysis process; (ii) the local biofilm porosity depends on the local biofilm composition. Both volume and surface detachment occur in biofilms and volume detachment extent depends on the biofilm local cohesion and thus on the local composition of biofilms for a given shear stress. The model is based on experimental trends and aims to represent these observations on the basis of biochemical and physical processes. Four case studies covering a wide range of contrasting growth conditions such as different COD/N ratios, applied SOLR and shear stresses are investigated. The model predicts how the biochemical and physical biofilm structures change as a result of contrasting growth conditions. More precisely simulation results are in good agreement with the main experimental observations reported in the literature, such as: (i) a strong nitrogen limitation of growth induces an important accumulation of polysaccharides leading to a more porous and homogenous biofilm, (ii) a high applied surface organic loading load allows to obtain a high biofilm thickness, (iii) a strong shear stress applied during the biofilm growth leads to a reduction of the biofilm thickness and to a consolidation of the biofilm structure. Overall, this model represents a relevant decision tool for the selection of appropriate enzymatic treatments in the context of negative biofilm control. From our results, it appears that protease based treatments should be more appropriate for biofilms developed under low COD/N ratios (about 20 gCOD/gN) whereas both glucosidases and proteases based treatments should be more appropriate for biofilms developed under high COD/N ratio (about 70 gCOD/gN). In addition, the model could be useful for other applications such as resource recovery in biofilms or granules, and help to better understand biological membrane fouling. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus and coagulase-negative staphylococci (CoNS) have become the main causative agents of medical device-related infections due to their biofilm-forming capability, which protects them from the host’s immune system and from the action of antimicrobials. This study evaluated the ability of RNA III inhibiting peptide (RIP) to inhibit biofilm formation in 10 strains isolated from clinical materials, including one S. aureus strain, two S. epidermidis, two S. haemolyticus, two S. lugdunensis, and one isolate each of the following species: S. warneri, S. hominis, and S. saprophyticus. The isolates were selected from a total of 200 strains evaluated regarding phenotypic biofilm production and the presence and expression of the ica operon. The isolates were cultured in trypticase soy broth with 2% glucose in 96-well polystyrene plates containing catheter segments in the presence and absence of RIP. The catheter segments were observed by scanning electron microscopy. The results showed inhibition of biofilm formation in the presence of RIP in all CoNS isolates; however, RIP did not interfere with biofilm formation by S. aureus. RIP is a promising tool that might be used in the future for the prevention of biofilm-related infections caused by CoNS.

Abstract Despite many decades of research, biofilm architecture and spreading mechanisms are still not clear because of the heterogenous 3D structure within biofilms. Here, patterned “slippery” lubricant‐infused porous surfaces are utilized to study biofilm structure of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Staphylococcus aureus. It is found that bacteria are able to spread over bacteria‐repellent lubricant‐infused regions by using a mechanism, termed “biofilm bridges”. Here, it is demonstrated that bacteria use bridges to form interconnected networks between distant biofilm colonies. Detailed structure of bridges shows a spatial distribution of bacteria with an accumulation of respiratory active bacteria and biomass in the bridges. The core–shell structure of bridges formed by two‐species mixed population is illustrated. It is demonstrated that eDNA and nutrients have a strong effect on biofilm bridges formation. Thus, it is believed that biofilm bridging is important to reveal the structure and communication within biofilms.

Since biofilm formation by microfoulers significantly contributes to the fouling process, it is important to evaluate the performance of marine surfaces to prevent biofilm formation, as well as understand their interactions with microfoulers and how these affect biofilm development and structure. In this study, the long-term performance of five surface materials—glass, perspex, polystyrene, epoxy-coated glass, and a silicone hydrogel coating—in inhibiting biofilm formation by cyanobacteria was evaluated. For this purpose, cyanobacterial biofilms were developed under controlled hydrodynamic conditions typically found in marine environments, and the biofilm cell number, wet weight, chlorophyll a content, and biofilm thickness and structure were assessed after 49 days. In order to obtain more insight into the effect of surface properties on biofilm formation, they were characterized concerning their hydrophobicity and roughness. Results demonstrated that silicone hydrogel surfaces were effective in inhibiting cyanobacterial biofilm formation. In fact, biofilms formed on these surfaces showed a lower number of biofilm cells, chlorophyll a content, biofilm thickness, and percentage and size of biofilm empty spaces compared to remaining surfaces. Additionally, our results demonstrated that the surface properties, together with the features of the fouling microorganisms, have a considerable impact on marine biofouling potential.

Surface contamination by Pseudomonas fluorescens biofilms was studied using pilot scale washing tanks with standard design features encountered in the fresh-cut vegetable processing industry. Consequences on the biofilms' resistance to the shear stress and to enzymatic cleaning were analysed. It was found that biofilm structures depended on the location in the tanks, which could range from discontinuous monolayer to large thick clusters, as observed for the wetting front areas corresponding to intermittently immersed areas. Biofilms grown in zones subjected to shear and at the wetting front were significantly more resistant to mechanical stress. Therefore, poor design features such as horizontal surfaces, right angles, welds and corners at the vat bottom appeared critical in terms of hygiene, being more resistant to enzymatic cleaning. Biofilms developed at the wetting front, in a priori hygienic areas, also appear resistant to enzymatic cleaning, constituting additional critical areas. • Biofilm formation and structure are strongly related to the equipment design. • Resistant biofilms are observed on surfaces at the wetting front. • Biofilms grown under mechanical stress are highly resistant to shear stress. • Biofilms grown on horizontal surfaces are difficult to clean by enzymes. [ABSTRACT FROM AUTHOR]

The ubiquitous divalent cations magnesium and calcium are important nutrients required by bacteria for growth and cell maintenance. Multi-faceted roles are shown both in bacterial initial attachment and biofilm maturation. The effects of calcium and magnesium can be highlighted in physio-chemical interactions, gene regulation and bio-macromolecular structural modification, which lead to either promotion or inhibition of biofilms. This review outlines recent research addressing phenotypic changes and mechanisms undertaken by calcium and magnesium in affecting bacterial biofilm formation. [ABSTRACT FROM AUTHOR]

Biofilms of Actinobacillus succinogenes have demonstrated exceptional capabilities as biocatalysts for high productivity, titre and yield production of succinic acid (SA). The paper presents a microscopic analysis of A. succinogenes biofilms developed under varied fermenter conditions. The concentration of excretion metabolites is controlled by operating the fermenter in a continuous mode where the liquid throughput is adjusted. It is clearly illustrated how the accumulation of excreted metabolites (concomitant with the sodium build-up due to base dosing) has a severe effect on the biofilm structure and physiology. Under high accumulation (HA) conditions, some cells exhibit severe elongation while maintaining a cross-sectional diameter like the rod/cocci-shaped cells predominantly found in low accumulation (LA) conditions. The elongated cells formed at high accumulation conditions were found to be more viable than the clusters of rod/cocci-shaped cells and appear to form connections between the clusters. The global microscopic structure of the HA biofilms also differed significantly from the LA biofilms. Although both exhibited shedding after 4 days of growth, the LA biofilms were more homogenous (less patchy), thicker and with high viability throughout the biofilm depth. The viability of the HA biofilms was threefold lower than the corresponding LA biofilms towards the end of the fermentation. Visual observations were supported by quantitative analysis of multiple biofilm samples and strengthened the main observations. The work presents valuable insights on the effect of metabolite accumulation on biofilm structure and growth. [ABSTRACT FROM AUTHOR]

Biofilms have permeable structures, which can be used as membranes for solids separation, e.g., dynamic membrane bioreactors (DMBRs) that relied on the biofilm developed on the support material with large apertures for filtration. This study found that the biofilm formed on the support material in DMBRs under different solids retention time (SRTs) had diverse structures, which in turn determined the hydraulic resistance and filtration performance. At 5-day SRT, a thin but compact gel-like biofilm layer (porosity = 27.5%) was formed on the support materials, which had a higher hydraulic resistance of approximately 4.9 × 1011 m−1. As a result, the transmembrane pressure (TMP) rose dramatically every 20–40 days' operation. A thick but porous biofilm layer (porosity > 60% and hydraulic resistance < 2.5 × 109 m−1) was formed at longer SRTs of 20 and 40 days and the TMP could keep consistently low (<20 Pa) for more than 180 days. Therefore, a longer SRT could facilitate the formation of a porous biofilm layer on the support material, which was of critical importance for achieving long-term and low-pressure filtration. The biofilm porosity was negatively correlated with the protein/polysaccharide ratio of its extracellular polymeric substance (EPS). Extending SRT would reduce the Live/Dead cell ratio of biofilm, thus lowering EPS production while increasing the porosity. Moreover, extending SRT promoted the growth of protozoa (e.g., Euglypha) that inhabited the biofilm, which could maintain the biofilm porous and mitigate the support layer biofouling though feeding and movement. Image 1 • Rapid rise in TMP occurred less frequently with increasing SRT in DMBR. • A compact and gel-like biofilm layer was formed at a short SRT. • Extending SRT increased biofilm porosity and decreased its hydraulic resistance. • Increasing SRT lowered the ratios of Live/Dead cell and PN/PS in biofilm. • Protozoa could maintain biofilm porous and mitigate support layer biofouling. [ABSTRACT FROM AUTHOR]

Abstract Biofouling severely impacts operational performance of membrane systems increasing the cost of water production. Understanding the effect of critical parameters of feed water such as biodegradable substrate concentration on the developed biofilm characteristics enables development of more effective biofouling control strategies. In this study, the effect of substrate concentration on the biofilm characteristics was examined using membrane fouling simulators (MFSs). A feed channel pressure drop (PD) increase of 200 mbar was used as a benchmark to study the developed biofilm. The amount and characteristics of the formed biofilm were analysed in relation to membrane performance indicators: feed channel pressure drop and permeate flux. The effect of the characteristics of the biofilm developed at three substrate concentrations on the removal efficiency of the different biofilms was evaluated applying acid/base cleaning. Results showed that a higher feed water substrate concentration caused a higher biomass amount, a faster PD increase, but a lower permeate flux decline. The permeate flux decline was affected by the spatial location and the physical characteristics of the biofilm rather than the total amount of biofilm. The slower growing biofilm developed at the lowest substrate concentration was harder to remove by NaOH/HCl cleanings than the biofilm developed at the higher substrate concentrations. Effective biofilm removal is essential to prevent a fast biofilm regrowth after cleaning. While substrate limitation is a generally accepted biofouling control strategy delaying biofouling, development of advanced cleaning methods to remove biofilms formed under substrate limited conditions is of paramount importance. Highlights • Faster flow channel pressure drop increase at higher substrate concentration. • Lower permeate flux decline at higher substrate concentration. • More biomass accumulated at higher substrate concentration. • Permeate flux decline not predictive for amount of accumulated biomass. • Slower growing, harder to remove biofilms at lower substrate concentration. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa, an opportunistic human pathogen, causes many biofilm-mediated chronic infections. In this study, biofilm structures of various clinical strains of P. aeruginosa isolated from hospitalized patients were examined and their influence on the biofilm-dispersing effects of chemicals was investigated. The clinical isolates formed structurally distinct biofilms that could be classified into three different groups: 1) mushroom-like, 2) thin flat, and 3) thick flat structures. A dispersion of these differently structured biofilms was induced using two biofilm-dispersing agents, anthranilate and sodium nitroprusside (SNP). Although both SNP and anthranilate could disperse all types of biofilms, the thick flat biofilms were dispersed less efficiently than the biofilms of other structures. This suggests that biofilm-dispersing agents have higher potency on the biofilms of porous structures than on densely packed biofilms. [ABSTRACT FROM AUTHOR]

The Cyanobacterium Oculatella subterranea Zammit, Billi, Albertano inhabits hypogea and stone caves and is a pioneer of different stone substrata. In this study, a strain isolated from the House of Marco Castricio (Archaeological Park of Pompeii, Italy) was identified by a polyphasic approach and used for an in vitro colonization test to verify the influence of the substrate on the biofilm architecture. Fine structure of O. subterranea microbial mats was revealed as well as filaments orientation toward light source. This aim has been achieved through confocal laser scanner microscope microscopy and computer image analysis. Moreover, bioreceptivity of five different substrates, commonly retrieved in archaeological sites of Campania, was assessed for O. subterranea. Our results show that the three-dimensional structure of O. subterranea microbial mats is poorly affected by physical and geochemical features of substrates: in fact, the porous architecture of its biofilm was preserved, independently of the materials. On the other hand, the area/perimeter ratio relative to the O. subterranea growth on tuff, brick, and porphyry showed significant differences, indicating dissimilar levels of bioreceptivity of the three substrates. [ABSTRACT FROM AUTHOR]

[Display omitted] • Aerotolerant bioanode was constructed with small external resistance or high shear stress. • Aerotolerant biofilm was flat and compact with a large laminar boundary layer for reducing oxygen diffusion. • Compact electrode assembly MFC with aerotolerant bioanode achieved a power density of 4300 mW/m2. Reducing electrode spacing is considered the most effective way to maximize the power density of microbial fuel cell (MFC) due to the effective reduction of internal resistance. However, reducing electrode spacing presents the challenge of diffusing oxygen from the cathode to the anode, thereby reducing anode performance. Constructing aerotolerant anode may be one of the effective ways to minimize the effect of oxygen on anode performance. Here, we report that using small external resistance or maintaining high nitrogen sparging rate during bioanode enrichment could significantly enhance the aerotolerant characteristic of the bioanode. The voltage loss affected by oxygen was significantly reduced and, as a result, the power density of the compact electrode assembly MFC increased by 85% reaching 4300 mW/m2. The aerotolerant bioanode had a flat and compact biofilm structure and a larger laminar boundary layer (over 300 μm) to reduce oxygen diffusion, while the aerointolerant bioanode had a rough and porous biofilm structure and a smaller laminar boundary layer (less than 80 μm). The microbial community of all the bioanodes was dominated by Geobacter (over 55%). Therefore, this study provides a sample way to construct an aerotolerant bioanode for high power generation in MFCs. [ABSTRACT FROM AUTHOR]