Characterization Of Biofilms Research Articles

Advanced characterization of natural biofilm on nanofiber scaffold

Nanofiber scaffolds provide numerous advantages over common carriers engineered for microorganisms. The most important advantage is an increased speed of primary surface colonization (up to four times faster), which shortens the time required for the areal biofilm formation and optimum performance of attached microorganisms (higher efficiency of biological activity of up to twice as fast). Image analysis predicts early formation of biofilm even in beginning stages; analysis of biofilm reveals the different structures of bacterial colonies on both scaffolds (higher porosity, size, and number of bacterial colonies on nanofiber's surface). The image analysis correlates well with determinations of dry matter (linear correlation of 0.96) and proteins (linear correlation of 0.89).

Towards mechanical characterization of granular biofilms by optical coherence elastography measurements of circumferential elastic waves.

Microbial granular biofilms are spherical, multi-layered aggregates composed of communities of bacterial cells encased in a complex matrix of hydrated extracellular polymeric substances (EPS). While granular aggregates are increasingly used for applications in industrial and municipal wastewater treatment, their underlying mechanical properties are poorly understood. The challenges of viscoelastic characterization for these structures are due to their spherical geometry, spatially heterogeneous properties, and their delicate nature. In this study, we report a model-based approach for nondestructive characterization of viscoelastic properties (shear modulus and shear viscosity) of alginate spheres with different concentrations, which was motivated by our measurements in granular biofilms. The characterization technique relies on experimental measurements of circumferential elastic wave speeds as a function of frequency in the samples using the Optical Coherence Elastography (OCE) technique. A theoretical model was developed to estimate the viscoelastic properties of the samples from OCE data through inverse analysis. This work represents the first attempt to explore elastic waves for mechanical characterization of granular biofilms. The combination of the OCE technique and the theoretical model presented in this paper provides a framework that can facilitate quantitative viscoelastic characterization of samples with curved geometries and the study of the relationships between morphology and mechanical properties in granular biofilms.

Towards the biofilm characterization and regulation in biological wastewater treatment

There is an increasing need for application of biofilm process in the upcycling of wastewater treatment plants all around the world in recent years, yet there are few literatures on summarizing wastewater biofilm during the life cycle. In particular, there is a vacancy on characterization at various stages of biofilm and its regulation. This review provided a whole look at biofilm formation and its development, accompanied by microbial physiology, ecology, and activity, where the initialization of biofilm formation and its characterization were stressed. The new progresses on biofilm physio-ecology analysis and methods on evaluating microbial activity were summarized, while it is worth mentioning that the concept of aging biofilm was also presented. Furthermore, regulations methods of biofilm were reviewed and future research trends on biofilm control were prospected, aiming at guiding biofilm control in biofilm-based wastewater treatment.

Spatial structuring of bacterial communities in epilithic biofilms in the Acquarossa river (Italy).

Epilithic river biofilms characterize the rock surfaces along the Acquarossa river (Viterbo, Italy); they are in part red and in part black colored, maintaining a well-defined borderline. This peculiarity has raised questions about the biotic and abiotic phenomena that might avoid the mixing of the two biofilms. In this study, the structuring of bacterial communities in black and red epilithic biofilm in the Acquarossa river has been investigated with both culture dependent and independent approaches. Data obtained highlighted a (very) different taxonomic composition of black and red epilithons bacterial communities, dominated by Acinetobacter sp. and iron-oxidizing bacteria, respectively. The chemical characterization of both river water and biofilms revealed a substantial heavy metals pollution of the environment; heavy metals were also differentially accumulated in red and black epilithons. Overall, our data revealed that the structuring of red and black epilithons might be affected mainly by the antagonistic interactions exhibited by bacterial genera dominating the two biofilms. These findings suggest that biotic factors might be responsible for the structuring of natural bacterial communities, suggesting that there is a selection of populations at very small scale, and that different populations might compete for different niches.

Interaction between wastewater microorganisms and geopolymer or cementitious materials: Biofilm characterization and deterioration characteristics of mortars

This paper compares the biofilm formation during field exposure to real municipal wastewaters on geopolymer and copper-doped geopolymer mortar, with plain Portland cement and calcium aluminate cement as controls. The samples were submerged in a wastewater treatment plant at three different stages for 35 days (primary clarifier, activated sludge and final effluent), which led to diverse chemical and biological exposure conditions due to the different organic load of the three environments. The deterioration characteristics of the four different mortars were analyzed using X-ray diffraction, thermogravimetric analysis and optical microscopy. The formed biofilm was characterized by measuring the protein concentration at the sample surface and bacterial respiration. After wastewater exposure, the protein concentration on the Portland cement samples was 50% higher as compared to the geopolymer mortars, while the respiration rates on the Portland/calcium aluminate cement samples was 2.5 times higher than on the geopolymer doped with copper, suggesting a lower bioreceptivity for the latter material. Scanning electron microscopy revealed the presence of biofilm on mortar surfaces, especially on the samples exposed to organic-rich wastewaters. After being exposed to wastewater, the degree of degradation of the geopolymer-based mortars was much lower than the two controls. This may suggest that these materials can be a potential alternative to conventional cement-based binders in order to limit the growth of microorganism on concrete surfaces as well as exhibiting an enhanced acid resistance.

Nanoplasmonics for Real-Time and Label-Free Monitoring of Microbial Biofilm Formation.

Microbial biofilms possess intrinsic resistance against conventional antibiotics and cleaning procedures; thus, a better understanding of their complex biological structures is crucial in both medical and industrial applications. Existing laboratory methodologies have focused on macroscopic and mostly indirect characterization of mechanical and microbiological properties of biofilms adhered on a given substrate. However, the kinetics underlying the biofilm formation is not well understood, while such information is critical to understanding how drugs and chemicals influence the biofilm formation. Herein, we report the use of localized surface plasmon resonance (LSPR) for real-time, label-free monitoring of E. coli biofilm assembly on a nanoplasmonic substrate consisting of gold mushroom-like structures. Our LSPR sensor is able to capture the signatures of biofilm formation in real-time by measuring the wavelength shift in the LSPR resonance peak with high temporal resolution. We employ this sensor feature to elucidate how biofilm formation is affected by different drugs, including conventional antibiotics (kanamycin and ampicillin) as well as rifapentine, a molecule preventing cell adhesion yet barely affecting bacterial viability and vitality. Due to its flexibility and simplicity, our LSPR based platform can be used on a wide variety of clinically relevant bacteria, thus representing a valuable tool in biofilm characterization and drug screening.

Biological and geological characterization of modern biofilms and microbial mats and comparison with similar lithified structures in Colombian Cretaceous formations

Microorganisms may play an important role in the aggregation of sediments and the formation of sedimentary structures. Biofilms are microbial aggregates that, in a mature stage, can develop into microbial mats, fibrillar networks that irreversibly bind filaments of cyanobacteria and sediments, inside which it has been identified a stratification with functional groups of microorganisms that coexist, generate symbiotic relationships and potentially modify the characteristics of sediments and sedimentary rocks, particularly in extreme environments. In this work, filamentous cyanobacteria from biofilms of a lacustrine environment with intervals of flooding/desiccation and a saline environment, and a microbial mat from the Agua Caliente Thermal, El Rosal, Cundinamarca, are identified. In the biofilms, most cyanobacteria were found to belong to the Orden Oscillatoriales, while in the microbial mat cyanobacteria of the order Orden Nostocales were also recognized. Two rock samples isolated from the thermal which genesis was possibly influenced by the activity of cyanobacteria are described and classified. One of them, named R-1, is a calcareous rock inside which it was possible to differentiate biolaminations and an apparent dominance of biomineralization processes. This sample was both classified as a travertine and a microbial framestone with stromatolitic and thrombolytic texture. The second one, called R-2, is a siliciclastic rock classified as a mudstone and a microbial boundstone. Finally, a comparison between the sedimentary structures identified in those rocks with similar structures in the formations La Luna, Paja and Tetuán and the microbially-induced sedimentary structures (MISS) described in the literature is performed. Based on morphological resemblance, fibrillar networks identified locally in those formations are interpreted as possible biolaminations originated from the activity of cyanobacteria.

Novel insights into the role of Pseudomonas quinolone signal in the control of reverse osmosis membrane biofouling

The role of the Pseudomonas quinolone signal (PQS) pathway in the control of biofouling in reverse osmosis membrane filtration was examined by inoculating Pseudomonas aeruginosa into feed water to simulate biofouling. Various concentrations of PQS (2-heptyl-3-hydroxy-4-quinolone) and an anthranilate analog (methyl anthranilate) were used as the activator and inhibitor of the PQS pathway, respectively. Membrane performance results showed that addition of exogenous PQS caused severe decreases in permeate flux and salt rejection and increase in irreversible membrane fouling. In contrast, the presence of methyl anthranilate mitigated the declines in permeate flux and salt rejection by biofouling and significantly reduced the total fouling resistance and hence shorten the membrane cleaning periods. Biofilm characterization demonstrated that PQS aggravated membrane biofouling by promoting initial bacterial attachment, increasing levels of the extracellular polymeric substances (EPS), total organic carbon (TOC) and extracellular DNA, and changing the mature biofilm structure from a mushroom shape to a denser flat shape. In contrast, addition of methyl anthranilate increased protein adsorption during the initial biofouling stage through electrostatic interactions, but significantly inhibited biofilm growth via reducing levels of EPS and TOC, as well as total cell number in the biofilm. These results provide evidence that PQS pathway plays an important role in biofilm formation and an anthranilate analog, methyl anthranilate, could be a novel agent to control RO membrane biofouling. Therefore, PQS synthetic pathway and precursors of quorum sensing signals may provide viable targets for biofouling control in practical RO membranes applications.

Characterization of Aquatic Biofilms with Flow Cytometry.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing. Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing. Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined. Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.

Development and characterization of an oral multispecies biofilm implant flow chamber model.

Peri-implant infections are the most common cause of implant failure in modern dental implantology. These are caused by the formation of biofilms on the implant surface and consist of oral commensal and pathogenic bacteria, which harm adjacent soft and hard tissues and may ultimately lead to implant loss. In order to improve the clinical situation, there has to be a better understanding of biofilm formation on abiotic surfaces. Therefore, we successfully developed a system to cultivate an oral multispecies biofilm model in a flow chamber system, optimized for the evaluation of biofilm formation on solid materials by direct microscopic investigation. The model contains four relevant oral bacterial species: Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis in ratios similar to the native situation. The reliability of the developed “Hanoverian Oral Multispecies Biofilm Implant Flow Chamber” (HOBIC) model was verified. Biofilm volume and live/dead distribution within biofilms were determined by fluorescence staining and confocal laser scanning microcopy (CLSM). The individual species distribution was analyzed using quantitative real time PCR with propidium monoazide pretreatment (PMA-qRT-PCR) and by urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH). This in vitro model may be used to analyze biofilm formation on dental implants in more detail and to develop future implant systems with improved material properties.

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

A Versatile Strategy for Characterization and Imaging of Drip Flow Microbial Biofilms.

The inherent architectural and chemical complexities of microbial biofilms mask our understanding of how these communities form, survive, propagate, and influence their surrounding environment. Here we describe a simple and versatile workflow for the cultivation and characterization of model flow-cell-based microbial ecosystems. A customized low-shear drip flow reactor was designed and employed to cultivate single and coculture flow-cell biofilms at the air-liquid interface of several metal surfaces. Pseudomonas putida F1 and Shewanella oneidensis MR-1 were selected as model organisms for this study. The utility and versatility of this platform was demonstrated via the application of several chemical and morphological imaging techniques-including matrix-assisted laser desorption/ionization mass spectrometry imaging, secondary ion mass spectrometry imaging, and scanning electron microscopy-and through the examination of model systems grown on iron substrates of varying compositions. Implementation of these techniques in combination with tandem mass spectrometry and a two-step imaging principal component analysis strategy resulted in the identification and characterization of 23 lipids and 3 oligosaccharides in P. putida F1 biofilms, the discovery of interaction-specific analytes, and the observation of several variations in cell and substrate morphology present during microbially influenced corrosion. The presented workflow is well-suited for examination of both single and multispecies drip flow biofilms and offers a platform for fundamental inquiries into biofilm formation, microbe-microbe interactions, and microbially influenced corrosion.

Isolation and screening of bacteria with biofilm formation ability and characterization with hydrolytic enzyme production for enhanced biogas production

Aims: Biofilm is a complex structure that provides protection towards the bacteria within the barrier. Enhanced biogas production from Palm Oil Mill Effluent (POME) can be achieved by applying biofilm based anaerobic digestion system. Methodology and results: Bacteria that produces biofilm were isolated and tested on its hydrolytic enzyme secretion. The biofilm produced were also characterized. Out of 120 strains isolated from POME, PKC and food waste compost, only 33 strains were producing biofilm and only 11 of them exhibited significant amount of biofilm produced at optical density of wavelength 595 nm (>0.01). In hydrolysis enzyme assay test, all strains were not able to secrete protease enzyme. The biofilms were extracted and characterized to show similar characteristic for all strains. Strain numbers of 11, 9C, 23C and 30C showed positive result for cellulase, amylase and lipase enzymes, to be tested as single strain bacteria and also mixed with other isolated bacterium for prospect research on effective hydrolysis towards enhanced biogas production. The composition of biofilms from different bacteria mixture also similar under the same incubation condition. Conclusion, significance and impact of study: Bacteria producing biofilm are very limited and does not secrete the same hydrolytic enzymes. Utilization of these bacteria may eliminate the problem of microbial instability in a system.

Options and Limitations in Clinical Investigation of Bacterial Biofilms.

Bacteria can form single- and multispecies biofilms exhibiting diverse features based upon the microbial composition of their community and microenvironment. The study of bacterial biofilm development has received great interest in the past 20 years and is motivated by the elegant complexity characteristic of these multicellular communities and their role in infectious diseases. Biofilms can thrive on virtually any surface and can be beneficial or detrimental based upon the community's interplay and the surface. Advances in the understanding of structural and functional variations and the roles that biofilms play in disease and host-pathogen interactions have been addressed through comprehensive literature searches. In this review article, a synopsis of the methodological landscape of biofilm analysis is provided, including an evaluation of the current trends in methodological research. We deem this worthwhile because a keyword-oriented bibliographical search reveals that less than 5% of the biofilm literature is devoted to methodology. In this report, we (i) summarize current methodologies for biofilm characterization, monitoring, and quantification; (ii) discuss advances in the discovery of effective imaging and sensing tools and modalities; (iii) provide an overview of tailored animal models that assess features of biofilm infections; and (iv) make recommendations defining the most appropriate methodological tools for clinical settings.

Ugly ducklings\u2014the dark side of plastic materials in contact with potable water

Bath toys pose an interesting link between flexible plastic materials, potable water, external microbial and nutrient contamination, and potentially vulnerable end-users. Here, we characterized biofilm communities inside 19 bath toys used under real conditions. In addition, some determinants for biofilm formation were assessed, using six identical bath toys under controlled conditions with either clean water prior to bathing or dirty water after bathing. All examined bath toys revealed notable biofilms on their inner surface, with average total bacterial numbers of 5.5 × 106 cells/cm2 (clean water controls), 9.5 × 106 cells/cm2 (real bath toys), and 7.3 × 107 cells/cm2 (dirty water controls). Bacterial community compositions were diverse, showing many rare taxa in real bath toys and rather distinct communities in control bath toys, with a noticeable difference between clean and dirty water control biofilms. Fungi were identified in 58% of all real bath toys and in all dirty water control toys. Based on the comparison of clean water and dirty water control bath toys, we argue that bath toy biofilms are influenced by (1) the organic carbon leaching from the flexible plastic material, (2) the chemical and biological tap water quality, (3) additional nutrients from care products and human body fluids in the bath water, as well as, (4) additional bacteria from dirt and/or the end-users’ microbiome. The present study gives a detailed characterization of bath toy biofilms and a better understanding of determinants for biofilm formation and development in systems comprising plastic materials in contact with potable water.

Bacterial Biofilm Characterization and Microscopic Evaluation of the Antibacterial Properties of a Photocatalytic Coating Protecting Building Material

Use of photocatalytic paint-like coatings may be a way to protect building materials from microbial colonization. Numerous studies have shown the antimicrobial efficiency of TiO 2 photocatalysis on various microorganisms. However, few have focused on easy-to-apply solutions and on photocatalysis under low irradiance. This paper focuses on (a) the antibacterial properties of a semi-transparent coating formulated using TiO 2 particles and (b) the microscopic investigations of bacterial biofilm development on TiO 2 -coated building materials under accelerated growth conditions. Results showed significant antibacterial activity after few hours of testing. The efficiency seemed limited by the confinement of the TiO 2 particles inside the coating binder. However, a pre-irradiation with UV light can improve efficiency. In addition, a significant effect against the formation of a bacterial biofilm was also observed. The epifluorescence approach, in which fluorescence is produced by reflect rather than transmitted light, could be applied in further studies of microbial growth on coatings and building materials.

Formation and Characterization of Early Bacterial Biofilms on Different Wood Typologies Applied in Dairy Production.

The main hypothesis of this work was that Sicilian forestry resources are suitable for the production of equipment to be used in cheese making and indigenous milk lactic acid bacteria (LAB) are able to develop stable biofilms providing starter and nonstarter cultures necessary for curd fermentation and cheese ripening, respectively. Hence, the present work was carried out with deproteinized whey to evaluate LAB biofilm formation on different woods derived from tree species grown in Sicily. Microbiological and scanning electron microscopy analyses showed minimal differences in microbial levels and compositions for the neoformed biofilms. The specific investigation of Salmonella spp., Listeria monocytogenes, Escherichia coli, coagulase-positive staphylococci (CPS), and sulfite-reducing anaerobes did not generate any colony for all vats before and after bacterial adhesion. LAB populations dominated all vat surfaces. The highest levels (7.63 log CFU/cm2) were registered for thermophilic cocci. Different colonies were characterized physiologically, biochemically, and genetically (at strain and species levels). Six species within the genera Enterococcus, Lactobacillus, Lactococcus, and Streptococcus were identified. The species most frequently present were Lactobacillus fermentum and Lactococcus lactis LAB found on the surfaces of the wooden vats in this study showed interesting characteristics important for dairy manufacture. To thoroughly investigate the safety of the wooden vat, a test of artificial contamination on new Calabrian chestnut (control wood) vats was carried out. The results showed that LAB represent efficient barriers to the adhesion of the main dairy pathogens, probably due to their acidity and bacteriocin generation.IMPORTANCE This study highlights the importance of using wooden vats for traditional cheese production and provides evidence for the valorization of the Sicilian forest wood resources via the production of dairy equipment.

Raman and Surface-Enhanced Raman Scattering for Biofilm Characterization

Biofilms are a communal way of living for microorganisms in which microorganism cells are surrounded by extracellular polymeric substances (EPS). Most microorganisms can live in biofilm form. Since microorganisms are everywhere, understanding biofilm structure and composition is crucial for making the world a better place to live, not only for humans but also for other living creatures. Raman spectroscopy is a nondestructive technique and provides fingerprint information about an analyte of interest. Surface-enhanced Raman spectroscopy is a form of this technique and provides enhanced scattering of the analyte that is in close vicinity of a nanostructured noble metal surface such as silver or gold. In this review, the applications of both techniques and their combination with other biofilm analysis techniques for characterization of composition and structure of biofilms are discussed.