Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm−2) or 5.34 ± 0.33 log (gu cm−2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to 300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation

From a thin film model for passive suspensions towards the description of osmotic biofilm spreading

Biofilms are ubiquitous macro-colonies of bacteria that develop at various interfaces (solid-liquid, solid-gas or liquid-gas). The formation of biofilms starts with the attachment of individual bacteria to an interface, where they proliferate and produce a slimy polymeric matrix - two processes that result in colony growth and spreading. Recent experiments on the growth of biofilms on agar substrates under air have shown that for certain bacterial strains, the production of the extracellular matrix and the resulting osmotic influx of nutrient-rich water from the agar into the biofilm are more crucial for the spreading behaviour of a biofilm than the motility of individual bacteria. We present a model which describes the biofilm evolution and the advancing biofilm edge for this spreading mechanism. The model is based on a gradient dynamics formulation for thin films of biologically passive liquid mixtures and suspensions, supplemented by bioactive processes which play a decisive role in the osmotic spreading of biofilms. It explicitly includes the wetting properties of the biofilm on the agar substrate via a disjoining pressure and can therefore give insight into the interplay between passive surface forces and bioactive growth processes

Effect of CSLM imaging rate on biofilms of P. aeruginosa and S. aureus

Biofilms are sessile communities of bacteria that can be found in an wide range of environments. Their inhabitants are phenotypically distinct from plank- tonic bacteria and are capable of forming complex, three-dimensional structures. Biofilms are studied using confocal scanning laser microscopy, or CSLM. This technique uses lasers and Novel Fluorescent Proteins (NFPs) to measure growth and structure formation of single- and multi-species biofilms in situ in three dimensions. We investigate the effects of slow and fast rates of image acquisition on mono- and co-cultures of biofilm forming bacteria: Pseudomonas aeruginosa and Staphylococcus aureus. After calculating growth rates and lag times, we find that fast scanning rates reduce the growth rate of P. aeruginosa in co-culture. Additionally, co-culture speeds up P. aeruginosa growth relative to monoculture when imaged at a slow rate, and fast scanning reverts co-culture growth to monoculture-like behavior. Additionally, a significant lag time is observed for P. aeruginosa grown in co-culture. The observed influence of confocal imaging rate on population dynamics should be considered in future studies to ensure accurate measurement of bacterial phenomena.Physic

Unraveling How Candida albicans Forms Sexual Biofilms.

Biofilms, structured and densely packed communities of microbial cells attached to surfaces, are considered to be the natural growth state for a vast majority of microorganisms. The ability to form biofilms is an important virulence factor for most pathogens, including the opportunistic human fungal pathogen Candida albicans. C. albicans is one of the most prevalent fungal species of the human microbiota that asymptomatically colonizes healthy individuals. However, C. albicans can also cause severe and life-threatening infections when host conditions permit (e.g., through alterations in the host immune system, pH, and resident microbiota). Like many other pathogens, this ability to cause infections depends, in part, on the ability to form biofilms. Once formed, C. albicans biofilms are often resistant to antifungal agents and the host immune response, and can act as reservoirs to maintain persistent infections as well as to seed new infections in a host. The majority of C. albicans clinical isolates are heterozygous (a/α) at the mating type-like (MTL) locus, which defines Candida mating types, and are capable of forming robust biofilms when cultured in vitro. These "conventional" biofilms, formed by MTL-heterozygous (a/α) cells, have been the primary focus of C. albicans biofilm research to date. Recent work in the field, however, has uncovered novel mechanisms through which biofilms are generated by C. albicans cells that are homozygous or hemizygous (a/a, a/Δ, α/α, or α/Δ) at the MTL locus. In these studies, the addition of pheromones of the opposite mating type can induce the formation of specialized "sexual" biofilms, either through the addition of synthetic peptide pheromones to the culture, or in response to co-culturing of cells of the opposite mating types. Although sexual biofilms are generally less robust than conventional biofilms, they could serve as a protective niche to support genetic exchange between mating-competent cells, and thus may represent an adaptive mechanism to increase population diversity in dynamic environments. Although conventional and sexual biofilms appear functionally distinct, both types of biofilms are structurally similar, containing yeast, pseudohyphal, and hyphal cells surrounded by an extracellular matrix. Despite their structural similarities, conventional and sexual biofilms appear to be governed by distinct transcriptional networks and signaling pathways, suggesting that they may be adapted for, and responsive to, distinct environmental conditions. Here we review sexual biofilms and compare and contrast them to conventional biofilms of C. albicans

Growth limiting conditions and denitrification govern extent and frequency of volume detachment of biofilms

This study aims at evaluating the mechanisms of biofilm detachment with regard of the physical properties of the biofilm. Biofilms were developed in Couette–Taylor reactor under controlled hydrodynamic conditions and under different environmental growth conditions. Five different conditions were tested and lead to the formation of two aerobic heterotrophic biofilms (aeHB1 and aeHB2), a mixed autotrophic and heterotrophic biofilm (MAHB) and two anoxic heterotrophic biofilms (anHB1 and anHB2). Biofilm detachment was evaluated by monitoring the size of the detached particles (using light-scattering) as well as the biofilm physical properties (using CCD camera and image analysis). Results indicate that volume erosion of large biofilm particles with size ranging from 50 to 500 lm dominated the biomass loss for all biofilms. Surface erosion of small particles with size lower than 20 lm dominates biofilm detachment in number. The extent of the volume detachment events was governed by the size of the biofilm surface heterogeneities (i.e., the absolute biofilm roughness) but never impacted more than 80% of the mean biofilm thickness due to the highly cohesive basal layer. Anoxic biofilms were smoother and thinner than aerobic biofilms and thus associated with the detachment of smaller particles. Our results contradict the simplifying assumption of surface detachment that is considered in many biofilm models and suggest that discrete volume events should be considered

Extracellular electron transfer mechanism in Shewanella loihica PV- 4 biofilms formed at indium tin oxide and graphite electrodes

Electroactive biofilms are capable of extracellular electron transfer to insoluble metal oxides and electrodes; such biofilms are relevant to biogeochemistry, bioremediation, and bioelectricity production. We investigated the extracellular electron transfer mechanisms in Shewanella loihica PV-4 viable biofilms grown at indium tin oxide (ITO) and graphite electrodes in potentiostat-controlled electrochemical cells poised at 0.2 V vs. Ag/AgCl. Chronoamperometry and confocal microscopy showed higher biofilm growth at graphite compared to the ITO electrode. Cyclic voltammetry, differential pulse voltammetry, along with fluorescence spectroscopy showed that direct electron transfer through outer membrane c type cytochromes (Omcs) prevailed at the biofilm/ITO interface, while biofilms formed at graphite electrode reduced the electrode also via secreted redox mediators, such as flavins and quinones. The biofilm age does not affect the prevalent transfer mechanism at ITO electrodes. On the other hand, secreted redox mediators accumulated at biofilm/graphite interface, thus increasing mediated electron transfer as the biofilm grows over five days. Our results showed that the electrode material determined the prevalent electron transfer mechanism and the dynamic of secreted redox mediators in S. loihica PV-4 biofilms. These observations have implications for the optimization of biofilm-based electrochemical systems, such as biosensors and microbial fuel cells

Turbulence accelerates the growth of drinking water biofilms

Biofilms are found at the inner surfaces of drinking water pipes and, therefore, it is essential to understand biofilm processes to control their formation. Hydrodynamics play a crucial role in shaping biofilms. Thus, knowing how biofilms form, develop and disperse under different flow conditions is critical in the successful management of these systems. Here, the development of biofilms after 4 weeks, the initial formation of biofilms within 10 h and finally, the response of already established biofilms within 24-h intervals in which the flow regime was changed, were studied using a rotating annular reactor under three different flow regimes: turbulent, transition and laminar. Using fluorescence microscopy, information about the number of microcolonies on the reactor slides, the surface area of biofilms and of extracellular polymeric substances and the biofilm structures was acquired. Gravimetric measurements were conducted to characterise the thickness and density of biofilms, and spatial statistics were used to characterise the heterogeneity and spatial correlation of biofilm structures. Contrary to the prevailing view, it was shown that turbulent flow did not correlate with a reduction in biofilms; turbulence was found to enhance both the initial formation and the development of biofilms on the accessible surfaces. Additionally, after 24-h changes of the flow regime it was indicated that biofilms responded to the quick changes of the flow regime. Overall, this work suggests that different flow conditions can cause substantial changes in biofilm morphology and growth and specifically that turbulent flow can accelerate biofilm growth in drinking water

Differential growth of wrinkled biofilms

Biofilms are antibiotic-resistant bacterial aggregates that grow on moist surfaces and can trigger hospital-acquired infections. They provide a classical example in biology where the dynamics of cellular communities may be observed and studied. Gene expression regulates cell division and differentiation, which affect the biofilm architecture. Mechanical and chemical processes shape the resulting structure. We gain insight into the interplay between cellular and mechanical processes during biofilm development on air-agar interfaces by means of a hybrid model. Cellular behavior is governed by stochastic rules informed by a cascade of concentration fields for nutrients, waste and autoinducers. Cellular differentiation and death alter the structure and the mechanical properties of the biofilm, which is deformed according to Foppl-Von Karman equations informed by cellular processes and the interaction with the substratum. Stiffness gradients due to growth and swelling produce wrinkle branching. We are able to reproduce wrinkled structures often formed by biofilms on air-agar interfaces, as well as spatial distributions of differentiated cells commonly observed with B. subtilis.Comment: 19 pages, 13 figure

Biofilm dynamics characterization using a novel DO-MEA sensor: mass transport and biokinetics

Biodegradation process modeling is an essential tool for the optimization of biotechnologies related to gaseous pollutant treatment. In these technologies, the predominant role of biofilm, particularly under conditions of no mass transfer limitations, results in a need to determine what processes are occurring within the same. By measuring the interior of the biofilms, an increased knowledge of mass transport and biodegradation processes may be attained. This information is useful in order to develop more reliable models that take biofilm heterogeneity into account. In this study, a new methodology, based on a novel dissolved oxygen (DO) and mass transport microelectronic array (MEA) sensor, is presented in order to characterize a biofilm. Utilizing the MEA sensor, designed to obtain DO and diffusivity profiles with a single measurement, it was possible to obtain distributions of oxygen diffusivity and biokinetic parameters along a biofilm grown in a flat plate bioreactor (FPB). The results obtained for oxygen diffusivity, estimated from oxygenation profiles and direct measurements, revealed that changes in its distribution were reduced when increasing the liquid flow rate. It was also possible to observe the effect of biofilm heterogeneity through biokinetic parameters, estimated using the DO profiles. Biokinetic parameters, including maximum specific growth rate, the Monod half-saturation coefficient of oxygen, and the maintenance coefficient for oxygen which showed a marked variation across the biofilm, suggest that a tool that considers the heterogeneity of biofilms is essential for the optimization of biotechnologies.Peer ReviewedPostprint (published version