Water in bacterial biofilms:pores and channels, storage and transport functions

Bacterial biofilms occur in many natural and industrial environments. Besides bacteria, biofilms comprise over 70 wt% water. Water in biofilms occurs as bound- or free-water. Bound-water is adsorbed to bacterial surfaces or biofilm (matrix) structures and possesses different Infra-red and Nuclear-Magnetic-Resonance signatures than free-water. Bound-water is different from intra-cellularly confined-water or water confined within biofilm structures and bacteria are actively involved in building water-filled structures by bacterial swimmers, dispersion or lytic self-sacrifice. Water-filled structures can be transient due to blocking, resulting from bacterial growth, compression or additional matrix formation and are generally referred to as "channels and pores." Channels and pores can be distinguished based on mechanism of formation, function and dimension. Channels allow transport of nutrients, waste-products, signalling molecules and antibiotics through a biofilm provided the cargo does not adsorb to channel walls and channels have a large length/width ratio. Pores serve a storage function for nutrients and dilute waste-products or antimicrobials and thus should have a length/width ratio close to unity. The understanding provided here on the role of water in biofilms, can be employed to artificially engineer by-pass channels or additional pores in industrial and environmental biofilms to increase production yields or enhance antimicrobial penetration in infectious biofilms

E coli Accumulation behind an Obstacle

This paper describes our findings regarding the accumulation of motile bacte-ria at the rear of a confined obstacle and the physical description of the me-chanisms at play. We found that the modification of flow due to the presence of the obstacle produces vorticity that favor the diffusion of bacteria towards the downstream stagnation point. By testing different flow rates, we deter-mined the range in which bacteria accumulate. More interestingly, we observe that hydrodynamic interaction between the bacteria and the top and bottom surface of the microfluidic chip maintain the bacteria in the region where the flow velocity is lower than their own velocity. In the case of non-motile bacte-ria, this effect is not observed because bacteria follow the streamlines as pas-sive tracers do.Fil: Miño, Gastón Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Entre Ríos. Universidad Nacional de Entre Ríos. Centro de Investigaciones y Transferencia de Entre Ríos; ArgentinaFil: Baabour, Magali Denise. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Física. Grupo de Medios Porosos; ArgentinaFil: Chertcoff, Ricardo Héctor. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Física. Grupo de Medios Porosos; ArgentinaFil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Clément, Eric. Centre National de la Recherche Scientifique; FranciaFil: Auradou, Harold. Universite de Paris Xi. Laboratoire Automatiques et Systeme Thermiques; FranciaFil: Ippolito, Irene Paula. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Física. Grupo de Medios Porosos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Interfacial Interactions and Dynamic Adhesion of Synthetic and Living Colloids in Flow

This thesis focuses on the interactions between flowing particles and a surface, where hydrodynamics couples with chemical interactions in order to modify the way they come into play. First this thesis shows how electrostatic chemical heterogeneities on a flowing particle affect the interactions with a wall, using a highly tunable electrostatically heterogenous system produced by adsorbing small amounts of cationic polyelectrolytes onto silica particles in suspension and studying their behavior in flow over the fixed surface. By comparing this behavior to a system with equivalent chemical heterogeneity on a channel wall it was shown that the rotation of a particle will produce a lower attempt frequency, resulting in chemical heterogeneity being less effective on a flowing particle then on a fixed surface. This establishes the importance of hydrodynamics in the chemical interactions of flowing colloids. Next this work shows how swimming of Escherichia coli increases both the frequency of bacteria encountering a surface and the durations of the resulting engagements, in an unconfined flowing environment, due to hydrodynamic interactions between bacteria and surfaces. This swimming effect was decoupled from the effect of flagella interactions. It was found that the presence of flagella, when not active, producing steric kicks, increasing the escape frequency and as a result reducing surface engagements length. Expansion of this work showed that the effects caused by morphological differences between bacteria strains can be significantly reduced by altering the mechanical properties of a surface coating. Finally, this thesis shows that rod shaped particles are able to diffuse through a concentration boundary layer and adhere to surfaces at a rate faster than possible with spherical particles, due to hydrodynamic interactions between the non-spherical particles and the surrounding fluid. Current literature contains a wide variety of experiential results and mathematical predictions for the behavior of rods in flow but the lack of consistent systems with well-studied spherical controls has led to many discrepancies in their results. This thesis addresses these apparent discrepancies using model rod-shaped silica particle suspensions and spherical controls. Overall, this thesis probes the effects of three hydrodynamic effects on interactions between particles and surfaces in the presence of flow

Microfluidics Expanding the Frontiers of Microbial Ecology

Microfluidics has significantly contributed to the expansion of the frontiers of microbial ecology over the past decade by allowing researchers to observe the behaviors of microbes in highly controlled microenvironments, across scales from a single cell to mixed communities. Spatially and temporally varying distributions of organisms and chemical cues that mimic natural microbial habitats can now be established by exploiting physics at the micrometer scale and by incorporating structures with specific geometries and materials. In this article, we review applications of microfluidics that have resulted in insightful discoveries on fundamental aspects of microbial life, ranging from growth and sensing to cell-cell interactions and population dynamics. We anticipate that this flexible multidisciplinary technology will continue to facilitate discoveries regarding the ecology of microorganisms and help uncover strategies to control microbial processes such as biofilm formation and antibiotic resistance.National Science Foundation (U.S.) (Grant OCE-0744641-CAREER)National Science Foundation (U.S.) (Grant IOS-1120200)National Science Foundation (U.S.) (Grant CBET-1066566)National Science Foundation (U.S.) (Grant CBET-0966000)National Institutes of Health (U.S.) (NIH grant 1R01GM100473-0)Human Frontier Science Program (Strasbourg, France)Human Frontier Science Program (Strasbourg, France) (award RGY0089)Gordon and Betty Moore Foundation (Microbial Initiative Investigator Award

Effect of motility on the transport of bacteria populations through a porous medium

The role of activity on the hydrodynamic dispersion of bacteria in a model porous medium is studied by tracking thousands of bacteria in a microfluidic chip containing randomly placed pillars. We first evaluate the spreading dynamics of two populations of motile and nonmotile bacteria injected at different flow rates. In both cases, we observe that the mean and the variance of the distances covered by the bacteria vary linearly with time and flow velocity, a result qualitatively consistent with the standard geometric dispersion picture. However, quantitatively, the motile bacteria display a systematic retardation effect when compared to the nonmotile ones. Furthermore, the shape of the traveled distance distribution in the flow direction differs significantly for both the motile and the nonmotile strains, hence probing a markedly different exploration process. For the nonmotile bacteria, the distribution is Gaussian, whereas for the motile ones, the distribution displays a positive skewness and spreads exponentially downstream akin to a Γ distribution. The detailed microscopic study of the trajectories reveals two salient effects characterizing the exploration process of motile bacteria: (1) the emergence of an "active" retention effect due to an extended exploration of the pore surfaces and (2) an enhanced spreading at the forefront due to the transport of bacteria along "fast tracks" where they acquire a velocity larger than the local flow velocity. We finally discuss the practical applications of these effects on the large-scale macroscopic transfer and contamination processes caused by microbes in natural environments.Fil: Creppy, Adama. Centre National de la Recherche Scientifique; FranciaFil: Clément, Eric. Université Paris Diderot - Paris 7; FranciaFil: Douarche, Carine. Centre National de la Recherche Scientifique; FranciaFil: D'angelo, María Verónica. Universidad de Buenos Aires. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Auradou, Harold. Centre National de la Recherche Scientifique; Franci

Pili: the microbes' Swiss army knifes

Surface attachment is the crucial first step for a single cell transitions from a planktonic to a surface associated state, which can lead to the development of multicellular communities called biofilms. Microbes extensively use pili for initial surface attachment. Pili are filamentous appendages that extend into the extracellular environment and can attach to a wide range of surfaces. This Thesis contributes to the understanding of how pili work and how bacteria transition from a planktonic to a surface bound life style. This will aid future development in creating new ways to prevent bacterial attachment and biofilm formation and thereby avoid the necessity for the removal of fully developed biofilms which often requires harsh physical and chemical treatments which can be impractical in a biomedical context. We used single cell studies, microfluidic methods and quantitative computational analysis to study in detail the mechanism of pili-mediated attachment in Caulobacter crescentus and Pseudomonas aeruginosa. In C. crescentus we confirm the recently described ability of pili to retract, which was previously considered not possible for this type of pili. We characterized this functionality in greater detail and our results highlight the importance of pili in reorienting cells and bringing the cell body closer to surfaces, whereby cells can promote long term attachment by secreting a glue-like substance called holdfast. We also investigated the role of the second messenger c-di-GMP during pilimediated cell attachment and biofilm formation. We show a novel role for c-di-GMP in directly regulating elongation and retraction of pili in C. crescentus and P. aeruginosa. In P aeruginosa a novel c-di-GMP effector, FimW, regulates surface attachment and walking behaviour, and how its asymmetric distribution drives surface colonization. In C. crescentus we show that c-di-GMP positively regulates attachment. We manipulated a key component of the secretion machinery, HfsK, and show that c-di-GMP not only regulates the timing of holdfast synthesis, but also its cohesion and adhesion properties. Lastly, we report a novel protein, PdeL, which is both a phosphodiesterase and a transcriptional factor that regulates the expression of biofilm related genes in Escherichia coli. In the appendixes we describe in detail the process for creating microfluidic devices, extensively used in the studies described in this thesis. Moreover, we include a manual for the use of WHISIT, a custom-made software program for the analysis of bacterial fluorescent signals in an automated and quantitative approach

Microbes in porous environments: From active interactions to emergent feedback

Microbes thrive in diverse porous environments -- from soil and riverbeds to human lungs and cancer tissues -- spanning multiple scales and conditions. Short- to long-term fluctuations in local factors induce spatio-temporal heterogeneities, often leading to physiologically stressful settings. How microbes respond and adapt to such biophysical constraints is an active field of research where considerable insight has been gained over the last decade and a half. With a focus on bacteria, here we review recent advances in microbial self-organization and dispersal in inorganic and organic porous settings, highlighting the role of active interactions and feedback which mediate their survival and fitness. We conclude by discussing open questions and opportunities for leveraging integrative cross-disciplinary approaches to advance our understanding of the biophysical strategies that microbes employ -- at both species and community scales -- to make porous settings habitable. Active and responsive behaviour is key to microbial survival in porous environments, with far-reaching ramifications for developing strategies to mitigate anthropogenic impacts, innovate subsurface storage solutions, and predict future ecological scenarios imposed by current climatic changes.R-AGR-3401 - A17/MS/11572821/MBRACE - part UL (15/05/2018 - 14/05/2023) - SENGUPTA AnupamR-AGR-3692 - C19/MS/13719464/TOPOFLUME (01/09/2020 - 31/08/2023) - SENGUPTA Anupa

Biomechanical Analysis of Infectious Biofilms.

The removal of infectious biofilms from tissues or implanted devices and their transmission through fluid transport systems depends in part of the mechanical properties of their polymeric matrix. Linking the various physical and chemical microscopic interactions to macroscopic deformation and failure modes promises to unveil design principles for novel therapeutic strategies targeting biofilm eradication, and provide a predictive capability to accelerate the development of devices, water lines, etc, that minimise microbial dispersal. Here, our current understanding of biofilm mechanics is appraised from the perspective of biophysics , with an emphasis on constitutive modelling that has been highly successful in soft matter. Fitting rheometric data to viscoelastic models has quantified linear and nonlinear stress relaxation mechanisms, how they vary between species and environments, and how candidate chemical treatments alter the mechanical response. The rich interplay between growth, mechanics and hydrodynamics is just becoming amenable to computational modelling and promises to provide unprecedented characterisation of infectious biofilms in their native state

Microbes in beach sands : integrating environment, ecology and public health

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Reviews in Environmental Science and Bio/Technology 13 (2014): 329-368, doi:10.1007/s11157-014-9340-8.Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.2015-05-0