Zwitterionic surface chemistry enhances detachment of bacteria under shear.

The ubiquitous nature of microorganisms, especially of biofilm-forming bacteria, makes biofouling a prevalent challenge in many settings, including medical and industrial environments immersed in liquid and subjected to shear forces. Recent studies have shown that zwitterionic groups are effective in suppressing bacteria and protein adhesion as well as biofilm growth. However, the effect of zwitterionic groups on the removal of surface-bound bacteria has not been extensively studied. Here we present a microfluidic approach to evaluate the effectiveness in facilitating bacteria detachment by shear of an antifouling surface treatment using (3-(dimethyl;(3-trimethoxysilyl)propyl)ammonia propane-1-sulfonate), a sulfobetaine silane (SBS). Control studies show that SBS-functionalized surfaces greatly increase protein (bovine serum albumin) removal upon rinsing. On the same surfaces, enhanced bacteria (Pseudomonas aeruginosa) removal is observed under shear. To quantify this enhancement a microfluidic shear device is employed to investigate how SBS-functionalized surfaces promote bacteria detachment under shear. By using a microfluidic channel with five shear zones, we compare the removal of bacteria from zwitterionic and glass surfaces under different shear rates. At times of 15 min, 30 min, and 60 min, bacteria adhesion on SBS-functionalized surfaces is reduced relative to the control surface (glass) under quiescent conditions. However, surface-associated bacteria on the SBS-functionalized glass and control show similar percentages of live cells, suggesting minimal intrinsic biocidal effect from the SBS-functionalized surface. Notably, when exposed to shear rates ranging from 104 to 105 s-1, significantly fewer bacteria remain on the SBS-functionalized surfaces. These results demonstrate the potential of zwitterionic sulfobetaine as effective antifouling coatings that facilitate the removal of bacteria under shear.

Osmotically-induced tension and the binding of N-BAR protein to lipid vesicles.

The binding affinity of a curvature-sensing protein domain (N-BAR) is measured as a function of applied osmotic stress while the membrane curvature is nearly constant. Varying the osmotic stress allows us to control membrane tension, which provides a probe of the mechanism of binding. We study the N-BAR domain of the Drosophila amphiphysin and monitor its binding on 50 nm-radius vesicles composed of 90 mol% DOPC and 10 mol% PIP. We find that the bound fraction of N-BAR is enhanced by a factor of approximately 6.5 when the tension increases from zero to 2.6 mN m(-1). This tension-induced response can be explained by the hydrophobic insertion mechanism. From the data we extract a hydrophobic domain area that is consistent with known structure. These results indicate that membrane stress and strain could play a major role in the previously reported curvature-affinity of N-BAR.

Single-cell control of initial spatial structure in biofilm development using laser trapping.

Biofilms are sessile communities of microbes that are spatially structured by an embedding matrix. Biofilm infections are notoriously intractable. This arises, in part, from changes in the bacterial phenotype that result from spatial structure. Understanding these interactions requires methods to control the spatial structure of biofilms. We present a method for growing biofilms from initiating cells whose positions are controlled with single-cell precision using laser trapping. The native growth, motility, and surface adhesion of positioned microbes are preserved, as we show for model organisms Pseudomonas aeruginosa and Staphylococcus aureus. We demonstrate that laser-trapping and placing bacteria on surfaces can reveal the effects of spatial structure on bacterial growth in early biofilm development.

Change of line tension in phase-separated vesicles upon protein binding.

We measured the effect of a model membrane-binding protein on line tension and morphology of phase-separated lipid-bilayer vesicles. We studied giant unilamellar vesicles composed of a cholesterol/dioleoylphosphatidylcholine/palmitoylsphingomyelin mixture and a controlled mole fraction of a Ni-chelating lipid. These vesicles exhibited two coexisting fluid-phase domains at room temperature. Owing to the line tension, σ, between the two phases, the boundary between them was pulled like a purse string so that the smaller domain formed a bud. While observing the vesicles in a microscope, histidine-tagged green fluorescent protein was added, which bound to the Ni-chelating lipid. As protein bound, the vesicle shape changed and the length of the phase boundary increased. The change in morphology was attributed to a reduction of σ between the two phases because of preferential accumulation of histidine-tagged green fluorescent protein-Ni-chelating lipid clusters at the domain boundary. Greater reductions of σ were found in samples with higher concentrations of Ni-chelating lipid; this trend provided an estimate of the binding energy at the boundary, approximately k(B)T. The results show how domain boundaries can lead to an accumulation of membrane-binding proteins at their boundaries and, in turn, how proteins can alter line tension and vesicle morphology.

The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms.

Extracellular polysaccharides are compounds secreted by microorganisms into the surrounding environment, and they are important for surface attachment and maintaining structural integrity within biofilms. The social nature of many extracellular polysaccharides remains unclear, and it has been suggested that they could function as either cooperative public goods or as traits that provide a competitive advantage. Here, we empirically tested the cooperative nature of the PSL polysaccharide, which is crucial for the formation of biofilms in Pseudomonas aeruginosa We show that (i) PSL is not metabolically costly to produce; (ii) PSL provides population-level benefits in biofilms, for both growth and antibiotic tolerance; (iii) the benefits of PSL production are social and are shared with other cells; (iv) the benefits of PSL production appear to be preferentially directed toward cells which produce PSL; (v) cells which do not produce PSL are unable to successfully exploit cells which produce PSL. Taken together, this suggests that PSL is a social but relatively nonexploitable trait and that growth within biofilms selects for PSL-producing strains, even when multiple strains are on a patch (low relatedness at the patch level).IMPORTANCE Many studies have shown that bacterial traits, such as siderophores and quorum sensing, are social in nature. This has led to an impression that secreted traits act as public goods, which are costly to produce but benefit both the producing cell and its surrounding neighbors. Theories and subsequent experiments have shown that such traits are exploitable by asocial cheats, but we show here that this does not always hold true. We demonstrate that the Pseudomonas aeruginosa exopolysaccharide PSL provides social benefits to populations but that it is nonexploitable, because most of the fitness benefits accrue to PSL-producing cells. Our work builds on an increasing body of work showing that secreted traits can have both private and public benefits to cells.

Shaping the Growth Behaviour of Biofilms Initiated from Bacterial Aggregates.

Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell aggregates. Here, we use agent-based computer simulations to investigate the role of pre-formed aggregates in biofilm development. Focusing on the initial shape the aggregate forms on the surface, we find that the degree of spreading of an aggregate on a surface can play an important role in determining its eventual fate during biofilm development. Specifically, initially spread aggregates perform better when competition with surrounding unaggregated bacterial cells is low, while initially rounded aggregates perform better when competition with surrounding unaggregated cells is high. These contrasting outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities.

Role of Multicellular Aggregates in Biofilm Formation.

UNLABELLED: In traditional models ofin vitrobiofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development ofPseudomonas aeruginosabiofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. IMPORTANCE: During the past decades, there has been a consensus around the model of development of a biofilm, involving attachment of single planktonic bacterial cells to a surface and the subsequent development of a mature biofilm. This study presents results that call for a modification of this rigorous model. We show how free floating biofilm aggregates can have a profound local effect on biofilm development when attaching to a surface. Our findings show that an aggregate landing on a surface will eventually outcompete the biofilm population arising from single cells attached around the aggregate and dominate the local biofilm development. These results point to a regime where preformed biofilm aggregates may have a fitness advantage over planktonic cells when it comes to accessing nutrients. Our findings add to the increasingly prominent comprehension that biofilm lifestyle is the default for bacteria and that planktonic single cells may be only a transition state at the most.