Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances, largely polysaccharides. Multiple types of extracellular polymeric substances can be produced by a single bacterial strain. The distinct polymer components of biofilms are known to provide chemical protection, but little is known about how distinct extracellular polysaccharides may also protect biofilms against mechanical stresses such as shear or phagocytic engulfment. Decades-long infections of Pseudomonas. aeruginosa biofilms in the lungs of cystic fibrosis patients are natural models for studies of biofilm fitness under pressure from antibiotics and the immune system. In cystic fibrosis infections, production of the extracellular polysaccharide alginate has long been known to increase with time and to chemically protect biofilms. More recently, it is being recognized that chronic cystic fibrosis infections also evolve to increase production of another extracellular polysaccharide, Psl; much less is known about Psl’s protective benefits to biofilms. We use oscillatory bulk rheology, on biofilms grown from longitudinal clinical isolates and from genetically-manipulated lab strains, to show that increased Psl stiffens biofilms and increases biofilm toughness, which is the energy cost to cause the biofilm to yield mechanically. Further, atomic force microscopy measurements reveal greater intercellular cohesion for higher Psl expression. Of the three types of extracellular polysaccharides produced by P. aeruginosa, only Psl increases the stiffness. Stiffening by Psl requires CdrA, a protein that binds to mannose groups on Psl and is a likely cross-linker for the Psl components of the biofilm matrix. We compare the elastic moduli of biofilms to the estimated stresses exerted by neutrophils during phagocytosis, and infer that increased Psl could confer a mechanical protection against phagocytic clearance.

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Biofilms are communities of bacteria embedded in a polymeric matrix which are found in infections and in environments outside the body. Breaking down the matrix renders biofilms more susceptible to physical disruption and to treatments such as antibiotics. Different species of bacteria, and different strains within the same species, produce different types of matrix polymers. This suggests that targeting specific polymers for disruption may be more effective than nonspecific approaches to disrupting biofilm matrixes. In this study, we treated Pseudomonas aeruginosa biofilms with enzymes that are specific to different matrix polymers. We measured the resulting alteration in biofilm mechanics using bulk rheology and changes in structure using electron microscopy. We find that, for biofilms grown in vitro, the effect of enzymatic treatment is greatest when the enzyme is specific to a dominant matrix polymer. Specifically matched enzymatic treatment tends to reduce yield strain and yield stress and increase the rate of biofilm drying, due to increased diffusivity as a result of network compromise. Electron micrographs qualitatively suggest that well-matched enzymatic treatments reduce long-range structure and shorten connecting network fibers. Previous work has shown that generic glycoside hydrolases can cause dispersal of bacteria from in vivo and ex vivo biofilms into a free-swimming state, and thereby make antibiotic treatment more effective. For biofilms grown in wounded mice, we find that well-matched treatments that result in the greatest mechanical compromise in vitro induce the least dispersal ex vivo. Moreover, we find that generic glycoside hydrolases have no measurable effect on the mechanics of biofilms grown in vitro, while previous work has shown them to be highly effective at inducing dispersal in vivo and ex vivo. This highlights the possibility that effective approaches to eradicating biofilms may depend strongly on the growth environment.

Biofilm infections can consist of bacterial aggregates that are an order of magnitude larger than neutrophils, phagocytic immune cells that densely surround aggregates but do not enter them. Because a neutrophil is too small to engulf the entire aggregate, it must be able to detach and engulf a few bacteria at a time if it is to use phagocytosis to clear the infection. Current research techniques do not provide a method for determining how the success of phagocytosis, here defined as the complete engulfment of a piece of foreign material, depends on the mechanical properties of a larger object from which the piece must be removed before being engulfed. This article presents a step toward such a method. By varying polymer concentration or cross-linking density, the elastic moduli of centimeter-sized gels are varied over the range that was previously measured for Pseudomonas aeruginosa biofilms grown from clinical bacterial isolates. Human neutrophils are isolated from blood freshly drawn from healthy adult volunteers, exposed to gel containing embedded beads for 1 h, and removed from the gel. The percentage of collected neutrophils that contain beads that had previously been within the gels is used to measure successful phagocytic engulfment. Both increased polymer concentration in agarose gels and increased cross-linking density in alginate gels are associated with a decreased success of phagocytic engulfment. Upon plotting the percentage of neutrophils showing successful engulfment as a function of the elastic modulus of the gel to which they were applied, it is found that data from both alginate and agarose gels collapse onto the same curve. This suggests that gel mechanics may be impacting the success of phagocytosis and demonstrates that this experiment is a step toward realizing methods for measuring how the mechanics of a large target, or a large structure in which smaller targets are embedded, impact the success of phagocytic engulfment.

Biofilms are communities of sessile microbes that are bound to each other by a matrix made of biopolymers and proteins. Spatial structure is present in biofilms on many lengthscales. These range from the nanometer scale of molecular motifs to the hundred-micron scale of multicellular aggregates. Spatial structure is a physical property that impacts the biology of biofilms in many ways. The molecular structure of matrix components controls their interaction with each other (thereby impacting biofilm mechanics) and with diffusing molecules such as antibiotics and immune factors (thereby impacting antibiotic tolerance and evasion of the immune system). The size and structure of multicellular aggregates, combined with microbial consumption of growth substrate, give rise to differentiated microenvironments with different patterns of metabolism and gene expression. Spatial association of more than one species can benefit one or both species, while distances between species can both determine and result from the transport of diffusible factors between species. Thus, a widespread theme in the biological importance of spatial structure in biofilms is the effect of structure on transport. We survey what is known about this and other effects of spatial structure in biofilms, from molecules up to multispecies ecosystems. We conclude with an overview of what experimental approaches have been developed to control spatial structure in biofilms and how these and other experiments can be complemented with computational work.

The increasing prevalence of carbon nanotubes (CNTs) as components of new functional materials has the unintended consequence of causing increases in CNT concentrations in aqueous environments. Aqueous systems are reservoirs for bacteria, including human and animal pathogens, that can form biofilms. At high concentrations, CNTs have been shown to display biocidal effects; however, at low concentrations, the interaction between CNTs and bacteria is more complicated, and antimicrobial action is highly dependent upon the properties of the CNTs in suspension. Here, impact of low concentrations of multiwalled CNTs (MWCNTs) on the biofilm-forming opportunistic human pathogen Pseudomonas aeruginosa is studied. Using phase contrast and confocal microscopy, flow cytometry, and antibiotic tolerance assays, it is found that sub-lethal concentrations (2 mg/L) of MWCNTs promote aggregation of P. aeruginosa into multicellular clusters. However, the antibiotic tolerance of these "young" bacterial-CNT aggregates is similar to that of CNT-free cultures. Overall, our results indicate that the co-occurrence of MWCNTs and P. aeruginosa in aqueous systems, which promotes the increased number and size of bacterial aggregates, could increase the dose to which humans or animals are exposed.

Methods for measuring the mechanics of surface-attached bacterial systems on a range of lengthscales enables enhanced understanding of their disease-causing properties.

12Biofilms are communities of bacteria embedded in an extracellular matrix of self-13 produced polymeric substances. This polymer matrix lends the bacteria protection against 14 a wide array of chemical and mechanical stresses that they may experience in their 15 environment, which might be a location in the human body in the case of a biofilm 16 infection, or a surface immersed in fluid in an industrial setting. Breaking down the matrix 17 network renders biofilms more susceptible to physical disruption and to treatments. 18 Different species of bacteria, and different strains within the same species, produce 19 different types of matrix polymersthis suggests that targeting specific polymers for 20 disruption may be more effective than non-specific approaches to disrupting biofilm 21 matrices. In this study, we treated Pseudomonas aeruginosa biofilms with enzymes that 22 are specific to different matrix polymers. We used bulk rheology to measure the resulting 23 approaches to eradicating biofilms in environmental or industrial settings may need to be 40 very different from effective treatments of infection. 41 42 and the importance of a specific polymer type varies with biofilm-forming species and 47 strain. For biofilm infections, the matrix protects the inhabitant bacteria chemically by 48 inhibiting the diffusion of antibiotics into the biofilm and by binding to antibacterial 49 chemicals produced by the host immune response. 1-3 Furthermore, the mechanical 50 integrity and structure of the bacterial biofilm conferred by the matrix gives rise to stable 51 microenvironments that contribute to phenotypic antibiotic tolerance. 4-5 Thus, both the 52 chemical and the structural properties of the biofilm matrix contribute to bacterial 53 tolerance of harsh environments. 54Many common interventions in P. aeruginosa infections, like antibiotic treatment, 55 are far more successful if the bacteria are in a planktonic state rather than in a biofilm. 6