Physiological concentrations of calcium interact with alginate and extracellular DNA in the matrices of Pseudomonas aeruginosa biofilms to impede phagocytosis by neutrophils.

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase and/or DNAse I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in non-trivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.

Incorporation of collagen into Pseudomonas aeruginosa and Staphylococcus aureus biofilms impedes phagocytosis by neutrophils.

Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances (EPS). Matrix components can be produced by biofilm organisms and can also originate from the environment and then be incorporated into the biofilm. For example, we have recently shown that collagen, a host-produced protein that is abundant in many different infection sites, can be taken up into the biofilm matrix, altering biofilm mechanics. The biofilm matrix protects bacteria from clearance by the immune system, and some of that protection likely arises from the mechanical properties of the biofilm. Pseudomonas aeruginosa and Staphylococcus aureus are common human pathogens notable for forming biofilm infections in anatomical sites rich in collagen. Here, we show that the incorporation of Type I collagen into P. aeruginosa and S. aureus biofilms significantly hinders phagocytosis of biofilm bacteria by human neutrophils. However, enzymatic treatment with collagenase, which breaks down collagen, can partly or entirely negate the protective effect of collagen and restore the ability of neutrophils to engulf biofilm bacteria. From these findings, we suggest that enzymatic degradation of host materials may be a potential way to compromise biofilm infections and enhance the efficacy of the host immune response without promoting antibiotic resistance. Such an approach might be beneficial both in cases where the infecting species is known and also in cases wherein biofilm components are not readily known, such as multispecies infections or infections by unknown species.

Physiological Concentrations of Calcium Interact with Alginate and Extracellular DNA in the Matrices of Pseudomonas aeruginosa Biofilms to Impede Phagocytosis by Neutrophils.

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa (P. aeruginosa) is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study, we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro, and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple-particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase or DNase I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in nontrivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.

High-throughput assays show the timescale for phagocytic success depends on the target toughness.

Phagocytic immune cells can clear pathogens from the body by engulfing them. Bacterial biofilms are communities of bacteria that are bound together in a matrix that gives biofilms viscoelastic mechanical properties that do not exist for free-swimming bacteria. Since a neutrophil is too small to engulf an entire biofilm, it must be able to detach and engulf a few bacteria at a time if it is to use phagocytosis to clear the infection. We recently found a negative correlation between the target elasticity and phagocytic success. That earlier work used time-consuming, manual analysis of micrographs of neutrophils and fluorescent beads. Here, we introduce and validate flow cytometry as a fast and high-throughput technique that increases the number of neutrophils analyzed per experiment by two orders of magnitude, while also reducing the time required to do so from hours to minutes. We also introduce the use of polyacrylamide gels in our assay for engulfment success. The tunability of polyacrylamide gels expands the mechanical parameter space we can study, and we find that high toughness and yield strain, even with low elasticity, also impact the phagocytic success as well as the timescale thereof. For stiff gels with low-yield strain, and consequent low toughness, phagocytic success is nearly four times greater when neutrophils are incubated with gels for 6 h than after only 1 h of incubation. In contrast, for soft gels with high-yield strain and consequent high toughness, successful engulfment is much less time-sensitive, increasing by less than a factor of two from 1 to 6 h incubation.

Specific Disruption of Established Pseudomonas aeruginosa Biofilms Using Polymer-Attacking Enzymes.

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.

A vision for doctoral research training in health behavior: a position paper from the American Academy of Health Behavior.

OBJECTIVE: To establish and disseminate the position of the American Academy of Health Behavior (The Academy) on doctoral research training. METHODS: A collaborative process involving the Work Group on Doctoral Research Training with input from The Academy membership led to the development of the guidelines described herein. RESULTS: A set of guidelines is provided that describe the process of learning to be a scholar/researcher and the outcomes of learning the practice of health behavior research. CONCLUSIONS: The doctoral students who are to become the stewards of our field should be prepared to engage in scholarship that creates new knowledge, uses research to transform practice, and effectively communicates research findings.