Assaying How Phagocytic Success Depends on the Elasticity of a Large Target Structure.

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.

Evolutionary adaptations of biofilms infecting cystic fibrosis lungs promote mechanical toughness by adjusting polysaccharide production.

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.

Effects of temperature and pH on the persistence of avian paramyxovirus-1 in water.

We evaluated the duration of infectivity of two isolates of avian paramyxovirus-1 using a model distilled water system. Both viruses remained infective for extended periods with 1-log10 reduction times ranging from 370 (4 C) to 6 (37 C) days. Minimal effects related to pH (5.4-8.4 at 28 C) were observed.