Specific Disruption of Established P. aeruginosa Biofilms Using Polymer-Attacking Enzymes
Preprint posted on April 05, 2019 https://www.biorxiv.org/content/10.1101/598979v1
Context and background: Biofilms are communities of microbes that live in a self-produced matrix. The matrix is responsible for the mechanical stability of biofilms, and its structure is determined mainly by matrix polymers and its numerous components (polysaccharides, proteins, nucleic acids, lipids); these play different roles in enabling adhesion to surface for biofilm attachment, as well as formation of a 3D intricate well connected structure, encasing the bacteria. The matrix also protects bacteria by preventing diffusion of antibiotics into the biofilm, pH changes, diffusion of other small molecules, and also protects from mechanical stresses (see  for a review on the biofilm matrix). The exact structure, role of a specific polymer type and its amount in the matrix vary with strains. Targeting the polymers within a matrix, which leads to weakening of the matrix network, shows promise as a biofilm reduction and removal therapy . Weakening of the biofilm and dispersal of bacteria from the biofilm into a planktonic state can lead to effective treatment of infections.
This study takes such an approach to study the effects of enzymes attacking specific polymers of Pseudomonas aeruginosa biofilms. P. aeruginosa is a robust biofilm forming pathogen, implicated in numerous infections, and some important polymers in its matrix are known (alginate, Pel and Psl).
Experimental setup: The study used 3 variants of the PAO1 strain – Psl+ (ΔwspF Δpel (overexpresses Psl)), Pel+ (ΔwspF Δpsl (overexpresses Pel)), and Alg+ (ΔmucA (overexpresses alginate)). For in vitro studies, biofilms were grown on LB agar plates and treated with enzymes, and biofilm mechanics were measured by rheology Enzyme treatments were tested in vivo against biofilms in a mouse model of chronic wound infection.
Specific Enzyme Treatment affects mechanical properties of biofilms – The authors initially tested if mechanical properties of P. aeruginosa biofilms are altered when treated with enzymes targeting specific matrix polysaccharides. Treatment with alginate lyase resulted in a 6x increase in elastic modulus for Alg+ biofilms and a 1.6x decrease in yield strain. Yield strain describes how large a deformation a particular material can withstand before it loses mechanical integrity. A decrease in yield strain for Alg+ biofilms upon treatment means the biofilm would be able to tolerate lesser deformation upon treatment with alginate lyase, indicating a weakening of the biofilm matrix. Similarly, Pel+ biofilms showed a 2.3x increase in elastic modulus and a 1.6x decrease in yield strain upon treatment with DNase I. Pel is thought to associate with extracellular DNA (eDNA) to form a strong network of polymers and thus, DNase I can degrade eDNA, leading to weakening of the polymer connections. These mechanical changes in the matrix polymers caused by enzyme treatment were also found to be dose-dependent.
The effects of these treatments on biofilms were visualized by Scanning Electron Microscopy (SEM). Upon treatment, networks within the Pel+ biofilm were seen to be reduced as compared to the control. However, the SEM was unable to visualize the changes observed for alginate lyase treatment. The authors attribute this to small amounts of Psl and Pel present in Alg+ biofilms as well as the sample preparation of SEM which involves agitation of biofilms, which could remove Alg+ biofilms that have been weakened upon treatment (since alginate can play an important role in surface attachment).
While this study looked at polymer-specific enzymes, other studies have shown generic enzymes (cellulase and α-amylase) to be successful in inhibiting and dispersing P. aeruginosa biofilms. When these enzymes were tested on P. aeruginosa biofilms in this study, the effects were not as drastic as observed with the polymer-specific enzymes.
Increase in elastic modulus an effect of less water retention in vitro – Cleavage of polymers by enzymes results in a weakening of the matrix network. This could thus enable water to diffuse more freely from the biofilm, thus escaping the biofilm matrix and leading to a ‘drying’ of the biofilm. Before and after treatment, the weights of biofilms revealed a significant loss as compared to control. Even visually, the biofilms appeared drier after treatment.
Polymer-specific enzyme treatment less effective in vivo –Three-day old biofilms grown in the mouse chronic wound infection model, were subjected to an enzyme treatment mixture (alginate lyase + DNase I) ex vivo. Here a 48% dispersal was seen for Psl+ biofilms and only 16% and 3% for Alg+ and Pel+ biofilms respectively. This did not mirror the results seen in vitro, where Alg+ biofilms had the maximum mechanical changes and Psl+ had the minimum. The authors give various explanations for this – for biofilm dispersal, changes are not primarily mechanical in nature, and the matrix produced in vivo may be different from that in vitro in terms of its polymers.
Interesting aspects of the study: This study shows an important aspect of mechanical alterations of the biofilm matrix – improved diffusion through a weakened biofilm matrix. Higher water diffusion through the matrix, upon cleavage by enzymes, can also mean higher diffusion of small molecules and antibiotics. While this seems like a promising aspect to utilize for treatment, it’s important to remember that this experiment was performed in vitro, where the surroundings of the biofilms are dry. However, within the body, the infecting biofilm is always surrounded by some fluids and thus, a biofilm in the clinical setting would likely not face this ‘drying’ phenomenon and thus may not see the change in mechanical properties we see here. This could also be a factor contributing to the inconsistent data between in vitro and in vivo studies here, where the in vivo biofilms could receive fluids from underlying tissue, preventing the drying process.
Questions for the authors: One of the explanations for the inconsistent data between in vitro and in vivo studies here could in fact be the difference in matrix proteins used by bacteria to form a biofilm in the two cases. However, even other conditions, like the wound depth and biofilm thickness, could play a role here. If the wounds were deep and thicker biofilms has been formed, the matrix could have a higher strength and the doses used may not have been enough. This can also be due to the difference in times of biofilm growth, where the in vitro studies used biofilms grown overnight, whereas in vivo studies used biofilms grown for 72 hours. These differences could itself cause changes in matrix population and thus the effect of the treatment.
 Flemming, Hans-Curt, and Jost Wingender. “The biofilm matrix.” Nature reviews microbiology 8.9 (2010): 623.
 Koo, Hyun, et al. “Targeting microbial biofilms: current and prospective therapeutic strategies.” Nature Reviews Microbiology 15.12 (2017): 740.
Posted on: 14th May 2019 , updated on: 15th May 2019Read preprint
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