Use of a 3D biofilm model to determine the antibiofilm properties of Klebsiella pneumoniae bacteriophages

This research aims to determine the antibiofilm activity of K. pneumoniae bacteriophages (phage) using an already established 3D biofilm model. Biofilm formation by antibiotic-resistant K. pneumoniae is a key virulence factor.

It allows the pathogen to persist on abiotic surfaces, such as indwelling medical devices and is a crucial step in the pathogenesis of many infections, especially urinary tract infections. Biofilm formation also contributes to the recalcitrant nature of many pathogens to clinically important antibiotics.

Growing concerns about the rise of antibiotic resistance have led to renewed interest in investigating alternatives such as phage therapy. Phages are viruses which infect and kill their bacterial hosts by lysis upon replication to release their progeny.

Phages also possess depolymerase enzymes which degrade polymeric substances, such as capsules and biofilms, so that they can dock with their bacterial host surface cell receptors. Thus, phages represent a promising approach to control bacterial biofilms.

Previous studies have shown that phages show promise in combating biofilms however these studies utilize 2D biofilms established over 24h-72h. Such models poorly represent the true nature of biofilm formation and structure. There is evidence that in vivo biofilms mature and acquire insusceptibility to antimicrobials over weeks.

In prior research, I successfully constructed a 3D biofilm model and have used this in antimicrobial susceptibility testing (under review).

The biofilm within the constructed 3D model showed similarity to that in vivo in terms of structure, maturity, and antimicrobial resistance.

My 3D model is therefore more clinically relevant and provides an ideal system for testing the efficacy of phage to disrupt biofilms in a realistic setting.

The host laboratory has a large collection of broad-spectrum phages that infect clinical, drug-resistant isolates of K. pneumoniae.

They have also recently characterized 8 phages-encoding capsular depolymerases, enzymes which have the capacity to degrade the polysaccharide capsule of K. pneumoniae.

Polysaccharides are a major component of biofilms and the ability of phages to degrade them is clear evidence of the ability of phages to destroy biofilms. I will also have access to approximately 150 clinical K. pneumoniae isolates. This project will therefore utilize my 3D biofilm model to examine the antibiofilm properties of these phages.

Methodology: First, K. pneumoniae isolates will be screened for biofilm forming ability. Then, the killing effect of phages will be examined against 2D biofilm culture (microtiter biofilm assay). Phages with high antibiofilm activity will be selected for further analysis using the 3D biofilm model.

Biofilm beads will be synthesized by combining 2D-biofilm culture with alginate and dispensing them through 25 G syringes in cross-linking solution.

The model will be designed as two layers of alginate matrix enclosing K. pneumoniae biofilm beads on a Thin insert chamber suspended on 12 well plates containing culture media where bacteriophages will be added. Scanning electron microscopy will be used to visualize morphological changes to biofilm due to phage activity.

Bactericidal activity will be assessed via live/dead assay and quantification of bacterial load within the matrix as well as measuring the optical density of the culture medium.