Biofilm is a three-dimensional structure formed by cells embedded in the extracellular matrix. Bacteria in a biofilm are more resistant to antibiotics, proteases released by host defence cells, and other environmental stress factors. Staphylococcus aureus is a major human pathogen causing a wide range of diseases including hospital-acquired infections. The persistence of chronic and medical device-related infections caused by S. aureus is related to its ability to form biofilm. Phage therapy is an alternative approach for the treatment of infections caused by antibiotic-resistant bacteria. However, the details of how the phage propagates through the structured biofilm are largely unknown.
We use light-sheet fluorescent microscopy (LSFM) with an integrated microfluidic system. To visualize biofilm-forming cells we labelled S. aureus by red fluorescent protein mCherry constitutively expressed from a plasmid. The components of the biofilm matrix are labelled by dyes fluorescent in non-overlapping spectra.
To study the propagation of phage in a biofilm we utilise different approaches to induce fluorescence in phage-infected cells. First, we cloned reporter plasmids containing a gene for green fluorescent protein (GFP) controlled by phage late promoters into S. aureus cells. Second, we are trying to modify phage genomes using CRISPR-Cas10 targeting specific parts of phage genome and recombination with a template containing GFP gene. Furthermore, we are also attempting to directly label the phage P68 capsid by a fusion of GFP to its head fibre protein. Taken together, we will be able to monitor the propagation of the phage through S. aureus biofilms in great detail using LSFM.