Development of an in-vivo high throughput assay to monitor biofilm development of the pathogenic microorganism Pseudomonas aeruginosa, in C. elegans.

Bacterial biofilms are clusters of bacteria attached to host tissues and abiotic surfaces, such as medical implants, imbedded in a self-generated matrix; this is characterised by extracellular polysaccharides, proteins and eDNA, providing nutrients and protection to the bacteria, which become resistant to both the host’s immune system and antimicrobials, therefore becoming extremely difficult to treat and eradicate. About 80% of recurring chronic microbial diseases in humans are caused by bacterial biofilms, recognition of which has led to an increased attention on identifying new treatments. Particularly, biofilms are difficult to detect in clinical settings, due to lack of biofilm-specific biomarkers. Therefore, further studies are essential to identify markers unique to bacterial biofilms. In this study, we mainly focused on Quorum Sensing (QS) signalling, a well-known system of communication found in many microbial species, involved in cell density regulation for biofilm formation. Using the biofilm- former Pseudomonas aeruginosa (P. aeruginosa), we targeted QS-related genes, to build a biofilm-specific reporter, to use for in-vivo studies in the nematode Caenorhabditis elegans (C. elegans). Particularly, we aimed to create a high-throughput C. elegans in-vivo biofilm assay for industrial research and to provide a cost and time-effective protocol for it. C. elegans is extremely advantageous to address many research questions, particularly due to its transparency, short generation time and low maintenance costs; moreover, it shares more than 80% of human genes and it is colonised by pathogenic bacteria, such as P. aeruginosa. Our results show that P. aeruginosa transgenic fluorescent reporters can be employed in a C. elegans biofilm infection model, which can provide a non-invasive host’s health readout and visualisation of bacterial tissue colonisation; detection of fluorescence signal by biofilm-competent bacteria, as opposed to lack of signal by mutant biofilm-incompetent bacteria, also suggested that C. elegans could be used to monitor biofilm progression in the animal. Further studies will be needed to complement this research, to prove evidence of biofilm detection and to address standardisation of this assay for a possible industrial use; overall, this was a first step which holds promise in the attempt to bridge the gap between in-vivo clinical diagnostics and in-vitro biofilm research.