The use of double-reporter Mycobacterium abscessus strains to improve anti-biofilm drug screening

Pulmonary infections caused by Mycobacterium abscessus are a pressing health issue due to the bacterium's high antibiotic resistance. Developing effective treatments is imperative, but conventional in vitro antibiotic susceptibility assays often do not correspond to clinical efficacy. M. abscessus easily aggregates and forms biofilms in various environments, where the protection conferred by an extracellular matrix, together with the mycobacteria's ability to enter a non-replicative persistent stage, highly hampers the activity of antibiotics. We developed a protocol to grow M. abscessus biofilms in a setup that allows high-throughput drug screening. The mycobacteria's luminescence is used as a readout of biofilm viability and its fluorescence as a measure of bacterial load, without the need for additional stains and maintaining the biofilm's integrity throughout the protocol. The use of imaging equipment allows a visual representation of the viability and bacterial load of each biofilm per well, and appropriate software can be used to quantify the luminescence and fluorescence signals. Quantification of biofilm mass can be done afterwards, using the same plate, by crystal violet staining. Although luminescent reporter assays have been used before, we believe this is the first time that a mycobacteria luminescent strain has been applied to assess drug activity against biofilms. This protocol enables the simultaneous screening of multiple compounds and identification of hits against M. abscessus biofilms in a fast, easy, and reliable manner. Most importantly, by mimicking the biofilm status that M. abscessus assumes in vivo, this assay will give more predictive information regarding compound efficacy.