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.

Waterborne pathogen detection is challenging, requiring effective target organisms enrichment to enable reliable and accurate monitoring of pathogens which could be present at low numbers. However, existing processes are manual, require operator expertise and are optimised for particular applications, e.g. a certain approach for viruses and another for bacteria etc. This study presents the design, development, and optimisation of an easy-to-use automated Rexeed™ dead-end filtration system tailored for concentrating multiple waterborne pathogens including viruses, bacteria and protozoa within a single unit. Knowing the variability in recovery rates across filtration technologies due to factors such as filter type, pre-treatment chemicals, backflush volumes, and flow rates, this system was engineered to optimise pathogen recovery and improve consistency in sample processing, generating a system that reduces manpower requirements and worked well across viruses, bacteria and protozoa. System parameters were investigated, including flow rates of filtration from 0 to 1200mL/min, backflush volumes ranging from 0 to 500mL, chemicals used for backflush water enhanced with surfactants and chemical dispersants. Additionally, pre-treatments with sodium polyphosphate (NaPP) and bovine serum albumin (BSA) were investigated using fluorescent beads as proxies for pathogens. Key results indicate that high flow rates (≥ 900mL/min), backflush volume of 250mL containing NaPP, Tween and antifoam achieved significantly higher recovery rates. Validation using fluorescent particle counting and turbidity measurements confirmed that this automated setup yields recovery rates in line with existing filtration methods. By enabling simultaneous concentration of diverse pathogens, this automated system offers a practical solution for comprehensive waterborne pathogen monitoring, demonstrating that one automated filtration unit can effectively concentrate multiple pathogens.