Exploiting Synthetic Biology and Phage Delivery Technologies to Tackle Antimicrobial Resistance

Antimicrobial resistance (AMR) is a health emergency spreading worldwide. The WHO identified the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., sometimes extended to ESKAPEE to include Escherichia coli) among frightens pathogens to be monitored. The request for developing and evaluating new therapies and treatment against AMR is massive and novel approaches must be explored to overcome the onset of new AMR strains. Synthetic biology, combining biological and engineering knowledge, can play a relevant role in this run. From this perspective, this works to exploit phage engineering and CRISPRi technologies to restore antimicrobial resistance in A. baumanii, K. pneumoniae, and P. aeruginosa. To achieve this, two main aspects have been explored: (I) the design and optimization of the delivery system, i.e. the engineered phage particles, and (ii) the actuator machinery, requiring the definition of a toolkit/library counting for genetic parts working on the desired strains, also involving the transformation protocols optimization (for decoupling parts characterization from delivery optimization) in non-conventional bacterial models. To assess the efficacy of the actuator machinery, the CRISPRi system, and electroporation protocols were optimized and performed in A. baumanii and K. pneumoniae obtaining a stable expression of red fluorescent protein (RFP). A transduction assay was performed to evaluate the ability of the engineered phage M13 particles to express the RFP. In this context, further optimization of the delivery platform is needed. In addition, the tropism of M13 phage particles must be studied and explored to infect the target strains.