Bacteriophages as Antimicrobial Agents and Synthetic Biology Scaffolds to Tackle Antimicrobial Resistance

Antimicrobial resistance (AMR) is a global health emergency, with the World Health Organization (WHO) identifying the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.; sometimes extended to ESKAPEE to include Escherichia coli) as priority threats. The urgent need for new antimicrobial strategies has motivated the exploration of phages and synthetic biology as alternative tools to combat resistant bacteria. In this work, two complementary approaches were pursued. First, lytic phages were studied as direct antimicrobial agents against carbapenem-resistant K. pneumoniae. Their activity was evaluated in gut-like environments, where resistance genes are often exchanged, and in urine, due to the clinical relevance of urinary tract infections. Both in vitro and in vivo assays were performed, and phage cocktails were tested for efficacy. In parallel, the molecular determinants of host specificity, such as phage co-receptors, were investigated as potential targets for engineering. Second, a non-lytic phage-based delivery system was developed to introduce clustered regularly interspaced short palindromic repeats interference (CRISPRi) into pathogenic bacteria. To achieve this, genetic parts including constitutive and inducible promoters were tested in A. baumannii. The CRISPRi system was characterized by first repressing a red fluorescent protein (RFP), and later targeting an outer membrane porin implicated in virulence and biofilm formation. Preliminary delivery assays were also performed in E. coli using engineered M13 particles, first to transfer a reporter cassette and then to silence resistance genes to colistin and meropenem. Together, these results demonstrate the feasibility of combining phage biology and synthetic biology to design novel antimicrobial strategies, highlighting both the therapeutic potential and the technical challenges of such approaches.