Phage-plasmid borne methionine tRNA ligase mediates epidemiologically relevant antimicrobial persistence

Antimicrobial resistance (AMR) is a global public health crisis with few options for control. As such early identification of emerging bacterial strains capable of rapidly evolving AMR is key. Although antimicrobial tolerance and persistence are precursor phenotypes for AMR, little evidence exists to support their importance in real-world settings. Here we used bacterial genome wide association on national genomic surveillance data of the diarrhoeal, and World Health Organisation AMR priority pathogen, Shigella sonnei (n=3745) to agnostically identify common genetic signatures among lineages convergently evolving toward AMR (n=15). This revealed an association of an AMR trajectory with a multi-and highly variable second copy of metG, borne by a phage-plasmid we called pWPMR2. Further analyses revealed that pWPMR2 was present across clinically relevant enteric pathogens globally, including past and contemporary outbreaks, and that the additional-metG mechanism was present across multiple bacterial phyla. Functional microbiology, experimental evolution, and single-cell physiology studies confirmed that the expression of auxiliary metG, particularly the mutated version on pWPMR2, created a sub population of cells predisposed to survival in, and evolving resistance against, third generation cephalosporins (3GC). Thus, we demonstrate a novel mechanism of auxiliary metG carriage that predisposes bacteria to AMR with real world impacts. Furthermore, our approach is a timely example of using genomic epidemiology to rapidly guide functional microbiology studies in the era of routine genomic surveillance and also highlights several deficiencies in current AMR surveillance practices.