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

Abstract 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. Competing Interest Statement This work was funded by BBSRC (BB/V009184/1) and MRC (MR/X000648/1) project grants. JPJH is supported by an MRC Career Development Award (MR/W02666X/1). The research in the Bakshi lab was supported by the Wellcome Trust Award (grant number RG89305), a University Startup Award for Lectureship in Synthetic Biology (grant number NKXY ISSF3/46), an EPSRC New Investigator Award (EP/W032813/1) and a seed fund from the School of Technology at University of Cambridge. CJ, CRB, KSB, LCEM and PR are affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Gastrointestinal Infections at University of Liverpool in partnership with the United Kingdom Health Security Agency (UKHSA), in collaboration with University of Warwick. SN and PR are affiliated with the NIHR HPRU in Genomics and Enabling Data at University of Warwick in partnership with the UKHSA, in collaboration with University of Cambridge and University of Oxford. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health and Social Care or UKHSA Funding Statement This work was funded by BBSRC (BB/V009184/1) and MRC (MR/X000648/1) project grants. JPJH is supported by an MRC Career Development Award (MR/W02666X/1). The research in the Bakshi lab was supported by the Wellcome Trust Award (grant number RG89305), a University Startup Award for Lectureship in Synthetic Biology (grant number NKXY ISSF3/46), an EPSRC New Investigator Award (EP/W032813/1) and a seed fund from the School of Technology at University of Cambridge. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes Revised version of manuscript with new data