Native CRISPR-Cas mediated in situ genome editing reveals the exquisite interplay of resistance mutations in clinical multidrug resistant Pseudomonas aeruginosa

Abstract

23Antimicrobial resistance (AMR) is imposing a global public health threat. Despite its 24 importance, resistance characterization in the native background of clinically isolated resistant 25 pathogens is frequently hindered by the lack of genome editing tools in these "non-model" 26 strains. Pseudomonas aeruginosa is both a prototypical multidrug resistant (MDR) pathogen 27 and a model species for understanding CRISPR-Cas functions. In this study, we report the 28 successful development of the first native type I-F CRISPR-Cas mediated, one-step genome 29 editing technique in a paradigmatic MDR strain PA154197. The technique is readily applicable 30 in additional type I-F CRISPR-containing, clinical/environmental P. aeruginosa isolates. A 31 two-step In-Del strategy is further developed to edit genomic locus lacking an effective PAM 32 (protospacer adjacent motif) or within an essential gene, which together principally allows any 33 type of non-lethal genomic manipulations in these strains. Exploiting these powerful 34 techniques, a series of reverse mutations are constructed and the key resistant determinants of 35 the MDR PA154197 are elucidated which include over-production of two multidrug efflux 36 pumps MexAB-OprM and MexEF-OprN, and a typical fluoroquinolone (FQ) resistance 37 mutation T83I in the drug target gene gyrA. Characterizing antimicrobial susceptibilities in 38 isogenic strains containing various combinations of single, double, or all three key resistance 39 determinants reveal that i) extensive synergy exists between the target mutation and over-40 production of efflux pumps, and between the two over-produced tripartite efflux pumps to 41 confer clinically significant FQ resistance; ii) while basal level MexAB-OprM confers 42 resistance only to penicillins, its over-production leads to substantial resistance to all 43 antipseudonmonal β-lactams and additional resistance to FQs; iii) despite the acquisition and 44 over-production of multiple resistant mutations, no obvious evolutionary trade-off of collateral 45 sensitivity is developed in PA154197. Together, these results provide new insights into 46 3 resistance development in clinical MDR P. aeruginosa strains and demonstrate the great 47 potentials of native CRISPR systems in AMR research. 48 49 Author Summary 50 Genome editing and manipulation can revolutionize the understanding, exploitation, and 51 control of microbial species. Despite the presence of well-established genetic manipulation 52 tools in various model strains, their applicability in the medically, environmentally, and 53 industrially important, "non-model" strains is often hampered owing to the vast diversity of 54 DNA homeostasis in these strains and the cytotoxicity of the heterologous CRISPR-Cas9/Cpf1 55 systems. Harnessing the native CRISPR-Cas systems broadly distributed in prokaryotes with 56 built-in genome targeting activity presents a promising and effective approach to resolve these 57 obstacles. We explored and exploited this methodology in the prototypical multi...