ABSTRACT
Antimicrobial resistance (AMR) is imposing a global public health threat. Despite its importance, characterizing drug resistance directly in the clinical isolates of resistant pathogens is frequently hindered by the lack of genome editing tools in these “non-model” strains. Pseudomonas aeruginosa is both a prototypical multidrug resistant (MDR) pathogen and a model species for CRISPR-Cas research. In this study, we report the successful development of a simple and efficient one-plasmid mediated, one-step genome editing approach in a paradigmatic MDR strain PA154197 by exploiting its native type I-F CRISPR-Cas system. The technique is readily applicable in two additional type I-F CRISPR-containing, clinical and environmental P. aeruginosa isolates. A two-step In-Del strategy involving insertion and subsequent deletion of a tag nearby the desired editing site is further developed to edit the genomic locus lacking an effective PAM (protospacer adjacent motif) or within an essential gene, which principally allows any non-lethal genomic manipulation in the strains. With these powerful techniques, the resistant determinants of PA154197 were delineated in its native genetic background. Moreover, relative contributions and extensive synergy of different resistance determinants previously unrecognized by using laboratory strains were disclosed. The two efflux pumps with different substrate preference, MexAB-OprM and MexEF-OprN, synergistically expel fluoroquinolones, trimethoprim and chloramphenicol. Among the three resistant mutations synergizing fluoroquinolones resistance, gyrA mutations elicit a greater resistance than drug efflux by MexAB-OprM or MexEF-OprN. These results advanced our understanding of the MDR development of clinical P. aeruginosa strains and demonstrated the great potentials of native CRISPR systems in AMR research.
IMPORTANCE Genome editing and manipulation often revolutionizes the understanding, exploitation, and control of microbial species. Despite the presence of well-established genetic manipulation tools in various model strains, their applicability in the medically, environmentally, and industrially significant, “non-model” strains is often hampered owing to the vast diversity of DNA homeostasis in these strains and the cytotoxicity of the heterologous CRISPR-Cas9/Cpf1 system. Harnessing the native CRISPR-Cas systems broadly distributed in prokaryotes with built-in genome targeting activity presents a promising and effective approach to resolve these obstacles. We explored and exploited this methodology in the prototypical multidrug resistant pathogen P. aeruginosa by exploiting the most common subtype of the native CRISPR systems in the species. Our successful development of the first type I-F CRISPR-mediated genome editing technique and its subsequent extension to additional clinical and environmental P. aeruginosa isolates opened a new avenue to the functional genomics of antimicrobial resistance in pathogens.