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. 2019 Oct 22;63(11):e01454-19. doi: 10.1128/AAC.01454-19

FIG 1.

FIG 1

CRISPR-Cas engineered conjugative pPD1 derivative plasmids target and eliminate antibiotic resistance genes in in vitro coculture. (A) Schematic of the pPD1 derivative pKH88. pKH88 encodes cas9 and a CRISPR guide RNA under the control of the constitutive enterococcal bacA promoter. The guide RNA consists of a CRISPR repeat region and a unique spacer sequence with complementarity to the ermB gene (pKH88[sp-ermB]) of pAM771 or the tetM gene (pKH88[sp-tetM]) of pCF10. pKH88 derivatives encode genes necessary for conjugative transfer, chloramphenicol resistance (cat), and biosynthesis of the bacteriocin bac-21. (B) Cartoon depicting the delivery of pKH88 derivatives to target recipient E. faecalis cells. Abundance of erythromycin-resistant (C) or tetracycline-resistant (D) E. faecalis OG1SSp recipients following acquisition of pKH88[sp-ermB] or pKH88[sp-tetM]. Removal of erythromycin and tetracycline resistance from the transconjugant population depends on the introduction of a cognate spacer targeting pKH88 derivative. Markers: R, rifampin; F, fusidic acid; Cam, chloramphenicol; Abx, antibiotic resistance. Error bars represent the geometric mean plus or minus the geometric standard deviation. **, P = 0.006 (for passage 0 in C); **, P = 0.003 (for passage 1 in C); **, P = 0.009 (for passage 0 in D).