Figure 2.

Targeting antibiotic resistance genes and plasmids in an MRSA strain. (a) Treatment of a mixed population of RN^Φ and USA300^Φ results in killing of the targeted USA300 MRSA strain and delivery of an immunizing phagemid to the rest of the population. (b) pDB121::mecA specifically kills USA300^Φ in a mixed population. Exponentially growing USA300^Φ and RN^Φ cells were mixed 1:1 and treated with pDB121 at an MOI of ~5. Cells were plated either on a non-selective medium, on chloramphenicol-containing medium to measure the proportion of cells receiving the phagemid treatment, or on oxacillin-containing medium to measure the proportion of USA300^Φ cells in the population (mean ± s.d.). (c) The CRISPR array sequence is programmed to target the pUSA01 and pUSA02 plasmids simultaneously. (d) USA300^Φ was treated with pDB121 lysates targeting each plasmid individually or in combination. Cells were plated either on a non-selective medium, on chloramphenicol-containing medium to measure the proportion of cells receiving the phagemid treatment, or on tetracycline-containing medium to measure the proportion of cells cured of pUSA02 (mean ± s.d.). (e) Plasmid curing was confirmed by the lack of PCR amplification with plasmid specific oligonucleotides in 8 independent CFUs after treatment with the double targeting construct. (f) A population of RN^Φ cells was immunized against plasmid horizontal transfer by treatment with the pUSA02-targeting pDB121 phagemid. 30 min after treatment, the population is transduced with a ΦNM1 stock grown on USA300. Cells are plated either without selection or on tetracycline to measure transduction efficiency of the pUSA02 plasmid (mean ± s.d.).