Fig 1.
Graphical abstract.
Table 1.
Description of bacterial strains in this study.
Fig 2.
Schematic presenting workflow of plasmid construction and antimicrobial re-sensitization by transformation.
(A) The candidate target sequence was inserted into CRISPR array on pCRISPR, and then CRISPR array was constructed into phagemid pRC319 that contained Cas9 and tracrRNA and the programmed CRISPR-Cas9 targeting blaCTX-M genes and the promoter (B) The modified pRC319 containing the CRISPR-Cas9 system was transformed into E. coli K-12 carrying blaCTX-M on pMBLe plasmid. The programmed CRISPR-Cas9 expressed functional Cas9-tracrRNA-crRNA complex to cleave pMBLe-blaCTX-M plasmid at the target site that resulted in antimicrobial re-sensitization and plasmid clearance. The figure was generated by BioRender web software (www.app.biorender.com).
Fig 3.
Plasmid map of phagemid and helper phage for phage particle production.
(A) Phagemid pRC319 containing modified CRISPR-Cas9 targeting blaCTX-M. (B) Modified helper phage pHP17_CO7 containing CmR gene.
Fig 4.
Schematic presenting workflow of phage particle production and antimicrobial re-sensitization by transduction.
(A) Construction of modified helper phage pHP17_CO7. (B) Phage ΦRC319-G1_II and ΦRC319-G9 were produced from co-transformation of helper phage pHP17_CO7 together with pRC319-G1_II or pRC319-G9. (C) Phage ΦRC319-G1_II and ΦRC319-G9 contained the programmed CRISPR-Cas9 system in the genome was delivered to E. coli K-12 that carrying blaCTX-M on pMBLe plasmid by transduction. The CRISPR-Cas9 expressed functional Cas9-tracrRNA-crRNA complex to cleave pMBLe-blaCTX-M plasmid at the target site that resulted in antimicrobial re-sensitization and plasmid clearance. The figure was generated by BioRender (www.app.biorender.com).
Table 2.
Characteristics of target nucleotide sequence selected from blaCTX-M group 1, group 9, and their promoter.
Fig 5.
Re-sensitization to cefotaxime (CTX) of E. coli K-12 carrying blaCTX-M on pMBLe (E. coli K-12: blaCTX-M) by transformation.
Colony-forming units (CFUs) of CTX-resistant (CTXR) E. coli K-12 was enumerated to calculate ratio of viable cells after re-sensitization to total transformants. Reduction of the ratio in treated groups acquiring programmed CRISPR-Cas9-containing pRC319 (white bar) compared with ratio (approximately 1) in the control groups acquiring unmodified pRC319 (black bar). (A) Ratio of CTXR cells to total transformants of E. coli K-12: blaCTX-M-15: pRC319-G1_I and E. coli K-12: blaCTX-M-55: pRC319-G1_I. (B) Ratio of CTXR cells to total transformants of E. coli K-12: blaCTX-M-15: pRC319-G1_II and E. coli K-12: blaCTX-M-55: pRC319-G1_II. (C) Ratio of CTXR cells to total transformants of E. coli K-12: blaCTX-M-14: pRC319-G9, E. coli K-12: blaCTX-M-27: pRC319-G9, E. coli K-12: blaCTX-M-65: pRC319-G9, and E. coli K-12: blaCTX-M-90: pRC319-G9. (D) Ratio of CTXR cells to total transformants of E. coli K-12 carrying blaCTX-M group 1: pRC319-P and blaCTX-M group 9: pRC319-P. Error bars represent results from three biological replicates. **P<0.01 was considered statistically significant that was calculated using the Student’s t-test, and bar charts were generated using GraphPad Prism version 8.0.
Fig 6.
Clearance of plasmid pMBLe: blaCTX-M in E. coli K-12 carrying blaCTX-M on pMBLe (E. coli K-12: blaCTX-M).
Colony-forming units (CFUs) of gentamicin-resistant (GmR) E. coli K-12 was enumerated to calculate ratio of viable cells after plasmid clearance to total transformant. Reduction of the ratio in treated groups acquiring programmed CRISPR-Cas9-containing pRC319 (white bar) compared with ratio (approximately 1) in the control groups acquiring unmodified pRC319 (black bar). (A) Ratio of CTXR cells to total transformants of E. coli K-12: pMBLe: pRC319-G1_I, E. coli K-12: blaCTX-M-15: pRC319-G1_I, and E. coli K-12: blaCTX-M-55: pRC319-G1_I. (B) Ratio of CTXR cells to total transformants of E. coli K-12: pMBLe: pRC319-G1_II, E. coli K-12: blaCTX-M-15: pRC319-G1_II, and E. coli K-12: blaCTX-M-55: pRC319-G1_II. (C) Ratio of CTXR cells to total transformants of E. coli K-12: pMBLe: pRC319-G9, E. coli K-12: blaCTX-M-14: pRC319-G9, E. coli K-12: blaCTX-M-27: pRC319-G9, E. coli K-12: blaCTX-M-65: pRC319-G9, and E. coli K-12: blaCTX-M-90: pRC319-G9. (D) Amplicon of blaCTX-M gene (1,100 bp) and CRISPR array (421 bp) amplified by colony PCR, the blaCTX-M amplicon was detected from pMBLe-blaCTX-M-carrying E. coli K-12 without modified pRC319 transformation but no amplification of blaCTX-M in each group with the modified pRC319 transformation. Moreover, CRISPR array from the modified pRC319 was detected in re-sensitized cells indicated successful transformation of modified pRC319. Error bars represent results from three biological replicates. **P<0.01 was considered statistically significant that was calculated using the Student’s t-test, and bar charts were generated using GraphPad Prism version 8.0.
Fig 7.
Dose-dependent reduction of viable cefotaxime-resistant (CTXR) E. coli K-12 carrying blaCTX-M on pMBLe (E. coli K-12: blaCTX-M) after re-sensitization by the programmed CRISPR-Cas9 delivered by phage transduction at multiplicity of infection (MOI) from 0.1 to 100.
(A) Viable CTXR cells of transductant E. coli K-12: blaCTX-M-15: ΦRC319-G1_II and E. coli K-12: blaCTX-M-55: ΦRC319-G1_II. (B) Viable CTXR cells of transductant E. coli K-12: blaCTX-M-14: ΦRC319-G9, E. coli K-12: blaCTX-M-27: ΦRC319-G9, E. coli K-12: blaCTX-M-65: ΦRC319-G9, and E. coli K-12: blaCTX-M-90: ΦRC319-G9. (C) Viable CTXR cells of transductant E. coli K-12: blaCTX-M-15: ΦRC319 and E. coli K-12: blaCTX-M-55: ΦRC319 (control group). (D) Viable CTXR cells of transductant E. coli K-12: blaCTX-M-14: ΦRC319, E. coli K-12: blaCTX-M-27: ΦRC319, E. coli K-12: blaCTX-M-65: ΦRC319, and E. coli K-12: blaCTX-M-90: ΦRC319 (control group). Error bars represent results from three biological replicates. The line chart was generated using GraphPad Prism version 8.0.