Figures & data
Figure 1. Changes in carbapenem resistance and conjugation frequency of evolved ECNX52 strains. A. Evolution diagram of ECNX52 under different concentrations of MEM pressure. B. Resistance changes of evolved ECNX52 under 1 μg/mL continuous MEM pressure. C. Resistance changes of evolved ECNX52 under 2 μg/mL continuous MEM pressure. D. Resistance changes of evolved ECNX52 under 4 μg/mL continuous MEM pressure. E. Conjugation frequency changes of evolved ECNX52 under different concentrations of MEM pressure. “**” means P < 0.01; “***” means P < 0.001.
Figure 2. Fitness assessment of evolved ECNX52 strains. A. Growth curve of evolved ECNX52 strains in antibiotic-free LB medium. B. Growth curve of evolved ECNX52 strains in LB medium containing 16 μg/mL MEM. C. Stability of pNX52-NDM-5 plasmid in evolved ECNX52 strains. D. Competition index between evolved ECNX52 strains and E. coli C600. E. Fitness assessment of blaNDM-5 resistance gene. pSTV28-NDM5 is a blaNDM-5 overexpression plasmid with pSTV28 as the vector.
Figure 3. Transcriptomic and genomic changes in resistance evolved strains. A. Venn diagrams of differentially expressed genes in resistance evolved strains. The diagrams on the upper left and upper right showed upregulated and downregulated genes in EM2N1 and EM2N3 compared to the ancestral strain, respectively. The diagram on the lower left and lower right showed upregulated and downregulated genes in EM4N1-EM4N4 compared to the ancestral strain, respectively. B, C. Bubble charts of GO functional enrichment analysis of differentially expressed bacterial host genes in resistance evolved strains. Panel B demonstrated the GO functional enrichment analysis of differentially expressed host genes in EM2N1 and EM2N3 in comparison to the ancestral strain ECNX52, while panel C depicted the same analysis for EM4N1-EM4N4. The y-axis represented GO terms. The x-axis represented the ratio of differentially expressed genes (the ratio reflected genes related to the GO term in the differentially expressed genes to the total number of differentially expressed genes). The size of the bubble represented the number of enriched genes. The colour of the bubble represented the value of P value, and the shape of the bubble represented the regulation direction of GO functional terms. D. Expression levels of resistance-related genes blaNDM-5, ompC, ompF, acrA, acrB, and tolC in resistance evolved strains using qRT-PCR. E. Changes in the copy number of plasmids in resistance evolved strains using qPCR. “*” means P < 0.05; “**” means P < 0.01; “***” means P < 0.001; “****” means P < 0.0001.
Figure 4. Plasmid or host factors mediated resistance evolution. A. MICs of evolved plasmids in the ancestral host strain. ECpM1N1-ECpM4N4 were recombinant strains constructed by transforming the evolved plasmids into the ancestral E. coli C600. B. MICs of ancestral plasmids in evolved host strains. HM1N1p0-HM4N4p0 were recombinant strains constructed by transforming the ancestral plasmid p0 into the evolved host strains. C. Conjugation frequency of evolved plasmids in the ancestral host strain. “**” means P < 0.01; “***” means P < 0.001; “****” means P < 0.0001. D. Conjugation frequency of ancestral plasmids in evolved host strains.
Figure 5. Functional validation of the replication protein repA D140Y (GAT→TAT) mutation in the pNX52-NDM-5 plasmid. A. Schematic diagram of the structure of the replication protein repA in the pNX52-NDM-5 plasmid. B. Schematic diagram of the lacZ reporter system used to validate the function of the repA D140Y (GAT→TAT) mutation. C. The repA D140Y (GAT→TAT) mutation reduced inhibition of promoter activity, leading to an increase in plasmid copy number. “**” means P < 0.01; “***” means P < 0.001.
Figure 6. Porin Deficiency or Plasmid Copy Number Increase Mediated Carbapenem-Resistant Escherichia coli Resistance Evolution. OM means outer membrane. IM means inner membrane. MEM means meropenem.
