Fig. 3

The knockout strains of CRISPR-Cas components show sensitivity towards antimicrobial peptides (AMP), and the complement system. (A) The strains were exposed to antimicrobials (i) AMPs- protamine sulfate (0.5 µg/mL), polymyxin B (0.5 µg/mL), and (ii) serum (20% FBS) for 1 h. For AMPs, the percentage survival was determined by analyzing CFU in both untreated and treated samples, the untreated samples were used as controls. Percentage survival in serum was determined by using heat-inactivated samples as control. (B) Total RNA isolated from late log-phase bacteria strains was used for cDNA synthesis, followed by qRT-PCR to assess the expression of polymyxin resistance (pmr) genes like pagB, pagD, pmrH, pmrE, pmrD. Relative expression of the gene was calculated using the 2 ^–ΔΔCt method, and normalized to reference gene rpoD. One-way ANOVA (Dunnett’s multiple comparison test) was used to determine significant differences between the WT and knockout strains, in at least three independent experiments, with at least 3 replicates in each. Error bars indicate SD. Statistical significance is shown as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p < 0.0001; and ns, not significant