Fig. 2: Testing individual TFI strains and postulated effector gene mutants for hyper susceptibility versus tolerance to rifampicin.

A–H Time-kill kinetics of individual TFI strains during steady state 10 X MIC rifampicin exposure. All rifampicin hypersusceptible strains (A–D) showed significantly faster killing compared to their uninduced counterparts. Conversely, rifampicin tolerant TFI strains (E–G) show significantly slower killing upon ATc induction compared to the vector control (H). Studies were performed in quadruplicate transwells and each dot represents one replicate. I Fold difference in (A–H) the CFU between ATc-induced and uninduced replicate cultures of TFI strains at four days of rifampicin exposure. J Determining which hypersusceptible or tolerant TFs reverse their phenotype when knocked down by CRISPRi. TF knockdown strains were pooled and their competitive time-kill kinetics was studied after exposure to steady state rifampicin concentration using TTR. Of the seven TFs, blaI and sigC showed a reversal of the rifampicin susceptible phenotype. The X-axis shows the relative (ATc induced versus uninduced) growth rate of the knockdowns in the absence of rifampicin exposure while the Y-axis shows the relative survival (induced versus uninduced) after four days of rifampicin exposure. K Phenotypes of postulated hypersusceptibility or tolerance effector genes when overexpressed. Each dot represents the mean relative abundance of a given mutant from triplicate culture based on the normalized read count frequency. L Growth versus kill kinetics of CRISPRi knockdown strains. Each dot represents the mean value of three replicates collected from different transwells. M Volcano plot of mRNA expression in ATc induced versus uninduced blaI TFI strains showing significant downregulation of the cydABDC operon (red dots). N Time-kill kinetics of the cydA (mutant 3) knockdown strain when exposed to rifampicin (10 X MIC) for up to 12 days using the TTR method (line indicates mean CFU from 3 biological replicates). Estimated CFU was generated by multiplying the relative abundance of each strain against the total observed CFU from replicates. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001, statistical significance estimated using a two-tailed unpaired t-test, GraphPad Prism 9.1. VC = vector control.