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[Preprint]. 2023 Oct 12:2023.10.12.561972. [Version 1] doi: 10.1101/2023.10.12.561972

Figure 4. Antibiotic tolerance is mediated by amino acid starvation leading to protein synthesis arrest.

Figure 4.

(A) Homogenized biofilm cultures were transferred to CDM broth or CDM broth lacking individual amino acids and antibiotics were added as indicated. The reduction in CFUs compared to the starting inoculum was calculated after 24 hours of antibiotic exposure. (B) Homogenized biofilm cultures were transferred to fresh CDM or CDM-R and protein synthesis inhibitors were added as indicated along with either ceftaroline or a vehicle control. The reduction in CFUs compared to the starting inoculum was calculated after 24 hours of antibiotic exposure. (C) Representative western blot showing the labelling of nascent protein by click chemistry 4 hours after the transfer of homogenized biofilm cultures to the indicated growth conditions. (D) A ratio of nascent protein to total protein was calculated using integrated density values obtained by analyzing the western blots in imageJ. Data represent technical replicates of biological triplicates. 2-way ANOVA with Tukey multiple comparisons test; *=p<0.05, **=p<0.005, ***=p<0.0005, ****=p<0.0001, ns=not significant, ABX=antibiotics, VAN=vancomycin, CPT=ceftaroline, DEL=delafloxacin, LZD=linezolid, DOX=doxycycline, CLD=clindamycin.