## 2 Results  
### Growth Rates Gradually Decreased during the Evolution of Resistance  
To investigate the effect of the stringent response and oxidative stress response on the fitness cost of resistant strains during the de novo acquisition of antibiotic resistance, growth rates were measured at each MIC value reached. Four (p)ppGpp-related (\(\Delta\)_hipA_, \(\Delta\)_hipB_, \(\Delta\)_relA_, and \(\Delta\)_rpoS_) and four ROS-related (\(\Delta\)_sodA_, \(\Delta\)_sodB_, \(\Delta\)_soxR_, and \(\Delta\)_soxS_) single-gene knockout \(E\). _coli_ strains and the wild-type MG1655 strain were exposed to stepwise increasing levels of amoxicillin, enrofloxacin, kanamycin, and tetracycline. When these strains evolved higher resistance, the growth rate in an antibiotic-free medium and the MIC were recorded, and it showed that, generally, the growth rate decreased with increasing MIC (Figure 1). Before the resistance evolution, the growth rates of all ancestor strains were tested, and no significant differences were found (Figure 1Q). The growth rates during the amoxicillin resistance evolution of \(\Delta\)_hipA_ and \(\Delta\)_hipB_ were consistently similar to that of the wild type (WT) (Figure 1A). At the early stage of amoxicillin resistance evolution, when the MIC was 8 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\), the growth rates of \(\Delta\)_rpoS_ and \(\Delta\)_rpoS_ were clearly lower than WT; however, when the MIC reached 2048 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\), the growth rate of \(\Delta\)_rpoS_ was equal to that of WT (Figure 1E). For ROS-related knockout strains, the growth rate began to decrease at the middle stage of the evolution when the MIC was 128 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\) but was similar to WT at the highest concentration (Figure 1I,M). When the MIC reached 128 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\), the growth rates of \(\Delta\)_relA_, \(\Delta\)_sodB_, \(\Delta\)_soxR_, and \(\Delta\)_soxS_ were significantly reduced compared with WT (Figure 1R). The growth rate of all amoxicillin-resistant strains decreased gradually with an increase in MIC. Among them, \(\Delta\)_soxR_ and \(\Delta\)_soxS_ decreased the fastest, and their slopes were 0.025 and 0.030, respectively, compared to 0.018 for the WT (Figure 1M). The evolution process of enrofloxacin resistance was relatively complicated. Some strains such as \(\Delta\)_hipA_, \(\Delta\)_relA_, and \(\Delta\)_rpoS_ cannot evolve to higher enrofloxacin concentrations, which makes them difficult to compare (Figure 1B,F). The other knockout strains had no significant difference compared with WT (Figure 1B,J,N). However, enrofloxacin-resistant strains showed a lower slope of decline in growth rate than amoxicillin throughout the evolution. During kanamycin resistance evolution, \(\Delta\)_hipA_ and \(\Delta\)_hipB_ had a higher growth rate than WT until reaching the middle phase, i.e., until the MIC reached 256 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\) (Figure 1C). Similarly, the growth rates of \(\Delta\)_sodA_, \(\Delta\)_sodB_, \(\Delta\)_soxR_, and \(\Delta\)_soxS_ were slightly higher than those of WT at MICs lower than 128 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\) (Figure 1K,O). However, their growth rates decreased significantly thereafter and reached the same level as WT at the highest concentration. Similar to the case of amoxicillin, the growth rate of \(\Delta\)_relA_ during the kanamycin resistance evolution was lower than that of the WT starting from MIC 16 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\) (Figure 1G). \(\Delta\)_rpoS_ showed a clear and gradual decreasing trend with an R-squared value of 0.957, indicating a high correlation between a decrease in growth rate and an increase in MIC (Figure 1G). Compared with WT, the growth rates of \(\Delta\)_relA_, \(\Delta\)_rpoS_, \(\Delta\)_sodA_, \(\Delta\)_sodB_, \(\Delta\)_soxR_, and \(\Delta\)_soxS_ were significantly lower than that of WT when the MIC was 256 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\) (Figure 1S).