## 3 Discussion  
In this study, we found that, as resistance increases during amoxicillin or kanamycin evolution, the growth rate of resistant strains gradually decreases. This is to be expected because bacteria must spend the additional energy on either resistance mutations or on additional metabolic activity [9]. We tried to address the question of whether there is a common pattern for this increase in fitness cost, and whether other stress responses have synergistic or antagonistic effects. The answer we propose is that two types of growth inhibition patterns of resistant strains related to these two stress responses exist and that both the stringent response and oxidative stress response have an impact on the fitness cost during the evolution of resistance. The reduced growth rates of ROS-related single-gene knockout strains \(\Delta\)_sodA_, \(\Delta\)_sodB_, \(\Delta\)_soxR_, and \(\Delta\)_soxS_ (Figure 5) are due to mutations related to antibiotic permeability and efflux pumps that occur halfway through the evolution process. This conclusion concurs with the observation that strains with increased levels of ROS production have a faster rate of resistance development [14]. During the evolution of bactericidal antibiotic resistance, drug-target interactions stimulate NADH oxidation within the TCA cycle-dependent electron transport chain (ETC) [16]. The excessively produced superoxide compound in the ETC damages iron-sulfur clusters through the Fenton reaction, causing hydroxyl radical formation [36]. These superoxide and hydroxyl radicals, called ROS, can damage DNA [37]. In particular, guanine on the genome is oxidized by ROS to 8-hydroxy-2\({}^{\prime}\)-deoxyguanosine (8-HOdG), resulting in cytosine to thymine substitutions during DNA replication [38]. However, the non-lethal dose of ROS produced during non-lethal antibiotic exposure can still damage DNA and induce the formation of mutations [39]. This mutagenic effect is caused by the activation of the cell's damage repair function through the SOS response [17]. The upregulation of the transcription level of error-prone DNA polymerase introduces errors during DNA damage repair [40]. These low-fidelity DNA polymerases can be considered a tool used by bacteria to increase their environmental adaptability by increasing their mutation rate [41]. Mutations beneficial to antibiotic resistance are ultimately retained through selection. The single-gene knockout of superoxide dismutase SodA or SodB, or superoxide response regulation, SoxR, or SoxS reduces the ability of cells to remove ROS but increases the speed of resistance acquisition [14]. These earlier-evolved mutations related to antibiotic permeability and efflux pumps all cost the cell more energy, ultimately leading to increased glucose consumption and reduced growth rate. This is, therefore, a general cellular reaction, similar in character but not identical to the ROS-mediated secondary killing mechanism of bactericidal antimicrobials. The growth rates of single-gene knockout strains related to the stringent response during the evolution process did not show any common features. This may be related to the different extents in which these strains trigger the alarmin (p)ppGpp under stringent responses. Only \(\Delta\)_relA_ showed significant changes in both growth rate and biomass yield during the evolution of amoxicillin and kanamycin resistance. When _relA_ is deleted, synthesis of (p)ppGpp is reduced, and the amino acid starvation caused by the synthesis of resistance-related proteins cannot be offset in a timely manner [42]. The result is that deacylated tRNAs bind to ribosomes and hinder the translation elongation process [22]. Bacteria cannot promptly upregulate amino acid synthesis or proteolysis to increase the level of aminoacylated tRNA [43]. This will, ultimately, lead to reduced growth and increased energy requirements [44]. Moreover, the fitness burden caused by _relA_ knockout is higher than that caused by specific resistance mutations.