## 3 Discussion
As a consequence, the growth rate of \(\Delta\)_relA_ decreases at lower MIC levels than the growth rate of the WT (Figure 5A,B). The wild-type and all single-gene knockout strains used in this study were derived from the _E. coli_ K12 strain of the same genetic background. Although some minor genetic differences exist between strains, no reports link these differences to resistance and fitness costs. Two typical types of gene mutations were observed in antibiotic-resistant strains: target-specific mutations, such as _ampC_ upstream mutations in amoxicillin-resistant strains and _fusA_ mutations in kanamycin-resistant strains; and non-target specific mutations, which mainly affect the intracellular antibiotic content, for example, reducing antibiotic uptake and increasing efflux. The mutations in the promoter region of _ampC_ increase beta-lactamase expression by upregulating its transcription level, which increases the energy consumption of bacteria and affects its growth rate [45]. Mutations of the EF-G encoding gene _fusA_ also decrease the growth rate of bacteria by reducing the protein synthesis rate [46]. Similarly, mutations that affect intracellular antibiotic levels can also increase bacterial metabolic costs. Mutations related to outer membrane porins that reduce antibiotic permeability can also block the absorption of other beneficial compounds, including nutrients [47]. In addition to consuming extra energy, the activated efflux pumps can also export beneficial compounds out of the cell, ultimately increasing the metabolic burden of bacteria [48; 49]. We speculate that this is why _ArelA_-resistant strains have not evolved mutations related to mechanisms such as antibiotic permeability and efflux pumps because these mutations will cause the already nutrient-starved strains to become even more nutrient deficient. During later stages of resistance evolution, almost all strains evolved mutations related to antibiotic permeability and efflux pumps. The final resistant strains all had significantly lower biomass yield on glucose compared to their ancestor. Moreover, the biomass yield of _ArelA_-resistant strains was significantly lower than that of other resistant strains. This further indicates that, in the _relA_ knockout, the ability to respond to stress is weakened due to limited synthesis of (p)ppGpp, resulting in a reduced growth rate and increased energy requirement to maintain reproduction. Furthermore, we found a slight increase in the growth rate of specific resistant strains during the later stages of amoxicillin, enrofloxacin, and kanamycin evolution. At this point, we found mutations that have not been reported to be directly associated with antibiotic resistance; therefore, we hypothesize that this is  
Figure 5: Schematic diagram of two processes causing reduced growth rates as resistance increases. As the MICs for amoxicillin (**A**) and kanamycin (**B**) increase, the growth rates of all strains gradually decrease. This relates to two different processes. ROS-related knockout strains develop mutations related to antibiotic permeability and efflux pumps earlier (in the mid-stage) than other strains. Because these mutations increase energy consumption, the fitness cost increases and the growth rate decreases. The other process is that, in the early stages of resistance evolution, _ArelA_ shows a decrease in growth rate, which may be related to it simulating starvation-induced growth arrest. Different colored dots represent the median of the growth rate of each strain at different MICs. Gray shading indicates the range of variation in WT growth rates. The green line connects the median of the growth rates of _ArelA_. the result of compensatory evolution, i.e., compensating for the metabolic burden caused by antibiotic-resistant adaptations [50; 51]. Compensatory evolution allows bacteria to maintain antimicrobial-resistant properties, which is one of the reasons why resistant strains are difficult to completely eliminate [4; 52; 53].