## 3 Results
In accordance with the decreased flagellin production detected in the \(\Delta\)lifq mutant strain, bacterial motility was significantly impaired in the absence of Hfq (Figure 2C). In addition, protein bands ranging in size from 35 to 43 kDa showed different intensities between strains. The \(\Delta\)fruR and \(\Delta\)ssrAB strains in particular increased the production of porin proteins, such as OmpA (37.5 kDa), OmpC (41.2 kDa), and OmpD (41.3 kDa). The altered production of porin proteins in these mutants might compromise the membrane integrity and affect bacterial resistance against antimicrobials [33; 34]. To test this  
Figure 1: Bacterial virulence of the \(\Delta\)_fruR, \(\Delta\)ssrAB, and \(\Delta\)lifq mutant strains in mice. Groups of 7-week-old female BALB/c mice (five mice per group) were injected intraperitoneally with the ST mutant strains (\(\Delta\)fruR, \(\Delta\)ssrAB, or \(\Delta\)lifq) at 10\({}^{4}\) CFU/ dose or with the wild-type (WT) strain at 10\({}^{3}\) CFU/ dose. At day 7 after infection, all mice were sacrificed and the body weight was measured (**A**). The spleens were isolated and weighed (**B**). Splenomegaly was not observed in three mutants of \(\Delta\)fruR, \(\Delta\)ssrAB, and \(\Delta\)lifq. Homogenized spleen tissues were diluted using PBS and plated onto SS agar plates to calculate the bacterial number (**C**). Bacterial numbers were significantly low in the infection with \(\Delta\)fruR or \(\Delta\)lifq. Statistical analysis was performed using one-way ANOVA with [PERSON]’s multiple comparison test in compared with WT strain. Asterisks (***) indicate a significant difference (\(p<0.001\)). possibility, bacterial strains were treated with 10 different antibiotics and the susceptibility to these agents was compared (Figure 2D). In comparison with WT _Salmonella_, the three mutants were more susceptible to gentamicin, kanamycin, and neomycin, all of which are aminoglycosotide antibiotics. A biochemical test using the API kit showed that the _\(\Delta\)fruR_, \(\Delta\)ssr\(AB\), and _\(\Delta\)hfq_ mutant strains shut down biochemical activities associated with arginine dihydrolase, citrate utilization, and lysine decarboxylase, while promoting amygdala and sucrose fermentation activities, indicating altered metabolic pathways associated with these compounds in these mutant strains (Table S3). Figure 2: Characterization of the _\(\Delta\)fruR_, \(\Delta\)ssr\(AB\), and \(\Delta\)hfq_ mutant strains in vivo and in vitro. (**A**) Intracellular survival of the ST wild-type (WT) and mutant (_\(\Delta\)fruR_, \(\Delta\)ssr\(AB\), and \(\Delta\)hfq_) strains in RAW264.7 murine macrophage cells. RAW264.7 cells were infected with each _Salmonella_ strain at an MOI of 100 and the number of intracellular bacteria was counted at 2 h (phagocytosis) and 10 h (survival) post-infection. The ratio of the number of mutant/WT bacterial cells is presented (* \(p<0.05\); ** \(p<0.01\); *** \(p<0.001\)). All three mutants showed attenuated intracellular survival at 10 h post-infection. (**B**) OMPs from WT and mutant strains were isolated and analyzed using 12% SDS–PAGE. Representative protein bands with altered production levels in comparison with WT were indicated with arrows. (**C**) Bacterial motility was compared between WT and mutant strains using semi-solid agar. The migration diameter (mm) of each bacterial strain was measured at 0.5, 2, 4, 6, 8, and 12 h, and analyzed by two-way analysis of variance (** \(p<0.01\)). Bacterial motility was significantly impaired in the absence of Hfq. (**D**) The bacterial resistance to 10 different antibiotics  
was compared between the WT and three mutant strains using the Kirby-Bauer disk diffusion assay. The diameter of the growth inhibition zone was measured to determine antibiotic susceptibility, and the susceptibility of WT _Salmonella_ to each antibiotic was set as 100%. In general, the three mutants were more susceptible to gentamicin, kanamycin, and neomycin, all of which are aminoglycosotide antibiotics.