## 3 Results
The antibiotics tested included amoxicillin/davulanic acid (AMC, 30 \(\upmu\)g), ampicillin (AM, 10 \(\upmu\)g), cephalothin (CF, 30 \(\upmu\)g), gentamicin (GM, 10 \(\upmu\)g), kanamycin (K, 30 \(\upmu\)g), nalidixic acid (NA, 30 \(\upmu\)g), neomycin (N, 30 \(\upmu\)g), ampicillin/sulbactam (SAM, 20 \(\upmu\)g), sulfamethoxazole/trimethoprim (SXT, 25 \(\upmu\)g), and tetracycline (TE, 30 \(\upmu\)g). The values are the averaged susceptibilities from triplicates. Taken together, these results suggest that the three regulators of FruR, SsrAB, and Hfq modulate a variety of cellular activities, including virulence, motility, antibiotic resistance, and metabolism. Evaluation of the Immune Responses Induced by the \(\Delta\)fruR, \(\Delta\)ssrAB, and \(\Delta\)fifq Mutants  
The immunogenicity of the three mutant strains was compared by measuring the levels of serum IgG, IgM, and IgG subclasses (IgG1 and IgG2a) after immunization of female BALB/c mice. Groups of mice were immunized (i.p.) with the WT and mutant strains and then orally infected with a WT \(S\). Typhimurium strain (ST2173) at 21 dpi (Figure 3A). Mouse sera were collected on a weekly basis and to measure the levels of _Salmonella_-specific antibodies. The \(\Delta\)fruR mutant caused rapid increments in IgG and IgM levels at 14 dpi compared with the WT and \(\Delta\)fifq mutant strains, whereas the \(\Delta\)ssrAB mutant strain remarkably increased IgG levels at 28 dpi (Figure 3B,C). However, the levels of IgG and IgM were comparable among strains at 35 dpi. The IgG2a/IgG1 ratio is indicative of the type of immune responses triggered by T-helper 1 (Th1) or Th2 effector cells. Mice immunized with the WT and \(\Delta\)fifq mutant strains did not exhibit significant changes in IgG1 levels up to 35 dpi, whereas immunization with the \(\Delta\)fruR and \(\Delta\)ssrAB mutant strains caused surges in IgG1 levels at 28 and 35 dpi, respectively (Figure 3D). In contrast with the IgG1 responses, the IgG2a levels increased gradually up to 35 dpi, regardless of the _Salmonella_ strains used in the immunization, although the \(\Delta\)fifq mutant strain stimulated IgG2a production to a lesser extent than did the remaining three strains (Figure 3E). Notably, the vigorous IgG2a production triggered by either the WT or mutant strains indicates that immunization with _Salmonella_ strains could elicit a Th1-mediated immune response proficiently. However, the lower IgG2a/IgG1 ratios afforded by the \(\Delta\)fruR (ranging from 2.7 at 28 dpi to 8.9 at 14 dpi) and \(\Delta\)ssrAB (ranging from 2.0 at 28 dpi to 6.5 at 35 dpi) mutants compared with WT _Salmonella_ (ranging from 5.8 at 14 dpi to 15.7 at 35 dpi) suggest that the \(\Delta\)fruR and \(\Delta\)ssrAB mutant strains were more competent in stimulating an IgG1-associated Th2 immune response as well as an IgG2a-associated Th1 immune response than was the WT _Salmonella_ strain (Figure 3F). Figure 3: Stimulation of _Salmonella_-specific antibodies in immunized mice. Groups of six BALB/c mice were inoculated intraperitoneally with the three mutant strains (10\({}^{4}\) CFU/mouse), the wild-type (WT) strain (10\({}^{3}\) CFU/mouse), or PBS (100 \(\mu\)L/mouse; N control). (**A**) At 21 days post-infection (dpi), all mice but the N control mice were orally challenged with the virulent ST2173 strain at a dose of LD50 (10\({}^{8}\) CFU/mouse). Positive (P) control mice were pre-inoculated with PBS similar to the N control and then challenged with ST2137 at the same dose. The antibody levels of (**B**) IgG, (**C**) IgM, and (**D** and **E**) IgG subclasses, including IgG1 and IgG2a, were measured using the sera collected from the infected mice at 0, 14, 28, and 35 dpi. Data pertaining to IgG, IgM, and IgG subclass levels are shown as the mean \(\pm\) SEM. In comparison with the WT, the \(\Delta f\)_r_t_R mutant caused rapid increments in IgG and IgM levels at 14 dpi, while the \(\Delta ssrAB\) mutant strain increased IgG levels at 28 dpi. (**F**) The levels of IgG1 and IgG2a are displayed as a ratio (IgG2a/IgG1).