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Ciprofloxacin is the choice treatment for infections caused by  Salmonella  Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn 5  transposon mutants of  S . Typhi were screened.  S.  Typhi  zxx ::EZ-Tn 5  mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn 5  transposon insertions were sequenced. Null mutants of interrupted genes, as well as inducible genetic constructs, were produced using site-directed mutagenesis, to corroborate phenotypes. SDS-PAGE and Real-time PCR were used to evaluate the expression of proteins and genes, respectively. Five mutants with increased ciprofloxacin susceptibility were found in the screening. The first confirmed mutant was the glutamine synthetase-coding gene  glnA . Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the  glnA  mutant. Expression of  ompF  increased four times in the  glnA  null mutant compared to WT strain. To understand the relationship between the expression of  glnA  and  ompF , a strain with the  glnA  gene under control of the tetracycline-inducible P tet  promoter was created, to modulate  glnA  expression. Induction of  glnA  decreased expression of  ompF , at the same time that reduced susceptibility to ciprofloxacin. Expression of sRNA MicF, a negative regulator of OmpF was reduced to one-fourth in the  glnA  mutant, compared to WT strain. In addition, expression of  glnL  and  glnG  genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the  glnA  mutant. Further studies indicate that deletion of  glnG  decreases susceptibility to CIP, while deletion of  micF  gene increases susceptibility CIP. Our findings indicate that  glnA  inactivation promotes  ompF  expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.

frontiers in microbiology

Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi