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Active efflux pump is a primary fluoroquinolone resistant mechanism of clinical isolates of  Salmonella enterica  serovar Typhimurium. RamA is an essential element in producing multidrug resistant (MDR)  S.enterica  serovar Typhimurium. The aim of the present study was to elucidate the roles of RamA on the development of ciprofloxacin, the first choice for the treatment of salmonellosis, resistance in  S. enterica  serovar Typhimurium. Spontaneous mutants were selected via several passages of  S. enterica  serovar Typhimurium CVCC541 susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin (CIP). Accumulation of ciprofloxacin was tested by the modified fluorometric method. The expression levels of MDR efflux pumps were determined by real time RT-PCR. In ST and its spontaneous mutants, the  ramA  gene was inactivated by insertion of the  kan  gene and compensated on a recombinant plasmid pGEXΦ( gst-ramA ). The mutant prevention concentration (MPC) and mutant frequencies of ciprofloxacin against ST and a spontaneous mutant in the presence, absence and overexpression of RamA were tested. Four spontaneous mutants (SI1-SI4) were obtained. The SI1 (CIP MICs, 0.1 mg/L) without any target site mutation in its quinolone resistant determining regions (QRDRs) and SI3 (CIP MICs, 16 mg/L) harboring the Ser83→Phe mutation in its QRDR of GyrA strains exhibited reduced susceptibility and resistance to multidrugs, respectively. In SI1, RamA was the main factor that controlled the susceptibility to ciprofloxacin by activating MdtK as well as increasing the expression level of  acrAB . In SI3, RamA played predominant role in ciprofloxacin resistance via increasing the expression level of  acrAB . Likewise, the deficiency of RamA decreased the MPCs and mutant frequencies of ST and SI2 to ciprofloxacin. In conclusion, the expression of RamA promoted the development of ciprofloxacin resistant mutants of  S. enterica  serovar Typhimurium. The inhibition of RamA could decrease the appearance of the ciprofloxacin resistant mutants.

The Role of RamA on the Development of Ciprofloxacin Resistance in Salmonella enterica Serovar Typhimurium