The Role Of Multidrug Resistant Efflux Pumps And Its Regulators In The Development Of Fluoroquinolone Resistance In Salmonella Enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is considered as the main food-borne pathogen. Fluoroquinolones are the main drugs for the treatment of salmonellosis. In the selection pressure of fluoroquinolones S. enterica serovar Typhimurium produce resistance to it. Today, target site mutation in quinolone resistant determining regions (QRDRs) and active multidrug resistant (MDR) efflux pump are main resistant mechanism of S. enterica serovar Typhimurium against fluoroquinolone. Among them, a single target site mutation in the QRDR of GyrA in a susceptible S. enterica serovar Typhimurium led to the strain exhibiting lower susceptibility to fluoroquinolones, wherease not resistance. However, a lot of clinical isolates harborig a target site mutation in the QRDR of GyrA produced resistance to fluoroquinolones. As a result, active MDR efflux pump plays a predominant role in the development of fluoroquinolone resistance of S. enterica serovar Typhimurium.In MDR S. enterica serovar Typhimurium AcrAB-TolC is an important MDR efflux pump which can prevent drugs from entering into cell. Likewise, it captures the substrates from the periplasm or the outer leaflet of the cytoplasmic membrane and the overexpression of single AcrAB in a susceptible S. enterica serovar Typhimurium lacking individual acrAB gene did not lead to the mutant exhibiting resistance to quinolones.This may indicate the contribution of some other efflux pumps in the fluoroquinolone resistance in S. enterica serovar Typhimurium. In the development of MDR S. enterica serovar Typhimurium some global regulators may participate in the regulation of the expression of MDR efflux pumps. However, no study clearly showed which regulator was the main protein controlling the expression of MDR efflux pumps extruding fluoroquinolones. Likewise, whether the main regulator played a role on the development of fluoroquinolone resistant mutants. In this experiment, spontaneous MDR mutants will be obtained from a susceptible S. enterica serovar Typhimurium in the selection pressure of ciprofloxacin. After that, active MDR efflux pumps extruding fluorowuinolones and its main regulator will be decided in the selcted spontaneous mutants. At last, the mutant prevention concentration (MPC) of parent strain and its first-step mutants agninat fluoroquinolone in the presence, absence and overexpression of the main regularor will be also tested. In this experiment some fresh knowledge will be provided in the field of MDR mechanism of S. enterica serovar Typhimurium. Likewise, some new theory basement will be drawn for preventing the development of clinical MDR isolates of S. enterica serovar Typhimurium.Spontaneous mutants were selected via several passages of S. enterica sreovar Typhimurium CVCC541susceptible strain (ST) on M-H agar with increasing concentrations of ciprofloxacin. The QRDRs of gyrA, gyrB, parC, and parE in the selected spontaneous mutants was amplified and sequenced. Accumulation of ciprofloxacin and enorofloxacin in the selected mutants in the presence and absence of efflux pump inhibitors (CCCP or PAβN) was measured with InfiniteTM200microplate readers by the modified fluorometric method. The expression level of MDR efflux pumps were determined by real time RT-PCR.The ramA or marA gene was inactivated by insertion of the kan gene in ST. After that, the deletions were transferred to the spontaneous mutants by P22HT105/int transductions. MICs of the strains with inactivated RamA or MarA to different drugs were tested. Likewise, the promoter regions of MDR efflux pumps (AcrAB and MdtK) and the sequences of the regulatory loci RamRA, MarRA, SoxRS and AcrR from ST and the selected spontaneous mutants were amplified and sequenced. At last, the RamA was overexpressed on a recombinant plasmid pGEXΦ (gst-ramA) in ST and a spontaneous mutant with inactivated RamA and the expression level of active efflux pumps were also tested.The growth speed of ST and SR (STramA::aph) were measured. The first-step mutants from ST and SR were selected on M-H agar containing different concentrations of of ciprofloxacin and then, tested their QRDR of gyrA. The RamA was overexpressed in the first-step mutants SR4-3(Ser83→Phe) and SRI-9(Asp87→Tyr) from SR. The MPCs of ciprofloxacin and enrofloxacin against ST, SR, their first-step mutants and the strains with the overproduction of RamA were tested. Likewise, the mutant frequencies of the strains to ciprofloxacin were also determined.Seven spontaneous mutants (SI1to SI7) were obtained which exhibited decreased susceptibility to multidrugs.The SI2(CIP:MIC0.1mg/1) strain without any target site mutation in its QRDRs exhibited dectreasd susceptibility to tetracycline, chloramphenicol, florfenicol as well as quinolones. The SI6(CIP:MIC16mg/1) strain harboring Ser83→Phe in the QRDR of GyrA exhibited high-level fluoroquinolones resistance and showed significantly increase in the MICs of chloramphenicol, florfenicol, tetracycline compared to that of SI2. The amounts of ciprofloxacin and enrofloxacin accumulated in SI2and SI6appeared to be lower than that in ST. After CCCP was added, the amounts of the drug accumulated in SI2slightly increased, whereas lower than that in ST. After the addition of PAβN, the accumulation of ciprofloxacin in SI2increased and was near to that in ST. The amount of ciprofloxacin accumulated in SI6in the presence of CCCP dramatically increased and was near to that in ST in the presence of CCCP. Nevertheless, the concentration of ciprofloxacin accumulated in SI6in the presence of PAPN was lower than that in ST in the presence of PAβN. On the other hand, the change trends of the concentrations of norfloxacin accumulated in SI2and SI6were similar to that of ciprofloxacin. The results of real-time RT-PCR showed that the expression level of acrA and mdtK in S12and S16increased6.08-,3.87-fold and30.1-,8.15-fold, respectively, compared to that in ST.When RamA was inactivated in ST, the susceptibility of SR (STramA::aph) to the tested drugs did not dramatically change. However, when RamA was inactivated in SI2, MICs of SI2R (SI2ramA::aph) to the tested drugs except for nalidixic acid decreased2-to8-fold. The MICs of SI2R to ciprofloxacin, sarafloxacin, enrofloxacin and nalidixic acid were the same as that of SR. On the other hand, when RamA was overexpressed in SR, MICs of STRA to the drugs tested increased2-to6-fold compared to that of SR. The MICs of STRA to ofloxacin and tetracycline were the same as that of SI2. The MICs of STRA to nalidixic acid was higher than that of SI2. The MICs of STRA to the remaining antimicrobial agents did not significantly change except that the MICs of STRA to ciprofloxacin, norfloxacin and enrofloxacin exhibited2-fold decrease compared to that of SI2. Likewise, the expression level of acrA in STRA was similar to that in SI6and MdtK expression in STRA was similar to SI2. Based on the abovel-mentioned results, it was obvious that RamA was the main factor that controled the susceptibility of SI2to ciprofloxacin by activating MdtK as well as increasing the expression level of acrAB.When RamA was inactivated in SI6, MICs of SI6R (SI6ramA::aph) to the tested drugs except for nalidixic acid exhibited2-to8-fold decrease compared to that of SI6. The MICs of SI6R to ciprofloxacin decreased8-fold (CIP:SI6,16mg/L; SI6R,2mg/L), which indicated that RamA played a predominant role in the ciprofloxacin resistance of SI6. However, the SI6R strain still exhibited resistance to the tested fluoroquinolones. When RamA was overexpressed in SI3R (SI3ramA::aph), the MICs of SI3RA to ciprofloxacin, norfloxacin, tetracycline and chloramphenicol increased2-to16-fold compared to that of SI3R. The susceptibility of SI3RA to ofloxacin, sarafloxacin, and florfenicol was similar to that of SI3R. Unexpectedly, the susceptibility of SI3RA to enrofloxacin, nalidixic acid and erythromycin all exhibited reduced MICs compared to that of SI3R. To be noticed, the MICs of SI3RA to ciprofloxacin was lower than that of SI6R. The above-mentioned results demonstrated that some other efflux pumps not regulated by RamA contributed in the ciprofloxacin resistance of SI6. Likewise, the MICs of SI6R to tetracycline, a good substrate of AcrAB-TolC, reverted to that of ST. The MICs of SI3RA to chloramphenicol, another good substrate of AcrAB-TolC, was the same as that of SI6. The expression level of acrAB in SI6was the same as that in STRA with the overproduction of RamA. Therefore, RamA was responsible for increasing the expression level of acrAB in SI6. The cooperation of AcrAB-TolC and the other efflux pumps contributed in ciprofloxacin resistance.Only the changes in RamR were found in SI2and SI6. When RamR was complemented in SI2, MICs of SI2RR to fluoroquinolones decreased2-to4-fold compared to that of SI2. However, the susceptibility of SI1RR did not revert to that of ST. Likewise, when MarA, another regulator of MDR efflux pumps was inactivated in ST, SI2and SI6, the susceptibility of the mutants to the tested drugs did not dramatically change.During the logarithmic phase growth the growth speed of SR (STramA::aph) was higher than that of ST. The deficiency of RamA diminished the appearance of the first-step mutants harboring a target site mutation in the QRDR of GyrA. The overproduction of RamA in the SR4-3(ramA::aph+Ser83→Phe in GyrA) or SRI-9(ramA::aph+Asp87→Tyr in GyrA) increased the MPC to ciprofloxacin, whereas decreased the MPC to enrofloxacin. However, the MPC and mutant selection window (MSW) of ciprofloxacin against the SR4-3and SR1-9strains from SR were all lower than that of the mutant harboring the same target site mutation (ST8-1or ST2-6) from ST. Under the selection pressure of the same concentration of ciprofloxacin, the mutant frequencies of the STRA,83RA, and87RA strains with the overproduction of RamA all significantly augmented compared to that of the SR, SR4-3and SRI-9strains, respectively. While the mutant frequencies of the SR, SR4-3and SRI-9strains were slightly lower than that of the ST, ST8-1and ST2-6strains, respectively. As a result, the overexpression of RamA promoted the development of fluoroquinolones-resistant S. enterica serovars Typhimurium. The inhibition of RamA could decrease the appearance of the fluoroquinolones-resistant mutants.In conclusion, the fellowing results were first proven in this experiment. At first, MDR efflux pump MdtK participated in the development of ciprofloxacin resistance of S. enterica serovars Typhimurium and the co-operation of it and AcrAB promoted the development of ciprofloxacin resistant mutants. The main regulator RamA could activate the expression of MdtK, whereas not lead to its overexpression. In the secand, RamR, a local repressor of RamA, removed from the promoter region of ramA due to the aminao acid deletion in the binding-substrate region in the selection pressure of ciprofloxacin, which led to the expression of RamA. At last, RamA was a mian regulator of MDR efflux pumps in S. enterica serovars Typhimurium. The expression of RamA promoted the development of ciprofloxacin resistant mutants and the inhibition of RamA could decrease the appearance of its. These new discoveries enriched the contents of MDR mechanism of S. enterica serovars Typhimurium. In addition, RamA may be a new target in the research and development of MDR efflux pump inhibitors.