【作者】 孙亚伟；

【导师】 袁宗辉；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2011， 博士

【摘要】 鼠伤寒沙门氏菌是一类主要食源性病原菌,氟喹诺酮类是治疗该菌感染的首选药物。在该类药物选择压力下,细菌产生耐药。目前,喹诺酮耐药决定区靶位点突变和活性多重耐药泵是鼠伤寒沙门氏菌对氟喹诺酮类主要耐药机制。其中,喹诺酮耐药决定区GyrA内单个靶位点突变仅使细菌对氟喹诺酮类敏感性降低,而不导致其对该类药物耐药。然而,多数临床分离在GyrA内携带单个靶位点突变的鼠伤寒沙门氏菌却表现对氟喹诺酮类耐药,所以活性多重耐药泵在鼠伤寒沙门氏菌对氟喹诺酮类耐药过程中具有重要作用。鼠伤寒沙门氏菌细胞膜中AcrAB-TolC是最重要的一个多重耐药泵,它可阻止药物进入到细胞内,但该泵蛋白在细胞质间隙或细胞内膜外层俘获药物,同时在敏感菌中仅过表达该多重耐药泵也不导致细菌对喹诺酮类耐药,故其它家族多重耐药泵必然参与细菌耐药过程。在多重耐药泵表达过程中,多个调控蛋白可能参与其表达调节,然而哪个调控蛋白是外排氟喹诺酮类多重耐药泵主要调控蛋白?该蛋白对氟喹诺酮类耐药突变株产生具有何种影响?本实验用环丙沙星体外诱导敏感鼠伤寒沙门氏菌,获得系列多重耐药自发突变株,随后在所选自发突变株中确定外排氟喹诺酮类多重耐药泵及其主要调控蛋白,最后研究主要调控蛋白对敏感菌及其首次耐药突变株突变抑制浓度影响。该研究是鼠伤寒沙门氏菌对氟喹诺酮类多重耐药理论的一次有益补充,也将为临床多重耐药鼠伤寒沙门氏菌防治提供新的理论基础。鼠伤寒沙门氏菌CVCC541(ST)在含有不同浓度环丙沙星MH平板上逐步筛选,获得对环丙沙星敏感性降低并表现多重耐药的系列诱导菌。所选诱导菌经PCR扩增、测序,检测其喹诺酮耐药决定区(Quinolone Resistant Determine Regios, QRDRs)内靶位点突变。随后,通过检测多重耐药泵抑制剂(CCCP或PAβN)对部份诱导菌细胞内环丙沙星和诺氟沙星蓄积浓度变化,初步阐明所选诱导菌中外排氟喹诺酮类多重耐药泵。对所选诱导菌提取总RNA并反转录,以体外合成cDNA为模板,检测诱导菌细胞内外排氟喹诺酮类多重耐药泵相对表达水平。在ST中,按照PCR产物一步失活染色体基因方法,逐个失活多重耐药泵调控蛋白(RamA和MarA)。然后,利用噬菌体将失活基因逐个传导至诱导菌中,对调控蛋白失活后ST及其诱导菌检测不同药物敏感性。另外,在RamA失活菌中互补RamA,同时检测多重耐药泵表达水平。检测诱导菌中,活性多重耐药泵启动子区和主要调控蛋白(RamRA, MarRAB, SoxRS, AcrR)基因序列并对发生突变的调控蛋白在诱导菌中互补,阐明诱导菌中多重耐药泵主要调控蛋白表达的分子机制。检测ST和SR (STramA::aph)菌生长速度,体外筛选ST和SR环丙沙星首次耐药突变株,随后检测其喹诺酮耐药决定区内GyrA靶位点突变。在SR突变株SR4-3(Ser83→Phe)或SR1-9(Asp87→Tyr)中过表达RamA。测定ST、ST8-1(Ser83→Phe)、ST2-6(Asp87→Tyr)、SR、SR4-3、SR1-9及其RamA过表达菌对环丙沙星和恩诺沙星突变抑制浓度,同时检测各菌对环丙沙星突变抑制率。在环丙沙星选择压力下,获得7个对环丙沙星敏感性逐步降低的诱导菌(SI1-SI7)。其中,SI2(CIP：MIC0.1mg/l)在QRQRs区内没有任何靶位点突变,但表现对所测药物敏感性下降；SI6(CIP：MIC16mg/l)在GyrA中携带Ser83→Phe突变,但表现多重耐药,同时四环素、氯酶素和氟苯尼考MIC值与SI7相比没有继续上升。SI2和SI6中环丙沙星和诺氟沙星蓄积浓度均低于ST菌。当加入CCCP后,SI2中环丙沙星蓄积浓度增加但仍低于无抑制剂存在时ST菌药物蓄积浓度；当加入PApN后,环丙沙星在SI2中蓄积浓度显著上升与ST菌药物蓄积浓度相似；在SI6中,加入CCCP后,环丙沙星蓄积浓度与ST菌药物蓄积浓度相似；然而加入PAβN后,环丙沙星蓄积浓度低于ST菌。另外,SI2和SI6在耐药泵抑制剂存在下,诺氟沙星蓄积浓度变化与环丙沙星蓄积浓度变化趋势相似。Real time RT-PCR结果表明,SI6中仅检测到AcrAB和MdtK表达且其表达量相对ST分别增加30.1和8.15倍：另外,SI2中AcrAB和MdtK表达量相对ST增加6.08和3.87倍。当调控蛋白RamA在ST (CIP:MIC0.0125mg/1)中失活后,SR菌对所测药物敏感性相对ST没有显著改变。当RamA在SI2中失活后,SI2R对所测药物除萘啶酸外下降2-8倍,其中SI2R对环丙沙星、沙拉沙星、恩诺沙星和萘啶酸MIC值与SR菌所测值相同；当RamA在SR中过表达后,STRA对所测药物MIC值相对SR菌上升2-6倍,其中STRA对氧氟沙星和四环素MIC值与SI2菌所测值相同,对环丙沙星、诺氟沙星和恩诺沙星MIC值是S12菌所测值0.5倍。另外,STRA中AcrAB表达水平与SI6中相似,MdtK表达水平与SI2中相似。所以,RamA是SI2中主要调控蛋白,它通过激活MdtK和增加AcrAB表达水平,使细菌对多个药物敏感性下降。在SI6(CIP：MIC16mg/l)中,RamA失活后,SI6R(CIP：MIC2mg/l)对所测药物除萘啶酸外MIC值下降2-8倍,但SI6R仍对所测氟喹诺酮类耐药。当RamA在SI3R中过表达后,SI3RA对环丙沙星、诺氟沙星、四环素和氯霉素MIC值增加2-16倍；对氧氟沙星、沙拉沙星和氟苯尼考敏感性保持稳定；对恩诺沙星、萘啶酸和红霉素MIC值反而下降。尽管SI3RA和SI6R携带有相同GyrA靶位点突变,SI3RA对喹诺酮类MIC值均低于SI6R所测值。另外,SI6R对四环素MIC值与ST相同；SI3RA对氯霉素MIC值与SI6相同；AcrAB表达水平在STRA(?)SI6中相似。所以,RamA主要控制AcrAB表达水平,AcrAB和其它多重耐药泵协同作用导致SI6对氟喹诺酮类耐药。SI2和SI6仅在ramR序列中发现突变。当RamR在SI2中互补后,SI2RR对氟喹诺酮类MIC值下降2-4倍,但未回复到ST敏感水平。另外,当调控蛋白MarA在ST、SI2和SI6中失活后,细菌对所测药物敏感性均没有明显改变。在对数生长期,SR (STramA::aph)菌生长速度快于ST菌。在不同环丙沙星浓度选择下,RamA缺失降低携带GyrA(?)位点突变株筛选几率。RamA过表达导致SR首次耐突变株SR4-3和SR1-9对环丙沙星MPC值增加,对恩诺沙星MPC值下降。SR4-3和SR1-9对环丙沙星MPC值、突变选择框均低于分别携带相同靶点突变的ST首次耐突变株ST8-1和ST2-6。在相同浓度环丙沙星作用下,RamA过表达菌83RA和87RA拥有最大耐药突变率；SR4-3和SR1-9耐药突变率分别低与ST8-1和ST2-6。所以,RamA表达是细菌对环丙沙星产生耐药的一个主要原因,抑制RamA表达能够减少耐药菌出现。总之,本研究首次证实：(1)多重耐药泵MdtK参与了鼠伤寒沙门氏菌对环丙沙星耐药过程,它与AcrAB协同作用促进细菌多重耐药突变株产生。多重耐药泵调控蛋白RamA可激活mdtK表达,但不能导致其高水平表达。(2)在环丙沙星选择压力下,敏感鼠伤寒沙门氏菌中多重耐药泵主要调控蛋白RamA的抑制蛋白RamR,由于底物结合区氨基酸缺失导致其从ramA启动子区解离,进而启动ramA表达。(3)RamA是一个主要多重耐药泵调控蛋白,其表达促进了环丙沙星耐药突变株蓄积、增值,抑制RamA表达水平可减低环丙沙星耐药突变株出现。本研究以上发现丰富了鼠伤寒沙门氏菌的多重耐药理论,同时预示RamA可能成为鼠伤寒沙门氏菌多重耐药泵抑制剂研发的一个新靶标。更多还原

【Abstract】 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.更多还原