铜绿假单胞菌对碳青霉烯类抗生素的耐药表型与外排泵表达水平的关系

目的 了解铜绿假单胞菌(Pseudomonas aeruginosa,Pa)临床菌株外排泵抑制剂对碳青霉烯类抗生素活性的影响;探索铜绿假单胞菌对亚胺培南(IMP)和美罗培南(MEP)的耐药性与其外排泵表达水平关系.方法 对IMP耐药的Pa采用琼脂对倍稀释法进行外排泵抑制剂carbanyl cyanide m-chlorophenylhydrazone(CCCP,107株)与Pile-Arg-β-naphthylamide(PAβN,71株)的抑制试验,观察IMP和MEP的MIC变化;对32株对IMP和MEP不同耐药表型的Pa,采用实时荧光定量PCR法检测3种外排泵基因(mexA、mexD、mexF)的表达量.结果 联合外排泵抑制剂后,IMP、MEP耐药率与之前相比差异均无统计学意义,其中IMP联合CCCP、PApN前后耐药率X2值分别为0.338和0.086,P＞0.05;MEP联合CCCP、PABN前后耐药率X2值分别为1.065和1.458,P＞0.05.MIC值没有变化的菌株占50%以上;仅8株P且的MIC值降至原MIC值的1/4.在27株碳青霉烯类耐药Pa株中,24株(88.9%)存在外排泵高表达;其中3种外排泵(MexAB-OprM、MexCD-OprJ、MexEF-OprN)均高表达的菌株数量最多,13株,占54.2%.MexAB-OprM和MexCD-OprJ同时高表达以及MexAB-OprM和MexEF-OprN同时高表达菌株分别有3株,各占12.5%.仅MexEF-OprN高表达及仅MexAB-OprM高表达的菌株分别为2株,各占8.3%;未见仅MexCD-OprJ高表达者.3种外排泵基因mexA、mexD、mexF在对IMP及MEP均敏感的菌株中表达水平分别为0.48±0.48、0.48±0.53和0.30±0.41,与碳青霉烯类耐药组之间差异均有统计学意义(P＜0.05),碳青霉烯类耐药组的表达水平高于对IMP及MEP均敏感组的表达水平.MexA的表达水平在IMP、MEP均耐药组和IMP耐药、MEP敏感组间差别有统计学意义,前者高于后者.结论 当外排泵抑制剂CCCP和PAβN浓度分别为5μg/ml和20μg/ml时,对碳青霉烯类抗Pa活性影响较小,不能明显增强其对耐药菌的抗菌活性.MexAB-OprM的高表达与MEP耐药性有关,而MexCD-OprJ和MexEF-OprN的高表达与IMP耐药性有关,与MEP耐药性的关系尚有待进一步研究.     

喹诺酮耐药基因qnrA的协同耐药分子机制研究

目的探讨喹诺酮类药物耐药基因qnrA介导的耐药性及其协同耐药分子机制。方法采用引物特异性PCR结合测序检测qnrA阳性株及gyraseA和parC基因变异情况,Phe-Aag-β-Naphthylamine(PAβN)抑制试验识别外排机制介导的耐药性,琼脂稀释法测定qnrA阳性株对喹诺酮类抗菌药的MIC值。结果QnrA阳性株对喹诺酮类抗菌药的耐药水平存在明显差异,gyraseA、parC基因变异或AcrAB-TolC外排泵等协同耐药机制存在与否与其关系密切。结论qnrA基因介导的耐药性存在协同机制,并加剧了qnrA阳性株的耐药性,给临床抗感染治疗带来严峻挑战。     

泛耐药鲍曼不动杆菌主动外排泵adeB基因表达及耐药性研究

目的：探讨细菌主动外排泵adeB基因在临床分离的泛耐药鲍曼不动杆菌耐药性中的作用。方法琼脂稀释法检测临床分离泛耐药鲍曼不动杆菌经外排泵抑制剂羰基氰氯苯腙(CCCP)处理前后对抗生素最小抑菌浓度(MIC)变化,以聚合酶链反应(PCR)、逆转录-聚合酶链反应(RT-PCR)检测外排泵基因adeB及其表达水平。结果50株泛耐药鲍曼不动杆菌临床株加入CCCP后,MIC值小于原值的1/4或1/4以下的四环素42株(84％)、氧氟沙星45株(90％)、亚胺培南45株(90％)、头孢噻肟50株(100％)、庆大霉素50株(100％)。50株泛耐药鲍曼不动杆菌临床株和4株敏感菌株均检测到adeB基因,但泛耐药株表达水平明显高于敏感株(P<0.01)。结论泛耐药鲍曼不动杆菌耐药性与外排泵adeB基因高表达密切相关。     

嗜麦芽窄食单胞菌对氟喹诺酮类药物主动外排机制的研究

目的 探讨嗜麦芽窄食单胞菌耐药株是否存在主动外排作用,以揭示嗜麦芽窄食单胞菌对氟喹诺酮类药物(FQS)的耐药机制.方法 应用琼脂稀释法检测嗜麦芽窄食单胞菌耐药株和敏感株在加入主动外排泵抑制剂羰基氰氯苯腙(CCCP)后对环丙沙星、左氧氟沙星的最低抑菌浓度(MIC) 变化情况.结果 55株嗜麦芽窄食单胞菌对FQS 耐药率较低,CCCP在体外能增强FQS抗菌活性,主动外排机制既存在于FQS 耐药株,也存在于FQS 敏感株, 对耐药株的影响更大.结论 嗜麦芽窄食单胞菌对FQS耐药与主动外排泵有关,泵抑制剂可部分降低这种耐药性.     

铜绿假单胞菌对喹诺酮类药物耐药机制的研究

目的研究30株铜绿假单胞菌对喹诺酮类药物的耐药机制。方法用PCR及测序法研究拓扑异构酶Ⅱ和Ⅳ的gyrA、gyrB、parC和parE基因;同时用琼脂稀释法测定环丙沙星、左氧氟沙星的MIC和加入羰基氢氯苯腙(CCCP)后的MIC,以确定存在外排机制。结果菌株中23株(76.7%)有gyrA突变,主要为Thr-83→Ile,第87位突变3株;10株(33.3%)parC基因突变,其中5株为Ser-87→Leu,3株第91位突变;gyrB和parE突变较少见。CCCP能显著降低环丙沙星和左氧氟沙星的MIC。结论铜绿假单胞菌的耐药性日趋严重,两类拓扑异构酶基因突变和外排泵机制是铜绿假单胞菌耐喹诺酮类药物的主要机制。     

嗜麦芽窄食单胞菌对氟喹诺酮类抗菌药的耐药机制研究

目的探讨嗜麦芽窄食单胞菌的DNA解旋酶和拓扑异构酶Ⅳ与氟喹诺酮类抗菌药物的耐药关系。方法选择氟喹诺酮类抗菌药物耐药且主动外排表型机制阴性的临床分离菌株19株和氟喹诺酮类抗菌药物敏感株10株,对其gyrA和parC的喹诺酮决定区域的基因进行PCR扩增,扩增产物进行测序,BLAST比对分析其氨基酸序列。采用SPSS软件进行统计学分析。结果 19株耐药菌的gyrA和parC基因各有7株菌核苷酸发生了碱基替换改变但均为同义突变,有2株菌在gyrA基因中124位氨基酸发生了突变由谷氨酸变为天冬氨酸(GAA→GAC),在氟喹诺酮类抗菌药物敏感组中也同样有1株发生同样的突变,两组比较差异无统计学意义(P>0.05)。结论嗜麦芽窄食单胞菌对氟喹诺酮类抗菌药物耐药与主动外排泵、外膜的通透性降低有关,但与解旋酶和拓谱异构酶Ⅳ的靶位点改变无显著关系。     

铜绿假单胞菌MexAB-OprM、MexXY-OprM主动外排系统的耐药作用

目的探讨MexAB-OprM、MexXY-OprM主动外排系统(外排泵)在多重耐药铜绿假单胞菌耐药机制中的作用。方法选取36株临床分离的多重耐药铜绿假单胞菌,采用琼脂二倍稀释法,测定环丙沙星对铜绿假单胞菌的MIC及进行泵抑制剂碳酰氰基-对-氯苯腙(CCCP)存在情况下的干预试验;用实时定量RT-PCR测定结构基因mexA、mexX的mRNA表达水平来判断MexAB-OprM、MexXY-OprM外排泵表达情况;用PCR法分别扩增其调控基因mexR、mexZ,并对其产物测序,用Blast软件在GenBank与已知序列比较,研究其过度表达的机制。结果在CCCP作用下,36株铜绿假单胞菌中24株菌对环丙沙星的敏感性(MIC)由20～320 mg/L提高到2.5～40 mg/L,主动外排表型阳性率为66.7%(24/36),15株菌高表达Mex-AB-OprM外排系统(41.7%),21株高表达MexXY-OprM外排系统(58.3%)。15株高表达MexAB-OprM外排系统铜绿假单胞菌同时表达MexXY-OprM外排系统;12株干预试验阴性的铜绿假单胞菌均无mexA、mexX高表达;随机选择mexA、mexX高表达的4株细菌均发生mexR、mexZ基因突变,出现氨基酸替代。结论主动外排系统MexAB-OprM、MexXY-OprM在多重耐药铜绿假单胞菌中过度表达是铜绿假单胞菌多重耐药的机制之一;外排泵MexAB-OprM、MexXY-OprM高表达分别与调控基因mexR、mexZ发生变异有关。     

外排泵adeB基因表达与鲍曼不动杆菌泛耐药的关系

目的探讨主动外排泵adeB基因在泛耐药鲍曼不动杆菌(Pandrug Resistance Acinetobacter Baumannii PRABA)耐药性中的作用。方法M-H琼脂稀释法检测临床分离PRABA对常用抗生素的最小抑菌浓度MIC,同时测定经外排泵抑制剂碳酰氰基-对-氯苯腙(CCCP)处理前后PRABA对常用抗生素的MIC值;PCR实时荧光RT-PCR检测外排泵adeB基因及其表达水平,PCR动力学经典算法:QR=(1+E)-△△CT,对基因表达进行相对定量分析。结果10株PRABA adeB基因阳性,2株全敏感鲍曼不动杆菌(PSABA)adeB基因阴性,PRABA菌株adeB基因mRNA的表达范围为2.5E+2~2.45E+3拷贝。结论PRABA菌株主动外排泵adeB基因高表达导致对常用抗生素泛耐药,甚至全部耐药。     

多重耐药鲍曼不动杆菌对替加环素的耐药机制研究

目的探讨多重耐药鲍曼不动杆菌(MDRAB)对替加环素敏感性降低的机制,为院内感染控制及临床合理用药提供理论依据。方法应用微量肉汤稀释法检测30株临床分离的非重复性MDRAB对替加环素的耐药性及应用外排泵抑制剂羰基氰化物间氯苯腙(CCCP)处理前后对替加环素的最低抑菌浓度(MIC);采用PCR技术扩增外排泵基因AdeABC、AdeFGH、AdeJ、AdeE、AbeM基因及外排泵调控基因AdeRS。结果 30株多重耐药鲍曼不动杆菌对替加环素的MIC值范围为0.25~8.00μg/L。其中替加环素敏感鲍曼不动杆菌(TSAB)共27株(90%),替加环素非敏感鲍曼不动杆菌(TNAB)共3株(10%);加入20μg/ml CCCP外排泵抑制剂进行处理,发现TNAB组菌株MIC值均下降≥4倍,为外排泵阳性菌株;PCR扩增外排泵基因:不敏感组(TNAB)菌株除ade E基因外均为阳性,敏感组(TSAB)中AdeA、AdeB、AdeC、AdeR基因检出率均为90%,Ade S基因检出率93.3%,AdeF、AdeG、AdeH基因检出率83.3%。所有菌株ade J和Abe M均为阳性,未发现ade E基因。结论外排泵系统Ade ABC和Ade FGH可能在多重耐药鲍曼不动杆菌对替加环素敏感性下降机制中起重要作用,而外排泵系统AdeIJK和AbeM可能与多重耐药鲍曼不动杆菌对替加环素耐药无关。     

I类整合子介导的铜绿假单胞菌耐药性分析

目的 检测铜绿假单胞菌(PA)的Ⅰ类整合子,探讨其与细菌耐药性的关系.方法以VITEK-AMS 微生物自动分析仪进行细菌鉴定和药敏试验;PCR扩增Ⅰ类整合酶基因,电泳分析PCR扩增产物.结果 116株PA中,Ⅰ类整合子检出率为25.9%(30/116).Ⅰ类整合子阳性PA对氨基糖苷类、喹诺酮类及头孢菌素类药物的耐药率较...     

美罗培南耐药铜绿假单胞菌多药外排泵研究

目的 研究美罗培南耐药铜绿假单胞菌的多药外排泵机制.方法 采用琼脂稀释法测定美罗培南对141株铜绿假单胞菌的最低抑菌浓度(MIC),同时观察加入泵抑制剂MC207110后的美罗培南对141株铜绿假单胞菌MIC变化,检测多药外排泵机制.实时定量PCR检测外排泵mRNA水平的表达.PCR扩增外排泵编码基因及调控基因并测序分...     

Identification and characterization of a novel efflux-related multidrug resistance phenotype in Staphylococcus aureus

Moxifloxacin is a C8-methoxy (C8-OMe) fluoroquinolone that is highly active against Staphylococcus aureus, including many strains resistant to older fluoroquinolones such as ciprofloxacin. Available data indicate that it is a poor substrate for the NorA multidrug efflux pump. We produced a mutant of S. aureus in vitro (SA-K2068) with a novel non-NorA-mediated multidrug resistance phenotype characterized by raised MICs of several fluoroquinolones, including the C8-OMe fluoroquinolones, moxifloxacin and gatifloxacin, and the organic cations ethidium and tetraphenylphosphonium. Reserpine reduced MIC increases by two- to eight-fold. SA-K2068 also demonstrated reduced accumulation of moxifloxacin, gatifloxacin and enoxacin, and increased efflux of ethidium, activities that were completely blocked by carbonyl cyanide m-chlorophenyl hydrazone (CCCP); competition experiments indicated that a single pump was responsible for the phenotype. The effect of CCCP and ionophores identified the proton motive force as the source of energy for efflux. These data, combined with previous work from our laboratory and genome sequence data, indicate that S. aureus possesses several multidrug efflux pump proteins and it is apparent that C8-OMe fluoroquinolones can be substrates for such pumps.     

Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa

Pseudomonas aeruginosa expresses a low level of the MexAB-OprM efflux pump and shows natural resistance to many structurally and functionally diverse antibiotics. The mutation that has been referred to previously as nfxC expresses an additional efflux pump, MexEF-OprN, exhibiting resistance to fluoroquinolones, imipenem, and chloramphenicol and hypersusceptibility to beta-lactam antibiotics. To address the antibiotic specificity of the MexEF-OprN efflux pump, we introduced a plasmid carrying the mexEF-oprN operon into P. aeruginosa lacking the mexAB-oprM operon. The transformants exhibited resistance to fluoroquinolones, trimethoprim, and chloramphenicol but, unlike most nfxC-type mutants, did not show beta-lactam hypersusceptibility. The transformants exhibited additional resistance to tetracycline. In the next experiment, we analyzed the MexEF-OprN pump subunit(s) responsible for substrate selectivity by expressing MexE, MexF, OprN, and MexEF in strains lacking MexA, MexB, OprM, and MexAB, respectively. The MexEF-OprM/DeltaMexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to beta-lactam antibiotics compared with the host strain. The MexAB-OprN/DeltaOprM, MexAF-OprM/DeltaMexB, and MexEB-OprM/DeltaMexA mutants exhibited antibiotic susceptibility indistinguishable from that in the mutant lacking both types of efflux pumps. The results imply that the MexEF-OprM pump selects substrates by a MexEF functional unit. Interestingly, OprN did not link functionally with the MexAB complex, despite the fact that OprM interacted functionally with MexEF.     

Involvement of an Active Efflux System in the Natural Resistance of Pseudomonas aeruginosa to Aminoglycosides

A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn501 insertion mutagenesis of Pseudomonas aeruginosa PAO1. Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB. This locus also contained a putative regulatory gene, mexZ, transcribed divergently from the efflux operon. Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host. Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides. Furthermore, this system appeared to function with an outer membrane protein, OprM. While the present paper was being written and reviewed, genes with a sequence identical to our pump genes, mexXY of P. aeruginosa, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations in Escherichia coli hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob. Agents Chemother. 43:415-417, 1999). Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of P. aeruginosa to aminoglycosides. Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of the mexXY operon in P. aeruginosa had no detectable effect on susceptibility to these agents.     

Clinical significance of overexpression of multiple RND-family efflux pumps in Bacteroides fragilis isolates

OBJECTIVES: The aim of the present study was to determine correlation between bmeB efflux pump overexpression and resistance to fluoroquinolones and beta-lactams in Bacteroides fragilis clinical isolates (n = 51) and the effects of broad-spectrum efflux pump inhibitors (EPIs) on the MICs of the test antibiotics. METHODS: Susceptibility to garenoxacin, levofloxacin, moxifloxacin, cefoxitin and faropenem +/- EPIs (CCCP, MC-207,110, reserpine and verapamil) was determined. Expression of bmeB efflux pumps was measured, topoisomerase genes were sequenced and beta-lactamase production was determined. RESULTS: Isolates were grouped into categories based on susceptibility patterns, topoisomerase sequence and efflux pump expression. Panel I isolates (19/51, 37.3%) were highly resistant to fluoroquinolones and cefoxitin (resistance to all agents was significantly reduced by EPIs, P < 0.05), had a point mutation in gyrA (C-->T) causing a Ser-82-->Phe substitution, and overexpressed bmeB4 and bmeB15. Panel II isolates (7/51; 13.7%) had intermediate-level resistance to fluoroquinolones and cefoxitin and a GyrA substitution. Panel IIIA isolates (21/51; 41.2%) had intermediate-level fluoroquinolone resistance and high-level cefoxitin resistance [resistance to all agents was significantly reduced by EPIs (P < 0.05)] and overexpressed bmeB4 and bmeB15. Panel IIIB isolates (4/51; 7.8%) had low-level fluoroquinolone resistance and high-level resistance to cefoxitin [cefoxitin resistance was significantly reduced by EPIs (P < 0.05)] and overexpressed bmeB4, bmeB6, bmeB10 and bmeB14. All isolates were beta-lactamase-positive. CONCLUSIONS: These data suggest that bmeB efflux pump overexpression can (i) cause low- to intermediate-level clinically relevant fluoroquinolone resistance; (ii) be coupled with GyrA substitutions to cause high-level fluoroquinolone resistance; (iii) contribute to high-level clinically relevant resistance to beta-lactams.     

Relative contributions of the AcrAB,MdfA and NorE efflux pumps to quinolone resistance in Escherichia coli

Quinolones are widely used, broad-spectrum antimicrobial agents. In screens for genes that, when overexpressed, allow Escherichia coli to grow on otherwise lethal concentrations of the fluoroquinolone norfloxacin, the ydhE gene was identified. We have shown that ydhE encodes a multidrug efflux pump with a narrower substrate range than that of its closest homologue, encoded by norM, and named the gene norE. The relative contributions to drug resistance of NorE compared with the two other known E. coli quinolone pumps, AcrAB and MdfA, have been defined. Overexpression of each of the three pumps separately resulted in roughly similar levels of quinolone resistance, whereas simultaneous overexpression of norE or mdfA in combination with acrAB gave synergic increases in quinolone resistance. The level of quinolone resistance mediated by efflux pumps seems to be constrained to an approximately 10-fold maximum, even with increased production of the pumps. We measured the drug resistance of an isogenic set of strains containing the various permutations of single, double and triple drug efflux pump mutants. The DeltanorE and DeltamdfA mutants were somewhat more susceptible to fluoroquinolones than the parent strain, and acrAB mutants were four- to six-fold more susceptible. Mutants lacking two or all three efflux pumps were not significantly more susceptible to fluoroquinolones than those lacking only one of the three pumps.     

Evidence of active efflux in resistance to ciprofloxacin and to ethidium bromide by Mycoplasma hominis

The uptake of fluoroquinolones was characterized for the fluoroquinolone-susceptible strain PG21 of Mycoplasma hominis. Accumulation of fluoroquinolones appeared to occur by passive diffusion. Addition of arginine as the energizer significantly reduced the uptake of fluoroquinolones, suggesting the presence of an energy-dependent efflux process. Reserpine and orthovanadate, two multidrug pump inhibitors, increased significantly the ciprofloxacin (CIP) uptake. In contrast, such a strong effect was not observed for moxifloxacin and pefloxacin uptakes. Two ethidium bromide (EtBr)-resistant strains, selected in vitro, showed a resistance profile compatible with a multidrug-resistant phenotype, with increased MICs for the hydrophilic fluoroquinolones, CIP and norfloxacin, EtBr, and acriflavine. Taking the EtBr-resistant strain RB1La as a model, a significant decrease of the CIP and EtBr uptakes was observed compared to the reference strain PG21. In the presence of reserpine and orthovanadate, both inhibitors of ATP-dependent efflux pumps, the CIP uptake increased significantly, reaching approximately the same level as that of the susceptible strain. Similar results were obtained with EtBr uptake and efflux experiments. Our data suggest the presence of an active efflux system, possibly an ABC-type efflux pump, implicated in the resistance to CIP and unrelated compounds like EtBr in the human mycoplasma M. hominis.     

The efflux pump inhibitor reserpine selects multidrug-resistant Streptococcus pneumoniae strains that overexpress the ABC transporters PatA and PatB

One way to combat multidrug-resistant microorganisms is the use of efflux pump inhibitors (EPIs). Spontaneous mutants resistant to the EPI reserpine selected from Streptococcus pneumoniae NCTC 7465 and R6 at a frequency suggestive of a single mutational event were also multidrug resistant. No mutations in pmrA (which encodes the efflux protein PmrA) were detected, and the expression of pmrA was unaltered in all mutants. In the reserpine-resistant multidrug-resistant mutants, the overexpression of both patA and patB, which encode ABC transporters, was associated with accumulation of low concentrations of antibiotics and dyes. The addition of sodium orthovanadate, an inhibitor of ABC efflux pumps, or the insertional inactivation of either gene restored wild-type antibiotic susceptibility and wild-type levels of accumulation. Only when patA was insertionally inactivated were both multidrug resistance and reserpine resistance lost. Strains in which patA was insertionally inactivated grew significantly more slowly than the wild type. These data indicate that the overexpression of both patA and patB confers multidrug resistance in S. pneumoniae but that only patA is involved in reserpine resistance. The selection of reserpine-resistant multidrug-resistant pneumococci has implications for analogous systems in other bacteria or in cancer.