碳青霉烯类抗生素耐药铜绿假单胞菌外排泵和外膜孔蛋白表达水平的研究

目的探讨我国临床分离的碳青霉烯类抗生素耐药铜绿假单胞菌外排泵MexAB—OprM和外膜孔蛋白OprD2表达水平及其与碳青霉烯类抗生素耐药性之间的关系。方法收集我国15个省市28所医院临床分离的铜绿假单胞菌655株，琼脂稀释法测定该菌加入外排泵抑制剂羟基氰氯苯腙（CCCP）前后对亚胺培南和美罗培南的MIC；RTPCR检测外排泵MexAB-OprM和孔蛋白OprD2的表达水平；十二烷基硫酸钠-聚丙酰胺凝胶电泳（SD-PAGE）和蛋白印迹（Western blot）检测外膜孔蛋白OprD2。结果收集到的655株铜绿假单胞菌菌株对亚胺培南和美罗培南敏感率分别为52．5％和59．2％，加入CCCP前后其对亚胺培南和美罗培南的敏感率无明显变化；52．0％（51／9B）美罗培南耐药株MexAB-OprM显著高表达（P〈0．05），43．7％（59／135）亚胺培南耐药株OprD2显著低表达（P〈0．05），14．8％（20／135）亚胺培南耐药株OprD2表达缺失；SDS-PAGE和Western—blot显示与标准菌株PAO1相比，OprD2表达水平减低菌株ZZ20显影减低，表达缺失菌株BJ1不显影。结论MexAB-OprM表达升高和OprD2表达降低甚至缺失是我国铜绿假单胞菌对美罗培南和亚胺培南耐药的重要机制。     

铜绿假单胞菌对碳青霉烯类抗生素的耐药机制——耐药株外膜蛋白OprD2缺失

目的：研究铜绿假单胞菌对碳青霉烯类抗生素的耐药机制。方法：以亚胺培南为代表,应用十二烷酸磺酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)外膜蛋白图谱、凝胶分光光度扫描分析方法,分别研究临床分离的铜绿假单胞菌亚胺培南耐药株和亚胺培南敏感株。结果：耐药组OprD2相对含量显低于敏感组。结论：OprD2的缺失是铜绿假单胞菌对以亚胺培南为代表碳青霉烯类抗生素耐药的主要原因。     

铜绿假单胞菌对碳青霉烯类抗生素的耐药机制——耐药株外膜蛋白OprD_2缺失

目的;研究铜绿假单胞菌对碳青霉烯类抗生素的耐药机制。方法:以亚胺培南为代表,应用十二烷酸磺酸钠－聚丙烯酞胺凝胶电泳(SDS-PAGE)外膜蛋白图谱、凝胶分光光度扫描分析方法,分别研究临床分离的铜绿假单胞菌亚胺培南耐药株和亚胺培南敏感株。结果:耐药组OprD2相对含量显著低于敏感组。结论:OprD2的缺失是铜绿假单胞菌对以亚胺培南为代表碳青霉烯类抗生素耐药的主要原因。     

耐碳青霉烯铜绿假单胞菌耐药表型及基因型分析

目的 了解深圳福田人民医院铜绿假单胞菌对碳青霉烯类抗生素的耐药机制,为临床合理使用抗生素提供参考依据.方法 用E-test试剂条检测铜绿假单胞菌对哌拉西林、头孢他啶、亚胺培南、美罗培南、庆大霉素、妥布霉素、环丙沙星7种抗生素的最小抑菌浓度,用三维实验对产AmpC、KPC酶菌株的耐药表型进行确认,金属酶的表型确证实验则采用EDTA双纸片扩散法.用PCR方法检测AmpC、VIM-1、VIM-2、IMP-1、SPM、KPC及OprD 7个基因型,并分析表型和基因型之间的关系,质粒接合实验用来证实耐药基因的传播性.结果 29例铜绿假单胞菌均为多重耐药菌株,且对亚胺培南、美罗培南及庆大霉素耐药率最高,3种药物最小抑菌浓度分别为32、32、256 μg/mL.26株携带有AmpC基因的铜绿假单胞菌仅有5株为持续高表达菌株,18株金属酶阳性菌中有15株与VIM-2基因型检测一致,其余3株VIM-2基因型阴性,并有1株为VIM-2基因型阳性但金属酶阴性.29株中检测出8株携带OprD基因,外膜蛋白缺失率为72%(21/29),其余基因型均阴性.结论 该院铜绿假单胞菌耐碳青霉烯类抗生素的耐药机制主要为产金属酶及外膜蛋白缺失,碳青霉烯类抗生素的不合理使用将加剧该类药物的耐药性.     

未经碳青霉烯类抗生素治疗患者中检出亚胺培南不敏感铜绿假单胞菌

目的 通过对未使用碳青霉烯类药物治疗患者中分离的亚胺培南耐药铜绿假单胞菌的分型分析,探讨铜绿假单胞菌对亚胺培南耐药性产生和传播的危险因素.方法 2006年4月至2008年3月西安交通大学医学院附属三二○一医院从未使用碳青霉烯治疗的住院患者中分离出37株亚胺培南不敏感铜绿假单胞菌,采用琼脂稀释法测定了菌株对11种抗生素的敏感性,并通过肉汤稀释法测试了外排泵抑制剂(PAβN)对亚胺培南和美罗培南耐药性的影响.通过PCR方法对金属酶编码基因及外膜孔蛋白编码基因oprD2进行了扩增和测序,用实时定量PCR( RT-PCR)方法对oprD2和ampC基因的表达水平进行测定,采用脉冲场电泳技术(PFGE)分析菌株的同源性.结果 37例患者中,体内分离出亚胺培南不敏感铜绿假单胞菌之前,使用两种或两种以上抗生素治疗的16例患者平均抗生素使用时间为(20.0±9.5)d,显著长于仅使用一种抗生素治疗的21例患者[(12.6±4.4)d;t=-2.9004,P＜0.01].在37株菌中,32株对3种以上抗生素耐药;仅有1株菌检出金属酶(IMP-9型),但该菌金属酶表型为阴性;29株菌的oprD2基因上均在同一位点正向插入序列ISpa1328;oprD2表达量检测有35株菌为零表达,2株菌过度表达ampC酶;37株菌PFGE分型分为6个脉冲型,其中26株菌为C2型;所有37株菌在外排泵抑制剂作用下,表现出外排功能.结论 氟喹诺酮类和头孢类抗生素是铜绿假单胞菌对碳青霉烯类抗生素不敏感的重要因素.     

铜绿假单胞菌外膜蛋白D2与亚胺培南耐药关系的研究

目的研究广西地区铜绿假单胞菌外膜蛋白D2（OprD2）的表达与亚胺培南耐药之间的关系。方法采用SDS-PAGE电泳法对提取出的106株耐亚胺培南铜绿假单胞菌的外膜蛋白进行电泳,并利用水杨酸盐抑制实验确定OprD2的位置,通过凝胶成像系统成像并将其与敏感株比较进行分析。结果 106株耐亚胺培南的铜绿假单胞菌中有99株细菌缺失OprD2。结论 OprD2缺失是铜绿假单胞菌对亚胺培南耐药的主要原因之一。     

耐碳青霉烯类铜绿假单胞菌耐药机制研究

目的分析本院重症监护病房(ICU)中连续分离到的多重耐药铜绿假单胞菌的同源性,并检测其对碳青霉烯类抗生素的耐药机制。方法用纸片扩散法测定铜绿假单胞菌对14种抗菌药的敏感性并进行表型分析;通过重复序列引物聚合酶链反应(rep-PCR)对铜绿假单胞菌进行分型;使用亚胺培南EDTA(乙二胺四乙酸)酶抑制试验检测菌株是否产生金属酶,同时用PCR检测OprD、IMP、VIM基因。结果抗菌药物敏感试验显示23株铜绿假单胞菌中19株同时对亚胺培南和美罗培南耐药,并且都至少对5种以上抗菌药物耐药;rep-PCR电泳结果显示19株耐药株基因表型基本一致,并且与敏感株有明显区别;亚胺培南-EDTA双纸片法筛得5株产金属酶(MBL)的铜绿假单胞菌;用特异性OprD、IMP、VIM引物扩增相应基因时,检测到1株发生OprD缺失,23株都未检测到blaIMP及blaVIM。结论ICU中19株多重耐药铜绿假单胞菌均来自同一克隆;其中1株发生OprD缺失突变,5株金属酶筛选试验阳性,但需分子生物学方法进一步证实。     

铜绿假单胞菌对亚胺培南耐药机制分析

目的铜绿假单胞菌对亚胺培南耐药机制分析研究。方法采用K-B法检测100株耐亚胺培南铜绿假单胞菌(IRPA),并用聚合酶链式反应(PCR)法检测分析耐亚胺培南铜绿假单胞菌相关金属酶IMP、VIM基因以及外膜通道蛋白OprD2基因。结果 70株耐亚胺培南铜绿假单胞菌为多重耐药菌,对阿米卡星、头孢哌酮/舒巴坦的敏感性较高。金属酶IMP基因阳性率为24.29%(17/70),vIM型金属酶基因阳性率为7.14%(5/70),外膜蛋白通道OprD2基因阳性率为68.57%(48/70),其中外膜蛋白通道OprD2基因缺失率为58.33%。结论 IRPA耐药情况严重。外膜蛋白通道OprD2基因缺失突变是本组铜绿假单胞菌对亚胺培南耐药的重要机制之一。     

头孢菌素敏感或中介的铜绿假单胞菌对亚胺培南耐药机制的研究

目的探讨临床新检出的对碳青霉烯类药物耐药、头孢他啶(CAZ)和/或头孢吡肟(FEP)不耐药的铜绿假单胞菌耐药机制。方法琼脂稀释法测定5种抗菌药物对铜绿假单胞菌的MIC。改良三维试验检测细菌耐药酶的产生。SDS-PAGE观察对亚胺培南(IPM)耐药的铜绿假单胞菌分子量(Mr)46 000外膜蛋白OprD的改变。PCR扩增oprD基因并进行序列分析。结果 29株分离菌对IPM均为中、低度耐药,对CAZ为敏感或者中介(除46号菌外)。改良三维试验显示29株细菌均未产碳青霉烯酶。SDS-PAGE显示外膜蛋白缺失可分为3种类型:7株为外膜蛋白整体缺失或者减少,4株外膜蛋白无明显变化,其他均为Mr 46 000外膜蛋白缺失或者减少。按3种缺失类型选择了12株菌测序,发现:其中1株不同位置有多个点突变,导致出现终止密码子;7株出现碱基的少量缺失;3株有新碱基插入;1株为大片段的碱基置换。结论由编码外膜蛋白的oprD基因缺失、突变以及插入导致的外膜蛋白缺失或改变是引起该表型铜绿假单胞菌对碳青酶烯类药物耐药的主要原因。     

淮北地区耐亚胺培南铜绿假单胞菌耐药机制

目的探讨淮北地区铜绿假单胞菌（pseudomonas aeruginosa,Pa）对亚胺培南的耐药机制。方法采用K-B法和MIC法检测25株临床分离亚胺培南耐药铜绿假单胞菌对12种临床常用抗生素的敏感性;采用PCR法检测外膜蛋白OprD2和金属β-内酰胺酶（MBLs）耐药相关基因（IMP、VIM、SPM、GIM）。结果 25株亚胺培南耐药铜绿假单胞菌中15株外膜通道蛋白OprD2基因缺失,缺失率60%;5株产金属酶,检出率20%（4株产VIM-2,1株产IMP-1）;未检出SPM-2、GIM基因。结论淮北地区铜绿假单胞菌亚胺培南耐药,外膜通道蛋白OprD2缺失与产金属酶均起重要作用。     

Resistance to pefloxacin in Pseudomonas aeruginosa.

Mechanisms of resistance to pefloxacin were investigated in four isogenic Pseudomonas aeruginosa strains: S (parent isolate; MIC, 2 micrograms/ml), PT1 and PT2 (posttherapy isolates obtained in animals; MICs, 32 and 128 micrograms/ml, respectively), and PT2-r (posttherapy isolate obtained after six in vitro subpassages of PT2; MIC, 32 micrograms/ml). [2-3H]adenine incorporation (indirect evidence of DNA gyrase activity) in EDTA-permeabilized cells was less affected by pefloxacin in PT2 and PT2-r (50% inhibitory concentration, 0.27 and 0.26 microgram/ml, respectively) than it was in S and PT1 (50% inhibitory concentration, 0.04 and 0.05 microgram/ml, respectively). Reduced [14C]pefloxacin labeling of intact cells in strains PT1 and PT2 correlated with more susceptibility to EDTA and the presence of more calcium (P less than 0.05) and phosphorus in the outer membrane fractions. Outer membrane protein analysis showed reduced expression of protein D2 (47 kDa) in strains PT1 and PT2. Other proteins were apparently similar in all strains. The addition of calcium chloride (2 mM) to the sodium dodecyl sulfate-solubilized samples of outer membrane proteins, before heating and Western blotting, probed with monoclonal antibody anti-OmpF showed electrophoretic mobility changes of OmpF in strains PT1 and PT2 which were not seen in strain S. Calcium-induced changes were reversed with ethyleneglycoltetraacetate. Decreased [14C]pefloxacin labeling was further correlated with an altered lipopolysaccharide pattern and increased 3-deoxy-D-mannooctulosonic acid concentration (P less than 0.01). These findings suggested that resistance to pefloxacin is associated with altered DNA gyrase in strain PT2-r, with altered permeability in PT1, and with both mechanisms in PT2. The decreased expression of protein D2 and the higher calcium and lipopolysaccharide contents of the outer membrane could be responsible for the permeability deficiency in P. aeruginosa.     

Purification of a 54-kilodalton protein (OprJ) produced in NfxB mutants of Pseudomonas aeruginosa and production of a monoclonal antibody specific to OprJ.

The 54-kDa outer membrane protein (designated OprJ) of a norfloxacin-resistant nfxB mutant of Pseudomonas aeruginosa PAO1 was purified by ion-exchange high-performance liquid chromatography. Mobility of OprJ in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was not affected by reduction and heating. A murine monoclonal antibody (MAb) against OprJ was prepared to investigate existence of this protein in fluoroquinolone-susceptible and -resistant strains of P. aeruginosa. Western blot (immunoblot) analysis with this MAb revealed a single band at the position corresponding to OprJ in outer membrane proteins of NfxB mutants derived from clinical isolates. However, the MAb did not react with any outer membrane proteins of the respective parent strains. Complementation of the NfxB mutation by transformation with plasmid pNF111, which contained the wild-type nfxB gene, led to disappearance of the single band corresponding to OprJ. The existence of OprJ was associated with fluoroquinolone resistance. Furthermore, the MAb did not react with any outer membrane proteins of other fluoroquinolone-resistant nalB and nfxC mutants. These results suggest that OprJ is newly produced in NfxB mutants of P. aeruginosa and is involved in fluoroquinolone resistance specific to NfxB, and it appears that the MAb to OprJ should aid in detection of the NfxB mutation in P. aeruginosa.     

Antibiotic susceptibility and outer membrane proteins of clinical Xanthomonas maltophilia isolates.

Twenty clinical Xanthomonas maltophilia isolates were studied for their susceptibility to various antibiotics and for their outer membrane protein profiles and compared to Pseudomonas aeruginosa isolates. Among the antibiotics studied, fleroxacin and ciprofloxacin exhibited the greatest activity (MIC90 4 micrograms/ml). All the X. maltophilia isolates exhibited similar outer membrane protein profiles with 40, 23 and 21 major proteins, whereas the outer membrane of P. aeruginosa revealed at least 7 major outer membrane proteins, as is known from the literature. Thus, it is assumed that the outer membrane profile of X. maltophilia contributes to the resistance of this nosocomial pathogen to most antibiotics.     

耐亚胺培南铜绿假单胞菌耐药机制研究

目的了解我院分离的耐亚胺培南铜绿假单胞菌(PA)耐药机制及同源性分型情况。方法收集临床分离耐亚胺培南PA,用K-B法检测28株耐亚胺培南PA对12种抗菌药的敏感性,用肠杆菌科细菌基因间重复序列(ERIC)进行同源性分析,并用PCR法检测外膜孔蛋白OprD2和金属β内酰胺酶(IMP、VIM)基因。结果28株耐亚胺培南PA对其他12种抗菌药的耐药率在14.8%~42.3%。经ERIC-PCR分析后共有11个基因型。其中A型为11株:A1型9株,A2型2株;B型为5株:B1型2株,B2型2株,B3型1株;C型3株;D型2株;E~K型各1株。PCR检测到OprD2基因仅4株,其缺失率为85.7%,28株PA中检测到1株VIM基因,经测序证实为VIM-2型。结论本院耐亚胺培南PA的主要耐药机制为外膜孔蛋白缺失。在外科和高干病房中出现多种基因型的耐亚胺培南PA传播。     

Decreases of the susceptibility to low molecular weight beta-lactam antibiotics in imipenem-resistant Pseudomonas aeruginosa mutants: role of outer membrane protein D2 in their diffusion

Decreased susceptibilities to two low Mr beta-lactam antibiotics, CS-533 (a carbapenem; Mr, 339) and CGP31608 (a penem; Mr, 262), were found in imipenem-resistant mutants of Pseudomonas aeruginosa PAO. The diffusion rates of several beta-lactam antibiotics including imipenem, CS-533 and CGP31608 across the proteoliposome membrane reconstituted from the outer membrane of the wild type strain or its imipenem-resistant mutants were determined by the liposome swelling technique. Diffusion rates of imipenem, CS-533 and CGP31608 in the proteoliposomes from outer membranes of the imipenem-resistant strain were found to be 27, 20 and 47%, respectively, of the diffusion rates in proteoliposomes reconstituted from outer membranes of the sensitive parent strain. The SDS-polyacrylamide gel electrophoretogram of outer membrane proteins of the imipenem-resistant strains indicated deletion of protein D2. These results suggested that decreased susceptibilities to imipenem, CS-533 and CGP31608 were due to decreased outer membrane permeability, and that D2 is a protein fraction constituting pores for diffusion of these antibiotics through the P. aeruginosa outer membrane.     

Transient carbapenem resistance induced by salicylate in Pseudomonas aeruginosa associated with suppression of outer membrane protein D2 synthesis.

Pseudomonas aeruginosa PAO1 showed increased phenotypic resistance to imipenem, panipenem, and biapenem specifically in the presence of salicylate. The antipseudomonal activity of carbapenems was reduced in proportion to the concentration of salicylate. This resistance was transient and nonheritable. The synthesis of the outer membrane protein D2 (OprD or OprD2) in P. aeruginosa PAO1 was inhibited by 4 to 32 mM salicylate in the bacterial growth medium, whereas no changes in any other outer membrane proteins were observed. These results indicate that salicylate suppresses the synthesis of OprD and therefore reduces the antipseudomonal activity of carbapenems. Under these conditions, one carbapenem--meropenem--is still active against P. aeruginosa, which indicates that meropenem can pass through the outer membrane via both the D2 channel and another undefined route(s).     

Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin.

Most strains of Pseudomonas aeruginosa are significantly more resistant, even in the absence of R plasmids, to many antimicrobial agents, including beta-lactams, tetracycline, chloramphenicol, and fluoroquinolones, than most other gram-negative rods. This broad-range resistance has so far been assumed to be mainly due to the low permeability of the P. aeruginosa outer membrane. The intrinsic-resistance phenotype becomes further enhanced in "intrinsically carbenicillin-resistant" isolates, which were often assumed to produce outer membranes of even lower permeability. It has been shown, however, that this hypothesis cannot explain the beta-lactam resistance of these isolates (D.M. Livermore and K.W.M. Davy, Antimicrob. Agents Chemother. 35:916-921, 1991). In this study, we examined the uptake of tetracycline, chloramphenicol, and norfloxacin by intact cells using strains showing widely different levels of intrinsic resistance. Their accumulation and the response to the addition of a proton conductor showed that even relatively susceptible strains of P. aeruginosa actively pump out these compounds from the cell and that the efflux activity becomes much stronger in strains showing higher levels of intrinsic resistance. We conclude that the efflux mechanism(s) are likely to contribute significantly to the intrinsic resistance of P. aeruginosa isolates to tetracycline, chloramphenicol, and fluoroquinolones, as does the low permeability of the outer membrane. This conclusion is supported by the observation that the hypersusceptibility to various agents of the mutant K799/61 (W. Zimmermann, Antimicrob. Agents Chemother. 18:94-100, 1980) was apparently caused by the lack of active efflux. Although the hypersusceptibility of this mutant has hitherto been assumed to be solely due to its higher outer membrane permeability, its outer membrane was shown to have a coefficient of permeability to cephaloridine that was not significantly different from that of the parent, resistant strain K799/WT. The strains with elevated intrinsic resistance overproduced two cytoplasmic membrane proteins and one outer membrane protein; at least two of these proteins appeared different from the proteins overproduced in the recently described mutant with a derepressed multidrug efflux system, MexA-MexB-OprK (K. Poole, K. Krebes, C. McNally, and S. Neshat, J. Bacteriol. 175:7363-7372, 1993).     

对碳青霉烯类抗生素耐药铜绿假单胞菌耐药机制及耐药性的研究

目的了解目前儿科临床分离对碳青霉烯类抗生素耐药铜绿假单胞菌外膜孔蛋白OprD2缺失和金属β内酰胺酶（MBI．）产生的情况，及具有不同耐药机制菌株的耐药特点。方法收集2004年1月--2006年12月从我院住院患儿中分离出的75株对碳青霉烯类抗生素耐药铜绿假单胞菌。用PCR方法进行oprD2基因、金属酶IMP、VIM、SPMT和GIM基因检测。用琼脂稀释法进行药敏试验，按照2006年CLSI推荐标准判断结果。使用WHONET5．3和SPSS13．0软件进行数据分析。结果检测75株对碳青霉烯类抗生素耐药的铜绿假单胞菌中外膜孔蛋白OprD2缺失者50株，占66．7％；产金属酶的46株．占61、3％，其中IMP阳性38株（82．6％），VIM阳性8株（17．4％）。外膜孔蛋白OprD2缺失同时产金属酶的34株，占45．3％，其对口内酰胺类抗生素的耐药率、MIC50和MIC90均高于其他菌株。结论目前从儿科临床分离对碳青霉烯类抗生素耐药铜绿假单胞菌外膜孔蛋白OprD2缺失和产生金属酶都十分严重，菌株的耐药性也很高，并且产MBI．菌株存在IMP和VIM2种基因型。     

Azithromycin exhibits bactericidal effects on Pseudomonas aeruginosa through interaction with the outer membrane

The aim of the present study was to elucidate the effect of the macrolide antibiotic azithromycin on Pseudomonas aeruginosa. We studied the susceptibility to azithromycin in P. aeruginosa PAO1 using a killing assay. PAO1 cells at the exponential growth phase were resistant to azithromycin. In contrast, PAO1 cells at the stationary growth phase were sensitive to azithromycin. The divalent cations Mg2+ and Ca2+ inhibited this activity, suggesting that the action of azithromycin is mediated by interaction with the outer membranes of the cells, since the divalent cations exist between adjacent lipopolysaccharides (LPSs) and stabilize the outer membrane. The divalent cation chelator EDTA behaved in a manner resembling that of azithromycin; EDTA killed more PAO1 in the stationary growth phase than in the exponential growth phase. A 1-N-phenylnaphthylamine assay showed that azithromycin interacted with the outer membrane of P. aeruginosa PAO1 and increased its permeability while Mg2+ and Ca2+ antagonized this action. Our results indicate that azithromycin directly interacts with the outer membrane of P. aeruginosa PAO1 by displacement of divalent cations from their binding sites on LPS. This action explains, at least in part, the effectiveness of sub-MICs of macrolide antibiotics in pseudomonal chronic airway infection.