铜绿假单胞菌phoQ基因敲除对氨基糖苷类抗生素耐药性的影响

目的 分析氨基糖苷类抗生素敏感或耐药铜绿假单胞菌菌株二元信号系统PhoQ/PhoP编码基因序列并确定该系统与耐约性关系.方法 采用PCR获得铜绿假单胞菌菌株全长phoQ和phoP基因片段,T-A克隆后测序.构建phoQ和phoP基因原核表达系统,Ni-NTA亲和层析法提纯目的 重组表达产物rPhoQ和rPhoP,皮内注射免疫法制备兔抗血清,免疫双扩散法测定抗血清效价.采用Red重组系统敲除氨基糖苷类抗生素耐药铜绿假单胞菌菌株phoQ基因,采用PCR、测序和Western blot对phoQ突变株进行鉴定.采用试管稀释法测定各铜绿假单胞菌野生株和突变株对4种氨基糖苷类抗生素的最低抑菌浓度(MIC).结果 与GenBank中相关序列比较,所克隆的phoP和phoQ基因核苷酸和氨基酸相似性分别为98.7%～99.6%和98.7～100%、98.4%～99.8%和99.1%～100%.采用pET-42a和E. coli BL21DE3系统成功地表达了rPhoQ和rPhoP.rPhoQ和rPhoP兔抗血清免疫双扩效价分别为1:4和1:8抗血清.经PCR、测序和Western blot鉴定,两株phoQ突变株phoQ基因及产物均缺失.上述phoQ突变株对4种氨基糖苷类抗生素的MIC值分别为其野生株的1/512～1/2048.结论 PhoQ/PhoP是序列保守的铜绿假单胞菌二元信号转导系统,该系统介导细菌对氨基精苷类抗生素的耐药性.     

焦磷酸测序检测铜绿假单胞菌的SHV基因点突变

目的:通过焦磷酸测序技术检测产超广谱β-内酰胺酶(ESBLs)铜绿假单胞菌的SHV基因点突变,探讨一种快速、准确的ESBLs基因分型方法。方法:双纸片法确定产ESBLs的铜绿假单胞菌,纸片扩散法(K-B法)进行药敏试验,聚合酶链反应(PCR)法扩增ESBLs的SHV基因片段,用焦磷酸测序法检测16株产ESBLs铜绿假单胞菌的SHV基因35位和43位密码子点突变。结果:焦磷酸测序发现,本地区分离出的16株产ESBLs铜绿假单胞菌有10株扩增出SHV基因片段,且在43位密码子基因没有突变,35位密码子有基因突变,核苷酸由T突变为A,亮氨酸变为谷氨酰胺,突变发生率达到70%(7/10)。16株产ESBLs的菌株对亚胺培南全部敏感。结论:焦磷酸测序技术可快速检测产ESBLs铜绿假单胞菌的SHV基因点突变,具有准确、快速、实时和高通量等优点,可应用于产ESBLs菌株的ESBLs基因分型。     

铜绿假单胞菌氨基糖苷类耐药性及其修饰酶基因的研究

铜绿假单胞菌是院内感染最常见的细菌之一,因其外膜通透性极低的特异性,表现出对多种抗生素的天然耐药.以往的研究多侧重于对β-内酰胺类和喹诺酮类抗生素的耐药机制,而临床工作中发现,铜绿假单胞菌对氨基糖苷类抗生素耐药现象亦非常严重.细菌对氨基糖苷类抗生素耐药主要是由于细菌产生的氨基糖苷类修饰酶（aminoglycosides-modifying enzymes,AME）对进入细胞内的药物分子进行修饰使之失去生物活性而耐药.AME基因型的分布带有明显的地区性和菌株差异,且不同基因型的细菌耐药和流行特征也不同.我们对2003年6月-2004年3月的26株铜绿假单胞菌进行AME基因型分布研究.     

临床分离耐喹诺酮类铜绿假单胞菌gyrA基因单点突变研究

目的 研究临床分离铜绿假单胞菌耐喹诺酮类药物的分子机制，对PCR－RFLP－SSCP分析铜绿假单胞菌gyrA基因突变的可行性评价。方法 以铜绿假单胞菌gyrA基因序列为靶序列，用PCR、PCR－SSCP、PCR－RFLP、DNA测序、OMIGA软件分析等方法，对铜绿假单胞菌gyrA基因突变进行研究。结果 在铜绿假单胞菌10株耐药突变株中，有8株的gyrA基因的83位表现出高频的单点突变，其突变方式全为ACC→ATC。gyrA的PCR扩增产物Sac Ⅱ酶切片段与测序结果一致。SSCP带谱与测序结果比较，除1株（PSA2）其SSCP带谱与标准株相同，但测序结果有点突变外，其余菌株与测序结果一致。结论 临床分离的铜绿假单胞菌耐喹诺酮类药物分子机制主要表现为gyrA基因83位氨基酸密码子突变（Thr-83→Ile)，利用PCR－SSPC－RFLP系统，可快速、准确地检测耐喹诺酮类药物的铜绿假单胞菌gyrA中至少1个碱基的差异。     

碳青霉烯类抗生素诱导耐药铜绿假单胞菌外膜蛋白D2表达与编码基因多点突变

目的 探讨对碳青霉烯类抗生素耐药的铜绿假单胞菌编码外膜蛋白D2(OprD2)基因变异与OprD2表达的关系.方法 由临床分离敏感铜绿假单胞菌,使用不同浓度亚胺培南与美罗培南含药琼脂平板法筛选耐药菌株.应用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、凝胶成像系统分析OprD2.应用聚合酶链反应及测序分析OprD2基因编码区.结果 人工诱导能产生出与临床分离株一样的对碳青霉烯类抗生素耐药的菌株.在SDS-PAGE图谱上亚胺培南与美罗培南耐药株与同源敏感株相比均有OprD2的减少.测序结果显示在多株耐药菌中,OprD2基因编码区3个基因位点同时出现碱基突变:在+84位点产生突变,胞嘧啶突变为胸腺嘧啶(C→T);在+401位点突变(C→A),造成第134位氨基酸苏氨酸突变为天冬氨酸(Thr→Asp);在+959位点突变(A→G),造成第320位氨基酸赖氨酸突变为精氨酸(Lys→Arg).结论 同时出现3个或更多的碱基点突变可能导致铜绿假单胞萧OprD2缺失或表达减少,可能是对碳青霉烯类抗生素产生耐药性的原因.     

多重耐药铜绿假单胞菌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25株外科监护室分离的多重耐药铜绿假单胞菌,用实时定量RTPCR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理.结果 25株多重耐药铜绿假单胞菌中,14株高表达MexAB-OprM系统（56%）,3株高表达MexXY-OprM系统（12%）,这3株菌同时高表达2种外排泵.在8株mexA高表达的菌株中,5株菌发生mexR基因突变,出现氨基酸替代,1株mexR提前出现终止密码子,2株菌未发现mexR基因突变.2株mexX高表达的菌株均发生基因mexZ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,是此菌多重耐药机制之一;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,同时存在其他调控机制.     

儿科对碳青霉烯类耐药铜绿假单胞菌产金属酶的研究

目的 了解目前儿科对碳青霉烯类抗生素耐药铜绿假单胞菌产金属酶的情况。方法 本研究收集了2003年12月至2005年11月，北京儿童医院住院患儿中分离出对碳青霉烯类抗生素耐药的铜绿假单胞菌59株。使用E试验法检测金属酶的耐药表型，PCR技术检测编码金属酶的IMP、VIM、SPM和GIM4种基因型。结果 本研究59株对碳青霉烯类抗生素耐药铜绿假单胞菌中，29株金属酶耐药表型结果阳性，占49．2％。39株金属酶基因型阳性，占66．1％，其中扩增出IMP型阳性35株，占89．7％，VIM型阳性4株，占10．3％。未检测出SPM和GIM型金属酶。对哌拉西林、哌拉西林-他唑巴坦产金属酶不敏感率高于非产金属酶菌株，差异有统计学意义（P〈0．05）。对产金属酶菌株β-内酰胺类抗生素头孢噻肟、头孢哌酮、头孢他啶、头孢吡肟、头孢哌酮-舒巴坦的MIC90均达到256μg／ml，MIC90均大于256μg／ml，高于非产金属酶菌株。结论 从本研究分离的菌株显示儿科铜绿假单胞菌产金属酶率高于成人报道，产金属酶是儿科分离对碳青霉烯类抗生素耐药铜绿假单胞菌的重要耐药机制。且以产IMP型金属酶为主，少部分产VIM型金属酶。产金属酶菌株对β-内酰胺类抗生素耐药比非产金属酶菌株更严重，尤其对哌拉西林和哌拉西林-他唑巴坦耐药性高。E试验法易用于铜绿假单胞菌产金属酶的初步筛选，但不能单独作为检测铜绿假单胞菌产生金属酶的确证性试验。     

我院重症监护室铜绿假单胞菌感染趋势及其耐药性分析

目的铜绿假单胞菌（Pseudomonas aeruginosa,PA）为院内感染的重要代表菌及临床分离率较高的菌株,在我院PA占所有菌株检出率的24.5%、占革兰阴性杆菌检出率的35.4%。通过对我院2006年1月-2009年6月期间4个重症监护室铜绿假单胞菌的感染情况及其耐药性进行分析,并与同期非重症监护室的病区进行比较,分析PA产碳青霉烯酶水解碳青霉烯类抗生素后同时携带多种耐药基因对其他药物敏感性的影响。方法采用Micro Scan全自动细菌鉴定和药敏分析仪,数据的分析采用WHONET软件。结果 4年间我院重症监护室铜绿假单胞菌检出数为559株,依次为：内科ICU272株、外科ICU149株、急诊ICU126株、儿科ICU12株。药敏结果显示：铜绿假单胞菌对大部分抗生素的敏感性呈逐年下降的趋势,尤其是重症监护室感染菌株。PA对碳青霉烯类抗菌素耐药的菌株产生多重甚至广泛耐药的几率较高。结论 4年间我院重症监护室铜绿假单胞菌对抗生素的耐药状况严重,尤其对头孢菌素,氨基糖苷类抗生素耐药率明显增加。对亚胺培南的耐药株同时携带多种耐药机制,产生多重耐药甚至泛耐药。多重耐药株和泛耐药株的检出亦呈逐年上升的趋势。     

64株大肠埃希菌对氨基糖苷类抗生素的耐药表型与钝化酶基因型的分析

目的 调查2004年北京地区临床收集的耐庆大霉素大肠埃希菌对氨基糖苷类抗生素的敏感性和氨基糖苷类钝化酶基因的流行状况.方法 采用微量肉汤稀释法检测64株大肠埃希菌对16种氨基糖苷类抗生素的最低抑菌浓度（MIC）值;利用巢式PCR方法对其可能含有的7种氨基糖苷类钝化酶基因进行检测和确证.结果 临床大肠埃希菌的耐药表型较为复杂,与钝化酶基因表达有关.测试菌株中存在aac（3）-Ⅱc、aac（6＇）-Ⅰ b、ant（2＂）-Ⅰ a和ant（3＂）-Ⅰ a 4种耐药基因,aac（3）-Ⅱc和aac（6′）-Ⅰ b是主要的产酶基因.约40%的耐药菌中存在2种或2种以上的钝化酶基因.结论 产生氨基糖苷类钝化酶是临床分离的大肠埃希菌对氨基糖苷类抗生素主要的耐药机制.采用巢式PCR方法检测耐药基因,特别是在缺少阳性对照菌株的情况下,可以保障检测结果的特异性和准确性.临床菌的耐药表型和钝化酶基因关系较为复杂,这可能与耐药菌中尚存在其他耐药基因有关.     

耐亚胺培南铜绿假单胞菌金属β-内酰胺酶和oprD2 基因的检测

目的: 了解我院临床分离下呼吸道感染的耐亚胺培南铜绿假单胞菌(IRPA) oprD2 基因和金属β-内酰胺酶(MBL)的分子流行病学情况。方法: 铜绿假单胞菌的鉴定和药敏采用VITEK-2系统进行药物的最低抑菌浓度(MIC)检测。应用双纸片增效法和聚合酶链反应(PCR)方法检测其产MBL的表型及相关的 oprD2 基因、IMP型和VIM型金属酶基因。结果: 157株亚胺培南铜绿假单胞菌对多种抗菌药物的耐药率均在40%以上,经双纸片增效法筛选出20株MBL阳性(12.7%),经PCR方法检测20株MBL阳性株中有13株IMP型基因阳性, oprD2 基因有67株阳性,其余90株缺失(57.3%),未发现VIM型基因。有10株IRPA既携带IMP型基因同时又有 oprD2 基因缺失。结论: 我院临床分离下呼吸道感染的耐亚胺培南铜绿假单胞菌呈较严重的多重耐药。IMP型基因是我院IRPA产MBL的主要基因型; oprD2 基因缺失可能是铜绿假单胞菌亚胺培南耐药的重要原因之一。     

PhoP/Q regulated genes inSalmonella typhi: identification of melittin sensitive mutants

Many of the genes (pags (phoPactivatedgenes) andprgs (phoPrepressedgenes) ) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessary for survival ofSalmonella typhimuriumin murine macrophages and pathogenesis in mice. Although a great deal is known about theS. typhimuriumphoP/Qregulon, little has been done with the human specific pathogenS. typhi, prompting us to investigateS. typhiphoP/Qregulated genes. IsogenicphoP12(null) andphoP24(constitutive) strains were constructed inS. typhiTy2 andS. typhimuriumC5 strains. Comparison of whole cell proteins from these strains by SDS-PAGE showed differences in both the number and molecular mass of PhoP/Q regulated proteins. This suggested thatS. typhiandS. typhimuriummay have different PhoP/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regulated genes. This involved random MudJ transposon mutagenesis of aphoP12mutant, creatinglacZ-gene fusions, and screening for Lac⁺ or Lac^- colonies. A mobilizable plasmid carrying thephoP24mutant gene was conjugated into these insertion mutants. Those that changed from Lac^- to Lac⁺ were inferred to bepag::MudJ insertions and those that changed from Lac⁺ to Lac^- were inferred to beprg::MudJ insertions. Five mutants with PhoP/Q regulated MudJ fusions were found by this scheme. The mutations were termedpqa(PhoPQ activated) andpqr(PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. Thepqa/pqr::MudJ mutations were transduced intoS. typhiphoP⁺ andphoP24strains by Vi-I phage transduction. Characterization of the mutants (Southern blot analysis, β-galactosidase activity on indicator plates and in liquid cultures) strongly suggested that their MudJ insertion mutations were in five different genes. Further characterization involved determining cationic peptide sensitivity and mouse virulence. Two mutants were found to be sensitive to the antimicrobial peptide melittin.     

Expression of the MexXY efflux pump in amikacin-resistant isolates of Pseudomonas aeruginosa

The MexZ-MexX-MexY multidrug efflux system in Pseudomonas aeruginosa was studied to determine its contribution to aminoglycoside resistance. Amikacin-resistant (AR) mutants were generated from P. aeruginosa strain PAO1, and clinical isolates of P. aeruginosa were collected from cystic fibrosis patients. The regulatory gene mexZ and the intergenic region (mexOZ) between mexZ and mexX were investigated for mutation by PCR and DNA sequence analysis. The results showed that 14 of 15 AR clinical isolates and one of ten laboratory mutants had at least one mutation in mexZ and/or mexOZ. To study the effect of mexZ and mexOZ mutations, the production of MexY mRNA was investigated quantitatively by real-time PCR. Seven of ten AR mutants (MIC 4-8 mg/L) produced 8-21-fold more MexY mRNA than PAO1. These isolates were sensitive to fluoroquinolones, carbapenems and ceftazidime. One AR mutant (MIC 64 mg/L) that produced > 200-fold more MexY mRNA than PAO1 was also resistant to fluoroquinolones, carbapenems and ceftazidime. Thirteen of 15 AR clinical isolates produced 3.4-727-fold more MexY mRNA. No evidence was found for the aminoglycoside-modifying enzymes 6'-N-acetyltransferase type Ib, 4'-O-nucleotidyltransferase type IIb or aminoglycoside 3'-phosphotransferase IIps in these strains. Nine AR mutants overproduced MexY without mutations in mexZ or mexOZ, suggesting that MexXY efflux is also regulated by gene(s) other than mexZ.     

The role of the phoPQ operon in the pathogenesis of the fully virulent CO92 strain of Yersinia pestis and the IP32953 strain of Yersinia pseudotuberculosis

At the genomic level, Yersinia pestis and Yersinia pseudotuberculosis are nearly identical but cause very different diseases. Y. pestis is the etiologic agent of plague; whereas Y. pseudotuberculosis causes a gastrointestinal infection primarily after the consumption of contaminated food. In many gram-negative pathogenic bacteria, PhoP is part of a two-component global regulatory system in which PhoQ serves as the sensor kinase, and PhoP is the response regulator. PhoP is known to activate a number of genes in many bacteria related to virulence. To determine the role of the PhoPQ proteins in Yersinia infections, primarily using aerosol challenge models, the phoP gene was deleted from the chromosome of the CO92 strain of Y. pestis and the IP32953 strain of Y. pseudotuberculosis, leading to a polar mutation of the phoPQ operon. We demonstrated that loss of phoPQ from both strains leads to a defect in intracellular growth and/or survival within macrophages. These in vitro data would suggest that the phoPQ mutants would be attenuated in vivo. However, the LD(50) for the Y. pestis mutant did not differ from the calculated LD(50) for the wild-type CO92 strain for either the bubonic or pneumonic murine models of infection. In contrast, mice challenged by aerosol with the Y. pseudotuberculosis mutant had a LD(50) value 40× higher than the wild-type strain. These results demonstrate that phoPQ are necessary for full virulence by aerosol infection with the IP32953 strain of Y. pseudotuberculosis. However, the PhoPQ proteins do not play a significant role in infection with a fully virulent strain of Y. pestis.     

The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence

Yersinia pestis and Yersinia pseudotuberculosis are closely related facultative intracellular pathogens. The response regulator PhoP was previously shown to be important for Y. pestis survival in macrophages and for virulence in a murine bubonic plague infection assay. Here the importance of PhoP for Y. pseudotuberculosis pathogenesis was investigated. Y. pseudotuberculosis phoP mutants were unable to replicate in low-Mg(2+) medium or in macrophages. phoP(+) Y. pseudotuberculosis strains initiated replication in macrophages after a lag period of approximately 5 h, as shown by fluorescence microscopy and viable count assays. Y. pseudotuberculosis phoP mutants died at a low rate in macrophages; there was no decrease in viability over the first 5 h of infection, and there was a 10-fold decrease in viability between 5 and 24 h of infection. Trafficking of phagosomes containing phoP(+) or phoP mutant Y. pseudotuberculosis was studied by using immunofluorescence microscopy and cathepsin D as a marker for lysosomes. Phagosomes containing phoP mutant Y. pseudotuberculosis acquired cathepsin D at a higher rate than phagosomes containing phoP(+) bacteria. However, the increased rate of marker acquisition for phagosomes containing mutant bacteria was only evident approximately 5 h after infection, suggesting that phoP mutants are able to retard phagosome maturation during the lag phase of intracellular growth. The results obtained with a Y. pestis phoP mutant were similar to those described above, except that the rates of intracellular killing and trafficking to cathepsin D-positive vacuoles were significantly higher. A Y. pseudotuberculosis phoP mutant was 100-fold less virulent than the wild-type strain in a murine intestinal infection model, suggesting that survival and replication in macrophages are important for Y. pseudotuberculosis pathogenesis.     

Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients

In total, 40 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients were included in this study. Twenty of these were collected in 1994 and 1997, from six CF patients, and the rest were collected from different CF patients in 2000 and 2001. The relative expression of mRNA for the efflux pump protein MexY was determined by real-time PCR and correlated with susceptibilities to amikacin and tobramycin. The chromosomal genes mexZ , rplY , galU , PA5471 and nuoG , which were found to have a role in the gradual increase in MICs of aminoglycoside antibiotics in laboratory mutants of P. aeruginosa , were analysed. MexY mRNA overproduction was found in 17/20 isolates collected in 1994 and 1997, and was correlated with decreased susceptibility to aminoglycosides. Alteration of the MexXY–OprM efflux system has been the main mechanism of resistance to aminoglycoside antibiotics in CF P. aeruginosa isolates over the 3-year period. In several isolates, expression of the PA5471 gene product might have some effect on elevated MICs of aminoglycosides. Inactivation of rplY , galU and/or nuoG may explain the gradual increase in MICs of aminoglycosides in laboratory mutants but probably not in the CF environment, as rplY and galU were unaltered in all isolates, and nuoG was not expressed in only one isolate. No 16S rRNA A-site mutations were found in any of the four copies of the gene in 13 investigated isolates.     

The Pseudomonas aeruginosa PhoP-PhoQ Two-Component Regulatory System Is Induced upon Interaction with Epithelial Cells and Controls Cytotoxicity and Inflammation

The adaptation of Pseudomonas aeruginosa to its environment, including the host, is tightly controlled by its network of regulatory systems. The two-component regulatory system PhoPQ has been shown to play a role in the virulence and polymyxin resistance of P. aeruginosa as well as several other Gram-negative species. Dysregulation of this system has been demonstrated in clinical isolates, yet how it affects virulence of P. aeruginosa is unknown. To investigate this, an assay was used whereby bacteria were cocultured with human bronchial epithelial cells. The interaction of wild-type (WT) bacteria that had adhered to epithelial cells led to a large upregulation of the expression of the oprH-phoP-phoQ operon and its target, the arn lipopolysaccharide (LPS) modification operon, in a PhoQ-dependent manner, compared to cells in the supernatant that had failed to adhere. Relative to the wild type, a phoQ mutant cocultured on epithelial cells produced less secreted protease and lipase and, like the phoQ mutant, piv, lipH, and lasB mutants demonstrated reduced cytotoxicity toward epithelial cells. Mutation in phoQ also resulted in alterations to lipid A and to increased inflammatory LPS. These data indicate that mutation of phoQ results in a phenotype that is similar to the less virulent but more inflammatory phenotype of clinical strains isolated from chronic-stage cystic fibrosis lung infections.     

Infection with an avirulent phoP mutant of Neisseria meningitidis confers broad cross-reactive immunity

Successful vaccines against serogroup A and C meningococcal strains have been developed, but current serogroup B vaccines provide protection against only a limited range of strains. The ideal meningococcal vaccine would provide cross-reactive immunity against the variety of strains that may be encountered in any community, but it is unclear whether the meningococcus possesses immune targets that have the necessary level of cross-reactivity. We have generated a phoP mutant of the meningococcus by allele exchange. PhoP is a component of a two-component regulatory system which in other bacteria is an important regulator of virulence gene expression. Inactivation of the PhoP-PhoQ system in Salmonella leads to avirulence, and phoP mutants have been shown to confer protection against virulent challenge. These mutants have been examined as potential live attenuated vaccines. We here show that a phoP mutant of the meningococcus is avirulent in a mouse model of infection. Moreover, infection of mice with the phoP mutant stimulated a bactericidal immune response that not only killed the infecting strain but also showed cross-reactive bactericidal activity against a range of strains with different serogroup, serotype, and serosubtyping antigens. Sera from the mutant-infected mice contained immunoglobulin G that bound to the surface of a range of meningococcal strains and mediated opsonophagocytosis of meningococci by human phagocytic cells. The meningococcal phoP mutant is thus a candidate live, attenuated vaccine strain and may also be used to identify cross-reactive protective antigens in the meningococcus.