绿脓杆菌ATCC27853外毒素PE40基因的扩增与测序

目的扩增绿脓杆菌ATCC27853外毒素基因PE40,并对PCR产物进行测序鉴定,为获得用于构建分子导向药物的毒素弹头基因PE40奠定基础。方法采用PCR技术,以绿脓杆菌ATCC27853基因组DNA为模板,扩增PE40基因并测序。用DNASTAR软件将测得的序列与GenBank中的国际标准产毒株PA103的PE40基因序列进行比较。结果扩增出目的基因片断,长度为1231bp。测序表明,ATCC27853与PA103核苷酸同源性为98%,产生了7个碱基的突变,突变碱基导致第364位的天冬酰胺变成丝氨酸,第506位的丝氨酸变成色氨酸,第515位的丝氨酸变成甘氨酸。但产生变化的3个氨基酸均不是文献报道中的重要活性位点。结论产生变化的3个氨基酸不会对PEA的酶活性及细胞毒性产生很大影响,绿脓杆菌ATCC27853的PE40基因可用作分子导向药物的弹头。     

Host resistance to lung infection mediated by major vault protein in epithelial cells

The airway epithelium plays an essential role in innate immunity to lung pathogens. Ribonucleoprotein particles primarily composed of major vault protein (MVP) are highly expressed in cells that encounter xenobiotics. However, a clear biologic function for MVP is not established. We report here that MVP is rapidly recruited to lipid rafts when human lung epithelial cells are infected with Pseudomonas aeruginosa, and maximal recruitment is dependent on bacterial binding to the cystic fibrosis transmembrane conductance regulator. MVP was also essential for optimal epithelial cell internalization and clearance of P. aeruginosa. These results suggest that MVP makes a substantial contribution to epithelial cell-mediated resistance to infection.     

铜绿假单胞菌呼吸机相关肺炎的临床特点与防治对策

目的：探讨铜绿假单胞菌(PA)呼吸机相关肺炎(VAP)的临床特点与防治对策。方法：对36例由PA所致VAP的患者进行回顾性分析。结果：PA—VAP的临床特点与其它肺炎相似，但病情较重，并发症多，易发生多器官功能衰竭(MSOF)．病死率高。PA对多种抗生素产生耐药，而对泰能、舒普深、头孢他啶仍有较高的敏感性。治疗好转率52．8％，病死率41．7％。结论：PA耐药菌株多，合理使用抗生素及加强各环节的严格消毒，对PA—VAP的防治十分有益。     

High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection

The lungs of cystic fibrosis (CF) patients are chronically infected for years by one or a few lineages of Pseudomonas aeruginosa. These bacterial populations adapt to the highly compartmentalized and anatomically deteriorating lung environment of CF patients, as well as to the challenges of the immune defenses and antibiotic therapy. These selective conditions are precisely those that recent theoretical studies predict for the evolution of mechanisms that augment the rate of variation. Determination of spontaneous mutation rates in 128 P. aeruginosa isolates from 30 CF patients revealed that 36% of the patients were colonized by a hypermutable (mutator) strain that persisted for years in most patients. Mutator strains were not found in 75 non-CF patients acutely infected with P. aeruginosa. This investigation also reveals a link between high mutation rates in vivo and the evolution of antibiotic resistance.     

A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus

Bacterial pathogens frequently use protein secretion to mediate interactions with their hosts. Here we found that a virulence locus (HSI-I) of Pseudomonas aeruginosa encodes a protein secretion apparatus. The apparatus assembled in discrete subcellular locations and exported Hcp1, a hexameric protein that forms rings with a 40 angstrom internal diameter. Regulatory patterns of HSI-I suggested that the apparatus functions during chronic infections. We detected Hcp1 in pulmonary secretions of cystic fibrosis (CF) patients and Hcp1-specific antibodies in their sera. Thus, HSI-I likely contributes to the pathogenesis of P. aeruginosa in CF patients. HSI-I-related loci are widely distributed among bacterial pathogens and may play a general role in mediating host interactions.     

水貂出血性肺炎流行病学调查及绿脓杆菌疫苗研究进展

由绿脓杆菌引起的水貂出血性肺炎近几年在我国各地养殖场频繁发病。总结了该病发病情况和流行特点,概述了水貂出血性肺炎疫苗的研究进展。调查发现,我国水貂出血性肺炎的死亡率为6%～54%,以沿海气候潮湿的地区发病较多,全国流行的血清型以G、B、C型为主,与国外流行的主要血清型相同。目前用于防治水貂出血性肺炎的疫苗产品以灭活疫苗为主,外膜蛋白(OMP)、脂多糖(LPS)和鞭毛等组分疫苗和基因工程疫苗在人医上尝试较多,兽医领域应用较少,但OMP疫苗和基因工程疫苗有提供共同保护性抗原的优点,在预防水貂出血性肺炎上有较好的应用前景。     

养殖鱼塘中铜绿假单胞菌致病性及耐药相关基因的研究

旨在分析养殖鱼塘水体中铜绿假单胞菌(Pseudomonas aeruginosa,PA)分离菌株的致病性、耐药性及其可能机制,为保障水产食品的安全提供科学依据。使用美国临床与实验室标准研究所的标准纸片扩散法,以及聚合酶链反应技术对PA分离菌株进行了抗菌素耐药性,以及毒性、固有和获得性耐药相关基因的检测与分析。结果显示,受试PA分离菌株的50%为exoS+/exoU-侵染型分子型,无临床分离菌株的exoS-/exoU+的细胞毒型分子型。PA菌株对6大类9种抗菌素的耐药性存在明显差异,其中甲氧胺苄嘧啶和利福平的耐药率最高为100%,其次是氨苄西林、卡那霉素和四环素,分别为90%、90%和80%,庆大霉素的耐药率最低为10%。多药抗性PA菌株均含有MexAB-OprM、MexXY-OprM和MexVW-OprM外排泵系统,其中20%菌株检测为β-内酰胺酶基因(ampC)阳性;而MexEF-OprN、MexJK-OprM、MexCD-OprJ和MexGHI-OpmD外排泵基因全部或部分缺失。此外,在PA菌株中均未检测到Ⅰ～Ⅲ类整合子的整合酶基因(comINT),但是整合接合元件(ICEs)保守模块功能基因(ICEint、soj、pilS2、pilD)均检测为阳性,提示PA菌株携带的ICEs具有潜在的转移活性,为进一步探讨PA多药抗性的散播奠定了基础。     

铜绿假单胞菌生物膜肺部感染大鼠血清IgG抗体及肺部IFN-γ水平的变化

目的 了解人工铜绿假单胞菌(PA)生物膜肺部感染对机体免疫功能的影响.方法 由支气管内直接注入PA(菌种为PAO579)藻酸盐微粒1×109CFU/mL),建立慢性PA生物膜肺部感染大鼠模型,以支气管内注入生理盐水作为对照,2周后评估血清PA特异性抗体IgG水平、肺部IFN-γ反应的变化及其与肺组织病理学的相关性.结果...     

Persistence of abnormal chloride conductance regulation in transformed cystic fibrosis epithelia

An airway epithelial cell line (CF/T43) was developed by infecting cultured airway epithelial cells from patients with cystic fibrosis (CF) with the pZIPneoSV(X)1/SV40T retrovirus and selecting for G418 resistance and ion transport properties. The distinctive chloride secretory phenotypes of the CF cell line CF/T43 and a normal cell line (NL/T4) were not perturbed by SV40T-induced cell transformation. Epithelial cell lines generated from CF cells with the SV40T gene can be used to test candidate CF genes and to evaluate the molecular mechanisms responsible for the CF phenotype.     

猪源绿脓杆菌的分离鉴定及16S rRNA基因同源性分析

从解剖病变为心包炎、纤维素性肺炎、肺脓肿的病死仔猪体内分离到1株细菌,通过形态特征观察、生化试验,将分离菌株初步鉴定为绿脓杆菌。动物致病性试验结果显示,分离菌株对小鼠具有很强的致病性。药敏试验结果显示,分离菌株对氧氟沙星、恩诺沙星高度敏感,对头孢噻肟、庆大霉素中度敏感,对青霉素、氟苯尼考、氨苄西林、阿莫西林、强力霉素、卡那霉素等耐药。将分离菌株的16S rRNA基因序列与Gen Bank中发表的绿脓杆菌序列进行Blast比对,结果表明,分离菌株与绿脓杆菌不同菌株的同源性高达99%以上,进一步确定分离菌株为绿脓杆菌。基于16S rRNA基因序列,使用MEGA 5.05软件,构建系统进化树,结果表明,分离菌株与病人痰液和腹泻婴儿分离菌株的同源性最高,与未加工水果分离菌株同源性最低。     

Effect of Lactobacillus acidophilus as a Dietary Supplement on Nonspecific Immune Response and Disease Resistance in Juvenile Common carp,Cyprinos carpio

Effects of diet fortified with Lactobacillus acidophilus at 0.0%, 0.2%, 0.4% and 0.6% on nonspecific immune response and disease resistant common carp juvenile infected with Pseudomonas aeruginosa and Aeromonas hydrophilla were examined. Immune response parameters(the total protein, albumin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea, creatinine and immunoglobulin), percentage mortality and relative protection level were determined on day 1 and days 14, 28, 42 and 56. Fish fed Lactobacilus acidophilus fortified diet significantly improved nonspecific immune parameters especially group treated with 0.4% inclusion with lower mortality rate indicating a better relative protection level against Pseudomonas aeruginosa and Aeromonas hydrophilla.     

固氮施氏假单胞菌全局性调控因子RsmA的进化分析及表达特性研究

RsmA是假单胞菌属高度保守的全局性调控因子,该蛋白可通过与靶基因的mRNA结合影响其稳定性或者抑制靶基因的翻译影响蛋白表达。固氮施氏假单胞菌A1501中发现一个rsmA同源基因及一个可能参与rsmA活性调节的非编码小RNA rsmZ基因。对A1501菌中RsmA及RsmZ的分子进化及表达特性进行了研究。分析结果表明,RsmA与铜绿假单胞菌同源性可达98%,RsmZ具有4个RsmA的结合位点。实时定量RT\|PCR结果显示,rsmA在生长初期具有高表达量,而在指数生长期以及平台稳定期表达量下降。非编码RNA rsmZ的转录水平在整个生长时期未发生显著变化。在碳匮乏条件下,rsmA和rsmZ的表达量都显著下调。在一般胁迫σ因子rpoS突变株中,rsmA的表达未受影响,但rsmZ表达上调了30多倍,说明rsmZ的表达受rpoS的负调控。进一步发现,A1501菌中,rsmA和rsmZ的转录不受转录调控因子gacA突变的影响,这与荧光假单胞菌CHAO和铜绿假单胞菌PAO1的调控模式区别,表明RsmA的转录调控模式在固氮施氏假单胞菌A1501中有其独特性。该结果为进一步探索固氮施氏假单胞中RsmA的功能及其特定条件下的调控机制奠定了理论基础。     

Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa

Endotoxic lipopolysaccharide (LPS) with potent immunostimulatory activity is recognized by the receptor complex of MD-2 and Toll-like receptor 4. Crystal structures of human MD-2 and its complex with the antiendotoxic tetra-acylated lipid A core of LPS have been determined at 2.0 and 2.2 angstrom resolutions, respectively. MD-2 shows a deep hydrophobic cavity sandwiched by two beta sheets, in which four acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. These structures suggest that MD-2 plays a principal role in endotoxin recognition and provide a basis for antiseptic drug development.     

Rapid detection of Pseudomonas aeruginosa by cross priming amplification

Pseudomonas aeruginosa(PA) is an opportunistic pathogen of humans and animals and a common source of nosocomial infections especially of the respiratory tract. Pseudomonas aeruginosa is also a major bacterial disease of poultry and in particular, eggs and newly hatched chicks. In this study, we developed a simple, accurate and rapid molecular detection method using cross priming amplification(CPA) with a nucleic acid test strip to detect P. aeruginosa. The assay efficiently amplified the target gene within 45 min at 62°C only using a simple water bath. The detection limit of the method was 1.18×10~2 copies μL~(–1) for plasmid DNA and 4.4 CFU mL~(–1) for bacteria in pure culture, and was 100 times more sensitive than conventional PCR. We screened 83 clinical samples from yellow-feather broiler breeder chickens and hospitalized/treated dogs and cats using CPA, PCR and traditional culture methods. The positive-sample ratios were 15.3%(13/83) by CPA, 13.3%(11/83) by PCR and 12.1%(10/83) by the culture method. The established CPA method has significant advantages for detecting P. aeruginosa. The method is easy to use and possesses high specificity and sensitivity without the requirements of complicated experimental equipment. The PA-CPA assay is especially fit for outdoor and primary medical units and is an ideal system for the rapid detection and monitoring of P. aeruginosa.     

Cl- channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase

Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.     

固氮施氏假单胞菌bifA基因的功能分析

环二鸟苷酸(c-di-GMP)是细菌中一种重要的第二信使,能够调控多种生理活动。C-di-GMP由两个GTP分子经环化酶(DGCs)合成,而被磷酸二酯酶(PDEs)降解,在铜绿假单胞菌中bifA基因为降解c-di-GMP的基因,参与了胞内c-di-GMP的浓度调节。以固氮施氏假单胞菌A1501中的bifA基因为研究对象,构建了bifA基因突变株;鉴定了bifA基因在c-di-GMP降解及生物膜形成中的功能。结果表明,bifA基因的突变造成了A1501菌中c-di-GMP的积累,同时增强了菌株的生物膜形成能力。此外bifA突变株运动能力大幅下降。由此推测bifA为c-di-GMP降解的关键基因,通过调节胞内c-di-GMP水平间接参与调控了生物膜形成以及菌体运动等生理活动。该结果为解析固氮微生物的信号传递及环境适应机制提供了理论基础。     

以生活污水中油脂为复合碳源的特性菌株筛选与降解效率研究

为了构建一套高效率的污水处理体系,从污水样品中分离、筛选出一株对动植物油脂均具有较强降解能力的菌株JZZ2.在生理生化特性和16S rDNA序列系统发育分析的基础上,评价了该菌株对不同类型动植物油脂的降解能力,同时对其脂肪酶和生物表面活性剂活性进行了分析.研究发现,该菌株属于Pseudomonas aeruginosa,在含1％油脂的MS培养基中,30℃培养24 h的Pseudomonas aeruginosa JZZ2对橄榄油、色拉油、芝麻油和牛油的降解率分别为72.4％、90.2％、82.3％和84.5％,同样条件下培养48 h的Pseudomonas aeruginosa JZZ2脂肪酶活性为2 200 U· L-1,研究进一步发现油脂的添加能够促使其生物表面活性物质的产生.由此可见,Pseudomonas aeruginosa JZZ2对动植物油脂均具有较强的降解能力,能生成脂肪酶和生物表面活性物质是其具有油脂降解特性的主要原因之一.     

Altered regulation of airway epithelial cell chloride channels in cystic fibrosis

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that beta-adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.     

生姜活性提取物对铜绿假单胞菌的抑制机理研究

[目的]探究生姜的活性提取物对铜绿假单胞菌抑制作用的机理。[方法]采用有机溶剂浸提法提取生姜中的活性物质,探讨生姜在不同条件下的活性提取物对铜绿假单胞菌的抑制效果及抑菌机理。[结果]生姜提取物对铜绿假单胞菌有明显的抑制作用,且生姜提取物的稳定性较好,不同因素处理下仍具有较强的抑菌性。通过对微生物的生长曲线和电导率的测定,进一步证明生姜的活性提取物抑制了铜绿假单胞菌的生长。[结论]生姜的活性提取物对铜绿假单胞菌有较好的抑制作用。     

玉米细菌性茎腐病组织中一株新的铜绿假单胞杆菌的分离鉴定

为了明确导致河南省郑州市玉米细菌性茎腐病的病原菌,对从玉米细菌性茎腐病样品中分离得到的病原菌进行了纯化培养、形态学观察、致病性测定、生理生化特征分析和16S rDNA的遗传关系分析等研究。结果表明,所分离的菌株（YM02）为革兰氏阴性致病菌,能利用多种糖（醇）化合物产酸,但可使阿拉伯半乳聚糖产碱。YM02与国内外分离的铜绿假单胞杆菌（Pseudomonas aeruginosa）菌株16S rDNA的序列相似性均在99%以上,故将其命名为Pseudomonas aeruginosa菌株YM02,该菌与Pseudomonas aeruginosa菌株ANSC、S164S、S167S（2）和CS\|2菌株遗传关系最近,该病原菌的分离和鉴定为玉米细菌性茎腐病的防治提供了基础资料。