生物膜铜绿假单胞菌对小白鼠的致病作用

目前生物膜菌感染的治疗是临床亟待解决的课题 ,因此对生物膜菌的致病机理需要深入的研究 ,才能有效的治疗生物膜菌引起的感染。对 6周龄、雄性清洁级小鼠 ,分别用铜绿假单胞菌悬液和生物膜状态的铜绿假单胞菌在小鼠气管内接种 ,进行 3项试验 :①以细菌浓度为 5× 10 6ＣＦＵ ｍｌ接种 2 0 μｌ 只测定鼠肺泡灌洗液 (ＢＡＬＦ)中白细胞数 ,结果见表 1。 2种细菌接种后ＢＡＬＦ中的白细胞数差异有显著性 (Ｐ <0 .0 1)。②测定 2组感染小鼠的存活率每组 30只小鼠分别将 10 6ＣＦＵ ｍｌ的 2组菌液 2 0 μｌ 只接种到鼠气管内 ,观察 10ｄ小鼠存…     

耐环丙沙星铜绿假单胞菌的耐药性分析

目的 了解本地区铜绿假单胞菌的耐药情况及超广谱β-内酰酶（ESBLS）的检出率，以指导临床合理用药。方法对90株临床分离菌用纸片扩散法（K-B法）进行药物敏感性试验；纸片扩散表型确证法进行ESBLS检侧。结果本地区铜绿假单胞菌对氨苄西林、萘啶酸、哌拉西林、头胞噻肟、头胞吡肟、阿米卡星、环丙沙星、左氧氟沙星、司帕沙星、加替沙星、氨曲南、头胞他啶、亚胺培南、头胞呋辛的耐药率分别是90．0％、83．3％、77．2％、29．4％、5％、8．9％、65．0％、60．0％、62．2％、50．0％、7．2％、18．3％、0、47．2％；ESBLS的检出率为24．4％。结论本地区铜绿假单胞菌耐药情况不容乐观且多重耐药菌株较多；氟喹诺酮药物之间交叉耐药明显；产ESBLS率较高。     

铜绿假单胞菌粘附对肠上皮细胞膜生物物理特性的影响

严重烧 (创 )伤后肠道细菌移位并致感染这一现象已被公认。细菌移位的第一步是其粘附于肠粘膜上皮细胞 ,进而定植于肠粘膜表面 ,随后侵入肠系膜淋巴结 ,最终播及全身。铜绿假单胞菌是肠道细菌移位的主要菌群之一。为了更好的了解铜绿假单胞菌粘附于肠上皮细胞表面后细胞膜的一系列变化 ,本研究拟从体外铜绿假单胞菌粘附肠上皮细胞模型入手 ,探索粘附后细胞膜生物物理特性的变化规律及可能的机制。研究采用大鼠肠上皮细胞株ＩＥＣ 6体外培养模型 ,实验分为铜绿假单胞菌粘附组和对照组 ;分别采用ＭＴＴ四唑盐法测定细胞活力、生化法检测细胞膜…     

临床分离的铜绿假单胞菌对头孢噻肟的耐药性分析

目的：测定铜绿假单胞菌对头孢噻肟的耐药性。方法：采用琼脂二倍稀释法测定铜铝假单胞菌对头孢噻肟的MIC值。用铜绿假单胞菌ATCC27853标准菌株作为质控标准。按照美国临床实验标准委员会（NCCLS）2004年颁布的标准操作和判定结果。结果：铜绿假单胞菌对头孢噻肟的耐药率为59．18％。结论：临床分离的铜绿假单胞菌对头孢噻肟高度耐药，应考虑通过进行细菌培养和药敏试验，合理选用抗生素，或选用有协同作用的二联疗法来提高抗铜绿假单胞菌感染的疗效。     

铜绿假单胞菌主动外排系统研究进展

铜绿假单胞菌(Pseudomonas aeruginosa，PA)是一种重要的院内感染条件致病菌，常常感染癌症病人、严重烧伤和AIDS患者等免疫力低下的病人，且由于它具有先天的和后天获得的多药耐药性(multidrug resistance．MDR)．临床治疗十分困难，一旦感染，死亡率很高．已经成为临床医生面临的治疗难题之一。近年来的研究发现，PA内存在着外排多种物质的的主动外排系统，在MDR中起着极其重要的作用。下面就对铜绿假单胞菌的主动外排系统研究进展进行综述。     

检测铜绿假单胞菌金属β-内酰胺酶方法比较

目的：探讨检测金属β-内酰胺酶的可靠方法。方法：收集临床分离耐亚胺培南的铜绿假单胞菌8株及产IMP-1金属争内酰胺酶标准株一株,分别采用纸片增效法和双纸片协同实验检测金属酶表型,比较其结果。结果和结论：纸片增效法容易出现假阳性结果,而纸片协同法是相对可靠的检测金属酶的方法。     

三种检测铜绿假单胞菌AmpC酶方法的评价

目的探讨一种简便易行，适用于临床微生物实验室常规开展检测AmpCβ内酰胺酶（简称AmpC酶）的方法。方法对临床分离的150株铜绿假单胞菌用表型筛选试验作AmpC酶测定，对AmpC酶阳性菌株同时进行氯唑西林双纸片协同试验、氟氯西林（FCC）双抑制剂扩散协同试验、PCR基因检测。用基因检测来评价比较三种测定方法的检出率及差异。结果表型筛选试验AmpC酶阳性37株同时进行氯唑西林双纸片协同试验、氟氯西林双抑制剂扩散协同试验、基因检测，检测出阳性株分别为15、14、11株，阳性率分别是40．54％（15／37）、37．84％（14／37）、29．73％（11／37）。表型筛选试验与后三种方法比较差异有统计学意义（P〈0．01），后三种方法结果比较差异无统计学意义（P〉0．05）。结论氯唑西林双纸片协同试验、氟氯西林双抑制剂扩散协同试验检测AmpC酶特异性较表型筛选试验高，且操作简便、结果可靠，适合临床实验室常规检测应用。     

铜绿假单胞菌的药物敏感试验结果分析

目的了解铜绿假单胞菌在本院的分布及对抗生素的敏感情况,为临床治疗提供科学依据。方法回顾性调查本院2008年1月至2009年10月住院及门诊患者各种标本分离的铜绿假单胞菌的分布及对抗生素的敏感情况。结果铜绿假单胞菌在呼吸道标本的检出率最高,占63.8%,其次是伤口分泌物标本,占18.3%,尿液标本占9.2%,血液标本占2.5%;其对11种抗生素的敏感率最高是妥布霉素,其次是哌拉西林/他唑巴坦,检出多重耐药株约占35%,未出现泛耐药株。结论根据药敏结果合理使用抗生素,并重视对多重耐药株的监测工作,可有效控制感染。     

铜绿假单胞菌的GM-PFGE分型的研究

目的研究全基因组ＤＮＡ稀有位点限制性内切酶酶切脉冲电场电泳图谱（ＧＭ－ＰＦＧＥ）在铜绿假单胞菌基因分型中的应用,并与表型分型比较。方法对１个月内来自两个医院的病人及环境的２０株铜绿假单胞菌进行了ＧＭ－ＰＦＧＥ图谱分析,同时进行３０种生化反应的统计分型、２３种药物的敏感性分型、胞外脂多糖的血清抗原分型;并利用数值分类软件包进行相关性比较研究。结果临床致病铜绿假单胞菌的生化表型基本稳定,但其药物抗性的获得与丢失较为明显。血清型、生化性状及药物抗性之间无明显对应关系。当２菌株ＧＭ－ＰＦＧＥ图谱条带相似系数大于８０％时,为同一菌株的不同克隆亚型,当相似系数在２５％～７０％之间时,则为不同菌株。结论ＧＭ－ＰＦＧＥ分析可显示染色体结构的区域多型性,其重复性好,分辨率明显高于表型分型,结果可靠,必将成为铜绿假单胞菌或其它病原微生物分子流行病学研究的有力工具。     

铜绿假单胞菌活菌对呼吸道上皮细胞表达IL-8的影响

目的:探讨铜绿假单胞菌活菌与人呼吸道上皮细胞的相互关系,细菌对呼吸道上皮炎症反应的影响。方法:采用PAO1及ATCC 27853两株铜绿假单胞菌,在体外与培养的呼吸道上皮细胞株A549及无血清培养的人支气管上皮原代细胞相互作用,收集细胞培养上清,ELISA检测上清IL-8浓度。结果:两株绿脓杆菌均能诱导呼吸道上皮细胞IL-8分泌增加,在细菌刺激下,A549细胞IL-8分泌比对照高出5倍(P<0.05),原代上皮细胞IL-8分泌比对照高出8倍(P<0.05)。结论:铜绿假单胞菌呼吸道感染的过程中,细菌与上皮细胞的直接作用可能是呼吸道炎症反应的重要原因。铜绿假单胞菌刺激上皮细胞炎症的分子机制和信号传导值得进一步探讨。     

Early detection of Pseudomonas aeruginosa in hemoculture media enriched with nitrates

Addition of a small amount of nitrates in blood culture media enhances growth of Pseudomonas aeruginosa, and permits earliest detection of that bacteria even in anaerobes media.     

Inhibitory activity on bacterial motility and in vivo protective activity of human monoclonal antibodies against flagella of Pseudomonas aeruginosa.

Three stable hybridoma cell lines, IN-2A8, IN-5D6, and ZI-3A8, that secrete human monoclonal antibodies (MAbs) specific for b-type flagella of Pseudomonas aeruginosa were established by fusing peripheral blood lymphocytes from healthy volunteers with murine myeloma P3X63-Ag8.653 cells. The immunoglobulin M MAbs reacted specifically with flagellin (Mr, 52,000) by Western blotting (immunoblotting) analysis and bound specifically to clinical isolates belonging to Homma serotypes A, B, H, I, and M at frequencies of 58, 50, 46, 30, and 35%, respectively, but did not bind to any serotype E or G isolates. Overall, the MAbs bound to 31% of the clinical isolates. MAb IN-2A8 strongly protected burned mice challenged with P. aeruginosa bearing b-type flagella from death following parenteral administration of 0.1 microgram per mouse. This MAb also inhibited P. aeruginosa colony spreading in soft agar at a concentration of more than 1 microgram/ml but only slightly enhanced opsonophagocytosis by human polymorphonuclear leukocytes. A line of evidence suggests that the potent in vivo activity of MAb IN-2A8 in the burned-mouse model is likely to be caused by its inhibition of bacterial motility after binding to flagella.     

Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance. The work described in this paper was carried out to determine the influence of the P. aeruginosa 1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilO gene of this organism. The isogenic knockout strain produced, 1244G7, was unable to glycosylate pilin but could produce pili normal in appearance and quantity. In addition, this strain had somewhat reduced twitching motility, was sensitive to pilus-specific bacteriophages, and could form a normal biofilm. Analysis of whole cells and isolated pili from wild-type P. aeruginosa strain 1244 by transmission electron microscopy with a glycan-specific immunogold label showed that this saccharide was distributed evenly over the fiber surface. The presence of the pilin glycan reduced the hydrophobicity of purified pili as well as whole cells. With regard to pathogenicity, P. aeruginosa strains producing glycosylated pili were commonly found among clinical isolates and particularly among those strains isolated from sputum. Competition index analysis using a mouse respiratory model comparing strains 1244 and 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. These results collectively suggest that the pilin glycan is a significant virulence factor and may aid in the establishment of infection.     

Influence of zinc on Pseudomonas aeruginosa susceptibilities to imipenem.

Serial dilution susceptibility testing of imipenem against 59 clinical isolates of Pseudomonas aeruginosa, conducted simultaneously on single lots of Difco and BBL Mueller-Hinton agar (MHA), resulted in MICs for 90% of strains tested of 8 and 16 micrograms/ml, respectively. MICs for Escherichia coli, Klebsiella pneumoniae, and Pseudomonas spp. were also higher on BBL MHA. Quantification of the cation content of the two MHAs by atomic absorption spectroscopy demonstrated that the zinc concentration in BBL MHA was 15 times greater than that measured in Difco MHA (2.61 and 0.17 micrograms/ml, respectively). Concentrations of calcium, magnesium, iron, manganese, and copper in the two agars were similar. Addition of zinc to Difco MHA resulted in increases in MICs of imipenem for P. aeruginosa but not in the MICs of ceftazidime or cefpirome for P. aeruginosa (P < 0.01). A lesser zinc effect was seen on the activity of imipenem against E. coli, K. pneumoniae, and Pseudomonas spp. The activities of ceftazidime and cefpirome were similar on both MHAs when tested against all gram-negative organisms in this study. Thus, the effect of zinc in MHA was clearly demonstrated by a significant increase in the MICs of imipenem for P. aeruginosa, and, to a lesser extent, for other gram-negative bacilli.     

Inhibition of Pseudomonas aeruginosa from cystic fibrosis by selective media.

Pseudomonas Isolation Agar (selective agent, Irgasan, 25 mg/1) and Pseudomonas Selective Agar (selective agents, cetrimide 200mg/1 and nalidixic acid 15 mg/1) inhibited some strains of P aeruginosa from cystic fibrosis sputum but did not inhibit isolates from other sources. Of 200 cystic fibrosis isolates, 22 were inhibited by 16 mg/1 Irgasan, 45 by 8 mg/1 nalidixic acid, and 15 by 128 mg/1 cetrimide. We recommend that cystic fibrosis sputum should be cultured on selective and non-selective media to maximise the isolation of P aeruginosa.     

Influence of Mucoid Coating on Clearance of Pseudomonas aeruginosa from Lungs

Pulmonary infection with mucoid strains of Pseudomonas aeruginosa in present in the majority of cystic fibrosis patients with chronic lung disease. It has been postulated that this mucoid coating may act to decrease lung clearance of Pseudomonas by limiting access of phagocytes, antibodies, and antibiotics to the bacteria. To determine whether mucoid coating of Pseudomonas might decrease intrapulmonary killing, groups of guinea pigs were infected with intrabronchial instillations of equivalent numbers of mucoid and nonmucoid Pseudomonas. For this study, mucoid strains of Pseudomonas were obtained from cystic fibrosis sputa and passaged on blood agar plates to obtain their nonmucoid revertants. Animals were then sacrificed at timed intervals after infection, and quantitative cultures were performed on lung homogenates. In all cases, mucoid challenge strains retained their mucoid morphology after passage in guinea pig lungs. No difference in killing of mucoid and nonmucoid Pseudomonas could be detected at 6, 24, or 48 h after lung infection. Further challenge studies used guinea pigs that were either prevaccinated with lipopolysaccharide P. aeruginosa vaccine or else treated with tobramycin sulfate after infection. Nonvaccinated or untreated controls had reduced intrapulmonary killing of Pseudomonas compared with vaccinees or treated groups (P < 0.02 and P < 0.01, respectively). However, there were no differences in pulmonary killing of mucoid and nonmucoid Pseudomonas in the presence of either specific antibodies or antibiotic. We conclude from these studies that mucoid coating of Pseudomonas does not selectively impede mechanisms of intrapulmonary killing in guinea pig lungs.     

Influence of macrolides on mucoid alginate biosynthetic enzyme from Pseudomonas aeruginosa

Objective: The long-term administration of erythromycin (EM), clarithromycin (CAM) or azithromycin (AZM) has generally resulted in a favorable outcome for patients with diffuse panbronchiolitis (DPB) infected with mucoid Pseudomonas aeruginosa. To elucidate the mechanism involved, the influence of macrolides on mucoid alginate production by P. aeruginosa was investigated in vitro.
Methods: The macrolides used in this study were EM with a 14-membered ring, AZM with a 15-membered ring, midecamycin (MDM) with a 16-membered ring, and CP-4305, which has had mycarose removed from MDM, The effects of macrolides on mucoid P. aeruginosa were investigated by quantitative assay of alginate production and inhibition of guanosine diphospho-D-mannose dehydrogenase activity.
Results: After incubation with EM, AZM and CP-4305, the structural material of P. aeruginosa biofilm was distorted, and the enzymatic activity of GDP-D-mannose dehydrogenase, the most important enzyme in mucoid alginate biosynthesis, was inhibited. However, these effects were not observed with the 16-membered macrolide MDM.
Conclusions: The basic mechanism of clinical efficacy seen characteristically in 14- or 15-membered macrolides for patients with airway biofilm disease depends on the ability of such macrolides to inhibit alginate production by P. aeruginosa. Furthermore, this suggests that the inhibitory effect observed with 14-, 15- and 16-membered macrolides may depend on the sugar chain connected with the macrolide ring.     

ELISA to determine immunoreactive Pseudomonas aeruginosa elastase in chronic suppurative otitis media.

A sensitive sandwich ELISA has been developed to measure the levels of Pseudomonas aeruginosa elastase (PE) in ear discharges from chronic suppurative otitis media (CSOM) patients. Preincubation of the sample with EDTA-2Na before ELISA was employed to inhibit PE activity which hydrolyzes the anti-PE IgG antibody into a smaller molecular form. The PE levels of 10 middle ear effusions (MEE) from chronic otitis media with effusion were also measured. In CSOM, 9 of 10 samples had significant PE levels, ranging from 6.8 to 62.1 micrograms/ml, which were significantly higher than those in MEE (p less than 0.01), the majority of which was below the detection limit. Two samples of CSOM with the P. aeruginosa infection showed high PE levels. This sandwich ELISA for the measurement of PE is a very sensitive method requiring only a small sample amount.     

Influence of growth rate and nutrient limitation on monobactam production and peptidoglycan synthesis in Pseudomonas aeruginosa

The effects of growth rate and nutrient limitation on monobactam production, peptidoglycan content and mean cell length in Pseudomonas aeruginosa was studied using continuous culture techniques. All three parameters increased progressively with growth rate, a greater response being shown under carbon limitation compared to that occurring under nitrogen limiting conditions. Interestingly, monobactum production mirrored peptidoglycan synthesis. In addition, the monobactam exhibited a broad range of antibacterial activity and bound preferentially to PBP 1A in the producing organism. Moreover, addition of the monobactam to a growing culture inhibited cell wall synthesis. These results are discussed in relation to the control and regulation of peptidoglycan synthesis.     

Influence of anti-slime glycolipoprotein serum on the interaction between Pseudomonas aeruginosa and macrophages.

Glycolipoprotein, a purified fraction of the exopolysaccharide slime of Pseudomonas aeruginosa, was identified as responsible for a number of the biological activities of viable cells, including toxicity and immunogenicity capable of stimulating protective antibody against the lethal effects of viable cells. Antiserum against glycolipoprotein also mediated the phagocytosis and subsequent killing of viable P. aeruginosa by unstimulated mouse peritoneal macrophages. In the absence of anti-glycolipoprotein serum, macrophages did not significantly reduce the number of bacteria. The presence of complement in the experimental mixture did not affect the reduction of bacteria by the macrophage in the presence of anti-glycolipoprotein serum. The limiting effect of antiserum concentration on macrophage activity was studied, and maximal activity was found at 2%, with no further increase in activity at 5% Preopsonization of the bacteria with anti-glycolipoprotein serum had little effect on the course of phagocytosis within the experimental conditions. Variations in bacterium-to-macrophage input ratios, ranging from 30:1 to 1:30, did not affect the capacity of the macrophages for phagocytosis.