呼吸道上皮细胞与致病菌相互作用--上皮细胞内吞细菌实验研究

目的：呼吸道上皮细胞在防御这类机会致病菌感染时发挥了重要的作用。本研究旨在探讨上皮细胞能否清除胞内的绿脓杆菌，胞内模式识别受体Nods蛋白家族是否参与胞内杀菌，防御素是否能通过促经克雷伯杆菌粘附到上皮细胞而被上皮细胞内在化而清除。方法：首先用绿脓杆菌活菌刺激支气管原代上皮细胞及A549细胞，活菌细胞共孵育两小时后庆大霉素杀死未进入胞内的细菌，继续孵育4小时，24小时后，用TritonX-100溶解细胞，采用平板菌落计数法计数胞内活菌数。绿脓杆菌与细胞按不同比例共孵育两小时后庆大霉素杀死未进入胞内的细菌，继续培养24小时后收集细胞培养上清，酶联免疫吸附法（ELISA）检测IL-8的表达量。为进一步研究Nods蛋白是否在胞内杀菌及活菌在胞内引起IL-8分泌中发挥作用，我们用超声处理的绿脓杆菌菌体成分刺激使细胞膜通透性增加的温和去污剂digitonin处理和未使用digitonin处理A549细胞，ELISA检测细胞IL-8表达水平。胞内模式识别受体Nod1，Nod2可以识别菌体成分，用RT-PCR检测肺上皮细胞Nodl，Nod2的表达。其次，     

HNP对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响

目的：探讨呼吸道抗感染防御机制，观察人中性粒细胞α-防御素（HNP）对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响。方法：从人中性粒细胞分离纯化HNP。A549细胞与HNP（20μg·ml^-1）及两株肺炎克雷伯杆菌临床分离株共同孵育4小时，用平板培养茵落计数法测定与细胞粘附的活茵数。结果：在HNP存在的情况下，Kp03.33株细菌对肺上皮细胞的粘附提高了12倍（P〈0.01），Kp03116株细菌对上皮细胞的粘附提高了5倍（p〈0.01）结论：HNP显著增强肺炎克雷伯杆菌粘附于呼吸道上皮细胞，可能有利于呼吸道清除细菌。     

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。     

肺炎克雷伯杆菌在T24细胞内存活的动态观察

目的：了解肺炎克雷伯杆菌与膀胱上皮细胞的相互关系，观察肺炎克雷伯杆菌在人膀胱上皮细胞抹T24中生存的动态变化。方法：采用肺炎克雷伯杆菌临床分离抹03138侵袭T24细胞，并用庆大霉素杀死细胞外的细菌，分别于细菌进入细胞后的4、24、48及72h裂解细胞，释放出细胞内的活细菌，用平板菌落计数法计数胞内活菌数。结果：T24细胞内的肺炎克雷伯杆菌03138抹在实验48h内有一定生长，试验72h细胞内活菌数量明显减少。加入细胞因子（TNF-αd和INF-γ）可以促进上皮细胞清除胞内细菌。结论：膀胱上皮细胞清除进入细胞内的肺炎克雷伯杆菌，可能是泌尿道天然免疫的一种防御机制，而细胞因子可以调控上皮细胞的抗菌作用。     

TNF及铜绿假单胞菌刺激呼吸道上皮细胞表达NOD2蛋白

目的：通过对TNF及铜绿假单胞菌（PAO1）刺激肺上皮细胞株（A549）表达胞浆蛋白NOD2的研究,进一步了解NOD2在机体天然免疫病原体识别过程中的作用。方法：培养呼吸道肺癌上皮细胞株A549,以TNF、铜绿假单胞菌PAO1刺激细胞,并以未刺激组做对照,通过半定量RT-PCR技术观察胞浆蛋白NOD2的表达变化。结果：与未刺激组比较,TNF、铜绿假单胞菌PAOI都能够上调A549细胞NOD2蛋白的表达。结论：TNFα与铜绿假单胞菌PAO1刺激能增强A549细胞胞装蛋白NOD2的表达,提示细胞内受体NOD2在呼吸道天然免疫病原体识别过程中可能起重要作用。     

TNF-α增强膀胱上皮细胞清除细胞内肺炎克雷伯杆菌

研究TNF-α对膀胱上皮细胞内肺炎克雷伯杆菌生存的影响。方法：以肺炎克雷伯杆菌侵入体外培养的人膀胱上皮细胞（T24细胞）为模型,观察在TNF-α等细胞因子处理条件下,不同时间点细胞内细菌数量变化。结果：单独使用TNF-α使T24细胞内的肺炎克雷伯杆菌K5株细菌数量明显减少,联合使用TNF-α与IFN-γ使胞内活菌数量更显著的减少。而IL-1β对细胞内活菌数量无明显影响。过氧化氢酶可以有效抑制TNF-α与IFN-γ刺激的＂1＂24细胞抗菌作用,而一氧化氮合酶抑制剂L-NAME无抑制作用。结论：TNF-α能够增强膀胱上皮细胞对抗细胞内肺炎克雷伯杆菌,抗菌机制与细胞产生活性氧（ROS）有关。     

MyD88缺去突变基因转染降低病原菌感染的人呼吸道上皮细胞IL-8的分泌

白细胞介素-8（1L-8）是呼吸道炎症反应的重要介质。本实验通过构建突变MyD88真核表达质粒（MyD88 DN），转染人呼吸道上皮细胞株A549及SPC-A-1，探讨其对病原菌感染上皮细胞IL-8表达的影响。结果显示：MyD88 DN转染可降低结核杆菌、绿脓杆菌培养上清诱导的IL-8释放；对肺炎克雷伯杆菌和绿脓杆菌活菌侵袭细胞所刺激的IL-8分泌也有明显的阻断作用。提示突变MyD88能够阻断细菌感染引起的呼吸道上皮细胞IL-8表达，可能成为呼吸道严重炎症反应基因治疗的新靶基因。     

膀胱上皮细胞清除胞内大肠杆菌

目的:了解大肠杆菌与膀胱上皮细胞的相互关系,观察大肠杆菌在人膀胱上皮细胞株T24中的动态变化。方法:采用大肠杆菌标准株K12和大肠杆菌临床分离株299侵袭T24细胞,并用庆大霉素杀死细胞外的细菌,分别于细菌侵袭细胞后的4、24及48 h用TritonX-100裂解细胞,释放出细胞内的活细菌,用平板菌落计数法计数胞内活菌数。结果:两株大肠杆菌在T24细胞内都不能生长,随着时间的推移,细胞内大肠杆菌活菌数量呈不断减少趋势。24 h细胞内活菌数下降为4 h活菌数的15%。48h细胞内活菌数进一步减少。结论:膀胱上皮细胞清除进入细胞内的大肠杆菌,这可能是泌尿道天然免疫防御的一个重要防御机     

腺病毒介导突变Rip2基因对膀胱上皮细胞清除克雷伯杆菌的影响

目的探讨受体相互作用蛋白2（Rip2）在克雷伯杆菌所致的尿路感染中的作用。方法使用含有突变Rip2基因（mRip2）、绿色荧光蛋白（GFP）基因的复制缺陷腺病毒（Ad-mRip2-GFP）感染人膀胱上皮癌细胞T24细胞，表达GFP为转染成功标志，观察转染率，同时感染含有GFP基因腺病毒做对照，利用克雷伯杆菌临床分离株Kp5与细胞37℃孵育4、24、48小时，用平板培养菌落计数活菌数。结臬刺激后，在感染Ad-mRip2-GFP的T24细胞清除胞内Kp5能力下降，与Ad-GFP组比较P〈0．01。结论Rip2可以阻断膀胱上皮细胞的清除克雷伯杆菌作用，提示Rip2在膀胱上皮细胞的天然免疫中可能起重要作用。     

肺炎克雷伯杆菌分泌因子及活菌诱导人肺上皮细胞表达分泌IL-8的研究

目的 :探讨肺炎克雷伯杆菌 (Klebsiellapneumoniae ,Kp)分泌因子及活菌诱导肺上皮细胞株表达和分泌IL 8的状况。方法 :用临床分离株Kp0 3 1 1 6、Kp0 3 1 83的细菌培养上清或活菌刺激肺上皮细胞株A5 49和SPC A 1 ,酶联免疫吸附实验 (ELISA)检测细胞IL 8表达量。结果 :①两株Kp培养上清分别刺激A5 49或SPC A 1后 ,IL 8表达量均有显著增高 (P <0 .0 1 ) ,且随上清刺激浓度增加而上升。②两株Kp及DH5 (活菌分别与SPC A 1共孵育 2h ,用庆大霉素杀死胞外菌 ,继续孵育 2 4h后两株Kp诱导细胞IL 8表达量均显著高于DH5α(P <0 .0 1 ) ,且随细菌数 /细胞数比例增大而上升。结论 :Kp分泌因子及活菌都能够诱导肺上皮细胞IL 8表达 ,而活菌上调IL 8的效应较培育上清分泌因子更显著 ,提示肺上皮细胞在Kp感染刺激的肺部炎症反应中起重要作用。     

Interpretive standards and quality control guidelines for imipenem susceptibility tests with 10-micrograms disks

Tests with 10-micrograms imipenem disks accurately categorized 98.5% of 551 bacterial isolates when interpretive breakpoints of less than or equal to 13 mm for resistant and greater than or equal to 16 mm for susceptible were used. Because a sufficient number of resistant or moderately susceptible strains were not available for testing, these interpretive standards must be considered tentative. Quality control limits for tests with Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 are 26 to 32 and 20 to 28 mm, respectively. Zones obtained with Staphylococcus aureus ATCC 25923 were too large and variable to be useful for quality control purposes.     

Quality control limits for microdilution susceptibility tests with norfloxacin.

A multilaboratory study was designed to define quality control limits for microdilution susceptibility tests with norfloxacin. The following limits were proposed: for Escherichia coli ATCC 25922, 0.03 to 0.125 micrograms/ml; for Pseudomonas aeruginosa ATCC 27853, 1.0 to 4.0 micrograms/ml; for Staphylococcus aureus ATCC 29213, 0.5 to 2.0 micrograms/ml; and for Streptococcus faecalis ATCC 29212, 2.0 to 8.0 micrograms/ml. The latter represents a change in the previously recommended control limits.     

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。     

Disk agar diffusion susceptibility testing with 30-micrograms ceftazidime disks: confirmation of interpretive breakpoints and quality control guidelines

Ceftazidime is a wide-spectrum, beta-lactamase-stable cephalosporin with remarkable potency against Pseudomonas spp., Enterobacteriaceae, and some gram-positive species. The reevaluation of the 30-micrograms ceftazidime disk diffusion tests with commercially prepared disks confirms the proposed susceptibility breakpoint zone of greater than or equal to 17 mm (minimal inhibitory concentration correlate, less than or equal to 8.0 micrograms/ml) and the resistance breakpoint zone of less than or equal to 13 mm (minimal inhibitory concentration correlate, greater than or equal to 32 micrograms/ml). Major and minor interpretive errors were only 4.4%, and these errors could be further reduced to 1.1% by not testing gram-positive organisms, particularly enterococci and Staphylococcus spp. On the basis of the results from a multilaboratory quality control study, the following zone diameter quality control guidelines are suggested: Escherichia coli ATCC 25922, 27 to 31 mm; Staphylococcus aureus, ATCC 25923, 16 to 20 mm; Pseudomonas aeruginosa ATCC 27853, 24 to 28 mm.     

Ofloxacin susceptibility testing quality control parameters for microdilution and disk diffusion, and confirmation of disk diffusion interpretive criteria.

The susceptibilities of 221 clinical isolates to ofloxacin were tested simultaneously by broth microdilution and disk diffusion methods with commercially prepared 5-micrograms ofloxacin disks. The acceptability of the following previously proposed zone diameter breakpoints was confirmed: greater than or equal to 16 mm, susceptible; 13 to 15 mm, intermediate; less than or equal to 12 mm, resistant. On the basis of a multilaboratory collaborative study, the following are proposed as acceptable ofloxacin MIC ranges for quality control organisms: Escherichia coli ATCC 25922, 0.03 to 0.06 micrograms/ml; Staphylococcus aureus ATCC 29213, 0.12 to 0.5 micrograms/ml; Pseudomonas aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212, 1.0 to 4.0 micrograms/ml. Ofloxacin quality control zone diameter ranges for the disk diffusion test are tentatively proposed, but variations in the performance of different lots of Mueller-Hinton agar may prove to be a serious problem for users.     

Disk diffusion susceptibility testing and broth microdilution quality control guidelines for BMY-28100, a new orally administered cephalosporin.

The BMY-28100 30-micrograms-disk test was evaluated by using 615 clinical isolates. Regression analyses and error rates were determined, leading to the recommendation of greater than or equal to 18-mm zone diameters (MIC correlate, greater than or equal to 8.0 micrograms/ml) for susceptibility and less than or equal to 14-mm zone diameters (MIC correlate, greater than or equal to 32 micrograms/ml) for resistance. Nearly all false-susceptible disk test results were among the Providencia spp. and the beta-lactamase-positive Haemophilus influenzae strains. Susceptibility disk test results for these species should be interpreted with caution. The following broth microdilution MIC quality control guidelines were determined from results of a multilaboratory trial: Escherichia coli ATCC 25922, 1.0 to 4.0 micrograms/ml; Enterococcus faecalis ATCC 29212, 4.0 to 16 micrograms/ml; Staphylococcus aureus ATCC 29213, 0.25 to 1.0 microgram/ml; and Pseudomonas aeruginosa ATCC 27853, greater than 32 micrograms/ml.     

Proposed quality control and interpretive criteria for disk diffusion susceptibility testing with enoxacin.

The standardized disk diffusion test, in which a 10-micrograms enoxacin disk is used, was performed and microbroth dilution MICs were determined to establish individual test control values with Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and S. aureus ATCC 29213. In addition, regression analysis correlating inhibitory zone diameter with MICs for approximately 400 gram-negative clinical isolates was performed. Based on linear regression and error rate-bounded analyses, criteria for the category calls of isolates are proposed.     

Evidence for different pyoverdine-mediated iron uptake systems among Pseudomonas aeruginosa strains.

Fourteen strains of Pseudomonas aeruginosa (P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, and 12 clinical isolates) were checked for the production of pyoverdine and for pyoverdine-mediated iron uptake. Under iron restriction, two isolates produced undetectable amounts of pyoverdine, but all the other strains produced a compound with physicochemical properties identical or close to those of the pyoverdine of P. aeruginosa ATCC 15692 (strain PAO1). The pyoverdines were purified and tested for their growth-promoting activity and for their ability to facilitate 59Fe uptake in homologous experiments involving each pyoverdine and its producing strain, as well as in heterologous systems involving all the other strains. The results of both types of experiments suggested the existence of three specificity groups. This was confirmed by analysis of the amino acid composition of the pyoverdines, which differed for each group. A partially purified polyclonal antiserum raised against a major 80-kilodalton (kDa) iron-regulated outer membrane protein (IROMP) of P. aeruginosa PAO1 recognized the 80-kDa IROMPs from P. aeruginosa PAO1 and the clinical isolates belonging to the same group, whereas the IROMPs from the strains belonging to the two other groups were not detected. A second antiserum raised against the P. aeruginosa ATCC 27853 80-kDa IROMP gave similar results by reacting specifically with the 80-kDa IROMP from the strains belonging to this group. Thus, together with the already known pyoverdine from P. aeruginosa PAO1, two new types of pyoverdines produced by strains belonging to this species were characterized.     

Quality control guidelines for testing gram-negative control strains with polymyxin B and colistin (polymyxin E) by standardized methods

An eight-laboratory study addressed the urgent need for quality control (QC) ranges for susceptibility determination when testing colistin (polymyxin E) and polymyxin B, two polycationic peptide antimicrobial agents, against multidrug-resistant gram-negative bacilli. For Escherichia coli ATCC 25922l, the QC ranges were as follows: for colistin, 0.25 to 1 microg/ml (11 to 17 mm), and for polymyxin B, 0.25 to 2 microg/ml (13 to 19 mm). For Pseudomonas aeruginosa ATCC 27853, the QC ranges were as follows: for colistin, 0.25 to 2 microg/ml (11 to 17 mm), and for polymyxin B, 0.25 to 2 mug/ml (14 to 18 mm). More than 97% of all reported QC results were within these proposed ranges.