住院患者革兰阴性杆菌的耐药性分析

目的了解临床分离的革兰阴性杆菌的分布及耐药性变迁。方法2001—2003年临床分离革兰阴性杆菌1710株。排除同一患者的重复菌株,按统一方案用K—B纸片扩散法,按美国国家临床实验室标准委员会（NCCLS）2000年版标准判读结果。结果住院患者最常见的临床分离菌株为铜绿假单胞菌（8．3％）、不动杆菌属（12．8％）、大肠埃希菌（9．5％）和沙雷菌属（13．6％）,革兰阴性杆菌占总分离率的62．4％。亚胺培南、头孢哌酮-舒巴坦和哌拉西林-他唑巴坦对革兰阴性杆菌抗菌活性好。产超广谱β-内酰胺酶（ESBLs）大肠埃希菌占其总株数的15．8％。结论近年来革兰阴性杆菌的耐药性呈上升趋势,特别对第3代头孢菌素耐药性上升最快,临床上应关注细菌耐药性变化,合理使用抗菌药物。     

细菌菌群的分布及耐药性分析

目的：了解本院临床分离致病菌菌群的分布及耐药情况,给临床经验用药提供可靠依据。方法：按《全国临床微生物检验操作规程》培养分离菌种,用美国BD公司的Sceptor半自动细菌鉴定仪或法国梅里埃公司的Vitek-60细菌鉴定仪进行鉴定及药物敏感实验,结果：临床分离 的致病菌中革兰阳性球菌占39．6％,革兰阴性杆菌占60．4％,前5位细菌分别为铜绿假单胞菌188株（17．0％）,大肠埃希菌143株（13．0％）,表皮葡萄球菌140株（12．7％）,肠球菌115株（10．7％）,金黄色葡萄球菌109株（9．9％）,药敏结果显示,苯唑西林耐药葡萄球菌（MRS）的耐药率明显高于苯唑西林敏感葡萄球菌109株（9．9％）,药敏结果显示,苯唑西林耐药葡萄球菌（MRS）的耐药率明显高于苯唑西林敏感葡萄球菌（MSS）,球菌中未分离出万古霉素耐药的菌株,但肠球菌中检测出有对万古霉素中介的菌株;肠杆菌科细菌对亚胺培南的耐药率最低,铜绿假单胞菌对头孢他啶,头孢哌酮,氨曲南,丁胺卡那霉素,亚胺培南的耐药率亦较低。结论：革兰阴性杆菌分离率高于革兰阳性球菌;革兰阳性球菌对万古霉素敏感率最高,肠杆菌科对亚胺培南敏感率最高,非发酵菌对亚胺培南,氨基糖甙类,头孢他啶,头孢哌酮药蓄亦较低。     

人工合成多肽检测抗HIV－1和HIV－2抗体的研究

采用固相法合成ＨＩＶ－１和ＨＩＶ－２两个多肽,建立了用混合多肽为包被抗原检测ＨＩＶ－１和ＨＩＶ－２感染的间接酶联免疫吸附法。检测４６份抗ＨＩＶ－１和ＨＩＶ－２抗体阳性血清标本以及９４份对照血清标本,与ＵＢＩ试剂比较,其阳性符合率为９７．８％,阴性符合率为１００％,总符合率为９９．３％。实验结果表明,此法可用于ＨＩＶ－１和ＨＩＶ－２感染的检测。     

新生儿与婴幼儿眼部菌群的调查

本文用分离培养法或聚合酶链反应检查了３６４例出生０－５岁的正常或患眼炎的新生儿及婴幼儿眼部菌群的种类分布，来源，药物敏感性及其同眼炎的关系。结果表明新生儿出生２４小时内眼部大多无菌或仅可检出革兰氏阴性细菌检出率为３３．３％，出生２天后可检出多种微生物，检出率达９４．２％，分离培养法检查的２２０例新生儿与婴幼儿中，眼部带菌者１９９例，感染率为９０．５％。     

7岁以下儿童细菌感染耐药分析

目的观察大连地区5家医院（大连医科大学附属一院、大连医科大学附属二院、大连儿童医院、大连市第六人民医院、大连开发区医院）2012年7岁以下儿童感染病原菌分布及其耐药情况。方法临床分离菌株,采用细菌分离鉴定方法（API、VITEK、Microsean系统）进行目标细菌的鉴定,药物敏感性试验用K—B纸片扩散法测定药物的敏感性。结果共收集细菌1235株,其中革兰阴性菌725株占58．7％,革兰阳性菌510株占41．3％;分离细菌前5位依次为大肠埃希菌占14．9％、凝固酶阴性葡萄球菌占13．6％、金黄色葡萄球菌占11．7％、肺炎克雷伯菌占10．8％、铜绿假单胞菌占7．4％。结论7岁以下儿童感染致病菌对抗生素均存在不同程度耐药情况,期望在临床治疗感染时有所帮助。     

Vitek-AMS鉴定临床细菌的应用研究

目的探讨Vitek-AMS对临床细菌鉴定的应用价值。方法对玉溪市人民医院1999年至2008年临床分离11 537株细菌(临床株)和省、部级临床检验中心下发微生物学室间质量评价鉴定菌种(参考株)的Vitek-AMS鉴定结果作对比分析。结果 11 537株临床分离菌中,不能鉴定细菌8株(0.07%);除外传染病因子,鉴定到属细菌114株(1.62%),鉴定到种(含亚种、物生型)细菌6 908株(98.38%)。共有64属193种。细菌类型的分布革兰阴性杆菌革兰阳性球菌酵母菌革兰阳性杆菌厌氧菌革兰阴性球菌。用参考菌种作比较,Vitek鉴定种的符合率为83.08%(54/65),属的符合率为98.46%(64/65);其中革兰阴性杆菌符合率最高(100%,24/24),酵母菌类符合率最低(66.67%,14/21)。7种鉴定卡菌种的阳性检出率平均为56.80%(234/412),其中GPI卡最高(100%),NHI卡最低(13.33%);但应用机会最多是GNI+卡。Vitek-AMS检测葡萄球菌产β-内酰胺酶阳性率为88.89%,大肠埃希菌和肺炎克雷伯菌产ESBLs阳性率分别为59.74%和32.20%。结论 Vitek-AMS的应用为临床细菌学检验提供了一种高效、快速、可靠的实验方法;但对于个别菌种、细菌酶的检测必要时应以参考方法确认。     

239株脑脊液标本非重复分离株病原学分布与药物敏感试验结果分析

目的通过回顾性分析脑脊液培养结果的病原菌分布与药物敏感试验结果,分析中枢神经系统感染相关细菌的耐药性及其变化趋势。方法选取西安交通大学医学院第一附属医院2012年1月至2014年9月期间从临床患者脑脊液标本中分离培养出的细菌,所有菌株均采用全自动微生物分析仪进行鉴定,仪器法与纸片法检测其对抗生素的敏感性。用WHONET 5.6软件进行数据统计分析。结果本研究共包含非重复分离菌株239株,其中革兰阳性菌175株(74%),革兰阴性菌59株(24%),真菌5株(2%)。革兰阳性菌主要为葡萄球菌属、肠球菌属与链球菌属细菌,其中表皮葡萄球菌最为常见。革兰阴性菌主要为肠杆菌科细菌与非发酵革兰阴性杆菌,其中鲍曼不动杆菌、肺炎克雷伯杆菌、大肠埃希菌最为常见。真菌均为新型隐球菌。分离菌株中革兰阳性菌与革兰阴性菌均有不同程度的耐药现象,革兰阳性菌仅对利奈唑胺、替考拉宁、万古霉素敏感率为100%,革兰阴性菌中肠杆菌仅对阿米卡星、美洛培南、哌拉西林/他唑巴坦、亚胺培南敏感率为90%以上,而鲍曼不动杆菌对各类抗生素耐药率均在80%以上。结论中枢神经系统感染以革兰阳性菌为主,表皮葡萄球菌为最常见的分离菌株。多重耐药菌的出现,使中枢神经系统感染的治疗面临巨大挑战,提示临床合理使用抗生素进行抗感染治疗至关重要。     

用ELISA一步法检测白色念珠菌

用白色念珠菌临床分离株免疫家兔并经纯化获得特异性抗体,建立了检测临床标本中白色念珠菌抗原的ＥＬＩＳＡ一步法,此法的敏感性为９９％,特异性为９８３％,与其它念珠菌和临床常见细菌均无交叉反应,整个反应只需１小时即可完成,可望成为一种取代培养法的快速诊断方法     

VITEK-2 Compact仪器法检测革兰阴性杆菌临床分离株的耐药性

目的 调查住院患者临床分离株中革兰阴性杆菌的分布及耐药状况,为革兰阴性菌的治疗提供参考.方法 收集浙江中医药大学附属第一医院2011年全年送检的标本,采用VITEK-2 compact全自动微生物鉴定仪,采用GNI鉴定卡、AST-GN13药敏卡进行菌株的鉴定和药敏,根据临床实验室标准化协会(CLSI 2011)制定的指导原则,判断细菌的耐药率.结果 共分离到革兰阴性菌972株.ICU分离到革兰阴性菌254株,其中非发酵菌占73.6％(187/254).非ICU分离到革兰阴性菌718株,其中非发酵菌246株占34.3％(246/718).亚胺培南对ICU分离菌的MIC50是非ICU的8倍,MIC90值相当.结论 ICU患者分离的革兰阴性菌以非发酵菌占大多数.非ICU患者分离的革兰阴性菌以肠杆菌科细菌为多.VITEK-2 Compact仪器药敏法是适合于临床常规使用的方法.     

革兰阴性杆菌ESBLs和AmpC酶的检测及耐药分析

目的检测引起医院感染的革兰阴性杆菌携带产ESBLs和去阻遏AmpC酶状况,探讨各菌对临床常用抗生素的主要耐药机制及耐药性,为临床制定合理使用抗生素策略提供依据。方法采用全自动微生物分析仪（VITEK-32）做细菌鉴定和药敏试验,用纸片扩散确证法检测超广谱β-内酰胺酶（ESBLs）,用三维法检测高水平表达染色体编码的AmpC酶。结果158株革兰阴性杆菌ESBLs检出率为26．6％,主要菌为大肠埃希菌（45．2％）、肺炎克雷伯菌（42．9％）、阴沟肠杆菌（11．9％）。AmpC酶检出率为10．1％,主要为鲍曼不动杆菌（43．8％）、阴沟肠杆菌（25％）;上述产酶细菌均对青霉素和一、二、三代头孢菌素、磺胺类、喹诺酮类、氨基糖苷类耐药,对亚胺培南敏感。结论革兰阴性杆菌耐药机制主要是产超广谱β-内酰胺酶和AmpC酶,这些产酶菌株均出现多重耐药。     

Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32 degrees C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.     

Microtiter Plate Assay for Assessment of Listeria monocytogenes Biofilm Formation

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32°C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.     

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.     

VITEK-2 compact全自动微生物鉴定仪对葡萄球菌鉴定能力的评价

目的评价VITEK-2 compact全自动微生物鉴定仪对葡萄球菌的鉴定能力。方法收集从我院病人标本中分离的葡萄球菌81株。常规细菌培养后,用VITEK-2 compact和API Staph系统进行检测,以API Staph系统为参照,评价VITEK-2 compact的优势和不足。结果 VITEK-2 compact和API Staph系统的总体鉴定符合率为95.1%,其中金黄色葡萄球菌的鉴定符合率为100%,凝固酶阴性葡萄球菌的鉴定符合率为90.5%。结论 VITEK-2 compact鉴定系统能够满足临床工作的需求,其中对金黄色葡萄球菌的鉴定率较高,在进行凝固酶阴性葡萄球菌鉴定时有一定的局限性,需要其他方法予以补充。     

A microtiter plate assay for protein kinase C

We report the development of a microtiter plate assay for protein kinase C. Reaction components and enzyme samples (protein kinase C purified by phosphatidylserine/cholesterol affinity or DEAE-Sephacel ion-exchange chromatography) were added to wells of a 96-well microtiter plate. The assay was started by the addition of [gamma-32P]ATP with a repeating pipet. After a 3-min incubation at 30 degrees C the wells were sampled six at a time with a 12-channel pipet and spotted onto phosphocellulose filter paper rectangles which were washed with tap water and acetone and counted for radioactivity. The microtiter plate method was more rapid than but gave results similar to those of a standard assay performed in plastic test tubes individually incubated in a 30 degrees C water bath. The microtiter plate procedure gave an intraassay (within one plate) variation of less than 9% and an interassay (between plates) variation of less than 5%. It was linear with time of incubation for 20 min and with amount of enzyme. This method can be used to expedite the assaying of column chromatography fractions for protein kinase C (and other kinase) activity.     

Fluorometric microassays for the determination of cathepsin L and cathepsin S activities in tissue extracts.

We established a continuous semi-microassay, and for large-scale studies both a stopped and a continuous microtiter plate assay for the fluorometric determination of cathepsin L and cathepsin S activities in body fluids, tissues or cell extracts in the presence of cathepsin B. For the detection of enzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathepsin B the specific inhibitor CA-074 for blocking interfering cathepsin B activities was applied. Furthermore, we took advantage of the stability of cathepsin S at pH 7.5 for further differentiation between cathepsin L and cathepsin S activities. The kinetic assays were characterized in terms of imprecision, analytical sensitivity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9%-7.2% for the continuous semi-microassay, 10.3%-11.7% for the stopped, and 4.5%-11.8% for the continuous microtiter plate assay. The between-days coefficients of variation for the continuous semi-microassay were 8.1%-8.9%, while for the stopped and continuous microtiter plate assays the coefficients were 11.2%-13.5% and 5.8%-12.2%, respectively. Compared to the continuous semi-microassay, the stopped and the continuous microtiter plate assays showed 3-fold and 11-fold higher sensitivity, respectively. Comparison between the continuous enzyme activity assays at substrate concentrations of 40 microM and 200 microM demonstrated a significant correlation of r = 0.97 and r = 0.99, respectively. The newly developed microtiter plate assay will allow efficient, sensitive and high precision determination of cathepsin L and cathepsin S activities in large-scale studies of cysteine-cathepsin dependent diseases.     

A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies.

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.     

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.     

Toward complete laser ablation of melanoma contaminant cells in a co-culture outgrowth model via image cytometry.

BACKGROUND: Contaminant cancer cells in autologous transplant tissue can cause relapse and the rates are unknown. A method capable of removing all contaminant cells with a high probability detected by cytomic analyses would be useful. Neither 100% cell purging nor techniques for measuring the probability of success have been developed. Here, we report a method for removing 100% of the cells under ideal staining conditions and quantify the probability of success. METHODS: Laser ablation was combined with previously reported automated microscopy to purge contaminant cells and evaluate 100% ablation in a co-culture model of prestained mouse melanoma cells mixed with mouse NIH-3T3 cells. Melanoma passage efficiency was measured by: (1) micropipetting single cells into microtiter wells and (2) ablating all but one melanoma cell in co-cultures. RESULTS: (74 +/- 5)% of single melanoma cells pipetted into microtiter plate wells divided at least once. With ablation of all but one contaminant cell in co-cultures, melanoma dominated in (62 +/- 8)% cultures in 21 days. With 100% ablation in six additional experiments, no melanoma outgrowth was observed, giving a >99.1% probability that all contaminant melanoma cells were purged. CONCLUSIONS: We successfully demonstrated a model for complete ablation within a defined probability using automated high-content image cytometry with ideal staining conditions. The results show that the instrumentation is capable of delivering 100% ablation at a defined probability and establishes the basis for further studies with clinical models wherein pretherapeutic cytomic analyses of unique cellular expression and/or morphological characteristics will be key for contaminant cancer cell identification.     

A Microtiter Plate Assay for Screening Antioxidant Activity in Extracts of Marine Organisms

A novel microtiter plate assay was developed to determine the total peroxyl radical-trapping activity of antioxidants extracted from marine organisms by measuring the inhibition rate of dye-substrate oxidation. We compared use of dihydrorhodamine-123, dihydrofluorescein, and dichlorodihydrofluorescein as reduced substrates for oxidation by peroxyl radicals generated from 2,2-azobis(2-amidinopropane) dihydrochloride. The oxidation products of these highly reactive substrates are intensely colored dyes that absorb maximally in the wavelength region, _max = 489 to 512 nm, and their concentrations were determined photometrically using a 96-well, microtiter plate reader. The microtiter plate method provides for concurrent multisample analysis with automated data storage, regression analyses, and calculation of oxidation inhibition rates. Dihydrorhodamine was selected as the preferred substrate for screening crude extracts, and typical assay results are presented. Novel lead antioxidants are selected from active extracts by chromatographic analysis with electrochemical detection.