微生物实验室的培养基质量控制浅析

在任何微生物实验室中培养基扮演着重要的角色。它们被广泛用于各种病原微生物分离、鉴定和敏感性试验,但许多实验室在日常工作中往往重视细菌实验的技术和结果而忽视了培养基的质量。因此,为了得到满意客观的实验结果和科研成果,需要进行必要的培养基质量控制。本文把影响培养     

替加环素治疗碳青霉烯类耐药肺炎克雷伯菌肺炎的临床疗效及预后分析

目的分析替加环素治疗碳青霉烯类耐药肺炎克雷伯菌（CRKP）肺炎患者的临床疗效及预后。方法回顾性分析21例CRKP肺炎患者的临床特征,采用Cox回归分析患者死亡相关因素和替加环素单药治疗或联合其他抗菌药物治疗的临床疗效及预后。结果基础疾病、年龄和病原菌不影响CRKP肺炎患者住院期间病死率（均P＞0.05）,可能与样本量较小有关。替加环素单药治疗9例,有效3例,有效率为33.3%;与其他抗菌药物联合治疗12例,有效9例,有效率为75.0%,治疗效果明显优于替加环素单药治疗（P＜0.05）,总临床有效率为52.4%。8例患者治疗后达到细菌学清除标准,清除率为38.0%。CRKP合并铜绿假单胞菌感染最常见,感染率为47.6%,铜绿假单胞菌的存在并未影响患者预后（P＞0.05）。结论替加环素能有效清除CRKP,可作为CRKP肺炎常规抗感染治疗用药,与其他抗菌药物联合使用能有效治疗其他条件致病菌引起的合并感染。     

问号钩端螺旋体T和B细胞表位联合基因的原核表达及其产物免疫性分析

目的 构建问号钩端螺旋体(简称钩体)LipL32、OmpL1和LipL21蛋白的优势T-和B-细胞联合表位融合基因及其原核表达系统,并对表达产物的免疫原性进行鉴定.方法 人工合成多表位联合基因并构建其原核表达系统.采用SDS-PAGE检测重组蛋白;采用MAT检测重组蛋白兔抗血清与我国钩体标准参考株的凝集效价;Western blot和ELISA检测重组蛋白的免疫原性.结果 获得了多表位融合基因并构建了原核表达系统.表达产物的相对分子质量约为23×103,且主要以可溶性形式存在;重组蛋白兔抗血清免疫双扩散效价为1∶8,该抗血清能与我国15群的钩体标准参考株发生凝集反应,ELISA证明该重组蛋白能检测不同群型钩体感染患者血清中的抗钩体抗体.结论 成功构建了包含钩体LipL32、OmpL1和LipL21蛋白的优势T和B细胞联合表位基因及其原核表达系统,表达产物具有良好的抗原性和交叉免疫反应性,可作为研制通用型问号钩体基因工程疫苗及血清学检测的抗原.     

钩端螺旋体溶血素Sph2表达模式及诱导巨噬细胞和肝细胞凋亡活性

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.     

尿路感染大肠埃希菌的耐药性分析

大肠埃希菌是尿路感染最常见的致病菌,耐药性大肠埃希菌的出现增加了临床治疗难度,产超广谱β-内酰胺酶(ESBLs)大肠埃希菌往往呈现多重耐药性。为了解大肠埃希菌的耐药情况,对我院临床送检的尿液标本中分离到的大肠埃希菌进行药物敏感性分析,以期为临床用药及医院感染控制提供参考。     

钩端螺旋体溶血素Sph2表达模式及诱导巨噬细胞和肝细胞凋亡活性

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.     

钩端螺旋体溶血素Sph2表达模式及诱导巨噬细胞和肝细胞凋亡活性

目的 了解问号钩端螺旋体(简称钩体)感染细胞前后sph2基因表达水平变化,确定鞘磷脂酶类溶血素Sph2及诱导细胞凋亡的活性.方法 采用PCR从黄疸出血群赖型赖株钩体基因组DNA中扩增全长sph2基因片段,T-A克隆后测序.构建sph2基因原核表达系统,采用SDS-PAGE检查重组Sph2(rSph2)的表达情况,Ni-NTA亲和层析法提纯rSph2.采用绵羊血平板溶血试验及血红蛋白分光光度法测定rSph2的溶血活性.采用流式细胞术检测rSph2诱导小鼠单核-巨噬样细胞株J774A.1和肝细胞株IAR20凋亡的活性,实时荧光定量PCR检测赖株钩体感染J774A.1和IAR20细胞前后sph2基因mRNA水平变化.结果 与GenBank中sph2基因比较,所克隆的sph2基因序列相似性为100%.所构建的原核表达系统能高效表达rSph2.rSph2以浓度依赖方式溶解绵羊红细胞.10 μg/ml rSph2可诱导J774A.1和IAR20细胞凋亡,凋亡率峰值分别为23.96%和32.92%.赖株钩体感染J774A.1和IAR20细胞后0.5～2 h内sph2基因mRNA水平显著升高,2 h后mRNA水平迅速下降.结论 钩体sph2基因呈宿主细胞接触式瞬时表达.rSph2有溶解绵羊红细胞及诱导巨噬细胞和肝细胞凋亡的活性,因而Sph2是钩体致病过程中重要的毒力因子.     

肺结核患者支气管肺泡灌洗液病原菌分布与耐药性分析

目的了解医院肺结核患者支气管肺泡灌洗液(BALF)病原菌分布及耐药性,为合理用药和医院感染控制提供参考。方法对医院2011-2012年肺结核患者BALF分离的病原菌进行回顾性分析,采用WHONET5.6软件进行统计分析。结果 443份BALF样本共分离出病原菌310株,其中革兰阴性菌占96.1%,主要为铜绿假单胞菌、鲍氏不动杆菌、褪色沙雷菌、肺炎克雷伯菌、奇异变形菌,革兰阳性菌占3.9%,主要为金黄色葡萄球菌、溶血葡萄球菌;主要革兰阴性杆菌对氨苄西林和头孢唑林的耐药率均>75.0%,对阿米卡星的耐药性较低,均<20.0%,铜绿假单胞菌和鲍氏不动杆菌多药耐药性严重,对亚胺培南的耐药率分别为71.1%和94.3%,主要革兰阳性菌对万古霉素、利奈唑胺完全敏感,耐甲氧西林金黄色葡萄球菌(MRSA)检出率为85.7%。结论肺结核患者BALF病原菌阳性率高,常见病原菌多为条件致病菌,其耐药性较为严重,应加强细菌耐药监测,以指导合理用药和医院感染控制。     

CISF-820血细胞分析仪一种新的排堵方法

我科自1997年引进日本产CISF-820血细胞分析仪以来,对日常使用过程中发生的检测器上的宝石孔和HB单元的管道堵塞现象,我们利用双缩脲试剂进行清堵,取得良好效果。现将处理方法介绍如下,供同行参考: 把浓缩的双缩脲试剂(宁波慈城产)用50-60℃的蒸馏水稀释5倍,用稀释杯取适量浸泡检测器上的宝石孔约15 min,然后用仪器附带的毛刷湿润后轻轻刷洗若干次,再分别按“WBC CLOG”和“RBC CLOG”两键连续清堵2次,最后换上干净的血细胞稀释液进行空白测定即可。HB单元的管道堵塞的排除应在仪器关机的情况下,拆开仪器的     

高龄骨折患者院内获得性肺炎分析及干预对策

随着我国迅速进入老龄化社会,高龄骨折患者住院率日益增加,除骨折外,并发症的发生往往成为影响患者预后的很大因素.医院获得性肺炎（hospital acquired pneumonia,HAP）是指患者入院时不存在、也不处感染潜伏期,而于入院48h后由细菌、真菌、支原体、病毒或原虫等病原体引起的各种类型的肺实质炎症.据文献报道,高龄患者发生院内获得性肺炎（HAP）感染是年龄＜65岁患者肺炎发生率的4倍[1-2],预后不佳,死亡率高,国内报道HAP引起的死亡率达34.2％[3].必须引起临床高度关注.笔者对486例高龄骨折患者发生HAP情况进行回顾分析,报道如下.