建立洁净区微生物数据库与无菌药品GMP生产过程控制的探讨

目的 建立洁净区微生物数据库,为追溯洁净区微生物污染来源提供依据,为无菌药品生产过程控制提供有力指导.方法 对乙型脑炎减毒活疫苗生产车间洁净区环境和操作人员进行微生物负载检测,并对该车间注射用水、纯化水系统样品进行微生物限度检测,鉴别研究相应分离菌,建立洁净区微生物数据库.同时对乙型脑炎减毒活疫苗中间产品进行无菌检查检测,并对阳性结果分离菌进行鉴别分析.然后根据建立的洁净区微生物数据库对阳性结果举例进行溯源探讨分析.结果 洁净区环境和人员主要存在里拉/藤黄微球菌、人葡萄球菌、表皮葡萄球菌以及科氏葡萄球菌科氏亚种等革兰阳性菌和蜡样芽胞杆菌等,水系统中则主要存在少动鞘氨醇单胞菌和铫子芽胞杆菌等.中间产品无菌阳性结果主要存在里拉/藤黄微球菌、蜡样芽胞杆菌以及奇异变形菌等,经调查分析,分别为操作过程中经环境偶然带入或试验动物操作不慎带入.结论 建立洁净区微生物数据库是追溯产品微生物污染来源的有效方法,能够为无菌药品GMP生产过程制定有效的控制措施提供依据,并使其更有针对性.     

无菌动物无菌检测的探讨

近年来,肠道微生物在人类的健康和疾病中的应用受到了广泛关注.无菌动物在肠道微生态中的模型应用越来越广泛,为人类的生理、病理研究提供了非常有效的研究工具.为保证实验动物的质量,必须对无菌动物频繁的进行无菌检测.但无菌检测受到诸多因素的干扰.目前传统的检测方法 主要是细菌、真菌培养及革兰染色镜检.随着高通量测序的发展,现在...     

无菌动物与微生态学专题教学改革实践

根据研究生教学特点,为适应肠道菌群与人类疾病关系研究成为新研究热点与前沿的形势,对其无菌动物与微生态学专题教学进行改革实践。根据菌群与疾病关系研究的最新研究进展、研究生科研能力培养规律,在研究生《医学实验动物学》课程的无菌动物与微生态学专题教学中设置微生态学理论与研究技术简介、无菌动物培育原理及特点、无菌动物实验设计方法、国内外无菌动物资源简介等4部分内容。力争紧跟学科发展动态,强调知识的前沿性、实用性、综合性和交叉性,促进研究生课题设计能力培养和科研思路的开拓,创建研究生无菌动物与微生态学专题教学的新模式。     

肝硬化患者无菌腹水中真菌DNA的检测及易位研究

目的检测和分析肝硬化患者无菌腹水中真菌DNA存在状况。方法选取118例无菌腹水的肝硬化患者为研究对象,分别采集患者刚入院时的腹水、血液和粪便标本。应用PCR技术直接检测标本的真菌DNA并克隆测序。并收集患者临床资料,对患者临床症状及检验结果进行分析比较。结果1名患者的腹水、血液、粪便中同时检测到真菌DNA,14名患者的腹水和粪便中同时检测到真菌DNA,3名患者的血液和粪便中同时检测到真菌DNA,大部分序列间的相似性高于99％。测序检测到最多的菌种为Candidaalbicans。结论肝硬化患者无菌腹水中能够检测到真菌DNA,并且提示在肝硬化患者腹水和血清中检测到的真菌DNA易位于肠道。     

盐酸氯丙嗪注射液无菌试验方法的改进

盐酸氯丙嗪,又称冬眠灵。临床用于治疗精神分裂症。中国药典各版均以正文收载了盐酸氯丙嗪注射液。其无菌试验法按常法加入供试液后,培养基立即出现白色浑浊。尤其是需、厌气培养基反应极为明显。霉菌培养,基也出现浑浊状态。经35℃、25℃培养,其浑浊程度更为严重。阳性对照菌液加入后难以观察对照菌是否生长。该浑浊液对阳性菌有抑制作用。所以     

斑马鱼胚胎的简易无菌培养体系及单增李斯特菌感染试验北大核心CSCD

【目的】斑马鱼的生活环境中微生物众多,会激活其先天免疫系统,从而影响相关试验结果,因此需建立适合于感染与免疫研究的斑马鱼无菌培养体系。【方法】建立了基于受精卵短时消毒和温控正压无菌隔离器的培养流程;根据无菌动物标准对斑马鱼胚胎及其生活环境进行微生物检测;并通过定量PCR检测无菌斑马鱼先天免疫相关基因(TLRs)表达水平;利用无菌斑马鱼胚胎模型进行了单增李斯特菌感染试验。【结果】本研究成功建立了斑马鱼培育的无菌操作系统,无菌检验结果显示其生活环境及体内不含病原微生物,先天免疫分子TLRs表达量较低或不表达,而常规培养斑马鱼以及浸泡感染单增李斯特菌的无菌斑马鱼中这些基因转录水平较高。无菌斑马鱼对单增李斯特菌强毒株EGDe静脉注射感染很敏感,100 CFU感染量能导致鱼在1周内全部死亡,而其mpl或plc B基因缺失株感染后致死率显著下降(分别为70%和40%)。巨噬细胞在亲本株EGDe浸泡感染的鱼肠道周围聚集,而mpl或plc B基因缺失株感染的鱼中几乎观察不到这一聚集现象。【结论】通过简易培养体系获得的无菌斑马鱼胚胎可用于先天免疫和病原感染机制等研究。     

血液或无菌体液培养阳性标本中病原菌检测结果分析

目的：了解血培养（包括血液、无菌体液培养,下同）阳性标本中病原菌的分布及阳性报警时间,为临床及时明确病原菌和正确用药提供参考依据。方法：无菌条件下采集血培养标本注人相应的培养瓶,经仪器扫描后放入血培养仪进行检测,报警后及时进行菌种鉴定和药敏试验。结果：364例阳性报警标本中真阳性标本为176例,其中革兰阳性菌占61．7％,革兰阴性菌占35．0％,真菌占3．3％;188例为假阳性标本,其中革兰阳性菌占54％,革兰阴性菌占41．9％,真菌占4．0％;新生儿科的感染阳性率最高;不同种类病原菌的阳性报警时间多重叠;临床医生经验用药正确或根据药敏结果更换用药的百分比为78．2％。结论：本院引起血液、无菌体液感染的病原菌以革兰阳性细菌为主,病原菌种类较多,存在一定的污染;当新生儿有局部感染时要警惕脓毒血症;单独靠血培养仪报警时间来鉴定区分病原菌与污染菌不一定可靠,及时了解血培养结果及标准药敏结果可以辅助找出感染性疾病的病因,尽早正确合理的使用抗菌药物,从而优化抗菌治疗。     

无菌检查的意外污染探讨

无菌检查法系指检查药物与敷料、器械是否无菌的一种方法。各国药典对无菌试验的具体操作 ,如所用的方法、培养基、培养时间、结果判断等均作了详细的规定。但未见到为了防止操作中的意外污染 ,应选用的仪器、设备和使用方法的规定。中国药典规定 :“无菌检查的全过程应严格遵守无菌操作 ,防止微生物的污染”。目前 ,我国无菌检查一般都在无菌室或超净工作台上操作 ,但国内的做法在检查过程中仍存在着较大意外污染的可能性 ;如样品容器外壁消毒不当 ,折颈时玻璃屑掉入 ,样品转移 ,滤膜过滤、淋法、滤膜转移等操作以及取样的针头或吸管均有意…     

无菌APPswe/PS1ΔE9双转基因小鼠模型建立及脑内斑块变化初步观察

目的 使用剖宫产净化方法建立无菌APPswe/PS1ΔE9(PAP)双转基因小鼠模型并对动物脑内斑块沉积情况进行初步观察,为研究肠道菌群与阿尔茨海默症关系提供新的动物模型。方法 选择阳性PAP雄性杂合子鼠与经产的C57野生型雌鼠1∶2进行交配。怀孕母鼠在超净工作台内行剖宫产手术,用无菌ICR小鼠代乳。术后每个月进行无菌状态检测;PCR方法检测剖宫产所得PAP仔鼠的基因型;免疫组化方法定量检测9月龄PAP小鼠脑内斑块变化情况。结果 实施剖宫产手术12例,获仔鼠66只,剖宫产存活率及离乳存活率分别为95.45%(63/66)和95.24%(60/63),净化后按国标检测无菌状态均为合格。免疫组化结果显示9月龄无菌PAP小鼠海马内斑块较同月龄SPF级动物减少。结论 通过剖宫产净化技术去除了PAP小鼠携带的菌群,9月龄无菌PAP小鼠脑内斑块减少。     

CD11b表达与人工髋关节置换术后无菌性松动的相关性分析

目的:分析髋关节周围滑膜组织CD11b表达水平与人工髋关节置换术后无菌性松动的相关性。方法:以2006年5月至2016年5月于西京医院接受人工髋关节置换的患者为研究对象,对其髋关节周围滑膜组织进行CD11b免疫组化染色,并随访术后5年和10年无菌性松动的发生情况,通过单因素分析及logistic回归分析讨论CD11b表达与无菌性松动之间的相关性。结果:共300例患者纳入研究,全部获得随访,CD11b表达阳性患者163例,阳性率为54.33%;术后5年松动患者29例,术后5年无菌性松动发生率为9.67%;术后10年无菌性松动患者49例,术后10年无菌性松动发生率为16.33%;单因素分析结果表明CD11b表达阳性患者5年及10年松动率均高于CD11b表达阴性患者(P0.05);logistic回归分析结果表明CD11b过表达是髋关节置换术后无菌性松动发生的危险因素。结论:髋关节周围滑膜组织CD11b过表达是人工髋关节置换术后无菌性松动发生的危险因素,该分子或可作为无菌性松动的辅助诊断指标及潜在治疗靶点。     

Transformation of blueberry without antibiotic selection

Transformation of the blueberry cultivar North Country (Vaccinium corymbosum × V. angustifolium) was achieved using the disarmed Agrobacterium tumefaciens isolate LBA4404, containing a binary vector with an intron-containing β-glucuronidase (GUS) marker gene. Plant regeneration was carried out in the absence of antibiotics due to their toxicity to blueberry explants. Agrobacterium contamination was controlled by dipping the explants in antibiotics and rinsing in sterile distilled water. Once regeneration had been achieved, the plantlets were placed on to medium containing the antibiotic ticaricillin at 250 mg litre^-1 to control and try to eliminate any remaining Agrobacterium. Selection of regenerating explants expressing GUS was achieved by growing the plant material for 2 days on a medium containing 4-methyl umbelliferyl glucuronide (MUG), and examining the medium under UV light to detect fluorescent activity. From the 19 explants showing signs of regeneration, seven produced fluorescent patches on MUG medium. From the selected explants, five plantlets were found to express the GUS gene as detected by fluorometric and histochemical analysis. PCR was used to confirm the presence of the GUS and/or NPTII marker genes after 2 years in culture. Bacterial contamination isolated from plant material (which appeared free of contamination) was examined for GUS activity and analysed using PCR with GUS and NPTII specific primers, but no positive results were obtained.     

Detection of Pseudomonas aeruginosa in water samples using a novel synthetic medium and impedimetric technology

AIMS: To investigate whether the use of a novel synthetic medium in conjunction with impedimetric technology could provide a rapid and automated detection of Pseudomonas aeruginosa in water samples. METHODS AND RESULTS: A selective synthetic medium (Z-broth) in which the only carbon and nitrogen source is acetamide was applied in direct impedimetric examination for the selective isolation of P. aeruginosa. A total of 1036 tap-water, well-water, swimming-pool water and dialysis water samples were investigated, and any P. aeruginosa contamination was detected in 7-24 h. Neither false-negative nor false-positive results were observed. CONCLUSIONS: The results of the present evaluation demonstrate that impedance measurement with the use of Z-broth is suitable for the rapid and automatic detection of P. aeruginosa in water. SIGNIFICANCE AND IMPACT OF THE STUDY: The main advantages of the method: 240 samples can be examined in one step, the procedure is fully automated, the results are obtained quickly and the labour and media requirements are low.     

Bacterial contamination of fetal fluids at the time of cesarean section in the cow

Complications after cesarean section delivery in cattle are mainly the result of infections. The bacteria responsible for this infection can be of exogenous or endogenous origin. In this investigation endogenous contamination was studied. Fetal fluid samples of 23 cows were collected from the uterine cavity during cesarean section just after the removal of the calf, by means of a sterile disposable plastic syringe. The uterine flora was cultured, quantitated and presumptively identified by using selective and elective agarplates. Nineteen samples were positive after culture. Eleven samples contained obligate anaerobic bacteria. When the amniotic sac was broken before the obstetrical examination, the total number of bacteria was significantly higher. Vaginal exploration by the farmer had no significant influence on the number of bacteria encountered. Cesarean section is considered a clean contaminated procedure. One must always take into account that the fetal fluids are contaminated with the endogenous vaginal flora. This leads inevitably to contamination of the wound and the peritoneal cavity. Properly antimicrobial prophylaxis is certainly indicated.     

Salmonella on pig carcasses: positive pigs and cross contamination in the slaughterhouse

AIMS: The purpose of this study was to investigate the prevalence of Salmonella in pigs at the moment of slaughter and in the slaughterhouse environment. METHODS AND RESULTS: In total, five different commercial slaughterhouses were sampled during eight slaughterhouse visits. Carcass swabs, colon content and mesenteric lymph nodes were taken to reflect the animal status and from the slaughterhouse environmental samples were taken. Salmonella was isolated from 37% of the carcass samples as a mean value. High variations were noticed between different slaughterhouses (between 0 and 70%) and sampling days in the same abattoir (between 3 and 52%). A correlation was found between the carcass contamination and the status of the delivered animals (P=0.01675). Cross contamination was estimated to account for 29% of the positive carcasses. The slaughterhouse environment was highly contaminated; before starting the slaughtering activities 25% of the samples were positive on average. The most prevalent serotypes isolated at the slaughterhouse environment and from the colon content were S. Typhimurium, S. Livingstone and S. Derby. On carcasses S. Typhimurium was predominately isolated (71%). The biggest variability of serotypes was found in the mesenteric lymph nodes. Serologically 56.3% of the pigs were found positive for Salmonella using a cut-off level of the optical density percentage higher than 10 (O.D.% > or = 10). While on individual pig level the correlation between the bacteriological and serological data was poor, because of recent Salmonella infections, a better correlation was found at the herd level on the moment of slaughtering. CONCLUSION: A high degree of carcass contamination is noticed after slaughtering. This contamination resulted from the delivery of Salmonella-positive pigs and cross-contamination from the slaughterhouse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: In pigs, Salmonella carriage is high, but it is obvious that slaughterhouse hygiene is a determinative factor for managing carcass contamination.     

The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing

Listeria monocytogenes was isolated in low numbers from a variety of environmental samples associated with the primary production of food, including vegetation, faeces and meat. The organism was rarely detected on growing grass and vegetables prior to processing. The excretion of L. monocytogenes by farm animals was linked to their diet, with animals fed entirely on hay or manufactured diets not excreting detectable levels of Listeria (i.e. absence in 25 g). However, animals fed on silage, which is frequently contaminated with L. monocytogenes , commonly excreted the organism. Transport of live animals over long distances (> 100 km) significantly increased the level of excretion of Listeria , but the contamination of carcasses of sheep and cattle was not high. Pigs and poultry faeces were free of Listeria prior to slaughter and pig carcasses were not found to have Listeria present. Frozen and chilled chicken did show detectable levels reflecting the greater potential for contamination during poultry processing. Samples of minced beef were tested and 21 of 23 samples were positive for L. monocytogenes , demonstrating that processing significantly increases the level of contamination compared to whole carcasses. Multilocus enzyme electrophoresis of a representative selection of the isolates showed that there was a wide range of electrophoretic types present in the primary production environment, relatively few of which have been linked to cases of human listeriosis. However, these types do arise on farms and occasional contamination of food raw material by potentially virulent strains may be sufficient to allow adaptable strains to become established in the processing environment and thus be responsible for more widespread contamination of the food available to the consumer.     

Directed neural differentiation of duck embryonic germ cells

Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.     

Identification, culture, and characterization of germline stem cell-like cells in chicken testes

We recently succeeded in inducing germline transmission by transferring chicken testicular cells into heterologous testes. This study was designed subsequently to identify pluripotent cells in the testicular cells, which would induce the germline transmission. Testicular cells retrieved from juvenile (4-wk-old) or adult (24-wk-old) White Leghorn (WL) chickens were stained with germ cell-specific markers anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-EMA1, anti-ITGA6, and anti-ITGB1 antibodies; 2C9; and lectin-Solanum tuberosum agglutinin (STA). The percentages of the cells that were positive for each marker were within the ranges of 0.33% -0.44% and 0.029%-0.072% of the total testicular cell population in the juvenile and adult, respectively, and significant (P < 0.0002) differences were detected between the ages. When 1 x 10(6) testicular cells were cultured in Dulbecco minimum essential medium-based medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF2), and/or insulinlike growth factor 1 (IGF1), colony formation was detected only in LIF++FGF2-containing or LIF+FGF2+IGF1-containing medium during primary culture, and the supplementation of LIF+FGF2+IGF1 was the most efficient for maintaining the colony-forming cells through subculture. The established cells retrieved at the end of the primary culture or the 20th subpassage were positive for chicken germ cell-specific periodic acid-Schiff (PAS), EMA1, 2C9, SSEA1, SSEA3, SSEA4, ITGA6, and ITGB1; and lectin-STA markers (evaluated after 11th subpassage). Double staining of lectin-STA with anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-ITGA6, and anti-ITGB1 also was possible. They differentiated spontaneously into embryoid bodies after being cultured in LIF-free medium. We conclude that germline stem cell-like cells are present in chicken testicular cells retrieved from both juvenile and adult testes, which can be identified with the specific markers for primordial germ cells or embryonic germ cells.     

Identification of Human and Animal Adenoviruses and Polyomaviruses for Determination of Sources of Fecal Contamination in the Environment

The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.     

Presence and Persistence of Coxiella burnetii in the Environments of Goat Farms Associated with a Q Fever Outbreak

Q fever is a zoonotic disease caused by inhalation of the bacterium Coxiella burnetii. Ruminant livestock are common reservoirs for C. burnetii, and bacteria present in aerosols derived from the waste of infected animals can infect humans. The significance of infection from material deposited in the environment versus transmission directly from infected animals is not known. In 2011, an outbreak of Q fever cases on farms in Washington and Montana was associated with infected goats. A study was undertaken to investigate the quantity and spatial distribution of C. burnetii in the environment of these goat farms. Soil, vacuum, and sponge samples collected on seven farms epidemiologically linked to the outbreak were tested for the presence of C. burnetii DNA by quantitative PCR. Overall, 70.1% of the samples were positive for C. burnetii. All farms had positive samples, but the quantity of C. burnetii varied widely between samples and between farms. High quantities of C. burnetii DNA were in goat housing/birthing areas, and only small quantities were found in samples collected more than 50 m from these areas. Follow-up sampling at one of the farms 1 year after the outbreak found small quantities of C. burnetii DNA in air samples and large quantities of C. burnetii persisting in soil and vacuum samples. The results suggest that the highest concentrations of environmental C. burnetii are found in goat birthing areas and that contamination of other areas is mostly associated with human movement.