医院空气环境微生物污染检测方法建立及其应用研究

发布时间：2018-05-05 06:23

本文选题：城市医院环境 + 空气微生物　；参考：《哈尔滨工业大学》2015年硕士论文

【摘要】：空气中微生物与抗生素抗性基因(AntibioticResistanceGenes,ARGs)污染得到人们越来越多的关注。医院因其场所的特殊性,存在着潜在的病原菌与ARGs污染问题。然而,目前对城市医院环境空气微生物与ARGs污染状况的研究较少,缺乏人群在医院环境暴露风险的基础数据。本论文基于非培养的研究手段,建立了一套适合医院空气环境的微生物污染的检测方法,并将该方法加以应用。论文研究了医院环境空气微生物群落结构、病原菌及ARGs污染状况,基于该研究结果对人群在医院环境的暴露状况进行评估,以了解医院空气生物污染状况,并初步探索了人群在医院的暴露风险。研究建立了一种基于非培养的医院空气环境微生物污染的检测方法。布点原则遵循空气微生物监测的标准,采样方法为大流量TSP(TotalSuspendedParticulate)采样器采集;采样时间在22~26h之间;采样膜预处理方法为酸性磁珠法;DNA样品浓度的定量采用Qubit荧光法。对医院空气环境DNA样品采用高通量测序技术和实时荧光定量PCR技术(qPCR)研究其空气微生物的组成特征,病原菌及抗生素抗性基因的污染状况。基于16SrRNA基因扩增片段的高通量测序结果表明:医院空气环境微生物的主导菌门为变形菌门、厚壁菌门等,空气中病原菌的平均比例为3.64%,主导病原菌为腐生葡萄球菌、棒状杆菌、大肠埃希氏菌等。典型医院空气的主导菌群和病原菌在冬夏季间差异明显,病原菌比例夏季稍高于冬季;主导菌群的分布在空气介质与尘土介质间略微不同,而病原菌比例尘土介质显著高于空气介质,表明尘土介质是医院环境病原菌的一个潜在存储库。qPCR定量实验的结果显示:医院空气中β-内酰胺类ARGs相对浓度均值最大,与β-内酰胺类抗生素的使用率较高有关;其中β-内酰胺类的blaTEM基因与大环内酯类的ermB基因是医院环境污染最严重的两种基因;典型医院四种ARGs(blaTEM、ermB、blaCTX-M、mecA)的污染在季节间差异不显著,空气介质中的相对浓度在103~107copies/ng范围内,医院环境空气介质ARGs污染不容忽视。采用估算呼吸吸入潜在剂量值的方法来评估医院环境空气生物污染物暴露风险,结果显示:病人每次输液就诊时可吸入细菌气溶胶高于433CFU(Colony-FormingUnits),其中可入肺细菌气溶胶高于145CFU,可入肺病原菌高于5CFU;门诊部的医护人员气溶胶暴露风险最大;医院环境ARGs暴露剂量为105~106copies/h,β-内酰胺类ARGs的暴露风险最大,且同种类ARGs暴露风险在不同部门间差别较小。
[Abstract]:More and more attention has been paid to the contamination of microorganism and antibiotic resistance gene antibiotic resilience genes in the air. Because of the particularity of the hospital, there are potential pathogens and ARGs pollution problems. However, there are few studies on microorganism and ARGs pollution in urban hospital environment, and lack of basic data on exposure risk of population in hospital environment. Based on the non-culture research methods, a set of methods for the detection of microbial contamination in hospital air environment were established and applied in this paper. The microbial community structure, pathogenic bacteria and ARGs pollution in hospital ambient air were studied in this paper. Based on the results of the study, the exposure status of the population in hospital environment was evaluated to understand the status of hospital air biological pollution. And preliminarily explored the exposure risk of the crowd in the hospital. A non-culture-based method for the detection of microbial contamination in hospital air environment was developed. The sampling method is a large flow rate TSP Total suspended Particulate sampler, the sampling time is between 22 ~ 26h, the sample film pretreatment method is the acid magnetic beads method and the Qubit fluorescence method is used to quantify the concentration of the sample. High-throughput sequencing technique and real-time fluorescence quantitative PCR technique were used to study the composition of airborne microbes and the contamination of pathogens and antibiotic resistant genes in hospital air environment DNA samples. The results of high throughput sequencing based on the amplified 16SrRNA gene showed that the dominant microbes in the air in hospital were Proteus, Thinacea, etc. The average proportion of pathogenic bacteria in the air was 3.64. The dominant pathogens were staphylococcus saprophyticus and Corynebacterium. Escherichia coli, etc. There were significant differences between the dominant bacteria and pathogenic bacteria in the air of typical hospitals during winter and summer, and the proportion of pathogenic bacteria in summer was slightly higher than that in winter, and the distribution of dominant bacteria was slightly different between air and dust. The proportion of pathogenic bacteria in dust medium was significantly higher than that in air medium, which indicated that dust medium was a potential repository of pathogenic bacteria in hospital environment. QPCR quantitative experiment showed that the average relative concentration of 尾-lactam ARGs in hospital air was the highest. Among them, 尾 -lactam blaTEM gene and macrolide ermB gene were two of the most serious environmental pollution genes in hospitals. The relative concentration of air media is in the range of 103~107copies/ng, and the ARGs pollution in hospital environment can not be ignored. The risk of exposure to biological pollutants in hospital ambient air was assessed by estimating the potential dose values of respiratory inhalation. The results showed that the inhalable bacterial aerosol was higher than 433 CFU colony in each transfusion visit, in which the pulmonary bacterial aerosol was higher than 145 CFU and the pulmonary pathogenic bacteria was higher than 5 CFU. The exposure dose of ARGs in hospital environment was 105 ~ 106copies-h. the exposure risk of 尾 -lactam ARGs was the highest, and the risk of exposure to the same type of ARGs was small among different departments.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X831

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