天津市水体中主要病原生物分布规律研究

【作者】 刘洪亮；

【导师】 陈学敏；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2005， 博士

【副题名】1.嗜肺军团菌分布规律及分子鉴定技术研究 2.引滦水源病原生物消长规律与控制对策研究

【摘要】 军团菌(legionella)是一种革兰氏阴性杆菌,广泛存在于自然环境和人工环境的水体、土壤中,可以通过含菌气溶胶的方式在空气中播散而被人体吸入导致人类感染,发生军团病。目前已发现作为军团菌肺炎(legionella pneumonia)病原体的军团菌属有42个种、64个血清型,其中至少有19个种与人类疾病有关,与人类疾病关系最密切的为嗜肺军团菌种(legionella pniumophila),已发现15个血清型。最近研究表明,在严重的社会获得性肺炎中,嗜肺军团菌为第二位病因,据统计军团病临床病例中由嗜肺军团菌所引起的病例占军团菌病的80%以上。军团菌可以污染社区内的水体,如自来水、空调系统冷却塔水、水井饮用水、洗浴用水等。目前尚未见到对自然环境、生活环境等各主要水体进行军团菌分布规律的系统研究。随着经济的发展和人民生活水平的提高以及国家采取大力发展第三产业的政策,中高层建筑迅速增加并普遍采用中央空调。而中央空调及建筑物相对封闭的内部环境都是引起军团病爆发的重要因素,并严重危害建筑物内人群身体健康。另外,环境中军团菌的广泛存在也有可能造成其他水体如喷泉、分体空调冷凝水等的污染。因此,急需建立一种快速、准确、简便的检验方法并对各种水体中嗜肺军团菌的时间、空间分布进行调查研究,为预防和控制军团菌传播提供科学依据。本研究对常规细菌培养方法的优化、常规PCR方法的优化、半套式PCR技术、简并随机引物扩增DNA多态性、扩增片段长度多态性、微卫星DNA多态性等分子鉴定技术在嗜肺军团菌鉴定及分型上的应用进行了比较深入的研究,分析对比了以上几种技术方法的优缺点,并根据环境大规模调查方法要求操作简便、费用较低、达到一定的敏感度和特异性的特点,选定用GVP培养基(加入万古霉素和多粘菌素B)对水样进行初次接种培养,如有阳性菌落再分别转种BCYE培养基培养。如在第2次培养阳性,挑取菌落进行PCR鉴定和血清学鉴定这一系列方法作为本次军团菌调查研究的鉴定方法,其特点是最大程度地降低交叉污染的机率和假阳性率。本研究以天津市城乡各种水体为研究对象进行采样检测,以求得到军团菌分布的空间和时间特点,掌握其分布规律,为进一步开展预防和控制军团菌病的措施研究打下基础。本研究在一年内连续采样,共设监测点138个,采集了444份水样,经检测其中军团菌阳性水样28份,嗜肺军团菌阳性水样25份,并对其中分离出的军团菌的种群进行了分型鉴定研究。研究发现自然水体、空调冷却水的监测点军团菌检出率分别为33.33%、27.78%;嗜肺军团菌阳性率分别达到28.6%和27.78%;另外,尚有PCR鉴定阳性、血清学鉴定阴性水样11份,PCR阴性、血清学鉴定阳性水样2份。而且在加湿器、淋浴喷头及人工喷泉等与人群生活密切相关水体中均有检出,提示嗜肺军团菌分布广泛,应尽快采取预防与控制措施以保护人群身心健康。1983年引滦入津作为天津市的饮用水源,改变了以前天津市一直以苦咸的海河水作为饮用水源的状况。但随着工农业生产的发展和人民生活水平的提高,引滦河道沿岸的生产企业通过排污、倒虹闸的泄漏使生活污水和工业废水进入河道内,引滦河水质也受到污染。如曾于1995年12月底至1996年1月19日发生宝坻县境内的窝头河倒虹管的损坏、污水渗入明渠河道及1998年12月至1999年1月发生秦庄子泵站污水泄漏事故。引滦入津二十年来对自尔王庄水库至宜兴埠泵站的26Km引滦河道定期进行水质常规检验的结果表明:生物污染较重(总大肠菌群达238000个/L),超过《生活饮用水源水质标准》(CJ3020-93)一级水源水标准的要求。鉴于此,本研究根据引滦河道的排污情况、河道的交叉情况及倒虹闸的分布情况确定采样点。采集水样以测定病原生物(包括细菌总数、总大肠菌群、粪大肠菌群、致病菌、f2噬菌体、隐孢子虫),并分析病原生物在河道各段的分布及变化规律,以便为制定引滦河水保护措施提供科学依据,所获结果如下:于桥水库至尔王庄水库段(明渠)的主要污染来源为沿途工业污水及生活污水的排污、涝水通过闸的泄漏。从时间变化趋势来看,细菌总数、总大肠菌群、粪大肠菌群、f2噬菌体在夏秋季较高,隐孢子虫除1-3月外,其他月份均有检出。从空间变化趋势来看,从于桥水库至尔王庄水库段均有生物污染现象,在洇流大桥和九王庄闸下两测点污染较严重。下游的尔王庄水库和宜兴埠泵站污染较轻。总大肠菌群超过《生活饮用水水源水质标准》(CJ3020-1993)对一级水质的要求,点次超标率为47.6%,水质较差。提出了切实可行的控制措施,为政府部门制定引滦水源保护措施提供科学依据,有的建议已经成为政府行为开始实施。预计这些建议被采纳并完成实施,必将产生巨大的社会效益、潜在的经济效益和一定的学术影响。更多还原

【Abstract】 Legionella is one of Gram-negative bacilli that distribute widely in the water and soil of natural and artificial environment. They can be inhaled by means of aerosol contaminated with the bacilli, resulting in infection and Legionnaire’s disease in humans. There are 42 species and 64 serum-type of legionella. Nineteen of them are related to human disease. Legionella pneumophila, the major pathogen of Legionnaire’s disease has 15 serum-type. It is the second important pathogen of serious social acquired pneumonia. Statistic data indicated that more than 80% Legionnaire’s disease cases were caused by Legionella pneumophila. Legionella pneumophila could pollute the water in community, such as tap water, cooling-tower water, well water, bathe water. There are no reports about the distribution of legionella in water mass of nature and live environment.With develop of economy and standard of living, more and more centre air-conditioner systems were installed in tall buildings. Center air-conditioner and close environment in tall buildings could lead to the breakout of Legionnaire’s disease and do harm to the health of people who live in tall building. Otherwise, legionella in environment probably pollute fountain and condensed water in fission air-conditioner. Therefore, it’s very imperative to develop a rapid, accurate, simple method to detect legionella in water bodies so that we could investigate the time and space distribution of legionella.Several molecular marker techniques were applied in identification of legionella, such as optimization of general bacteria incubation, optimization of general PCR, D-RAPD, AFLP, and MPD. The advantages and disadvantages of above methods were compared and analyzed. The large scale investigation of legionella in environment requires the detection method with the characteristic of handy, low-expense, high sensitivity and high specificity. Therefore, the selected procedure for detection and identification of legionella were described as following: the water samples were incubated with GVP culture medium at first. If the positive legionella clones were found, the second incubation was done with BYCE culture medium. If the candidate clones still grow after second incubation, PCR and serologic test were applied to identify legionella. This series of tests could avoid cross contamination and false positive farthest.We collected every kind of water samples in Tianjin to determine the distribution of Legionella pneumophila in all kinds of water sources and provide scientific evidence for preventing and controlling water-borne transmission of legionella.Twenty-eight representative water samples from the 126 sampling sites were tested positive for Legionella. In the yearly surveillance, 36 samples of the 430 samples collected over a year were positive for legionella, of which 23 samples were Lp positive. Therefore, the legionella positive rates for the ambient and recreational water bodies were 32.1% and 22.2%, respectively, and Lp positive rates were 21.4% and 16.7%, respectively. Overall, the positive rates were higher in the summer and autumn seasons as well as in air-conditioner cooling tower water, shower water, and lake water in public parks.Lps are widely distributed in all kinds of ambient and recreational water sources. The ambient temperature appeared to be an important factor for Lp’s contamination, because the summer and autumn are the seasons with high detectable rates of Legionella contamination and Legionellosis spread. However, Lps could also be found in the spring and winter seasons. People who have a close contact with the contaminated water, such as park water, air-conditioner cooling tower water, shower water etc., are at high risks of legionella and legionellosis infection and transmission. Necessary measures should be taken promptly to protect the public from health hazard of Lps in water supplies. Luan River-diverted channel became the drinking water source of Tianjin City in 1983. This project changed the quality of drinking water in Tianjin completely. With the development of industry and agriculture and the improvement of people life, living sewage and industry waste water were poured into Luan River-diverted channel through leakage and sluice by enterprise that located along Luan River-diverted waterway. The water quality of Luan River-diverted got worse and worse. For example, waste water was discharged into uncovered ditch because of the damage of floodpipe at Woutou River in Baodi County since Dec. 1995 to Jan. 1996. The leakage incident occurred at Qinzhuangzi pump station since Dec. 1998 to Jan. 1999.The results of routine water quality monitor indicated that the biological pollution status was very serious at the 26 km-longth Luan River-diverted waterway from Erwangzhuang reservoir to Yixingfu pump station in the recent 20 years. Total coliform is over 238000 cfu/L that exceed the first grade standard depend on "Life drinking water source standard" （CJ3020-93）. In this study the sample dots were selected according to pollution status, crossing of waterway and distribution of floodgates. Pathogenys in water sample were detected （including Total bacteria count, total coliform, fecal coliform, pathogenic bacteria, f₂ phage, cryptosporidium）. The distribution and wave law were studies either. The safeguards were determined according to the above data. The results as following:Major pollution source for the waterway for Yuqiao reservoir to Erwangzhuang reservoir （uncovered ditchs） were industrial waste water, sewage and leakage of floodgate.Total bacteria count, total coliform, fecal coliform and f₂ phage were detected frequently in summer and autumn. Cryptosporidium oocysts were detected from April to December.Biological contamination was detected from Yuqiao reservoir to Erwangzhuang reservoir. The contamination status was rather serious at Yinliu Bridge and Jiuwangzhuang Gate and rather slight at Erwangzhuang reservoir and Yixingfu Pumping Station.This study provided the available control strategy for safegjuard to prevention of the source water diverted from Luan River. Some suggestions come into effect already. These suggestions will bring huge social benefit, potential economic benefit and definite academic influence.更多还原