3种方法对供水系统嗜肺军团菌、阿米巴原虫及生物膜消毒效果的比较

目的评估嗜肺军团菌(LEP)在供水系统中的生长和定植,建立试水循环系统以模拟真实供水环境,比较不同消毒方法对LEP、阿米巴原虫和生物膜的清除作用。方法中试水循环系统包括4套循环管道和盲端管道系统,试验中,对3套系统分别连续施加铜/银离子(0.4/0.04 mg/L)、二氧化氯(0.5 mg/L)和热力消毒(55℃),持续75 d;第4套作为对照,采用培养、荧光原位杂交和LIVE/DEAD染色后共聚焦激光扫描显微镜观察等技术监测浮游和生物膜内菌群变化。结果干预前LEP高度污染中试系统(水∶3.4×103/ml和生物膜:2.4×104/cm2),二氧化氯和热力消毒对生物群体(包括LEP)的消毒效果明显好于铜银离子;铜银离子对HPC的影响不明显,也不能清除生物膜,二氧化氯可有效清除盲端管内生物群体,但干预终止后LEP很快恢复至干预前水平;终止干预后2个月,热力消毒组的循环水和管壁生物膜内均未检出LEP,铜银离子组则再次出现低浓度LEP。结论本研究中,连续加热处理是控制中试水循环系统中LEP最有效的方法 ,一旦LEP定植于环境,将其清除非常困难。     

上海市8所医院供水系统军团菌属污染调查及危险因素分析

目的 了解上海市医院供水系统管道中军团菌属的污染情况,并分析军团菌属污染的危险因素.方法 在2009年5-9月采集上海市8所医院供水管道末端水样共193份,分别进行军团菌和阿米巴的培养,应用军团菌乳胶凝集试验将所分离的军团菌属进行血清分型,同时收集相关资料进行危险因素分析.结果 在采样的8所医院中,7所(87.5%)医院有军团菌属的污染,2所医院水管出口嗜肺军团菌污染率>30.0%;在采集的193份水样中,83份(43.0%)分离出军团菌属,其中33份(17.1%)分离到嗜肺军团菌,63份水样中军团菌属的浓度≥10 3CFU/L;在军团菌属污染危险因素分析中,阿米巴的定植、无水管消毒、水管类型为镀锌钢管及水箱间接供水都是独立的危险因素.结论 上海市医院供水系统中军团菌属污染常见,且污染浓度高,存在发生医院内军团菌肺炎甚至暴发流行的严重隐患,建议对医院供水系统军团菌属污染情况和医院获得性军团菌病进行监测,并研究具有可行性的供水系统去除军团菌污染的有效方法.     

漳州市2012年星级宾馆集中空调通风系统污染情况调查

目的了解漳州市三星级以上宾馆集中空调通风系统卫生状况及污染情况,为卫生管理提供依据。方法按《公共场所集中空调通风系统卫生规范》,对集中空调风管内表面积尘量、细菌总数、真菌总数,冷却塔冷却水和冷凝水中的嗜肺军团菌进行检测。结果风管内表面积尘量0.11～90.51g/m2,合格率84.0%;细菌总数〈1～2.4×103CFU/cm2,合格率80.0%;真菌总数〈1～1.3×104 CFU/cm2,合格率62.0%;空调系统冷却水、冷凝水均未检出嗜肺军团菌。结论漳州市星级宾馆集中空调通风系统存在一定程度的微生物污染,应定期清洗消毒,加强卫生管理。     

工业循环冷却塔水嗜肺军团菌分离株分子生学特性研究

目的 了解钢铁企业工业循环冷却塔水中的嗜肺军团菌污染状况及菌株分子生物学特性.方法 于2011年3月-2012年9月对邯郸某钢铁厂相对固定的车间冷却塔水进行连续监测,进行嗜肺军团菌的定量分离培养,对分离到的菌株进行血清分型、脉冲场凝胶电泳分型、基因序列分型、毒力基因(lvh、rtxA)检测.结果 共检测117份水样,其中20份水样检出嗜肺军团菌,检出率为17.1％;菌落总数为100～50000 CFU/L,中位数为12000 CFU/L;共分离到嗜肺军团菌23株,血清型包括Lp1、Lp3、Lp5、Lp6、Lp8,并以Lp1为主(占36.1％,9/23).23株嗜肺军团菌PFGE分型得到19种不同带型,其中15种带型为国内首次发现.23株嗜肺军团菌分成20个SBT型别,有12个型别为本研究新发现.23株菌中,lvh、rtxA基因阳性的分别有20、22株,两种毒力基因全部阳性的有19株.结论 钢铁企业的冷却塔水存在嗜肺军团菌污染,菌型呈现基因多态性,同时具有独特的基因组成,并普遍具有毒力.     

工业循环冷却塔水嗜肺军团菌分离株分子生物学特性研究

目的了解钢铁企业工业循环冷却塔水中的嗜肺军团菌污染状况及菌株分子生物学特性。方法于2011年3月—2012年9月对邯郸某钢铁厂相对固定的车间冷却塔水进行连续监测,进行嗜肺军团菌的定量分离培养,对分离到的菌株进行血清分型、脉冲场凝胶电泳分型、基因序列分型、毒力基因(lvh、rtx A)检测。结果共检测117份水样,其中20份水样检出嗜肺军团菌,检出率为17.1%;菌落总数为100~50 000 CFU/L,中位数为12 000 CFU/L;共分离到嗜肺军团菌23株,血清型包括Lp1、Lp3、Lp5、Lp6、Lp8,并以Lp1为主(占36.1%,9/23)。23株嗜肺军团菌PFGE分型得到19种不同带型,其中15种带型为国内首次发现。23株嗜肺军团菌分成20个SBT型别,有12个型别为本研究新发现。23株菌中,lvh、rtx A基因阳性的分别有20、22株,两种毒力基因全部阳性的有19株。结论钢铁企业的冷却塔水存在嗜肺军团菌污染,菌型呈现基因多态性,同时具有独特的基因组成,并普遍具有毒力。     

水中嗜肺军团菌检测基础培养基的质量分析

目的为保证水中嗜肺军团菌检测的准确性,对市售的4家厂商的BCYE培养基进行质量分析。方法选取嗜肺军团菌标准株和环境分离株,经过不同处理方式(酸处理、热处理)后分别接种4家厂商的BCYE培养基,对菌落结果进行计数,参考ISO 11133:2014(E)计算生长率,评估检出限。结果嗜肺军团菌标准株和环境分离株在不同处理方式、不同厂商的BCYE培养基的生长率均大于0.5,符合ISO 11133:2014(E)对合格培养基生长率的规定,经Levene方差齐性检验,生长率间比较,差异无统计学意义(P0.05)。除1家厂商的BCYE培养基对嗜肺军团菌标准株的检出限为100 CFU/ml外,其他家厂商的检出限可达到10 CFU/ml。结论 4家厂商的BCYE培养基在生长率评定中均为合格,但有2家对嗜肺军团菌标准株的检出限较高,其可能对活性较弱的嗜肺军团菌检出有一定影响。     

常州市公共浴池水中嗜肺军团菌的调查

目的了解常州市公共浴池水中嗜肺军团菌的污染状况。方法于2016年,选择常州市2家温泉场所和12家热水浴室进行浴池水样采集,采用实时荧光PCR方法检测水样中的嗜肺军团菌,并对分离菌株进行血清学鉴定。结果共采集公共浴池水样72件,嗜肺军团菌的阳性率为20.8%(15/72),其中,温泉浴池水中嗜肺军团菌的阳性率为30.0%(12/40),高于热水浴池水[9.4%(3/32)],差异有统计学意义(P=0.032)。共分离出11株嗜肺军团菌,血清型以LP1型为主,占比54.5%(6/11)。水样中嗜肺军团菌浓度范围为3.19×10^5-2.18×10^7,copies/ml。结论常州市公共浴池水中存在不同程度的嗜肺军团菌污染,尤其温泉浴池可能是公共场所军团菌污染的一个重要来源。     

快速检测空调冷却水中Lp1型嗜肺军团菌实时荧光定量PCR方法的建立

目的建立嗜肺军团菌Lp1型实时荧光定量PCR检测方法,以实现对中央空调冷却水中嗜肺军团菌Lp1型的快速检测。方法基于Lp1型嗜肺军团菌基因组ORF11区域设计特异性的引物和探针,建立体系,验证方法的灵敏度、特异性和重复性,并对模拟的中央空调冷却水水样进行检测。结果该方法的检测灵敏度达3.8×10 cfu/ml,具有良好的特异性,仅嗜肺军团菌Lp1型菌株呈阳性结果,重复检测平均Ct值变异系数。对模拟的水样进行检测,与方法的灵敏度相一致,且具有良好的重复性。结论该方法可实现对嗜肺军团菌Lp1型的快速检测。     

以发热为主要症状的儿童嗜肺军团菌感染77例临床分析

【目的】 探讨以发热为主要症状的儿童嗜肺军团菌感染临床特点。 【方法】 回顾性分析了5年中以不明原因发热收入住院最终确诊为嗜肺军团菌感染的77例患儿临床资料。 【结果】 60例诊断为嗜肺军团菌感染,14例为嗜肺军团菌肺炎,3例合并多器官功能障碍;除11例有轻微咳嗽外,其余病例无特异性症状;38例无任何阳性体征,4例患儿双肺闻及中细湿啰音,21例呼吸音粗;53例血常规白细胞在4~10×10⁹/L内,2例＜4×10⁹/L,22例＞10×10⁹/L,36例CRP＜10 mg/L,38例＞10 mg/L;X线检查:双肺斑片影14例,双肺纹理稍粗21例,其余无异常。 【结论】 临床中遇不明原因持续发热的患儿,尤其是经青霉素类或头孢类抗生素治疗效果不佳的病例,除考虑常见的发热性疾病外,应警惕嗜肺军团菌感染可能。     

公共场所相关土壤样品中嗜肺军团菌的调查

目的采用巢式PCR和荧光定量PCR方法检测公共场所相关土壤样品中嗜肺军团菌的污染状况。方法于2012年8—10月,在我国南北方2个城市共选取3种类型的公共场所16家,采集空调风管积尘、场所内花卉土和场所周边景观土;同时选取3个居民区,采集小区内的浅层地面土作为环境背景土;分别应用巢式PCR和荧光定量PCR方法检测样品中的嗜肺军团菌。结果公共场所空调积尘、场所内花卉土和周边景观土嗜肺军团菌巢式PCR的阳性检出率分别为45.8%(11/24),34.5%(10/29)和44.4%(4/9),荧光定量PCR测定嗜肺军团菌的浓度水平为1.1×103~8.1×105 copies/g。结论通过本次调查,公共场所空调积尘、场所内花卉土和周边景观土存在嗜肺军团菌污染。     

壳聚糖对大肠埃希菌生物被膜形成的作用

目的探讨壳聚糖对大肠埃希菌生物被膜形成的作用。方法以空白导管(对照组)、包被分子量5000、30万、100万壳聚糖的导管为载体,构建形成7d的大肠埃希菌生物被膜,并依次设为第1、2、3、4实验组;然后细菌计数、生物被膜定量、扫描电镜(SEM)观察。结果第1、2、3和4组的细菌计数依次是2.29×107CFU/ml、1.19×107CFU/ml、1.45、1.99×107CFU/ml;吸光度为0.137、0.050、0.080和0.135;扫描电镜下,第1组的细菌密集、均匀,细胞壁较光滑、菌体规则;第2、3组的细菌少,呈聚集状,菌体有凹陷、扭曲现象;第4组细菌密集。结论分子量5000和30万的壳聚糖对形成7d的大肠埃希菌生物被膜有抑制作用,分子量5000的壳聚糖作用更强;分子量100万壳聚糖没有抑制作用。     

口腔综合治疗台水路生物膜观察与消毒干预

目的评价口腔综合治疗台独立水源水路经次氯酸钠消毒前、后生物膜结构和菌落数情况。方法选择10台独立水源口腔综合治疗台,采集经525 mg/L次氯酸钠消毒水路前、后治疗台工作端水管内壁生物膜,计算总菌落数,并应用激光共聚焦扫描显微镜和扫描电镜观察生物膜结构。结果口腔综合治疗台水路消毒前、后生物膜菌落数几何均值分别为1.7×103 CFU/cm2和0 CFU/cm2,两者比较,差异有显著性（t=12.03,P=0.02）。激光共聚焦扫描显微镜及扫描电镜观察结果显示,消毒前口腔综合治疗台水路内壁存在生物膜,呈特征性结构,杆菌与球菌分布于基质中;消毒后,其生物膜结构受到一定程度破坏,但基质仍然存在。结论口腔综合治疗台水路存在微生物污染,其内壁有生物膜,并含有高浓度细菌,存在引起医患医院感染的潜在风险。525 mg/L的次氯酸钠消毒剂对口腔综合治疗台水路具有较好的消毒效果,可常规应用于临床独立水源口腔综合治疗台水路的消毒处理。     

深圳公共场所水系统嗜肺军团菌污染研究

目的了解深圳公共场所水系统嗜肺军团菌污染现状,为卫生监督管理提供依据。方法依照《公共场所集中空调通风系统卫生规范》附录A方法,随机采集本市177家公共场所冷却水、冷凝水、淋浴水、泳池水、生活饮用水共500份水样,检测嗜肺军团菌及其菌型。结果冷却水、冷凝水、淋浴水嗜肺军团菌检出率分别为11.0%(28/255)、1.1%(2/176)、30.0%(12/40),泳池水、生活饮用水未检出嗜肺军团菌;酒店、公共浴室、候诊室、地铁站冷却水嗜肺军团菌检出率分别为18.1%(19/105)、4.0%(1/25)、31.8%(7/22)、0.1%(1/103),差异有统计学意义(χ2=11.74,P<0.05);分离出44株嗜肺军团菌,菌型以LP1型为主,占70.5%(31/44),LP3型、LP6型分别占2.3%(1/44)、27.2(12/44)。结论本市公共场所空调冷却水和冷凝水、淋浴水受嗜肺军团菌污染,存在健康隐患,应加强空调系统和淋浴-热水系统的清洗消毒。     

空调循环水系统军团菌微生态与其存活力研究

[目的]探讨冬季军团菌在空调循环水系统中的微生态环境与其存活力的关系。寻找军团菌在空调循环水系统中的越冬场所及其赖以生存的相关因素,为阻断军团菌在空调冷却塔中繁殖、传播提供依据。[方法]选择8月份军团菌数高的空调冷却塔作现场调查,同时模拟空调冷却塔条件,构建藻类减少和藻类增加两种不同的军团菌生态模型。定期分别采集水样和沉积在容器表面的地衣一藻类混合体作军团菌检测和扫描电镜观察,并对水样中的藻体数、总有机碳和硝酸盐氮等进行检测。[结果]10月下旬,空调机关机后,冷却塔水样中无军团菌检出,但在地衣一藻类混合体中检测到高达10^4CFU／4cm^2的军团菌,扫描电镜观察发现藻类混合体的外膜上附着有较多的杆菌和军团菌。12月底,藻类减少模型无军团菌检出,但冷却塔和藻类增加模型均有检出。次年2月下旬,冷却塔和藻类减少模型中均无检出,只有藻类增加模型有检出。扫描电镜观察发现,12月底可观察到有节的丝状藻类,个别的军团菌附着于藻壁上。次年2月下旬,则绝大多数为地衣的纤维状组织、霉菌及其菌丝体。[结论]空调冷却塔底部地衣一藻类混合体是军团菌越冬的场所。四棘藻和地中尖头藻这类丝状藻类的细胞壁可能是军团菌越冬赖以生存的营养物。空调器关机时地衣和藻类数越少,循环水不流动的时间越长,军团菌在循环水管道系统中度过冬季的可能性越少。     

嗜肺军团菌聚合酶链反应检测方法及其应用研究

根据嗜肺军团菌基因组ＤＮＡ的种特异性ＤＮＡ片段序列，合成一对引物进行聚合酶链反应（ＰＣＲ）。经琼脂糖凝胶电泳、ＥＢ染色结果表明，一条８７０ｂｐ的核苷酸区带为嗜肺军团菌１～１４血清型菌株所共有。ＰＣＲ检测水中军团菌，其敏感性为３５０ｃｆｕ／ｍｌ，而用同位素标记探针、斑点杂交法检测，其敏感性为４３ｃｆｕ／ｍｌ。ＰＣＲ检测人工感染嗜肺军团菌的豚鼠组织标本，阳性率为８３．３％，而细菌分离培养的阳性率仅为２６．６％。在现场调查中，用ＰＣＲ法初步验证了一起由Ｌｐ１０引起的军团菌病爆发。上述结果表明，ＰＣＲ法能快速、敏感、特异性检测嗜肺军团菌感染。     

Legionella on board trains: effectiveness of environmental surveillance and decontamination

ABSTRACT: BACKGROUND: Legionella pneumophila is increasingly recognised as a significant cause of sporadic and epidemic community-acquired and nosocomial pneumonia. Many studies describe the frequency and severity of Legionella spp. contamination in spa pools, natural pools, hotels and ships, but there is no study analysing the environmental monitoring of Legionella on board trains. The aims of the present study were to conduct periodic and precise environmental surveillance of Legionella spp. in water systems and water tanks that supply the toilet systems on trains, to assess the degree of contamination of such structures and to determine the effectiveness of decontamination. METHODS: A comparative pre-post ecological study was conducted from September 2006 to January 2011. A total of 1,245 water samples were collected from plumbing and toilet water tanks on passenger trains. The prevalence proportion of all positive samples was calculated. The unpaired t-test was performed to evaluate statistically significant differences between the mean load values before and after the decontamination procedures; statistical significance was set at p [less than or equal to]0.05. RESULTS: In the pre-decontamination period, 58% of the water samples were positive for Legionella. Only Legionella pneumophila was identified: 55.84% were serogroup 1, 19.03% were serogroups 2-14 and 25.13% contained both serogroups. The mean bacterial load value was 2.14 x 103 CFU/L. During the post-decontamination period, 42.75% of water samples were positive for Legionella spp.; 98.76% were positive for Legionella pneumophila: 74.06% contained serogroup 1, 16.32 % contained serogroups 2-14 and 9.62% contained both. The mean bacterial load in the post-decontamination period was 1.72 x 103 CFU/L. According to the t-test, there was a statistically significant decrease in total bacterial load until approximately one and a half year after beginning the decontamination programme (p =0.0097). CONCLUSIONS: This study indicates that systematic environmental surveillance could be a useful approach for assessing the risk of exposure to Legionella bacteria, which still represents a public health threat. According to the study results, an environmental surveillance programme, followed by decontamination procedures where necessary, would decrease the total bacterial count, protecting the health of travellers and workers.     

Detection, integration and persistence of aeromonads in water distribution pipe biofilms

The occurrence of Aeromonas spp. within biofilms formed on stainless steel (SS), unplasticized polyvinyl chloride (uPVC) and glass (GL) substrata was investigated in modified Robbins Devices (MRD) in potable (MRD-p) and recycled (MRD-r) water systems, a Biofilm Reactor (BR) and a laboratory-scale pipe loop (PL) receiving simulated recycled wastewater. No aeromonads were isolated from the MRD-p whereas 3-10% of SS and uPVC coupons (mean 3.85 CFU cm(-2) and 12.8 CFU cm(-2), respectively) were aeromonad-positive in the MRD-r. Aeromonads were isolated from six SS coupons (67%) (mean 63.4 CFU cm(-2)) and nine uPVC coupons (100%) (mean 6.50x 10(2) CFU cm(-2)) in the BR fed with recycled water and from all coupons (100%) in the simulated recycled water system (PL). Mean numbers of aeromonads on GL and SS coupons were 5.83 x 10(2) CFU cm(-2) and 8.73 x 10(2) CFU cm(-2), respectively. No isolate was of known human health significance (i.e. Aeromonas caviae, A. hydrophila or A. veronii), though they were confirmed as Aeromonas spp. by PCR and fluorescence in situ hybridization (FISH). Challenging the PL biofilms with a slug dose of A. hydrophila (ATCC 14715) showed that biofilm in the PL accumulated in the order of 10(3)-10(4) A. hydrophila cm(-2), the number of which decreased over time, though could not be explained in terms of conventional 1st order decay kinetics. A sub-population of FISH-positive A. hydrophila became established within the biofilm, thereby demonstrating their ability to incorporate and persist in biofilms formed within distribution pipe systems. A similar observation was not made for culturable aeromonads, though the exact human health significance of this remains unknown. These findings, however, further question the adequacy of culture-based techniques and their often anomalous discrepancy with direct techniques for the enumeration of bacterial pathogens in environmental samples.     

Sampling and detection of Legionella pneumophila aerosols generated from an industrial cooling tower

Cooling tower water has frequently been cited as a source of infection in outbreaks of Legionnaires' disease. However, there have been few reports on the presence of legionellae in aerosols from cooling towers. This paper describes our use of an impinger or a six-stage microbial impactor for detecting legionellae in air around a cooling tower contaminated with L. pneumophila (1.2+/-0.3x10(5) CFU/100 ml). Phosphate-buffered saline, Page's saline, 2% yeast extract solution and buffered yeast extract (BYE) broth were tested to evaluate their collection efficiency. These solutions were compared in laboratory experiments using an aerosol of L. pneumophila serogroup (SG) 1. Because BYE broth was the most efficient and storable collecting fluid among them, it was used for outdoor air sampling. In the outdoor air sampling, aerosolized L. pneumophila SG 6 was detected in the air around the cooling tower by the impinger (0.09 CFU/l. air). No legionellae were detected by the impactor with Legionella-selective agar plates (WYOalpha) because the plates were overgrown with fungi. Repetitive element PCR (rep-PCR) and arbitrarily primed PCR (AP-PCR) were employed to assess the epidemiological relationship among Legionella isolates from the air sample and the cooling tower water samples. L. pneumophila SG 6 isolated from the aerosols produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 6 strains from the cooling tower water, suggesting that the bacterium was aerosolized from the cooling tower.     

中央空调系统嗜肺军团菌传统细菌培养与荧光PCR结果比较

目的中央空调系统嗜肺军团菌传统细菌培养与TaqMan荧光定量PCR检测结果比较。方法对9家商场、宾馆、医院、办公楼公共场所中央空调系统的冷却塔水、生物膜、淤泥三环节样本进行军团菌检测,用传统细菌分离培养法分离鉴定,并用TaqMan荧光定量PCR法加以验证。结果本次对9家单位的水冷式中央空调系统三环节采样18份,其中传统细菌培养法检出4份军团菌,检出率为22.2%;冷却塔水、生物膜、淤泥检出率分别为22.2%、25.0%、20.0%。用荧光PCR法检出9份,检出率为50.0%;冷却塔水、生物膜、淤泥检出率分别为44.4%、50.0%、60.0%。结论金华市中央空调系统中存在嗜肺军团菌,荧光PCR检出率高于传统细菌培养法。