Legionella species and serogroups in Malaysian water cooling towers: identification by latex agglutination and PCR-DNA sequencing of isolates

In this study, we investigated the distribution of Legionella species in water cooling towers located in different parts of Malaysia to obtain information that may inform public health policies for the prevention of legionellosis. A total of 20 water samples were collected from 11 cooling towers located in three different states in east, west and south Malaysia. The samples were concentrated by filtration and treated with an acid buffer before plating on to BCYE agar. Legionella viable counts in these samples ranged from 100 to 2,000 CFU ml^-1; 28 isolates from the 24 samples were examined by latex agglutination as well as 16S rRNA and rpoB PCR-DNA sequencing. These isolates were identified as Legionella pneumophila serogroup 1 (35.7%), L. pneumophila serogroup 2–14 (39%), L. pneumophila non-groupable (10.7%), L. busanensis, L. gormanii, L. anisa and L. gresilensis. L. pneumophila was clearly the predominant species at all sampling sites. Repeat sampling from the same cooling tower and testing different colonies from the same water sample showed concurrent colonization by different serogroups and different species of Legionella in some of the cooling towers.     

Studies on the efficacy of Chloramine T trihydrate (N-chloro-p-toluene sulfonamide) against planktonic and sessile populations of different Legionella pneumophila strains

Effectiveness of Chloramine T trihydrate (N-chloro-p-toluene sulfonamide) on both planktonic and sessile populations of different Legionella pneumophila strains was assessed. Although Chloramine T is a recommended commercial formulation for disinfecting cooling towers, there is a lack of published data about the efficacy of this compound against both planktonic and sessile populations of L. pneumophila. Planktonic L. pneumophila strains were suspended in tap water and sessile L. pneumophila strains were grown on stainless steel which is used in the construction of cooling towers, followed by exposure to the biocide. The sensitivity of both planktonic and sessile populations of L. pneumophila strains was different. The biocide was found effective below recommended dosages (0.1-0.3%) against planktonic populations of L. pneumophila, whereas it was determined that higher dosages than those recommended were required for sessile populations of L. pneumophila. The results indicated that studying only the planktonic populations of L. pneumophila for biocide tests might not be sufficient to provide information about the optimum dosage and contact time. Therefore, efficacy has to be tested on both planktonic and sessile bacteria.     

Investigation and control of a cluster of cases of Legionnaires disease in western Sydney.

Three cases of Legionella pneumophila infection were identified in Sydney's west in November 1998. Epidemiological investigations identified an association with one workplace. Environmental sampling revealed that the cooling towers in the workplace, and at 2 other premises within a 1 km radius of the workplace, were positive for L. pneumophila serogroup 1 (LP1) which was indistinguishable from clinical isolates of 2 of the cases on DNA fingerprinting. Our report highlights limitations of the current control program for Legionella in cooling towers, including the finding of unregistered cooling towers, cooling towers positive for LP1 despite satisfactory results on inspection, and cooling towers potentially linked to disease with counts of LP1 below the current protocol requirements for immediate decontamination.     

Prevalence of antibodies to Legionella pneumophila among workers exposed to a contaminated cooling tower

A serosurvey for antibodies to Legionella pneumophila was conducted among 206 employees of a power-generating plant. L. pneumophila serotype 6 and a non-typable L. pneumophila organism were isolated from cooling-tower water specimens at the plant, and antibody titers in workers were measured using homologous antigens prepared from these isolates. For the serotype 6 water isolate, none of workers with low cooling tower exposure, 4.6% with intermediate exposure, and 7.6% with high exposure levels had titers equal to or greater than 1:128 (P less than .05, Kruskal-Wallis test). For the non-typable L. pneumophila isolate, a similar trend was observed, but differences among workers in the three exposure groups were not statistically significant. No association was observed between antibodies to L. pneumophila serotypes isolated from cooling water and workers' age, race, smoking status, or duration of job assignment. None of the study employees had findings suggesting the occurrence of L. pneumophila pneumonia since the plant had been in operation. Use of respiratory protection devices by workers exposed to aerosols from cooling towers was not recommended.     

Isolation of Legionella species/serogroups from water cooling systems compared with potable water systems in Spanish healthcare facilities

Surveillance of Legionella spp. in hospital water systems was performed in forty-four inpatient healthcare facilities in Spain during 2005-2006. A total of 2,341 samples were collected: 470 from cooling systems (cooling towers) and 1,871 from potable water systems. The latter included 211 from cold-water tanks and 260 from hot-water tanks, totalling 471 from central water reservoirs 136 from showers, 1,172 from unfiltered taps and 92 from filtered taps, totalling 1,400 from peripheral points. Temperature, chlorine levels and the presence of Legionella spp. were determined. In all, 373 (15.9%) samples yielded Legionella spp. Significantly higher isolation rates were obtained from cooling towers (23.8%) versus cold- and hot-water tanks (approximately 4.7%), due to the significantly higher number of samples positive for serogroup 1 (19.4 vs 0.9-3.5%). In potable water systems, no differences were found between central water tanks and showers, but significant differences in isolation rates between central water tanks and unfiltered taps were observed (4.7 vs 19.6%) due to differences in non-serogroup 1 L. pneumophila. Filters significantly decreased isolation rates of these serotypes (11 vs 0%). Some seasonal differences were noted, with higher isolation rates in summer for legionella serogroup 1 in cooling systems and for L. pneumophila serogroups 2-14 in potable water systems. In regression models, higher temperatures were associated with colonisation in cooling systems, while lower chlorine levels were associated with colonisation in potable water systems.     

南京市土壤中嗜肺军团菌污染状况调查

目的了解南京市土壤被嗜肺军团菌污染情况。方法分别从南京市冷却塔水嗜肺军团菌培养法阳性的12个公共场所中,采集冷却塔2m范围内及距离冷却塔>2km居民区土壤样品,用PCR方法检测军团菌的5srRNA和Mip基因。结果冷却塔旁土壤样品中军团菌阳性率为75.0%(36/48),居民区土壤样品军团菌阳性率为70.8%(34/48),差异无统计学意义。冷却塔旁军团菌阳性土壤样品中嗜肺军团菌阳性率为50.0%(18/36),居民区军团菌阳性土壤样品中未检出。结论冷却塔旁土壤中嗜肺军团菌阳性率较高,存在导致军团病散发流行或暴发的潜在危险。     

Strengths and limitations of molecular subtyping in a community outbreak of Legionnaires' disease

An epidemiological and microbiological investigation of a cluster of eight cases of Legionnaires' disease in Los Angeles County in November 1997 yielded conflicting results. The epidemiological part of the investigation implicated one of several mobile cooling towers used by a film studio in the centre of the outbreak area. However, water sampled from these cooling towers contained L. pneumophila serogroup 1 of another subtype than the strain that was recovered from case-patients in the outbreak. Samples from two cooling towers located downwind from all of the case-patients contained a Legionella strain that was indistinguishable from the outbreak strain by four subtyping techniques (AP-PCR, PFGE, MAb, and MLEE). It is unlikely that these cooling towers were the source of infection for all the case-patients, and they were not associated with risk of disease in the case-control study. The outbreak strain also was not distinguishable, by three subtyping techniques (AP-PCR, PFGE, and MAb), from a L. pneumophila strain that had caused an outbreak in Providence, RI, in 1993. Laboratory cross-contamination was unlikely because the initial subtyping was done in different laboratories. In this investigation, microbiology was helpful for distinguishing the outbreak cluster from unrelated cases of Legionnaires' disease occurring elsewhere. However, multiple subtyping techniques failed to distinguish environmental sources that were probably not associated with the outbreak. Persons investigating Legionnaires' disease outbreaks should be aware that microbiological subtyping does not always identify a source with absolute certainty.     

Prevalence study of Legionella spp. contamination in Greek hospitals

Water and swab samples were collected from 13 hospitals and analyzed for Legionella counts. Legionella was detected in eight out of 13 hospitals and in 22 of 130 water and swab-collected samples. A total of 72.7% of the strains were L. pneumophila ser. 1, 22.7% were L. pneumophila ser. 2-14, and 4.5% did not belong to any of these groups. AFLP typing of the L. pneumophila ser. 1 strains generated two distinguishable AFLP types. There was no significant correlation to the sample type with Legionella recovery. Legionella isolation was more likely to occur in the cooling towers than the water system. Water temperatures of 30-40 degrees C seem to favor Legionella growth. Of the 265 serum samples taken from the medical and technical staff for the control of IgG titre, 89.4% were negative, 7.2% were positive, and for 3.4% the result was doubtful. No association between IgG titre and maximum observed level of Legionella occurrence was detected.     

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.     

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.     

2006年武汉市公共场所集中空调通风系统卫生状况分析

目的了解武汉市公共场所集中空调通风系统污染状况，为制定防治策略提供科学依据。方法对武汉市38家公共场所的集中空调通风系统进行采样，共检测164个出风口，38个冷却塔。检测的污染物指标为可吸入颗粒物（PM10）、送风口细菌总数、风管内表面积尘量、风管内表面细菌总数、嗜肺军团菌。结果164个出风口样品中，PM10送风口细菌总数、风管内表面积尘量、风管内表面细菌总数4个指标均合格的占70．12％；38件冷却塔水样品中，嗜肺军团菌检出率为63．16％。结论武汉市公共场所集中空调通风系统仍存在一定的污染，应加强卫生监督和监测。     

北京市东城区公共场所冷却塔使用状况及健康风险评估

目的了解北京市东城区公共场所冷却塔使用及卫生状况,评估各场所冷却塔军团菌病健康风险。方法2018年5—9月,采集东城区内三类共26家公共场所冷却塔冷却水,填写相关调查问卷,通过冷却塔军团菌病健康风险指数模型计算各场所健康风险。结果本次研究中各场所冷却水样本嗜肺军团菌检测均为阴性,合格率为100%。游离性余氯平均为0.15 mg/L。酒店类场所冷却塔规模、加药方式和频率与其他场所有明显差别。26家公共场所健康综合风险平均为61.49,其中军团菌增殖风险、含菌气溶胶传播风险、暴露人群易感风险分别为16.13、22.66和22.70。不同场所冷却塔军团菌病健康风险评估结果存在一定差异,医院类场所健康风险指数高于酒店和商场(F=3.78,P<0.05)。结论冷却塔军团菌病健康风险高低主要受公共场所周围易感人群影响,该地区相关卫生部门应加强对相关易感人群的保护,对各类场所相关政策法规进行制定、完善和实施。     

Decreasing trend of Legionella isolation in a long-term microbial monitoring program in Greek hospitals

Legionnaires' disease is a known cause of severe pneumonia in a nosocomial setting. This study examined Legionella isolation in Greek hospitals. Water samples and swabs of showerheads from 41 hospitals were collected over a four-year period (2004-2007) from hot and cold water systems and cooling towers in Greece. In total, 1058 samples were examined for the presence of Legionella. From the hot water samples examined, 166 out of 607 (27.3%) were positive for Legionella, including 111 (18.3%) positive for Legionella pneumophila sg1 samples. The temperature of hot water samples less than 55°C was statistically significant, associated with Legionella spp. isolation (RR 4.01, 95%CI 2.33-6.92). Ten out of 17 (58.8%) hospital cooling towers required remedial actions due to Legionella colonization. Reemergence of Legionella spp. colonization was evident in more than half of the hospitals where frequent monitoring and appropriate risk assessment plans were absent or lacking. Hospitals that were monitored continuously over the study period presented a decreasing trend of Legionella colonization. Environmental risk assessment together with Legionella isolation should be enforced systematically in hospitals.     

杭州市区公共场所空调冷却水中嗜肺军团菌的调查和分析

目的：了解杭州市公共场所大型中央空调冷却水中嗜肺军团菌的污染情况及主要血清型。方法：于2005年9月～10月，采集杭州市13家商场和宾馆、2家医院和1家福利中心等中央空调冷却塔冷却水29件，应用GVPC、BCYE、BCY培养基进行嗜肺军团菌分离培养、进行血清分型及种特异性PCR鉴定。结果：冷却塔冷却水中嗜肺军团菌的污染率达到44．83％（13／29），共分离到嗜肺军团菌16株（有2份水样中同时检出二个血清型以上菌株，其中一份有Lp1、Lp2、Lp3三个血清型，另一份有Lp3及未定型二个血清型）。血清型以LP1为主达37．50％（6／16）。结论：杭州市公共场所大型中央率调冷却水中嗜肺军团菌检出率较高，对市民健康构成了威胁。     

公共场所集中空调冷却水水质及其与嗜肺军团菌污染的关系

[目的]分析集中空调冷却水水质及其与嗜肺军团菌污染间的相关性,为预防和控制军团菌病提供科学依据。[方法]对深圳市南山区安装有集中空调的36家公共场所冷却水进行检测,检测指标有冷却水嗜肺军团菌微生物指标和水质理化指标。[结果]集中空调冷却水水温较一般自来水高,中位数为30．0℃;细菌总数中位数为6．8CFU／mL,浊度中位数为3．5NTU,均超过生活饮用水卫生标准。多因素logistic回归分析显示,浊度、溶解性总固形物是冷却水嗜肺军团菌阳性率的影响因素,其比值比（OR及95％CI）分别为1．300（1．011—1．672）和1．102（1．016～1．196）。[结论]冷却水浊度和溶解性总固形物可能是影响嗜肺军团菌生长繁殖的重要因素,加强冷却水水质管理对预防军团菌病有重要公共卫生学意义。     

上海市16个冷却塔水中军团菌脉冲场凝胶电泳分型及基因型监测

目的 研究上海市公共场所16个空调冷却塔水中军团菌的脉冲场凝胶电泳(PFGE)分型,并连续监测该菌基因型变化.方法 2007年5-10月连续6个月以每月1次的频率采样,从16个公共场所空调冷却塔水中分离军团菌,经血清学凝集试验、胶乳凝集试验分型后,使用PFGE技术对酶切后的军团菌全染色体DNA进行电泳获得指纹图谱,利用BioNumerics软件进行聚类分析.结果 16个冷却塔水中共分离出131株军团菌,包括嗜肺军团菌、博杰曼军团菌、米克戴德军团菌和茴香军团菌,分为52个PFGE型别,其中37个PFGE型别(71.15%)仅分布于1个冷却塔中,即为该冷却塔所特有;15个PFGE型别(28.85%)分布于2个或以上冷却塔中.16个冷却塔具有2个或以上的PFGE型,13个冷却塔(81.25%)中多次出现相同的PFGE型别.2007年6-10月连续5个月从6个冷却塔中分离出18株PFGE型为LPAs.SH0078型的军团菌.结论 冷却塔水中的军团菌基因型具有多样性和复杂性,81.25%的冷却塔水中PFGE型具有持续性,且LPAs.SH0078型广泛分布,可能为优势PFGE型.     

大连市集中空调场所军团菌污染及人群健康状况影响调查

目的调查大连市集中空调场所冷却塔水军团菌污染状况及暴露人群中军团菌隐性感染状况,研究军团菌对人群健康的影响因素。方法2005年8～9月,随机采集大连市使用集中空调的32所公共场所集中空调冷却塔水,采用分离病原培养法进行军团菌检测;随机抽取使用集中空调场所的工作人员血样,采用微量凝集试验法,进行血清军团菌抗体检测。结果大连市公共场所集中空调冷却塔水军团菌检测的场所阳性率为53.13%,军团菌血清型分别为嗜肺军团菌(Legionella pneumophila,Lp)Lp7、Lp8、Lp9和Lp10型。其中,Lp9型28株,占总菌株的77.78%,为优势菌株。人群血清学抗体检测显示,使用集中空调的公共场所人群Lp10感染率较高。结论大连市使用集中空调的场所存在军团菌污染,人群中存在军团菌隐性感染。     

Legionella pneumophila in cooling water systems. Report of a survey of cooling towers in London and a pilot trial of selected biocides.

Fourteen recirculating cooling water systems were surveyed during the summer, 1981, to see what factors might influence the prevalence of Legionella pneumophila. The effect on the organism of three anti-microbials was studied, each in two systems, by intermittent treatment at two week intervals. L. pneumophila was isolated from six of the 14 cooling systems at the beginning of the trial but by the end was present in ten. An association was found between the presence of the organism and the concentration of dissolved solids, and chlorides and the pH. There also appeared to be associations with exclusion of light and higher water temperatures. Repeated tests on eight untreated systems showed that two were consistently infected, three became and remained infected, one was infected on a single occasion and two were never infected with L. pneumophila. Treatment of a contaminated system, either with a 10 p.p.m mixture of a quaternary ammonium compound and tributyltinoxide or slow release chlorine briquettes (maximum recorded free chlorine level 1.2 p.p.m.), did not eliminated legionellae. Treatment of two infected towers with a chlorinated phenol (100 p.p.m.) eliminated legionellae for at least three days, but after 14 days the organism was again found.     

Legionnaires' disease: the epidemiology of two outbreaks in Burlington, Vermont, 1980

Eighty-five cases of Legionnaires' disease were diagnosed in two major outbreaks at a large regional medical center in Burlington, Vermont, in the summer of 1980. Cases in both outbreaks were positive for Legionella pneumophila, serogroup 1 by culture, serology, or direct fluorescent antibody tests. All cases had spent time in the city of Burlington in the 10 days before the onset of symptoms. Cases in both outbreaks were both hospital- and community-acquired. A case-control study identified no common in-hospital exposure, including shower use, that was associated with illness. Cases without previous exposure to the hospital were more likely to occur in persons with residences in neighborhoods just downwind of cooling tower A, but not throughout the municipal water system. Epidemiologic and environmental studies supported the association of this cooling tower, located 150 m from the hospital, with both outbreaks. Maintenance employees who worked with tower A had higher Legionella titers than those who worked with a comparison tower located 1.6 km away. Aerosolization of L. pneumophila by tower A and airborne spread to the hospital and community are postulated. The distance of airborne transmission of L. pneumophila in these consecutive outbreaks is greater than previously reported.     

集中空调冷却塔军团菌污染对宾馆从业人员血清军团菌抗体的影响

目的尽管很多公共场所的冷却塔的进行定期消毒,但是我们江苏常州市进行的调查依然在89.6%冷却塔水样品中检出了的军团菌。本研究的目的是探索这些军团菌污染的冷却塔对从业人员健康的影响。方法从公共场所从业人员中随机选取364名对象进行血清抗体测试和问卷调查。暴露组来自1型嗜肺军团菌污染的冷却塔所在场所的从业人员,对照组对象来自没有使用的集中空调的场所。血清军团菌抗体的检测采用间接免疫荧光(IFA)的方法。结果暴露组对象的1型嗜肺军团菌抗体的阳性率显著性高于对照组(OR=2.845,P=0.019)。自来水和淋浴水中的军团菌污染可能是混杂因素。其中暴露组人群感冒症状的的发生频率显著性高于对照组,室内空气质量的满意度也与1型嗜肺军团菌抗体的阳性率相关。结论嗜肺军团菌污染的冷却塔与场所从业人员血清军团菌抗体的阳性率相关,自来水和淋浴水中的军团菌也是可能的污染源。