关键环境因子对嗜肺军团菌(Legionella pneumophila)在嗜热四膜虫(Tetrahymena thermophila)细胞内增殖的影响

嗜肺军团菌是一种生活在人工水环境中的条件性致病菌,环境条件对该菌的存活和胞内增殖具有重要的影响.本文用作者建立的军团菌-四膜虫模型,研究了空调水池水、Fe3+和感染温度3种关键性环境因子对嗜肺军团菌在嗜热四膜虫宿主细胞内增殖的影响.结果发现,尽管空调水中Fe3+浓度大大低于培养基中的Fe3+浓度,但适合嗜肺军团菌在嗜热四膜虫宿主胞内的存活和增殖;感染缓冲液中的Fe3+能够被嗜热四膜虫富集,并被胞内的军团菌加以利用,缓冲液中浓度0.05×10-6的Fe3+即可显著促进胞内军团菌的增殖,但0.1×10-6和0.5×10-6的Fe3+促进军团菌胞内增殖的效果差异不显著;较高的温度对军团菌的胞内增殖也有明显促进作用,35℃时,即使感染复数等于10,军团菌也可进行胞内复制扩增,并最终裂解宿主细胞.

An Investigation on the Molecular Characteristics and Intracellular Growth Ability among Environmental and Clinical Isolates of Legionella pneumophila in Sichuan Province, China

Objective To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila(L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. Methods Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16 S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing(SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci(MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue(lvh) and repeats in structural toxin(rtx A). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. Results All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types(STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtx A loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. Conclusion L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.

Pneumocystis jirovecii and Legionella pneumophila coinfection in a patient with diffuse large B-cell lymphoma:A case report

BACKGROUND Diffuse large B-cell lymphoma(DLBCL)is a common non-Hodgkin's lymphoma.R-CHOP is a protocol for long-term chemotherapy for DLBCL patients.Longterm chemotherapy can lead to low immunity and increase the risk of opportunistic pathogen infections in immunocompromised patients.CASE SUMMARY We report a case of coinfection with Pneumocystis jirovecii(P.jirovecii)and Legionella pneumophila(L.pneumophila)in a patient with DLBCL.The patient was a 40-year-old female who was diagnosed with DLBCL and was admitted due to pulmonary infection.P.jirovecii and L.pneumophila were detected in her bronchoalveolar lavage fluid by hexamine silver staining,isothermal amplification and metagenomic sequencing.CONCLUSION To the best of our knowledge,this is the first case of P.jirovecii and L.pneumophila coinfection found in a DLBCL patient.Clinicians should be aware of the risk of complicated infection in patients undergoing long-term chemotherapy.

A Case of Comorbidity of Complicated Infective Endocarditis and Severe Pneumonia Due to Legionella Pneumophila

Legionella pneumophila infection can cause Legionnaires’disease,a severe form of pneumonia.Extrapulmonary manifestations of Legionella infections include myocarditis,pericarditis,and endocarditis.We present a rare case of pneumonia caused by Legionella pneumophila with a possible etiologic link to a recently recovered culture-negative infective endocarditis.

Isolation,identification,characterization and antibiotic sensitivity profile of pathogenic Legionella pneumophila isolates from different water sources

Objective:To investigate the prevalence,isolation,identification,characterization,antibiotic profile and pathogenicity of Legionellae isolated from various set of waters.Methods:A total of 400 water samples were collected from different water sources.Water samples were pretreated using acid treatment followed by concentration and culture on buffered charcoal yeast extract agar.Parameters like ability of Legionella isolates to grow in various p H range,effect of different concentrations of chlorine and effect of different temperature optima were set up.Biochemical tests were performed to separate Legionellae into species.Antibiotic sensitivity tests and test for pathogenicity were also conducted on isolated strains.Results:The rates of isolation of Legionella pneumophila(L.pneumophila) in different water sources were found to be 20%(lakes),10%(ponds),8%(water-tanks) and 1%(rivers).Most of the isolates could grow in variable p H 6–8 and it could also survive the normal level of chlorination and even at temperature of 42 C.Isolated species of Legionellae resulted in identification of 5 different species,L.pneumophila being the dominant one.Strains of L.pneumophila were resistant to many antibiotics.Inoculation of Legionellae into intracerebral route of suckling mice revealed that L.pneumophila was the most virulent.Conclusions:Serious and fatal L.pneumophila infections may be transmitted through water.Legionella can survive under various conditions in various water sources.L.pneumophila is the important pathogen causing human disease.Great challenge prevails to health care professionals because these Legionellae acquired antibiotic resistance to many routinely prescribed antibiotics.

Fatal Legionella pneumophila serogroup 1 pleural empyema:A case report

BACKGROUND Legionella pneumophila(L.pneumophila)is a gram-negative intracellular bacillus composed of sixteen different serogroups.It is mostly known to cause pneumonia in individuals with known risk factors as immunocompromised status,tobacco use,chronic organ failure or age older than 50 years.Although parapneumonic pleural effusion is frequent in legionellosis,pleural empyema is very uncommon.In this study,we report a case of fatal pleural empyema caused by L.pneumophila serogroup 1 in an 81-year-old man with multiple risk factors.CASE SUMMARY An 81-year-old man presented to the emergency with a 3 wk dyspnea,fever and left chest pain.His previous medical conditions were chronic lymphocytic leukemia,diabetes mellitus,chronic kidney failure,hypertension and hyperlipidemia,without tobacco use.Chest X-ray and comouted tomographyscan confirmed a large left pleural effusion,which puncture showed a citrine exudate with negative standard bacterial cultures.Despite intravenous cefotaxime antibiotherapy,patient’s worsening condition after 10 d led to thoracocentesis and evacuation of 2 liters of pus.The patient progressively developed severe hypoxemia and multiorgan failure occurred.The patient was treated by antibiotherapy with cefepime and amikacin and with adequate symptomatic shock treatment,but died of uncontrolled sepsis.The next day,cultures of the surgical pleural liquid samples yielded L.pneumophila serogroup 1,consistent with the diagnosis of pleural legionellosis.CONCLUSION L.pneumophila should be considered in patients with multiple risk factors and undiagnosed pleural empyema unresponsive to conventional antibiotherapy.

Occurrence of Legionella pneumophila in Hot Potable Water in Latvia

Legionellae are found in freshwater environments or wet soil, but the major reservoirs are man-made aquatic environments, particularly warm water systems. Our aim was to study the occurrence of Legionella contamination in hot potable water supply systems in apartment and public buildings. Correlations between Legionella spp. positive cases and water source, sampling point and temperature of hot water at the point of consumption were studied. A total of 149 hot water samples were collected in 16 cities and districts of Latvia. Only Legionella pneumophila was isolated. L. pneumophila was found in 63 out of 149 （42%） hot water samples. From all Legionella spp. positive samples, 17% represented L. pneumophila serogroup 1 and 78% L. pneumophila serogroups 2-14 （15）. In 5% of Legionella spp. positive samples, both serogroups 1 and 2-14（15） were isolated. Statistically significant differences were observed in the distribution of L. pneumophila serogroup 1 in samples from various water sources. L. pneumophila was found in 45% of samples from private apartments and in 27% of samples from public buildings. Data analysis confirmed that the temperature of hot water significantly affects the frequency of L. pneumophila positive cases （p 〈 0.05）. Data showed that 85% of consumers received hot water at a temperature below 50°C.

Detection of <i>Legionellae pneumophila</i>in Waters by Culture and Polymerase Chain Reaction

Legionella pneumophila (L. pneumophila) is the most common causative agents for all outbreaks of Legionnaires’ disease. Prevention and control of Legionellosis requires surveying and monitoring of Legionella in the environment using conventional and modern technologies. The present study aims to compare detection of L. pneumophila in water samples using both culture and PCR techniques. A pre-enriched contaminated water sample was split into 13 subsamples. Culture and PCR tests were done from the subsamples after different intervals. The results showed a positive PCR result for L. pneumophila after 8 h of incubation. Also, L. pneumophila was detected by culture on non-selective BCYNE agar and selective GPVC agar after 5 and 6 days of incubation respectively. There was no significant difference between the non-selective BCYE- and the selective GVPC method. The PCR procedure was found more sensitive and differed significantly from the conventional selective GVPC method in isolation of L. pneumophila from water samples. It was concluded that pre-enrichment incubation allows the detection of L. pneumophila by PCR within a maximum of 12 h from the collection of water samples.

Legionella pneumophila-mediated host posttranslational modifications

Legionella pneumophila is a Gram-negative bacterium ubiquitously present in freshwater environments and causes a serious type of pneumonia called Legionnaires’disease.During infections,L.pneumophila releases over 300 effector proteins into host cells through an Icm/Dot type IV secretion system to manipulate the host defense system for survival within the host.Notably,certain effector proteins mediate posttranslational modifications(PTMs),serving as useful approaches exploited by L.pneumophila to modify host proteins.Some effectors catalyze the addition of host protein PTMs,while others mediate the removal of PTMs from host proteins.In this review,we summarize L.pneumophila effector-mediated PTMs of host proteins,including phosphorylation,ubiquitination,glycosylation,AMPylation,phosphocholination,methylation,and ADP-ribosylation,as well as dephosphorylation,deubiquitination,deAMPylation,deADP-ribosylation,dephosphocholination,and delipidation.We describe their molecular mechanisms and biological functions in the regulation of bacterial growth and Legionella-containing vacuole biosynthesis and in the disruption of host immune and defense machinery.

Detection of Legionella pneumophila from domestic water and their antibiotic resistance profiles

Objective:To investigate the presence of Legionella pneumophila(L.pneumophila)in domestic water in Bitlis province and to determine the in vitro susceptibility of the isolates against several antibiotics.Methods:A total of 320 tap water samples were collected from the urban areas and villages of Bitlis province during the period from May to December 2010.All samples were cultured on plates of buffered charcoal yeast extract agar.L.pneumophila strains were tested for antimicrobial susceptibility by the disk diffusion method.Results:L.pneumophila strains were isolated from six(1.9%)domestic water samples.All isolates were typed as L.pneumophila serogroup 1 by latex agglutination test.Four of strains were isolated in July and two of them were detected in August.Antibiotic susceptibility testing was carried out on six L.pneumophila serogroup 1 isolates.Of the six strains,two was resistant to erythromycin and streptomycin,four were resistant to ampicillin and gentamicin,but all were sensitive to chloramphenicol and doxycycline.Conclusions:Our results indicate that L.pneumophila serogroup 1 is the most common type in the domestic water samples and threats public health.This is the first report of L.pneumophila in domestic water samples from Bitlis province.

脂多糖生物探针法与传统培养法检测水中嗜肺军团菌的比较

目的比较脂多糖生物探针法和传统培养法(包括ISO 11731-2017法即“ISO法”、GB/T 18204.5-2013法即“国标法”)对水中嗜肺军团菌(Legionella pneumophila)的检测能力。方法分别采用脂多糖生物探针法和传统培养法对58份加标水样(其中50份为实际水样加标用于以上两类方法比对测试,8份为纯水加标用于脂多糖生物探针法的方法学性能测试)和50份实际水样进行检测,确定脂多糖生物探针法的检出限、准确度、精密度、特异性和灵敏度,并比较两类方法对50份实际水样加标前的定性结果和50份实际水样加标前后的定量结果。结果脂多糖生物探针法检出限为1.55 CFU/L,准确度为102.10%~139.29%,精密度为0.14%~1.78%,特异性为95.12%,灵敏度为88.89%。50份实际水样加标前脂多糖生物探针法和国标法两种方法(检出或未检出)的定性结果无统计学差异(P>0.05);50份实际水样加标前后脂多糖生物探针法定量结果的中位数(M)高于ISO法测定结果,差异具有统计学差异(P<0.001),两类方法测定结果存在相关(r=0.981)。脂多糖生物探针法检出限(1.55 CFU/L)低于ISO法的(15.74 CFU/L),检测时间(2 d)短于ISO法(10 d)。结论与传统培养法相比,脂多糖生物探针法耗时短,检测性能更优。

2023年中南某市夏季公共场所嗜肺军团菌污染状况

目的了解2023年中南某市三类公共场所嗜肺军团菌污染状况,分析健康风险。方法于2023年7月采取非随机抽样方法在在中南某市三类公共场所采集集中空调水样、室内空气样本,以及公共设施物体表面样本进行检测和分析。采用培养法对集中空调冷却水和冷凝水嗜肺军团菌进行检测;采用荧光定量PCR对室内空气以及物体表面嗜肺军团菌进行检测。利用Fisher确切概率法用于评估水样类型、场所类型与健康风险等级的相关性。结果调查中共采集集中空调水样24份、室内空气样本213份以及公共设施物体表面样本282份。培养法检测结果显示,集中空调水样的总体阳性率为37.5%,其中冷却水的阳性率为55.6%(5/9),冷凝水的阳性率为26.7%(4/15)。其中,地铁站的集中空调水样阳性率为27.3%(3/11),宾馆的阳性率为66.7%(6/9),火车站水样中未检测出嗜肺军团菌(0/4)。风险分析发现,冷却水的高风险率为55.6%(5/9),冷凝水的高风险率为20.0%(3/15);地铁和宾馆水样的高风险率分别为27.3%(3/11)和55.6%(5/9)。荧光定量PCR结果显示,公共场所的室内空气和物体表面的检测结果均为阴性,中高风险率为0%。结论三类公共场所中,地铁和宾馆嗜肺军团菌阳性率较高,冷却水和冷凝水中均有较高检出率。

嗜肺军团菌效应蛋白Lpg1972与CNOT7相互作用研究

嗜肺军团菌是一种寄生在真核细胞内的革兰氏阴性致病菌,其致病性主要是依靠IVB型分泌系统分泌的330多种效应蛋白,效应蛋白之间彼此协作共同干扰真核宿主细胞的各种细胞活动.嗜肺军团菌效应蛋白Lpg1972与真核细胞Ccr4-Not复合物中的CNOT7亚基存在相互作用,而Ccr4-Not复合物是真核细胞中调控mRNA降解的关键蛋白。基于此通过分子生物学的手段验证了它们之间的相互作用,设计共表达载体纯化了复合物蛋白,并利用AlphaFold2对复合物的结构和相互作用进行了预测分析,为后续研究奠定了基础。

城市污水处理厂病原菌多样性及健康风险评价

污水中普遍存在大量病原菌,污水处理厂作为接收和处理污水的主要场所,在控制病原菌污染和保障再生水安全中发挥重要作用。研究利用16S r RNA测序技术和实时荧光定量PCR技术对中国北方一座污水处理厂夏、冬两季的细菌群落结构和病原细菌存在情况进行分析。研究结果表明,共检出27个病原菌菌属,其中假单胞菌属(Pseudomonas)、不动杆菌属(Acinetobacter)是主要病原菌属。经过一系列工艺处理,出水中含有病原菌属数目和含量明显降低,但发现常规出水检测指标大肠杆菌不能指示其他病原菌的存在情况和风险水平,提示仍需引入新的检测指标减少再生水回用的病原菌风险。

广东地区环境水源和临床标本嗜肺军团菌培养与基因快速鉴定

目的建立嗜肺军团菌培养与基因快速鉴定方法,探讨广东地区环境水源和非典型肺炎患者嗜肺军团菌感染状况。方法对广东地区采集的57份中央空调冷凝塔水、30份湖泊水和53份临床标本,用本室改良BCYE-α培养基作培养和PCR试验。结果57份中央空调冷凝塔水培养检出嗜肺军团菌20株(35.1%),PCR检测LP-DNA阳性38份(66.7%);30份湖泊水细菌培养均阴性,PCR阳性4份(13.3%);53例非典型肺炎患者支气管洗液和痰液细菌培养均阴性,PCR阳性5例(9.4%);本实验设计的PCR扩增引物,只和标准嗜肺军团菌和本地分离株产生反应;不与其他病原菌扩增,而显示其良好特异性;本PCR敏感性可检出检体中0.6个细菌DNA含量。结论广东地区环境水源中普遍存在嗜肺军团菌,是引起非典型肺炎的重要病源之一。

建筑热水系统中的军团菌赋存水平及影响因素

建筑热水系统水质微生物安全,对公众健康至关重要。文中介绍了我国华东地区某宾馆建筑热水系统研究结果,采用常规水质指标检测及q-PCR定量分析,了解建筑热水系统中军团菌(Legionella spp.)的存在水平、时空分布特征及其影响因素。建筑热水系统的军团菌检出率为100%,含量为1.57×10^(4)~3.55×10^(5)copies/mL;嗜肺军团菌(Legionella pneumophila)检出率为50%,含量为33.45~259.00 copies/mL,其中热交换器中含量最高;宾馆热水温度低、浑浊度高是造成军团菌含量增加、嗜肺军团菌检出的重要原因;磷可能是军团菌的限制性生长因素。因此,通过适当提高热交换器热水供应温度、降低水的浑浊度、控制水中磷的含量等措施可有效控制军团菌生长繁殖,保障水质生物安全。

军团菌选择性分离培养基及其实用性比较研究

目的探讨与改进嗜肺军团菌选择性分离培养基及其临床实用性评价。方法在BCYEα基础培养基中添加3种不同抗生素组合：CCCV、DGVP（含自配特种染料）和GVPC，以不含抗生素的BCYEα平板作对照，采用菌落计数法比较添加3种不同抗生素和染料的选择性分离平板对嗜肺军团菌生长的影响，同时比较3种选择性分离平板对空调冷凝塔水中嗜肺军团菌的分离率。结果在BCYEα＋DGVP和BCYEα＋GVPC平板上菌落计数（菌液稀释度在10^5～10^7之间）和BCYEα平板结果相类似，差异无统计学意义（P〉0．05）。在BCYEα＋CCCV平板上生长菌落数只有前3种平板的1／2，差异有统计学意义（P〈．05）。从9个中央空调冷凝塔水中分离嗜肺军团菌，用BCYEα＋DGVP和BCYEα＋GVPC平板分离出5株嗜肺军团菌，而BCYEα＋CCCV平板仅分离出2株，不加抗生素的BCYEα平板未分离出军团菌。结论常规用BCYEα＋CCCV平皿对杂菌有较强的抑制作用，但对嗜肺军团菌亦有一定抑制作用，因而在实际应用中降低了嗜肺军团菌的分离率；BCYEα＋DGVP和BCYEα＋GVPC平皿（自制）对嗜肺军团菌的抑制作用微弱，而对杂菌有较强的抑制作用，提高了嗜肺军团菌的分离率。

空调冷却水军团菌微生态环境与繁殖传播研究

目的 研究含氯消毒剂不能持久控制空调冷却塔水中军团菌繁殖、传播的原因 ,为寻找持久、稳固控制军团菌的方法提供依据。方法 定期跟踪调查 7个大型中央空调冷却塔水中军团菌消长情况及其有机物、细菌、藻类构成 ;模拟空调冷却塔环境条件 ,构建藻类减少和藻类增加 2种不同的军团菌生态模型 ,观察军团菌在不同微生态环境中的繁殖速率 ,同时检测空气中的军团菌。结果 空调冷却塔水中军团菌数与水中总有机碳、硝酸盐氮及藻体数(主要是绿藻门中的盘星藻、四棘藻和蓝藻门中的地中尖头藻这类体积较大的藻类 )成正比 ,含氯消毒剂不能持久杀灭军团菌。军团菌在模拟的最佳空调冷却塔水环境 (藻类增加模型 )中 ,繁殖速率可达 1h增加 1倍 ,并随藻体数增加维持指数生长状态 ;在模拟藻类减少空调冷却塔水环境中 ,加入军团菌液 1h后 ,军团菌数也可增加 1倍 ,但以后繁殖速率越来越低 ,3d后军团菌数为藻类增加生态模型的 1/ 16 0。当水中军团菌数达 4 2× 10 4/ml时 ,在空气中可检测到军团菌。结论 军团菌在 2 0℃以下的空调冷却水中不是处于休眠状态 ,而是处于失活状态。军团菌的繁殖速率和持续时间主要与空调冷却水中的藻体种类及数量密切相关 ,其次与总有机碳、硝酸盐氮有关。改变军团菌的微生态环境 ,去除冷却?

水中嗜肺军团菌分布规律研究

目的 探讨水环境中嗜肺军团菌分布规律及分型特征。方法 采集自然环境及生活环境中各主要水体样品 ,浓缩后经常规细菌培养后的疑似菌株 ,用血清学鉴定与 1 6SrRNA半套式PCR技术双重鉴定进行军团菌和嗜肺军团菌的分型。结果 全部 1 2 6个监测点水样有 2 8个出现军团菌阳性 (其中嗜肺军团菌 2 1个 ) ;自然环境及生活环境监测点军团菌阳性率分别为 32 1 %、2 2 2 % (其中嗜肺军团菌阳性率分别为2 1 4 %、1 6 7% ) ;全年 4 30份水样有 36份军团菌阳性 (其中 2 3份为嗜肺军团菌 ) ,夏、秋季检出率较高 ,春、冬季也有检出 ;空调冷却塔水、淋浴喷头水、公园湖水检出率较高。结论 嗜肺军团菌在自然环境及生活环境各水体中分布广泛。环境温度是军团菌污染的重要因素 ,夏、秋季是军团菌污染和军团病传播的高发季节 ,春、冬季也可发生。公园娱乐水体、空调冷却水、淋浴喷头水等与人群密切接触水体是军团菌污染和军团病传播的高危水体。

广东地区嗜肺军团菌的扩增片段长度多态性分析

【目的】了解广东部分地区2002～2007年环境水中嗜肺军团菌的基因型及遗传学关系。【方法】参考欧洲军团菌感染工作组(the European Working Group for Legionella Infections,EWGLI)制定的分型草案(1.2版),采用PstⅠ酶对我室保存的2株标准菌株及41株不同时间、不同地点的嗜肺军团菌环境分离株进行扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分析,电泳图谱与EWGLI进行比对。【结果】AFLP分型结果稳定,重复性好;43株嗜肺军团菌,其不同来源菌株呈现多态性分布,经AFLP分析得到33个基因型,辨别力指数为99.79%,其中Kingmed AFLP 011为优势基因型。按Dice系数≥0.8,可分为18个群,Kingmed AFLPD群为优势群,占总菌株数的34.88%。由电泳图谱的相似性,初步推断存在EWGLI 001 Lugano型、002 Lugano、012 Rome、013 London、014 London型、015 Dresden型、021 Lyon等7个基因型,以及多个未报道的新基因型。【结论】广东地区嗜肺军团菌的基因型十分丰富,AFLP技术是其分子流行病学研究的有效手段。