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.

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。

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.

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.

酶底物定量法检测水中嗜肺军团菌

目的建立水中嗜肺军团菌定量检测法—酶底物定量法。方法通过加标比对试验确定酶底物定量法的样品保存条件以及试验条件;通过酶底物定量法与传统培养法对采集的水样加标前后测定结果的比对,获得该方法的准确度、精密度、特异性和灵敏度等技术指标,确定该方法的检测性能。结果酶底物定量法的样品应在含硫代硫酸钠的无菌采样容器中,常温条件下送往实验室完成检测;试验最适培养条件为36℃下培养7 d;准确度(回收率)范围在109%~113%;精密度RSD为0.37%~2.46%;方法特异性为94.59%,灵敏度为92.31%;50份实际样品测定时,酶底物定量法结果与真值的一致性为94%,准确度和灵敏度均高于传统培养法。结论酶底物定量法具有较好的准确度、精密度、特异性和灵敏度,适用于批量样品筛查以及较低水平嗜肺军团菌污染时的测定,是水中嗜肺军团菌标准检验方法的有益补充,同时为国标方法修订提供技术储备。

乙酰化修饰在嗜肺军团菌致病过程中的作用

乙酰化修饰是由乙酰基转移酶、去乙酰化酶介导的可逆的蛋白质翻译后修饰。其中,乙酰基转移酶将乙酰辅酶A的乙酰基团转移至底物蛋白的氨基酸残基,而乙酰基团的去除由去乙酰化酶完成。乙酰化修饰参与许多基本生物学过程的调节作用,越来越多的研究表明,蛋白质乙酰化修饰在病原菌的致病过程中具有重要作用。病原菌,如引起非典型性肺炎的嗜肺军团菌,可以通过分泌具有乙酰基转移酶活性的效应蛋白靶向宿主细胞信号通路的关键蛋白质因子,干扰宿主细胞信号通路及免疫反应。本文主要从嗜肺军团菌的致病机制、乙酰化修饰及乙酰化修饰在病原体致病过程中的调控作用进行综述,突出已知的乙酰化毒力蛋白的例子,并讨论它们如何影响与宿主的相互作用,为理解乙酰化修饰在嗜肺军团菌致病过程中的作用机制提供参考。

组蛋白去乙酰化酶6调控鞭毛重组蛋白A干扰巨噬细胞自噬

目的观察组蛋白去乙酰化酶6(HDAC6)调控嗜肺军团菌鞭毛重组蛋白A(rflaA)对小鼠巨噬细胞自噬相关因子表达的影响,分析其可能的作用机制。方法采用支气管肺泡灌洗法提取C57BL/6J与HDAC6-/-C57BL/6J两种小鼠巨噬细胞,用小鼠巨噬细胞表面标记物F4/80鉴定;纯化rflaA,CCK-8法检测其对小鼠巨噬细胞半数抑制浓度(IC50),筛选最佳作用浓度用于后续实验;rflaA分别干预C57BL/6J及HDAC6-/-C57BL/6J小鼠巨噬细胞6、12和24 h,RT-qPCR、Western blot及免疫荧光检测各组自噬相关因子自噬效应蛋白1(Beclin1)、自噬相关蛋白5(ATG5)、自噬微管相关蛋白轻链3(LC3)、SQSTM1蛋白(P62)的表达水平。结果根据CCK-8结果计算,IC50为0.41μg·μL^(-1),最佳作用浓度为0.041μg·μL^(-1)用于后续实验;RT-qPCR、Western blot、免疫荧光结果显示,rflaA干预C57BL/6J小鼠巨噬细胞后,与0 h相比,6、12、24 h HDAC6的表达水平升高,LC3、ATG5表达水平均降低,P62的表达水平升高,Beclin1的表达水平先降低后升高(P均<0.05);rflaA干预HDAC6-/-C57BL/6J小鼠巨噬细胞后,与0 h相比,6、12、24 h LC3、ATG5、Beclin1表达水平均升高,P62的表达水平降低(P均<0.05);在相同的时间点,与C57BL/6J小鼠巨噬细胞各组相比,HDAC6敲除后各组细胞ATG5、LC3表达水平升高,Beclin1先升高后降低,P62降低(P均<0.05)。结论HDAC6促进rflaA抑制小鼠巨噬细胞自噬,HDAC6可能通过影响微管蛋白乙酰化干扰巨噬细胞自噬。

2019年北京市海淀区家庭空调及淋浴水健康危险因素调查

目的了解北京市海淀区家庭空调及淋浴水卫生状况,为改善室内空气质量提供参考。方法在海淀区选取5个社区,入户采集家庭淋浴水水样检测嗜肺军团菌,采集空调过滤网涂抹样品检测细菌总数和真菌总数,并完成家庭空调及淋浴水健康危险因素问卷调查。结果116件家庭淋浴水水样嗜肺军团菌检出率为2.59%,热水器定期清洗率为14.66%,清洗消毒率为0,社区居民对淋浴水可引起军团菌病的知晓率为42.24%。116件空调过滤网涂抹样品细菌总数合格60件,合格率51.72%(95%CI:42.49%~60.95%)。真菌总数合格61件,合格率52.58%(95%CI:43.36%~61.81%)。空调清洗率为63.79%(95%CI:54.92%~72.67%),清洗消毒率为24.32%(95%CI:14.32%~34.33%)。空调过滤网不洁会引起鼻炎相关症状或疾病的知晓率为60.24%(95%CI:51.31%~69.38%)。结论海淀区家庭空调及淋浴水卫生状况值得关注,建议加强社区健康宣教工作,减少居民在家庭生活中对嗜肺军团菌以及空调致病微生物的暴露。

基于酶底物法对饮用水和非饮用水中嗜肺军团菌的定量测定

研究采用酶底物法对饮用水和非饮用水中的嗜肺嗜肺军团菌进行定量检测。结果表明,酶底物法对检测嗜肺嗜肺军团菌的质控样品有较高的准确度和稳定性。对饮用水实际样品中嗜肺军团菌进行检测,在30份管网水中未检出阳性结果,在60份二次供水样品中检出率达5%,其中3份阳性样品均来自公共场所建筑自来水龙头,阳性样品的检出浓度为1.0~5.2MPN/(100mL)。对非饮用水实际样品中嗜肺军团菌进行检测,在全部30份水样中检出率达67%,阳性样品的检出浓度为2.2~1992.6MPN/(100mL)。对酶底物法检测出的阳性样品均进行了菌落验证试验,无假阳性现象,表明酶底物法检测饮用水和非饮用水中实际水样的嗜肺军团菌特异度为100%。其结果表明,在非饮用水和饮用水末端公共场所区域水体中,仍存在嗜肺军团菌污染风险,对人群构成潜在威胁。

嗜肺军团菌Dot/Icm分泌系统的结构基础与工作机制

嗜肺军团菌(Legionella pneumophila)是一种能引起人类军团病肺炎的革兰氏阴性致病菌,它独特的IVB型分泌系统Dot/Icm(defective for organelle trafficking/intracellular multi-plication defect)对其致病性至关重要。作为嗜肺军团菌的毒力输出器,Dot/Icm分泌系统向宿主细胞内转运约330种效应蛋白质,并通过这些效应蛋白质调控宿主的多种细胞生命过程。本文总结了嗜肺军团菌Dot/Icm系统结构的最新研究进展,以及其底物识别和转运机制的最新认识。Dot/Icm分泌系统的结构分为外膜核心复合物(outer membrane core complex,OMCC)、内膜复合物(inner membrane complex,IMC)和Ⅳ型偶联复合物(type IV coupling complex,T4CC)3个组件。其中,OMCC和IMC共同组成一个跨细菌胞膜的通道以供效应蛋白质的向外输送。而T4CC利用自身不同的结构组件识别并捕获具有不同特征序列的效应蛋白质,这些效应蛋白质通过由IMC或T4CC形成的内膜通道最后穿过OMCC的中央通道分泌出去。本文将为深入理解嗜肺军团菌的致病机制提供帮助,也将为寻找更加有效地治疗微生物疾病的方法和开发生物技术和生物医学应用提供帮助。

乌鲁木齐市某中医院2018-2020年门诊部就诊1000例呼吸道感染患者病原体检测结果分析

目的分析乌鲁木齐市某中医院2018年-2020年门诊部就诊1000例呼吸道感染患者病原体检测结果。方法选取乌鲁木齐市某中医院2019年-2020年门诊部就诊1000例呼吸道感染患者,采集患者血清标本,用间接荧光免疫法测定血清标本病原体,分析病原体检测结果。结果1000例呼吸道感染患者中肺炎支原体(MP)病原体感染率最高,占比为34.5%,其次为嗜肺军团菌(LP),占比为23.2%;不同年龄呼吸道感染患者MP、甲型流感病毒(FluA)、乙型流感病毒(FluB)、LP病原体感染率比较,差异有统计学意义(P<0.05);不同年龄呼吸道感染患者腺病毒(AD)、呼吸道合胞病毒(RSV)、副流感病毒(PIV)、Q热立克次氏体(COX)、肺炎衣原体(CP)病原体感染率比较,差异无统计学意义(P>0.05);呼吸道感染患者病原体多重感染主要以MP+LP为主,占比为9.5%,其次为MP+FluB,占比为2.1%。结论1000例呼吸道感染患者主要感染病原体为MP、LP,不同年龄患者病原体易感性不同,多重感染多为MP合并其他病原体,临床应高度重视。

嗜肺军团菌与非嗜肺军团菌所致社区获得性肺炎的临床对比分析

目的探讨嗜肺军团菌与非嗜肺军团菌感染所致社区获得性肺炎的临床特点,提高对军团菌肺炎的认识。方法采用双重聚合酶链反应(DPCR)法对根据临床表现及血清抗体结果,临床诊断为军团菌肺炎患者的痰标本进行军团菌DNA检测。选择痰DPCR和血清抗体结果呈一致阳性的患者并根据检测结果将其分为嗜肺军团菌肺炎组(42例)和非嗜肺军团菌肺炎组(18例)。将2组患者的临床资料进行对比分析。结果痰标本DPCR结果与血清特异性抗体结果呈一致阳性,嗜肺军团菌肺炎与非嗜肺军团菌肺炎诊断明确。嗜肺军团菌肺炎多发于既往体健的青壮年,主要发病于夏秋季;非嗜肺军团菌倾向于四季散发,多发于有基础疾病的老年人。与非嗜肺军团菌肺炎相比,嗜肺军团菌肺炎患者的肺脏受累面积较大,胸腔积液及ARDS发病率高,同时全身感染中毒症状和肺外表现更为明显(P<0.05)。结论嗜肺与非嗜肺军团菌感染所致社区获得性肺炎临床特点有所不同,早期予大环内酯类/喹诺酮类抗生素治疗是改善预后的关键手段。

1株非嗜肺军团菌的鉴定

目的鉴定非嗜肺军团菌。方法用常规分离培养军团菌,进行生化和血清学试验,进一步运用PCR法进行相关基因的分子诊断。结果BCYE-α平板上生长,而BCYE-CYS及血平板上均不生长;触酶试验阳性、明胶液化阳性,与非嗜肺军团菌乳胶凝集;5SrRNA基因阳性、16SrRNA基因阳性,mip基因阴性。结论运用常规分离培养技术结合分子诊断方法,鉴定出1株非嗜肺军团菌。

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?

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。