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.

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.

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.

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.

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.

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.

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.

Rapid Identification of Legionella Pathogenicity by Surface-Enhanced Raman Spectroscopy

Objective To establish Surface-enhanced Raman Spectroscopy（SERS） can be used as a rapid and reliable method to distinguish virulent strain and mild strain of L. pneumophila. Methods We isolated and characterized of bacterial strains from ATCC and water samples strains, while we analyzed data from SERS technology using gold nanoparticles as a base and cell infections were employed to rapidly detect L. pneumophila strains. Origin 8.0 was used to collect Raman spectra, smooth and homogenize data, and to contrast spectra. Principal component analysis（PCA） was conducted to discriminate differences between groups using the multivariate analysis package Py Chem 3.0.5. Results Our results indicated that the peaks of high virulence strains reached ≥4000. This criterion was verified by subsequent cell experiments. In addition, we also conducted SERS rapid identification on the virulence of several collected clinical strains and obtained accurate results. Conclusion The present study indicates that the established SERS protocol can be used as a rapid and reliable method to distinguish virulent and mildly virulent strains of L. pneumophila, which can be further used in clinical samples.

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。

Staphylococcus haemolyticus superinfection of legionella pneumonia during infliximab therapy

We present the case of a 42-year-old man affected by psoriasis with Staphylococcus Haemolyticus superin-fection of Legionella pneumonia during infliximab therapy. The introduction of compounds that block TNF-α has yielded great benefits for patients affected by selected autoimmune diseases that fail to respond to classic anti-inflammatory agents, but, on the other hand, has led to an increased susceptibility to infec-tions, in particular of those caused by intracellular pathogens, such as L. Pneumophila. Emerging evi-dence suggests that legionellosis can be complicated by superinfection with other agents, including sap-rophytic microorganisms, among which coagulase- negative staphylococci. To our knowledge, this is the first report of systemic legionellosis with superinfec-tion by S. Haemolyticus, an emerging nosocomial multi-resistant pathogen that commonly causes sep-ticemia, osteomyelitis or endocarditis, but has not so far been associated with necrotizing pneumonia. De-spite the optimal antimicrobial therapy for Staphylo-coccus spp. pneumonia is still controversial, evidence suggests that in patients with confirmed positivity for methicillin resistant strains, particularly if sensitivity to vancomycin is suboptimal, linezolid should be the first choice therapy, being superior to vancomycin and teicoplanin.

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。

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。

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。