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。

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.

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.

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LP1株MompS抗原表位的预测与验证

目的探讨以嗜肺军团菌LP1株MompS抗原表位为基础的合成肽的免疫作用。方法在GenBank中检索LP1株MompS基因信息,利用在线生物信息学软件,结合DNA Star软件的蛋白质二级结构预测理论方案,预测其B表位抗原位点,设计合成具有LP1血清型特异性氨基酸序列的多肽,通过肽结合实验和动物免疫学效果评价检测合成肽对LP1免疫血清的结合能力。结果筛选得到3条多肽GP-15、NY-17、AA-19,多肽肽结合实验表明GP-15 D(450)反应值显著高于NY-17和AA-19片段(P<0.05),动物免疫学效果评价显示其各时间段的IgG抗体几何平均滴度显著高于NY-17和AA-19(P<0.05)。结论证实3条多肽均具备了MompS表位属性,GP-15肽结合实验和动物免疫效果评价均提示其可作为嗜肺军团菌多肽疫苗的候选肽段。

四川省嗜肺军团菌血清Ⅰ型(LP1)基因分型研究

目的为了解四川省嗜肺军团菌(Legionella pneumophila,LP)基因特征,采用多位点串联重复序列分析(MLVA)和基因序列分型(SBT)两种方法对四川省1989—2016年分离到的42株嗜肺军团菌血清I型(LP1)进行基因分型研究。方法根据文献报道的军团菌8个VNTR位点和SBT 7个管家基因对42株嗜肺军团1型菌(LP1)分别进行PCR扩增。VNTR结果经毛细管电泳分析后,得到MLVA分型。SBT结果测序后上传至欧洲军团菌感染组(EWGLI)数据库得到ST分型。结果 42株嗜肺军团菌血清I型(LP1)分为8个MLVA型,优势型别为M08型(47.6%)和M07型(23.8%)。SBT分为12个ST型,其中有2个ST型(ST2359,ST2360)为首次发现。ST1(52.3%)、ST630(14.2%)为优势型别。12个ST型分为3个克隆群(Clonal Complex,CCs)和2个singleton。结论 MLVA方法和SBT方法均能用于LP1型分子流行病学监测及分子遗传学特征的研究。四川省LP1型具备较高的遗传多样性和地区特异性,有感染人的风险,应加强对公共卫生环境中军团菌的监测工作。

嗜肺军团菌SBT、PFGE、AFLP分子分型方法的比较

比较SBT、PFGE、AFLP三种分子分型方法在嗜肺军团菌分型研究中的分辨力,探讨SBT方法在嗜肺军团菌分型中的可应用性。收集石家庄市6所医院冷却塔水中分离的32株嗜肺军团菌,对其中的24株血清I型嗜肺军团菌进行SBT分型研究,并与PFGE和AFLP分型结果进行了比较。24株LP1型嗜肺军团菌共分为4个ST型,分辨系数为0.239 1。PFGE方法将32株菌株共分为15个PFGE型,分辨系数为0.925 4。AFLP方法将32株菌株分为23个AFLP型,分辨系数为0.973 7。通过比对EWGLI网站SBT数据库,ST1021型和ST345型为本地区独特型别且属于同一克隆系;ST1型为优势型别并在我国长期流行;由于缺失neuA而未分型的嗜肺军团菌株与其他23株菌分属于不同的克隆系。SBT方法的分型能力不及PFGE方法和AFLP方法。但SBT分型方法能够通过全球比对数据库得到更多的关于菌株遗传进化和流行分布的资料,在研究菌株分子流行病学及进化方面优于PFGE和AFLP方法。

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。

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.

Legionella and legionnaires' disease: An overview

Legionellosis is the generic term used to describe infections caused by different varieties of Legionella spp.,including Legionnaires'disease(LD),a severe and potentially fatal form of pneumonia,and Pontiac fever,a self-limited flu-like illness.Legionellosis is usually acquired through inhalation or aspiration of aerosols containing Legionella spp.These bacteria can cause acute consolidating pneumonia in susceptible patients who are at an advanced age,have underlying debilitating diseases,or are immunodeficient.The main natural reservoir for Legionella is water and this pathogen colonizes many different natural and man-made freshwater environments such as water networks,cooling towers,and water systems in buildings and hospitals.In recent years,various laboratory diagnostic tests for Legionella infections have changed significantly.Although the sequencing method is nowadays considered the fastest and most reliable method for differentiation and detection of different Legionella species,the isolation of these bacteria from clinical specimens is the golden standard for diagnosis of Legionnaires'disease.Today the urinary antigen test as the most rapid and inexpensive method is routinely used for diagnosis of LD caused by Legionella pneumophila serogroup 1.The macrolides and fluoroquinolones are still the mainstays for the treatment of Legionella infections.For the prevention of spreading the contaminated water aerosols and controlling Legionella infections,an effective water treatment procedure is necessary.This review describs and summarizes the latest available information about all aspects of Legionella and Legionnaires'disease.

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.

Legionella from Environmental and Clinical Homes for the Mentally Disabled and Comparison of Their Sequence Types

In this study the authors compared the sequence types （STs） designed by sequence based typing （SBT） of 4 clinical and 12 environmental strains of Legionella pneumophila serogroup 1 （LP1） which were isolated from hospital facilities for the mentally disabled. The strains were selected after a retrospective surveillance of 565 clinical records （2002-2009） and investigations of water circuit. It was possible to correlate two clinical strains with the corresponding environment, which were collected from showers that had exposed the patients （ST685, ST16） and two clinical strains present in the same structure （STI）. The other environmental strains were isolated from water in the department with confirmed or suspected clinical cases. All the strains （seven） from the first structure had ST188; two from the second structure had ST34; and the last from the third structure, gave an ST694. The results were compared with the European Working Group for Legionella Infections （EWGLI） database： the ST 1, 16, 34 and 188 were already known in literature, among clinical and nosocomial cases, especially for ST 1, the most distributed worldwide. Two STs were new to the database. ST685 was isolated both from a patient and from the water; ST694, which was found exclusively in the environmental compartment of a control structure （no cases of legionellosis and low number of nosocomial pneumonia）, was unknown in the literature and the authors could only speculate on its possible minor virulence and/or distribution. The implementation of SBT and international comparisons may be useful to gain genotypic knowledge of circulating environmental strains, also verifying their presence in the clinical setting.

Bacterial Colonization and Occurrence of Legionella in Cooling Towers of Southern General Company for Fertilizer Production in Basrah City

Sixty six water samples were collected from inlets, inside and outlets of cooling towers （22 samples each, once a week）. Samples were screened for bacterial colonization with special focus on Legionella. Percentage occurrence of predominant bacteria present in water samples collected from inlets included： Klebsiella （95.45%）, Enterobacter （90.9%）, Pseudomonas （86.36%）, Escherichia coli （81.81%）, and Legionella （72.72%）. While those predominant in water inside cooling towers were： Pseudomonas （100%）, Staphylococci （81.81%）, Legionella （81.81%） and Bacillus （72.72%）. Incidence of Legionella recovered from inside and outlets of cooling towers sites was similar. Four species of Legionella were identified in water samples collected from inlets, inside and outlets of cooling towers, in the following consecutive frequencies L. pneumophila （72.72%, 81.81% and 81.81%）, L. jordans （18.18%, 36.36% and 27.27%）, L. dumffii （9.09%, 22.72% and 22.72%） and L. oekidegenes （4.54%, 13.63% and 18.18%）. The increased percentage occurrence of Legionella in water collected from inlets and inside cooling towers, as well as those of Pseudomonas and Staphylococci suggest that Legionella is also a hardy organism, being potentially survive as free organism despite water disinfection.

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。