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.

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.

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.

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.

Evaluation of the Effectiveness of a 4-Months Continuous Injection of a Gas Mixture (CO₂and Inert Gases) on <i>Legionella</i>Contamination of a Hot Water Distribution System

Within the biofilm and scales Legionella is less far susceptible to the effects of the most frequently used biocides. The objective of this study was to evaluate the effect of a 4-months continuous injection of a gas mixture (CO2 and inert gas) in the hot water distribution system of a large hotel colonized by L. pneumophila sg3 on limiting biofilm formation and scales and in turn Legionella growth. Before the continuous injection of the gas mixture, out of the 15 sampling points examined every month 60% were colonized by Legionella (mean concentrations of 102 cfu/L in the boilers and the return loop, and 104 cfu/L in taps and showers). One week after the injection of the gas mixture and daily fluxing of the distal outlets, the level of colonization decreased (3 cfu/L). When it was decided to flux all the distal outlets only 1 day per week the mean concentration of Legionella increased again (>104 cfu/L) in all the sampling points. Thus, cleaning of the boilers was performed and distal outlets were again fluxed daily. One week after the level of contamination decreased again (2 cfu/L). Nonetheless, the colonization was not eliminated and when fluxing of the distal outlets was not performed every day the mean concentrations of Legionella raised up to >104 cfu/L. Results indicate that the gas mixture was able to reduce the level of colonization by Legionella only because associated to the fluxing of the distal outlets.

Monochloramine for Remediation of <i>Legionella</i>Only in Domestic Hot Water Systems: An Iron Fist in a Velvet Glove

Monochloramine is a well-known disinfectant for drinking water with several advantages over chlorine and chlorine dioxide. With these two biocides, monochloramine is included in the US-EPA list of disinfectants for drinking water. At the beginning of XXI century, epidemiological studies demonstrated its superior ability to control Legionella in hot water health-care premises. In 2005 a research program started to set up a reliable and effective method to produce monochloramine directly in domestic hot water systems, at the safest concentration and without accumulation of by-products. Results of these researches, which has been carried out with the collaboration of Italian and American Institutions showed that monochloramine can be safely and reliably prepared and that it is the best approach to Legionella remediation in health-care facilities.

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.

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.

Preliminary Data on Legionella Detection in Water Distribution Systems in Cameroon

After the declaration of the first case of Legionnaire＇s disease in Cameroon in 2007, the Centre Pasteur of Cameroon implemented the detection method for Legionella. The introduction of this new method was put in places in order to investigate Legionella spp. colonization of water distribution systems （WDS） of large buildings including hospitals, hotels and Off Shore Exploitations Sites （OSES） in an attempt to identify risk factors for Legionella spp. Water systems of 6 hotels, 6 hospitals and 6 ships were investigated for the presence of Legionella spp.. A total of 130 samples were collected, 77 from hotels, 27 from hospitals and 26 from ships. 51 Legionella spp. were isolated from 41 （31.54%） water samples. Of a total of 51 positive isolates, 40/51 （78.4%） were L. pneumophila with 21 （52.5%） Legionellapneumophila serogroup （sg） 1, 16 （40%） L. pneumophila sg 5, 2 （5%） L. pneumophila sg 6, 1 （2.5%） L. pneumophila sg 7 and 11/51 Legionella spp. with 10 （90%） L. anisa, 1 （10%） Legionella dumoffii. 5 L. pneumophila sg 1 were associated with 5 L. pneumophila sg 5 and 4 L. pneumophila sg 1 were associated with 4 L. anisa. These results showed that WDS of hospitals, hotels and ships can be heavily colonized by Legionella spp. and may present a risk of Legionnaires＇ disease. Based on these preliminary results, we have just put in place a Legionella survey protocol in Cameroon.

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.

Continuous Disinfection by Monochloramine on Domestic Hot Water System of Health-care Facilities for the Control of Legionella Contamination in Italy

Backgroud： The prevention of Legionella spp. colonization of water distribution systems is a critical issue in healthcare settings and only an effective disinfection of water systems and appropriate environmental surveillance strategies allow to prevent nosocomial legionellosis. Methods： Due to the temporary effectiveness （increase of the temperature of water in boilers and shock hyperchlorination）, the high costs （point-of-use water filters） or the ineffectiveness （hydrogen peroxide, H202） of the previous control procedures, 3 devices （one for each hot water loop） continuously injecting monochloramine have been installed in two different Italian hospitals heavily contaminated by Legionellapneumophila SG3 and SG6. Aim： To evaluate the efficacy of continuous disinfection by monochloramine for control of Legionella on domestic hot water （DHW） distribution system of health-care facilities. Findings： One month after the disinfection of DHW with monochloramine, the load ofL. pneumophila SG3 and SG6 （previous mean count ranging from 103 to 105 CFU/L）, as well as the Heterotrophic Plate Count （HPC） （previous mean count ranging from 〉 10^2 to 〉 10^4 CFU/mL）, decreased at undetectable levels in 100% of the sampling points in the two hospitals. Conclusion： The results suggest that continuous injection of monochloramine on DHW systems can fully control L. pneumophila and HPC in contaminated hospitals since the beginning of its application.

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.

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.

Three-dimensional structures of virulence proteins of Legionella establish targets for new antibacterials

Legionella pneumophila,the causative agent of Legionnaires' disease,has been recognized as a major health problem responsible for an estimated number of 15 000-30 000 cases of severe pneumonia per year in Germany alone.Despite of the high clinical relevance,many aspects of the intracellular life cycle of Legionella,especially details on interactions with host cells,are not well understood.Structural information on virulence proteins helps unravel basal pathogenicity mechanisms and is a prerequisite for the rational development of effective drug molecules.Here we discuss structures of three important virulence proteins of Legionella that have been determined in our laboratory.The structure of the macrophage infectivity potentiator(Mip) protein of Legionella pneumophila is the first of a novel subgroup within the family of FK506-binding protein(FKBP) peptidyl-prolyl cis/trans isomerases.On the basis of the Mip structure,promising antibacterial agents are being designed.Recently,structures of two equally exciting Legionella proteins have been reported.The ferrous iron transport protein FeoB is a transmembrane protein responsible for Fe2+ aquisition after entry of the pathogen into the host cell.The structure of the cytoplasmic domain of FeoB provides insights into the family of prokaryotic G proteins and allows a detailed comparison with structures of related FeoBs.Furthermore,the characterization of DegQ,a periplasmatic chaperone-protease involved in protein quality control represents an intriguing example of how enzymatic activity is regulated by oligomerization as well as by an intrinsic loop activation cascade,depending on subtle conformational rearrangements.

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.

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。