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.

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。

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.

Comparison of Anolyte and Chlorine Dioxide for a Continuous Hot Water Disinfection in Nursing Home: A Two Years Legionnaires’ Disease Prevention

Worldwide epidemiological reports assert that drinking water is a source for infections and Legionella control represents a critical issue in healthcare settings. Chemical disinfections of water networks are control measures that need to be fine-tuned to obtain satisfactory results in large buildings over prolonged time periods. Aim of study is the evaluation of the effect of anolyte and chlorine dioxide, applied in two different hot water networks of a nursing home to manage Legionella risk. Nursing home has two buildings (A and B), with the same point of aqueduct water entrance. From June 2016, following a shock chlorination, the continuous disinfections with chlorine dioxide and anolyte were applied in hot networks of building A and B, respectively. Hot water was sampled at the central heating system and at two points of use for Legionella research, while chemical tests of manganese (Mn), iron (Fe), zinc (Zn) and trihalomethanes compounds (THM) were implemented to evaluate the disinfection by-products presence. Before chlorination Legionella pneumophila sg1 was recovered with a mean count of 2.4 × 104 CFU/L, while chemical compounds concentrations were within the law limits (Directive 98/83/EC). Then the disinfections Legionella was not recovered in both hot water plants. After the disinfection with chlorine dioxide (from June 2016 to May 2018), a statistically significant increase of iron, zinc and THM concentrations was detected in building A (p = 0.012;p = 0.004;p = 0.008). Both disinfectants appear effective against Legionella spp. growth in water network, but anolyte ensures a lower disinfection by-products release.

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.

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.

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.

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.

The bacterial effector SidN/Lpg1083 promotes cell death by targeting Lamin-B2

To facilitate survival,replication,and dissemination,the intracellular pathogen Legionella pneumophila relies on its unique type IVB secretion system(T4SS)to deliver over 330 effectors to hijack host cell pathways in a spatiotemporal manner.The effectors and their host targets are largely unexplored due to their low sequence identity to the known proteins and functional redundancy.The T4SS effector SidN(Lpg1083)is secreted into host cells during the late infection period.However,to the best of our knowledge,the molecular characterization of SidN has not been studied.Herein,we identified SidN as a nuclear envelope-localized effector.Its structure adopts a novel fold,and the N-terminal domain is crucial for its specific subcellular localization.Furthermore,we found that SidN is transported by eukaryotic karyopherin Importin-13 into the nucleus,where it attaches to the N-terminal region of Lamin-B2 to interfere with the integrity of the nuclear envelope,causing nuclear membrane disruption and eventually cell death.Our work provides new insights into the structure and function of an L.pneumophila effector protein,and suggests a potential strategy utilized by the pathogen to promote host cell death and then escape from the host for secondary infection.

Sensing bacterial infections by NAIP receptors in NLRC4 inflammasome activation

The inflammasome is an emerging new pathway in innate immune defense against microbial infection or endogenous danger signals.The inflammasome stimulates activation of inflammatory caspases,mainly caspase-1.Caspase-1 activation is responsible for processing and secretion of IL-1βand IL-18 as well as for inducing macrophage pyroptotic death.Assembly of the large cytoplasmic inflammasome complex is thought to be mediated by members of NOD-like receptor(NLR)family.While functions of most of the NLR proteins remain to be defined,several NLR proteins including NLRC4 have been shown to assemble distinct inflammasome complexes.These inflammasome pathways,particularly the NLRC4 inflammasome,play a critical role in sensing and restricting diverse types of bacterial infections.Here we review recent advances in defining the exact bacterial ligands and the ligand-binding receptors involved in NLRC4 inflammasome activation.Implications of the discovery of the NAIP family of inflammasome receptors for bacterial flagellin and type III secretion apparatus on future inflammasome and bacterial infection studies are also discussed.

Chest radiographic characteristics of community-acquired Legionella pneumonia in the elderly

Background Legionella is an important community-acquired pneumonia pathogen.Although the elderly are especially susceptible to Legionella,few studies have looked at comparative radiographic features of Legionella pneumonia in this population.The aim of this study was to explore the chest radiographic characteristics of community-acquired Legionella pneumonia in the elderly.Methods Serial chest radiographs obtained in 34 patients hospitalized with serologically proven Legionella pneumonia were retrospectively reviewed.Chest x-ray features of an aged group of ≥65 years were assessed and compared with a non-aged group of ＜65 years old with regard to initial patterns and distributions of pulmonary abnormalities,accompanying signs,and progression.Results The most common initial presentation was a patchy alveolar infiltrate involving a single lobe,most often the lower lobe.There was no middle or lingular lobe involvement in the aged group patients,but bilateral pleural effusion was significantly more common in this group.In the aged group patients,radiographic progression following adequate therapy,despite a clinical response,was more often noted and the radiographs were less likely to have returned to the premorbid state at discharge,but the differences were not significant between the two groups.Conclusion The discrepancy between imaging findings and clinical symptoms seems more prominent in community-acquired Legionella pneumonia in the elderly.

Aerosol exposure assessment during reclaimed water utilization in China and risk evaluation in case of Legionella

Reclaimed water utilization provides an effective way to alleviate water shortage.However,the residual pathogens in the recycled water like Legionella,could be spread into the air as aerosols through water-to-air transmission process.Inhaling the aerosols by the people nearby increases their susceptibility to diseases.For estimating the health risks associated with the potential exposure of airborne Legionella emitted from the urban use of reclaimed water in China,nationwide questionnaire was designed to investigate the exposure habits of Chinese population in different scenarios.Quantitative microbial risk assessment(QMRA)served as the suitable explanatory tool to estimate the risk.The results indicated that annual infection probability of populations exposed to Legionella for three scenarios,0.0764(95%CI:0.0032−0.6880)for road cleaning,1.0000(95%CI:0.1883−1.0000)for greenfield irrigation,0.9981(95%CI:0.0784−1.0000)for landscape fountain,were markedly higher than the threshold recommended by WHO(10^(−4) per person per year(pppy))according to the concentration distribution of Legionella in the reclaimed water.An age-,educational background-,region-and gender-specific data in annual infection probability also showed different tendencies for some subpopulations.This study provides some detailed information on the health risks from the water reuse in China and will be useful to promote the safe application of reclaimed water in water-deficient areas.

Informatic analysis reveals Legionella as a source of novel natural products

Microbial natural products are a crucial source of bioactive molecules and unique chemical scaffolds.Despite their importance,rediscovery of known natural products from established productive microbes has led to declining interest,even while emergent genomic data suggest that the majority of microbial natural products remain to be discovered.Now,new sources of microbial natural products must be defined in order to provide chemical scaffolds for the next generation of small molecules for therapeutic,agricultural,and industrial purposes.In this work,we use specialized bioinformatic programs,genetic knockouts,and comparative metabolomics to define the genus Legionella as a new source of novel natural products.We show that Legionella spp.hold a diverse collection of biosynthetic gene clusters for the production of polyketide and nonribosomal peptide natural products.To confirm this bioinformatic survey,we create targeted mutants of L.pneumophila and use comparative metabolomics to identify a novel polyketide surfactant.Using spectroscopic techniques,we show that this polyketide possesses a new chemical scaffold,and firmly demonstrate that this unexplored genus is a source for novel natural products.

Research advances of Legionella and Legionnaires’ disease

Legionella is a causative agent of legionellosis or Legionnaires’disease.Infection with Legionella spp.is an important cause of community and hospital-acquired pneumonia.Moreover,infection with Legionella is one of the most common causes of severe pneumonia in community settings,and Legionella is isolated in 40%of hospital-acquired pneumonia cases.The clinical features of legionellosis or Legionnaires’disease are not unique,which makes the diagnosis challenging.Macrolides andfluoroquinolones are the most widely used drugs in treatment.Early recognition,treatment and effective measures for prevention and control of the disease are important and required.Although there have been a lot of methods for diagnosis of Legionella infection,they were often time-consuming or complex,not suitable for clinical purposes.This review summarizes available information regarding the microbiology,taxonomy,epidemiology,pathogenesis and diagnosis of Legionnaires’disease,with an emphasis on the laboratory diagnosis of infection with Legionella spp.

Identification of Legionella species by the composition of cellular fatty acids

One hundred andfifty-three isolates from the environment and 36 reference strains of the Legionella were studied with regards to their composition of cellular fatty acids as determined by gas chromatography,and then were classified into 41 groups by numerical analysis.Most reference strains formed only a single group,except L.micdadei,L.jamestowniensis,L.parisiensis,L.jorda-nis,L.feeleii and L.longbeachae,which were clustered into two or three groups.Even serological types of L.pneumophila could be clearly identified.Therefore,in this study,numerical analysis of cellular fatty acid composition is an effective method for identifying Legionella species.