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Sample records for legionella pneumophila hijacks

  1. Legionella pneumophila type IV effectors hijack the transcription and translation machinery of the host cell.

    PubMed

    Rolando, Monica; Buchrieser, Carmen

    2014-12-01

    Intracellular bacterial pathogens modulate the host response to persist and replicate inside a eukaryotic cell and cause disease. Legionella pneumophila, the causative agent of Legionnaires' disease, is present in freshwater environments and represents one of these pathogens. During coevolution with protozoan cells, L. pneumophila has acquired highly sophisticated and diverse strategies to hijack host cell processes. It secretes hundreds of effectors into the host cell, and these manipulate host signaling pathways and key cellular processes. Recently it has been shown that L. pneumophila is also able to alter the transcription and translation machinery of the host and to exploit epigenetic mechanisms in the cells it resides in to counteract host responses.

  2. Caspase Exploitation by Legionella pneumophila

    PubMed Central

    Krause, Kathrin; Amer, Amal O.

    2016-01-01

    Legionella pneumophila remains a major health concern, especially for hospitalized patients. L. pneumophila in the environment can survive extracellular or as protozoan parasite within amoeba. After human infection it efficiently replicates in alveolar macrophages without activating inflammasome assembly and cleavage of caspase-1. In contrast murine macrophages actively recognize intracellular L. pneumophila via inflammasome components which initiate pro-inflammatory cytokine secretion, phagosomal maturation and pyroptotic cell death thereby leading to bacterial restriction. During this process flagellin-dependent and -independent signaling pathways trigger the canonical as well as the non-canonical inflammasome. This review describes the current knowledge about L. pneumophila-induced inflammasome pathways in permissive and restrictive host cells. PMID:27148204

  3. Cytotoxic glucosyltransferases of Legionella pneumophila.

    PubMed

    Belyi, Yury; Jank, Thomas; Aktories, Klaus

    2013-01-01

    Legionella is a gram-negative bacterium and the causative pathogen of legionellosis-a severe pneumonia in humans. A large number of Legionella effectors interfere with numerous host cell functions, including intracellular vacuole trafficking and maturation, phospholipid metabolism, protein ubiquitination, pro-/anti-apoptotic balances or inflammatory responses. Moreover, eukaryotic protein synthesis is affected by L. pneumophila glucosyltransferases Lgt1, Lgt2, and Lgt3. Structurally, these enzymes are similar to large clostridial cytotoxins, use UDP-glucose as a co-substrate and modify a conserved serine residue (Ser-53) in elongation factor 1A (eEF1A). The ternary complex consisting of eEF1A, GTP, and aminoacylated-tRNA seems to be the substrate for Lgts. Studies with Saccharomyces cerevisiae corroborated that eEF1A is the major target responsible for Lgt-induced cytotoxic activity. In addition to Lgt proteins, Legionella produces other effector glycosyltransferase, including the modularly composed protein SetA, which displays tropism for early endosomal compartments, subverts host cell vesicle trafficking and demonstrates toxic activities toward yeast and mammalian cells. Here, our current knowledge about both groups of L. pneumophila glycosylating effectors is reviewed.

  4. Ecological distribution of Legionella pneumophila.

    PubMed Central

    Fliermans, C B; Cherry, W B; Orrison, L H; Smith, S J; Tison, D L; Pope, D H

    1981-01-01

    Bacteria were concentrated 500-fold from 20-liter water samples collected from 67 different lakes and rivers in the United States. The data suggest that Legionella pneumophila is part of the natural aquatic environment and that the bacterium is capable of surviving extreme ranges of environmental conditions. The data further demonstrate the effectiveness of the direct fluorescent-antibody technique for detecting L. pneumophila in natural aquatic systems. Smears of the concentrated samples were screened microscopically for serogroups of L. pneumophila by the direct fluorescent-antibody technique. Virtually all of the 793 samples were found to be positive by this method. The 318 samples containing the largest numbers of positive bacteria which were morphologically consistent with L. pneumophila were injected into guinea pigs for attempted isolations. Isolates were obtained from habitats with a wide range of physical, chemical, and biological parameters. Samples collected monthly from a thermally altered lake and injected into guinea pigs demonstrated a seasonality of infection, with the highest frequency of infection occurring during the summer months. PMID:7013702

  5. A Plasmid in Legionella pneumophila

    DTIC Science & Technology

    1980-09-01

    13). which they were isolated and the number of the isolate The Legionnaires disease bacterium, L. pneu. from that city. The following 16... Legionnaires disease bacterium. .1. (un. Micro. biol. 8:320-:t25. appears reasonable that this organism could sup- 1:l. Fraser, D5. W., and J. F. McI~ade. 1979...INFECTION AND IMMUNITY, Sept. 1980, p. 1(92-1095 Vol. 29, No. :1 0I 9-9567/A)/- 1092/14$02.00/0. A Plasmid in Legionella pneumophila_ ( (/’ )GREGORY

  6. Plasmid Isolation in Legionella pneumophila and Legionella-like Organisms.

    DTIC Science & Technology

    1980-08-22

    AD-A090 844 ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FR-ETC FIG 6/5 PLASMID ISOLATI ON IN LEGIONELLA PNEUMOPHILA AND LEGIONELLA -LIKE--ETC(U... Legionella -like Organisms PERRY MIKESELL, J. W. EZZELL AND GREGORY B. KNUDSON United States Army Medical Research Institute of Infectious Diseases Fort... Hospital , Pittsburgh, Pa.). Growth conditions. Legionella -like bacteria were cultured on charcoal yeast extract agar (5), yeast extract broth (20) or

  7. Hartmannella vermiformis inhibition of Legionella pneumophila cultivability

    EPA Science Inventory

    Hartmannella vermiformis is frequently isolated from drinking water (DW) and is permissive to Legionella pneumophila intracellular replication. Thus, H. vermiformis may play an important role in the growth and survival potential of such environmental pathogens. In this study, Pag...

  8. Hartmannella vermiformis inhibition of Legionella pneumophila cultivability

    EPA Science Inventory

    Hartmannella vermiformis is frequently isolated from drinking water (DW) and is permissive to Legionella pneumophila intracellular replication. Thus, H. vermiformis may play an important role in the growth and survival potential of such environmental pathogens. In this study, Pag...

  9. Serospecific antigens of Legionella pneumophila.

    PubMed Central

    Otten, S; Iyer, S; Johnson, W; Montgomery, R

    1986-01-01

    Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria. Images PMID:3017918

  10. From amoeba to macrophages: exploring the molecular mechanisms of Legionella pneumophila infection in both hosts.

    PubMed

    Escoll, Pedro; Rolando, Monica; Gomez-Valero, Laura; Buchrieser, Carmen

    2013-01-01

    Legionella pneumophila is a Gram-negative bacterium and the causative agent of Legionnaires' disease. It replicates within amoeba and infects accidentally human macrophages. Several similarities are seen in the L. pneumophila-infection cycle in both hosts, suggesting that the tools necessary for macrophage infection may have evolved during co-evolution of L. pneumophila and amoeba. The establishment of the Legionella-containing vacuole (LCV) within the host cytoplasm requires the remodeling of the LCV surface and the hijacking of vesicles and organelles. Then L. pneumophila replicates in a safe intracellular niche in amoeba and macrophages. In this review we will summarize the existing knowledge of the L. pneumophila infection cycle in both hosts at the molecular level and compare the factors involved within amoeba and macrophages. This knowledge will be discussed in the light of recent findings from the Acanthamoeba castellanii genome analyses suggesting the existence of a primitive immune-like system in amoeba.

  11. Biofilms: The Stronghold of Legionella pneumophila

    PubMed Central

    Abdel-Nour, Mena; Duncan, Carla; Low, Donald E.; Guyard, Cyril

    2013-01-01

    Legionellosis is mostly caused by Legionella pneumophila and is defined as a severe respiratory illness with a case fatality rate ranging from 5% to 80%. L. pneumophila is ubiquitous in natural and anthropogenic water systems. L. pneumophila is transmitted by inhalation of contaminated aerosols produced by a variety of devices. While L. pneumophila replicates within environmental protozoa, colonization and persistence in its natural environment are also mediated by biofilm formation and colonization within multispecies microbial communities. There is now evidence that some legionellosis outbreaks are correlated with the presence of biofilms. Thus, preventing biofilm formation appears as one of the strategies to reduce water system contamination. However, we lack information about the chemical and biophysical conditions, as well as the molecular mechanisms that allow the production of biofilms by L. pneumophila. Here, we discuss the molecular basis of biofilm formation by L. pneumophila and the roles of other microbial species in L. pneumophila biofilm colonization. In addition, we discuss the protective roles of biofilms against current L. pneumophila sanitation strategies along with the initial data available on the regulation of L. pneumophila biofilm formation. PMID:24185913

  12. First isolation of Legionella pneumophila in Sri Lanka.

    PubMed

    Wahala, W M; Wickramasinghe, R S

    2000-12-01

    To determine the presence of legionellae and species composition of the genus Legionella in Sri Lankan hotel cooling towers, and to determine the previous exposure of hotel workers to Legionella pneumophila. Collection of water samples from 16 cooling towers of air conditioning plants from 7 representative hotels, and blood samples from hotel workers. Department of Bacteriology, Medical Research Institute, Colombo 8. Water samples from 4 (57.4%) hotels selected were positive for legionellae. Five (38.4%) selected cooling towers yielded legionellae with viable counts ranging from 1 to 5 colony forming units (CFU)/ml. 93.7% of the isolates were Legionella pneumophila. Only one hotel worker had significant antibody levels denoting past infection to Legionella pneumophila. Legionella does occur in the Sri Lankan hotel environment and Legionella pneumophila appears to be the most common species.

  13. Plaque assay for virulent Legionella pneumophila.

    PubMed Central

    Fernandez, R C; Lee, S H; Haldane, D; Sumarah, R; Rozee, K R

    1989-01-01

    Methods of assessing virulence of Legionella pneumophila, the etiologic agent of Legionnaires disease, include the infection of guinea pigs, fertile chicken eggs, and mammalian and protozoan cell cultures. Guinea pig assays, in particular, are expensive, laborious, or unsuitable for routine screening of Legionella isolates. We have developed a virulence assay that requires the enumeration of viruslike plaques which are the result of virulent L. pneumophila infecting mouse L929 cells. Each plaque is the consequence of the initial infection of an L cell with a single bacterium. A nonvirulent mutant derived from the serial passage of virulent L. pneumophila on Mueller-Hinton agar fails to survive within L cells and consequently fails to produce plaques. Images PMID:2674192

  14. DNA probe specific for Legionella pneumophila.

    PubMed Central

    Grimont, P A; Grimont, F; Desplaces, N; Tchen, P

    1985-01-01

    A procedure for preparing a DNA probe to be used in the specific detection of Legionella pneumophila by dot or colony hybridization has been devised. When total DNA from L. pneumophila was used as a radioactive probe, cross-hybridization occurred with DNA from many other species belonging to various families (including Legionellaceae, Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae). Cross-hybridizing restriction fragments in L. pneumophila ATCC 33152 DNA were identified on Southern blots. When unlabeled DNA from strain ATCC 33152 was cleaved by endonuclease BamHI, the DNA fragments cross-hybridizing with the labeled DNA from all of the other species and genera tested (or with Escherichia coli 16 + 23 S RNA) had a size of 21.4 and 16.2 kilobase pairs (major bands) and 28.0, 12.8, and 10.1 kilobase pairs (minor bands). BamHI restriction fragments of L. pneumophila DNA deprived of the cross-hybridizing fragments were pooled and used as a probe for the detection of L. pneumophila. This probe proved to be specific for L. pneumophila in colony and dot hybridization. It can potentially be used for the detection of L. pneumophila in clinical and water samples. The procedure described can be readily applied to the preparation of probes specific for phylogenetically isolated bacterial species other than L. pneumophila. Images PMID:3980693

  15. Isolation of Legionella pneumophila from pluvial floods by amoebal coculture.

    PubMed

    Schalk, J A C; Docters van Leeuwen, A E; Lodder, W J; de Man, H; Euser, S; den Boer, J W; de Roda Husman, A M

    2012-06-01

    Viable Legionella pneumophila bacteria were isolated by amoebal coculture from pluvial floods after intense rainfall and from water collected at sewage treatment plants. Several isolated L. pneumophila strains belonged to sequence types that have been previously identified in patients.

  16. Isolation of Legionella pneumophila from hotels of Greece.

    PubMed

    Alexiou, S D; Antoniadis, A; Papapaganagiotou, J; Stefanou, T

    1989-03-01

    Twenty water samples collected from 6 hotels situated in various areas of Greece were examined for the presence of Legionella pneumophila and Legionella-like organisms. Five of the six hotels included in this investigation were associated with cases of legionellosis. Legionella pneumophila serogroups 1 and 8 were isolated from four of six hotels, mainly from the hot water supply system. This is the first isolation and identification of L. pneumophila in Greece.

  17. Chemically Defined Medium for Legionella pneumophila Growth

    DTIC Science & Technology

    1981-01-01

    for Legionnaires disease bacteriuii I t ’In. Mi- L.egionnaire. dioa bal’teriutn ( Legionella pneumii crobiol. 8:320-32.5. phi/u n to heiiaIly defined... Legionella pneumophila Growth. W 6) JOSElPH D/HISTROPH’ KENNETH W./HEI)LUN. AND SRINIVAS/GOWIA nited States Army Medical Research Institute of Infectious... Diseases . Fort Detrick Frederick,~~Maryland 21701 " A chemically defined medium containing 18 amino acids, inorganic salts, E rhamnose, choline, and

  18. Genetic structure of populations of Legionella pneumophila.

    PubMed Central

    Selander, R K; McKinney, R M; Whittam, T S; Bibb, W F; Brenner, D J; Nolte, F S; Pattison, P E

    1985-01-01

    The genetic structure of populations of Legionella pneumophila was defined by an analysis of electrophoretically demonstrable allelic variation at structural genes encoding 22 enzymes in 292 isolates from clinical and environmental sources. Nineteen of the loci were polymorphic, and 62 distinctive electrophoretic types (ETs), representing multilocus genotypes, were identified. Principal coordinates and clustering analyses demonstrated that isolates received as L. pneumophila were a heterogeneous array of genotypes that included two previously undescribed species. For 50 ETs of L. pneumophila (strict sense), mean genetic diversity per locus was 0.312, and diversity was equivalent in ETs represented by isolates recovered from clinical sources and those collected from environmental sources. Cluster analysis revealed four major groups or lineages of ETs in L. pneumophila. Genetic diversity among ETs of the same serotype was, on average, 93% of that in the total sample of ETs. Isolates marked by particular patterns of reactivity to a panel of nine monoclonal antibodies were also genetically heterogeneous, mean diversity within patterns being about 75% of the total. Both Pontiac fever and the pneumonic form of legionellosis may be caused by isolates of the same ET. The genetic structure of L. pneumophila is clonal, and many clones apparently are worldwide in distribution. The fact that L. pneumophila is only 60% as variable as Escherichia coli raises the possibility that isolates recovered from clinical cases and man-made environments are a restricted subset of all clones in the species as a whole. PMID:4030689

  19. Legionella pneumophila Toxin, Isolation and Purification

    DTIC Science & Technology

    1981-01-01

    Fort Detrick, Frederick, Maryland, U.S.A. INTRODUCTION We are all aware of the dramatic outbreak of what is now termed Legionnaires ’ Disease that...bacteria now termed Legionella pneumophila was isolated and identified as the etiologic agent by Drs. Fraser (1) and McDade (2). Although Legionnaires ...recognize two distinct clinical syndromes associated with this Ot organism. The first termed Legionnaires ’ Disease , involves patients ranging in age from 3

  20. Ecological niche of Legionella pneumophila

    SciTech Connect

    Fliermans, C.B.

    1983-01-01

    This paper discusses the ecological niches, relationships and controls of Legionella derived from environmental sources. Only as clinical cases and studies relate directly to the ecological understanding of the bacterium will they be discussed. This review seeks to separate the ecological parameters associated with Legionella that are often incorporated into the medical literature as well as to highlight specific ecological studies. A series of ecological studies demonstrates the niches of Legionella, the ecological parameters that allow the bacterium to survive, grow and to be disseminated. Relationships among given habitats are explored along with biological relationships within a given habitat.

  1. Molecular epidemiology of Legionella pneumophila serogroup 1.

    PubMed Central

    van Ketel, R J; ter Schegget, J; Zanen, H C

    1984-01-01

    The DNA of patient and environmental isolates of Legionella pneumophila serogroup 1 was analyzed by restriction endonuclease cleavage. The electrophoretic patterns of the DNA digests of isolates from a group of patients with Legionnaires disease acquired in a hospital were indistinguishable from one another and were identical to the DNA pattern of a strain isolated from the hot water supply of the hospital. On the other hand, they were easily differentiated from strains isolated from patients and hot water supplies in other hospitals in the same city. The homogeneity of populations of L. pneumophila serogroup 1 colonizing plumbing systems was also investigated by DNA restriction endonuclease analysis in three hospitals. We distinguished two subtypes in one hospital; the two other hospitals had homogeneous populations. Restriction endonuclease digest analysis of L. pneumophila serogroup 1 DNA enables subtyping and appears to be a useful method for examining the epidemiology of outbreaks of Legionnaires disease. Images PMID:6092423

  2. Amoebae and Legionella pneumophila in saline environments.

    PubMed

    Gast, Rebecca J; Moran, Dawn M; Dennett, Mark R; Wurtsbaugh, Wayne A; Amaral-Zettler, Linda A

    2011-03-01

    Amoeboid protists that harbor bacterial pathogens are of significant interest as potential reservoirs of disease-causing organisms in the environment, but little is known about them in marine and other saline environments. We enriched amoeba cultures from sediments from four sites in the New England estuarine system of Mt. Hope Bay, Massachusetts and from sediments from six sites in the Great Salt Lake, Utah. Cultures of amoebae were enriched using both minimal- and non-nutrient agar plates, made with fresh water, brackish water or saltwater. Recovered amoeba cultures were assayed for the presence of Legionella species using nested polymerase chain reactions (PCR) and primers specific for the genus. Positive samples were then screened with nested amplification using primers specific for the macrophage infectivity potentiator surface protein (mip) gene from L. pneumophila. Forty-eight percent (185 out of 388) of isolated amoeba cultures were positive for the presence of Legionella species. Legionella pneumophila was detected by PCR in 4% of the amoeba cultures (17 out of 388), and most of these amoebae were growing on marine media. Our results show that amoebae capable of growing in saline environments may harbor not only a diverse collection of Legionella species, but also species potentially pathogenic to humans.

  3. Clinical application of a multiplex real-time PCR assay for simultaneous detection of Legionella species, Legionella pneumophila, and Legionella pneumophila serogroup 1.

    PubMed

    Benitez, Alvaro J; Winchell, Jonas M

    2013-01-01

    We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.

  4. Fatal coinfection with Legionella pneumophila serogroup 8 and Aspergillus fumigatus.

    PubMed

    Guillouzouic, Aurélie; Bemer, Pascale; Gay-Andrieu, Françoise; Bretonnière, Cédric; Lepelletier, Didier; Mahé, Pierre-Joachim; Villers, Daniel; Jarraud, Sophie; Reynaud, Alain; Corvec, Stéphane

    2008-02-01

    Legionella pneumophila is an important cause of community-acquired and nosocomial pneumonia. We report on a patient who simultaneously developed L. pneumophila serogroup 8 pneumonia and Aspergillus fumigatus lung abscesses. Despite appropriate treatments, Aspergillus disease progressed with metastasis. Coinfections caused by L. pneumophila and A. fumigatus remain exceptional. In apparently immunocompetent patients, corticosteroid therapy is a key risk factor for aspergillosis.

  5. Cocultivation of Legionella pneumophila and free-living amoebae.

    PubMed Central

    Tyndall, R L; Domingue, E L

    1982-01-01

    Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth. Images PMID:7149720

  6. Legionella pneumophila in coal miners.

    PubMed Central

    Davies, D H; Hill, E C; Howells, C H; Ribeiro, C D

    1985-01-01

    Legionnaires' disease was diagnosed in three mineworkers at a colliery. Investigation of water samples from various sites at the colliery did not discover a source of the infection. Results of serological surveys undertaken on the workmates of the patients and other miners showed only one additional positive Legionella indirect fluorescent antibody test. There was, therefore, no justification for any alteration in the water supply or the ventilation at the colliery. Images PMID:3890934

  7. Cocultivation of Legionella pneumophila and free-living amoebae

    SciTech Connect

    Tyndall, R.L.; Domingue, E.L.

    1982-10-01

    Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophia as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. roryba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth. 20 references, 1 figure, 4 tables.

  8. Community-Acquired Legionella pneumophila Pneumonia

    PubMed Central

    Viasus, Diego; Di Yacovo, Silvana; Garcia-Vidal, Carolina; Verdaguer, Ricard; Manresa, Frederic; Dorca, Jordi; Gudiol, Francesc; Carratalà, Jordi

    2013-01-01

    Abstract Legionella pneumophila has been increasingly recognized as a cause of community-acquired pneumonia (CAP) and an important public health problem worldwide. We conducted the present study to assess trends in epidemiology, diagnosis, clinical features, treatment, and outcomes of sporadic community-acquired L. pneumophila pneumonia requiring hospitalization at a university hospital over a 15-year period (1995–2010). Among 3934 nonimmunosuppressed hospitalized patients with CAP, 214 (5.4%) had L. pneumophila pneumonia (16 cases were categorized as travel-associated pneumonia, and 21 were part of small clusters). Since the introduction of the urinary antigen test, the diagnosis of L. pneumophila using this method remained stable over the years (p = 0.42); however, diagnosis by means of seroconversion and culture decreased (p < 0.001 and p = 0.001, respectively). The median age of patients with L. pneumophila pneumonia was 58.2 years (SD 13.8), and 76.4% were male. At least 1 comorbid condition was present in 119 (55.6%) patients with L. pneumophila pneumonia, mainly chronic heart disease, diabetes mellitus, and chronic pulmonary disease. The frequency of older patients (aged >65 yr) and comorbidities among patients with L. pneumophila pneumonia increased over the years (p = 0.06 and p = 0.02, respectively). In addition, 100 (46.9%) patients were classified into high-risk classes according to the Pneumonia Severity Index (groups IV–V). Twenty-four (11.2%) patients with L. pneumophila pneumonia received inappropriate empirical antibiotic therapy at hospital admission. Compared with patients who received appropriate empirical antibiotic, patients who received inappropriate therapy more frequently had acute onset of illness (p = 0.004), pleuritic chest pain (p = 0.03), and pleural effusion (p = 0.05). The number of patients who received macrolides decreased over the study period (p < 0.001), whereas the number of patients who received levofloxacin increased (p

  9. Isolation of Legionella pneumophila from hospital water systems in Victoria.

    PubMed

    Swann, R A; Peel, M M; Rouch, G J

    1982-09-04

    A survey of water symptoms in 12 Victorian hospitals was undertaken to establish the prevalence of Legionella pneumophila in buildings not known to be associated with cases of Legionnaires' disease. Samples of hot, cold and shower water were taken, together with water from cooling towers, and isolation was attempted by guinea-pig inoculation. Legionella pneumophila was not isolated from any of the hot, cold or shower samples, but six strains were isolated from five of the cooling towers sampled.

  10. Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species.

    PubMed Central

    Knudson, G B

    1985-01-01

    Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light. Images PMID:4004227

  11. Isolation of Legionella pneumophila from Pluvial Floods by Amoebal Coculture

    PubMed Central

    Docters van Leeuwen, A. E.; Lodder, W. J.; de Man, H.; Euser, S.; den Boer, J. W.; de Roda Husman, A. M.

    2012-01-01

    Viable Legionella pneumophila bacteria were isolated by amoebal coculture from pluvial floods after intense rainfall and from water collected at sewage treatment plants. Several isolated L. pneumophila strains belonged to sequence types that have been previously identified in patients. PMID:22467504

  12. Identification of Legionella pneumophila serogroups and other Legionella species by mip gene sequencing.

    PubMed

    Haroon, Attiya; Koide, Michio; Higa, Futoshi; Tateyama, Masao; Fujita, Jiro

    2012-04-01

    The virulence factor known as the macrophage infectivity potentiator (mip) is responsible for the intracellular survival of Legionella species. In this study, we investigated the potential of the mip gene sequence to differentiate isolates of different species of Legionella and different serogroups of Legionella pneumophila. We used 35 clinical L. pneumophila isolates and one clinical isolate each of Legionella micdadei, Legionella longbeachae, and Legionella dumoffii (collected from hospitals all over Japan between 1980 and 2007). We used 19 environmental Legionella anisa isolates (collected in the Okinawa, Nara, Osaka, and Hyogo prefectures between 1987 and 2007) and two Legionella type strains. We extracted bacterial genomic DNA and amplified out the mip gene by PCR. PCR products were purified by agarose gel electrophoresis and the mip gene was then sequenced. The L. pneumophila isolates could be divided into two groups: one group was very similar to the type strain and was composed of serogroup (SG) 1 isolates only; the second group had more sequence variations and was composed of SG1 isolates as well as SG2, SG3, SG5, and SG10 isolates. Phylogenetic analysis displayed one cluster for L. anisa isolates, while other Legionella species were present at discrete levels. Our findings show that mip gene sequencing is an effective technique for differentiating L. pneumophila strains from other Legionella species.

  13. Ciliate Paramecium is a natural reservoir of Legionella pneumophila.

    PubMed

    Watanabe, Kenta; Nakao, Ryo; Fujishima, Masahiro; Tachibana, Masato; Shimizu, Takashi; Watarai, Masahisa

    2016-04-15

    Legionella pneumophila, the causative agent of Legionnaires' disease, replicates within alveolar macrophages and free-living amoebae. However, the lifestyle of L. pneumophila in the environment remains largely unknown. Here we established a novel natural host model of L. pneumophila endosymbiosis using the ciliate Paramecium caudatum. We also identified Legionella endosymbiosis-modulating factor A (LefA), which contributes to the change in life stage from endosymbiosis to host lysis, enabling escape to the environment. We isolated L. pneumophila strains from the environment, and they exhibited cytotoxicity toward P. caudatum and induced host lysis. Acidification of the Legionella-containing vacuole (LCV) was inhibited, and enlarged LCVs including numerous bacteria were observed in P. caudatum infected with L. pneumophila. An isogenic L. pneumophila lefA mutant exhibited decreased cytotoxicity toward P. caudatum and impaired the modification of LCVs, resulting in the establishment of endosymbiosis between them. Our results suggest that L. pneumophila may have a mechanism to switch their endosymbiosis in protistan hosts in the environment.

  14. Ciliate Paramecium is a natural reservoir of Legionella pneumophila

    NASA Astrophysics Data System (ADS)

    Watanabe, Kenta; Nakao, Ryo; Fujishima, Masahiro; Tachibana, Masato; Shimizu, Takashi; Watarai, Masahisa

    2016-04-01

    Legionella pneumophila, the causative agent of Legionnaires’ disease, replicates within alveolar macrophages and free-living amoebae. However, the lifestyle of L. pneumophila in the environment remains largely unknown. Here we established a novel natural host model of L. pneumophila endosymbiosis using the ciliate Paramecium caudatum. We also identified Legionella endosymbiosis-modulating factor A (LefA), which contributes to the change in life stage from endosymbiosis to host lysis, enabling escape to the environment. We isolated L. pneumophila strains from the environment, and they exhibited cytotoxicity toward P. caudatum and induced host lysis. Acidification of the Legionella-containing vacuole (LCV) was inhibited, and enlarged LCVs including numerous bacteria were observed in P. caudatum infected with L. pneumophila. An isogenic L. pneumophila lefA mutant exhibited decreased cytotoxicity toward P. caudatum and impaired the modification of LCVs, resulting in the establishment of endosymbiosis between them. Our results suggest that L. pneumophila may have a mechanism to switch their endosymbiosis in protistan hosts in the environment.

  15. Ciliate Paramecium is a natural reservoir of Legionella pneumophila

    PubMed Central

    Watanabe, Kenta; Nakao, Ryo; Fujishima, Masahiro; Tachibana, Masato; Shimizu, Takashi; Watarai, Masahisa

    2016-01-01

    Legionella pneumophila, the causative agent of Legionnaires’ disease, replicates within alveolar macrophages and free-living amoebae. However, the lifestyle of L. pneumophila in the environment remains largely unknown. Here we established a novel natural host model of L. pneumophila endosymbiosis using the ciliate Paramecium caudatum. We also identified Legionella endosymbiosis-modulating factor A (LefA), which contributes to the change in life stage from endosymbiosis to host lysis, enabling escape to the environment. We isolated L. pneumophila strains from the environment, and they exhibited cytotoxicity toward P. caudatum and induced host lysis. Acidification of the Legionella-containing vacuole (LCV) was inhibited, and enlarged LCVs including numerous bacteria were observed in P. caudatum infected with L. pneumophila. An isogenic L. pneumophila lefA mutant exhibited decreased cytotoxicity toward P. caudatum and impaired the modification of LCVs, resulting in the establishment of endosymbiosis between them. Our results suggest that L. pneumophila may have a mechanism to switch their endosymbiosis in protistan hosts in the environment. PMID:27079173

  16. Effect of copper and silver ionization on Legionella pneumophila eradication

    SciTech Connect

    Lin, Y.E.; Vidic, R.D.; Stout, J.E.; Yu, V.L.

    1995-11-01

    The presence of Legionella pneumophila in water distribution systems has been epidemiologically linked to hospital-acquired Legionnaires` disease. The objective of this study was to evaluate the efficiency of copper and silver ions for inactivation of Legionella pneumophila. Experimental results showed that L. pneumophila was completely inactivated at copper concentration of 0.1 mg/L within the period of 2.5 hours while 6-log reduction requires a Ct value of 0.8 mg/L*hour. On the other hand, more than 24 hours was required to completely eradicate L. pneumophila at the highest silver ion concentration (0.08 mg/L) tested and only 4-log reduction is observed for Ct value of 0.8 mg/L*hour. The effective synergism of these ions in eradicating L. pneumophila was observed for copper concentrations of 0.05 and silver concentration of 0.04 mg/L. One approach for the control of L. pneumophila in water distribution systems is to initiate copper/silver ion concentrations at 0.4/0.04 mg/L to achieve complete eradication of L. pneumophila already present in the water distribution system (as established in previous studies) followed by a lower residual (0.05/0.04 mg/L) protection against L. pneumophila in the incoming water.

  17. Inactivation of Legionella pneumophila by hypochlorite and an organic chloramine.

    PubMed Central

    Swango, L J; Wilt, G R; Killen, A D; Williams, D E; Worley, S D

    1987-01-01

    The susceptibility of a strain of Legionella pneumophila to disinfection by an organic halamine, free chlorine, and a mixture of the organic halamine and free chlorine was assessed. The organic halamine was found to have superior stability in solution and to exhibit adequate disinfectant potential over a period of 1 month of repeated reinoculations of fresh bacteria. The combined halamine exhibited great potential for use in maintaining closed-cycle cooling water systems free of L. pneumophila. PMID:3435150

  18. Lgt: a family of cytotoxic glucosyltransferases produced by Legionella pneumophila.

    PubMed

    Belyi, Yury; Tabakova, Irina; Stahl, Michael; Aktories, Klaus

    2008-04-01

    Legionella pneumophila is a facultative intracellular pathogen responsible for severe lung disease in humans, known as legionellosis or Legionnaires' disease. Previously, we reported on the approximately 60-kDa glucosyltransferase (Lgt1) from Legionella pneumophila, which modified eukaryotic elongation factor 1A. In the present study, using L. pneumophila Philadelphia-1, Lens, Paris, and Corby genome databases, we identified several genes coding for proteins with considerable sequence homology to Lgt1. These new enzymes form three subfamilies, termed Lgt1 to -3, glucosylate mammalian elongation factor eEF1A at serine-53, inhibit its activity, and subsequently kill target eukaryotic cells. Expression studies on L. pneumophila grown in broth medium or in Acanthamoeba castellanii revealed that production of Lgt1 was maximal at stationary phase of broth culture or during the late phase of Legionella-host cell interaction, respectively. In contrast, synthesis of Lgt3 peaked during the lag phase of liquid culture and at early steps of bacterium-amoeba interaction. Thus, the data indicate that members of the L. pneumophila glucosyltransferase family are differentially regulated, affect protein synthesis of host cells, and represent potential virulence factors of Legionella.

  19. Involvement of minerals in adherence of Legionella pneumophila to surfaces.

    PubMed

    Koubar, Mohamad; Rodier, Marie-Hélène; Frère, Jacques

    2013-05-01

    Legionella pneumophila is the causative agent of 90 % of Legionnaires' disease cases. This bacterium lives naturally in fresh water and can colonize biofilms, which play an important role in the protection of Legionella against environmental stress factors. Relationship between the presence of minerals in water and Legionella adherence to surfaces is not well-known. In this study, we studied influence of minerals on bacterial adherence. For the first time, to our knowledge, this report shows that calcium and magnesium in a less extent, enhances the adherence of Legionella to surfaces compared to the bacteria behavior in distilled water. Treatment with proteinase K of live cells showed that surface proteins do not seem to play a crucial role in bacteria adherence to surfaces. Our results represent a first step in understanding effect of ions on Legionella adherence to surfaces. Such field of research could be helpful to better understand biofilm colonization by this bacterium to improve Legionella risk management in water networks.

  20. Isolation of Plasmids in Legionella pneumophila and Legionella-Like Organisms

    DTIC Science & Technology

    1981-03-01

    isolates examined. Recent reports have established that certain University Hospital , Pittsburgh, Pa.). Pseudom- strains of bacteria, designated Legionella ...McI~ade, J1. E., D. .J. Brenner and F. MW. Bozenman. microorganisms. 19.79. Legionnaires ’ disease bacterium tsolatedl in 1947. Ann. Intern. Med. 90...INtF1(-r1iN ANt11;MM NITY. St.?? NAI 1 1271 2. Voj . NO .j toNO TES (Isolation of Plasmids in Legionella pneumophila and Legionella -Like Organisms

  1. Effect of bacterial interference on biofilm development by Legionella pneumophila.

    PubMed

    Guerrieri, Elisa; Bondi, Moreno; Sabia, Carla; de Niederhäusern, Simona; Borella, Paola; Messi, Patrizia

    2008-12-01

    In the ecology of Legionella pneumophila a crucial role may be played by its relationship with the natural flora; thus we investigated the interactions between Legionella and other aquatic bacteria, particularly within biofilms. Among 80 aquatic bacteria screened for the production of bacteriocin-like substances (BLSs), 66.2% of them were active against L. pneumophila. The possible effect of some of these aquatic bacteria on the development and stability of L. pneumophila biofilms was studied. Pseudomonas fluorescens, the best BLS producer, showed the greatest negative effect on biofilm formation and strongly enhanced the detachment of Legionella. Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Aeromonas hydrophila, and Stenotrophomonas maltophilia, although producing BLSs at different levels, were less active in the biofilm experiments. Acinetobacter lwoffii did not produce any antagonistic compound and was the only one able to strongly enhance L. pneumophila biofilm. Our results highlight that BLS production may contribute to determining the fate of L. pneumophila within ecological niches. The interactions observed in this study are important features of L. pneumophila ecology, which knowledge may lead to more effective measures to control the persistence of the germ in the environment.

  2. Legionella pneumophila serogroup 12 isolated from human and environmental sources.

    PubMed Central

    Thacker, W L; Wilkinson, H W; Benson, R F; Brenner, D J

    1987-01-01

    A Legionella-like organism (strain 570-CO-H [= ATCC 43290]) isolated from the lung tissue of a patient with pneumonia was shown by growth, as well as physiological, serological, and genetic characteristics, to belong to a new Legionella pneumophila serogroup, serogroup 12. Two additional strains were detected with antiserum specific for strain 570-CO-H. These strains were isolated from environmental sources. PMID:3571461

  3. Viable Legionella Pneumophila Not Detectable by Culture on Agar Media

    DTIC Science & Technology

    1987-09-01

    UNIT ELEMENT NO. NO. NO. ACCESSION NO. , N/A 1 1. TITLE (JIncJuue Security Ciaisuicarlon) Viable Legionella Pneumophila not Detectable by Culture on...COSATICODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number) GROUP SUB-GROUP LEGIONELLA FLUORESCENT ANTIBODY...LEGIONNMIRES’-- DISEASE VIABLE NON-CULTURABLE CELLS co mntmue on reverse it necessary and identify by block number) ,-cesio(ri o NTIS CRA&I OTU AP- 0IDTIC

  4. Innate immunity against Legionella pneumophila during pulmonary infections in mice.

    PubMed

    Park, Bonggoo; Park, Gayoung; Kim, Jiyoung; Lim, Seon Ah; Lee, Kyung-Mi

    2017-02-01

    Legionella pneumophila is an etiological agent of the severe pneumonia known as Legionnaires' disease (LD). This gram-negative bacterium is thought to replicate naturally in various freshwater amoebae, but also replicates in human alveolar macrophages. Inside host cells, legionella induce the production of non-endosomal replicative phagosomes by injecting effector proteins into the cytosol. Innate immune responses are first line defenses against legionella during early phases of infection, and distinguish between legionella and host cells using germline-encoded pattern recognition receptors such as Toll-like receptors , NOD-like receptors, and RIG-I-like receptors, which sense pathogen-associated molecular patterns that are absent in host cells. During pulmonary legionella infections, various inflammatory cells such as macrophages, neutrophils, natural killer (NK) cells, large mononuclear cells, B cells, and CD4+ and CD8+ T cells are recruited into infected lungs, and predominantly occupy interstitial areas to control legionella. During pulmonary legionella infections, the interplay between distinct cytokines and chemokines also modulates innate host responses to clear legionella from the lungs. Recognition by NK cell receptors triggers effector functions including secretion of cytokines and chemokines, and leads to lysis of target cells. Crosstalk between NK cells and dendritic cells, monocytes, and macrophages provides a major first-line defense against legionella infection, whereas activation of T and B cells resolves the infection and mounts legionella-specific memory in the host.

  5. Growth temperature reversibly modulates the virulence of Legionella pneumophila.

    PubMed Central

    Mauchline, W S; James, B W; Fitzgeorge, R B; Dennis, P J; Keevil, C W

    1994-01-01

    In chemostat culture, the virulence of two strains of Legionella pneumophila was shown to be significantly (P < 0.05) reduced when the culture temperature was lowered from 37 to 24 degrees C. This modulation was reversed by returning the temperature to 37 degrees C, which resulted in a statistically significant (P < 0.05) increase in virulence. PMID:8005687

  6. Susceptibilities of Algae and Legionella pneumophila to Cooling Tower Biocides

    PubMed Central

    Soracco, Reginald J.; Gill, Helen K.; Fliermans, Carl B.; Pope, Daniel H.

    1983-01-01

    Nine algal strains and nine Legionella pneumophila strains were tested in laboratory culture for their susceptibility to inhibition by a variety of commercially available microbiocides. The responses ranged from ineffective to effective at 1/100 the manufacturers' recommended pulse doses. Tests were also performed to determine whether the action of the microbiocide was bacteriostatic or bacteriocidal. PMID:6859846

  7. Multiplication of Legionella pneumophila in unsterilized tap water.

    PubMed Central

    Yee, R B; Wadowsky, R M

    1982-01-01

    Naturally occurring Legionella pneumophila, an environmental isolate which had not been grown on artificial medium, was tested for the ability to multiply in tap water. A showerhead containing L. pneumophila and non-Legionellaceae bacteria was immersed in nonsterile tap water supplying this fixture. Also L. pneumophila and non-Legionellaceae bacteria were sedimented from tap water from a surgical intensive care unit. This bacterial suspension was inoculated into tap water from our laboratory. The legionellae in both suspensions multiplied in the tap water at 32, 37, and 42 degrees C. The non-Legionellaceae bacteria multiplied at 25, 32, and 37 degrees C. A water sample which was collected from the bottom of a hot water tank was found to contain L. pneumophila and non-Legionellaceae bacteria. These legionellae also multiplied when the water sample was incubated at 37 degrees C. These results indicate that L. pneumophila may multiply in warm water environments such as hot water plumbing fixtures, hot water tanks, and cooling towers. PMID:7103487

  8. Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila.

    PubMed

    Fonseca, Maris V; Swanson, Michele S

    2014-01-01

    The Gram-negative bacterium Legionella pneumophila is ubiquitous in freshwater environments as a free-swimming organism, resident of biofilms, or parasite of protozoa. If the bacterium is aerosolized and inhaled by a susceptible human host, it can infect alveolar macrophages and cause a severe pneumonia known as Legionnaires' disease. A sophisticated cell differentiation program equips L. pneumophila to persist in both extracellular and intracellular niches. During its life cycle, L. pneumophila alternates between at least two distinct forms: a transmissive form equipped to infect host cells and evade lysosomal degradation, and a replicative form that multiplies within a phagosomal compartment that it has retooled to its advantage. The efficient changeover between transmissive and replicative states is fundamental to L. pneumophila's fitness as an intracellular pathogen. The transmission and replication programs of L. pneumophila are governed by a number of metabolic cues that signal whether conditions are favorable for replication or instead trigger escape from a spent host. Several lines of experimental evidence gathered over the past decade establish strong links between metabolism, cellular differentiation, and virulence of L. pneumophila. Herein, we focus on current knowledge of the metabolic components employed by intracellular L. pneumophila for cell differentiation, nutrient salvaging and utilization of host factors. Specifically, we highlight the metabolic cues that are coupled to bacterial differentiation, nutrient acquisition systems, and the strategies utilized by L. pneumophila to exploit host metabolites for intracellular replication.

  9. Comparison of liquid growth media for Legionella pneumophila.

    PubMed Central

    Saito, A; Rolfe, R D; Edelstein, P H; Finegold, S M

    1981-01-01

    Ten liquid media were compared under standard conditions for their ability to support the growth of Legionella pneumophila. Modified gonococcal-ferric cysteine broth (without sodium chloride) supplemented with 1% yeast extract yielded the best overall growth of the one strain of L. pneumophila examined. Growth rates were independent of pH changes which occurred during incubation. The growth rates of 10 different strains of L.pneumophila were compared in this medium. There appeared to be little difference in the growth rates of strains passaged frequently or infrequently, or between environmental and clinical isolates. Moderate aeration resulted in a faster growth rate and in approximately a 1 log10 higher final cell concentration as compared to a static broth culture. These experiments demonstrate that there are moderate to marked differences among the various media described in the literature and that no liquid medium yet developed supports rapid growth of L. pneumophila incubated without shaking. PMID:7037831

  10. Presence and Persistence of Viable, Clinically Relevant Legionella pneumophila Bacteria in Garden Soil in the Netherlands

    PubMed Central

    van Heijnsbergen, E.; van Deursen, A.; Bouwknegt, M.; Bruin, J. P.; Schalk, J. A. C.

    2016-01-01

    ABSTRACT Garden soils were investigated as reservoirs and potential sources of pathogenic Legionella bacteria. Legionella bacteria were detected in 22 of 177 garden soil samples (12%) by amoebal coculture. Of these 22 Legionella-positive soil samples, seven contained Legionella pneumophila. Several other species were found, including the pathogenic Legionella longbeachae (4 gardens) and Legionella sainthelensi (9 gardens). The L. pneumophila isolates comprised 15 different sequence types (STs), and eight of these STs were previously isolated from patients according to the European Working Group for Legionella Infections (EWGLI) database. Six gardens that were found to be positive for L. pneumophila were resampled after several months, and in three gardens, L. pneumophila was again isolated. One of these gardens was resampled four times throughout the year and was found to be positive for L. pneumophila on all occasions. IMPORTANCE Tracking the source of infection for sporadic cases of Legionnaires' disease (LD) has proven to be hard. L. pneumophila ST47, the sequence type that is most frequently isolated from LD patients in the Netherlands, is rarely found in potential environmental sources. As L. pneumophila ST47 was previously isolated from a garden soil sample during an outbreak investigation, garden soils were investigated as reservoirs and potential sources of pathogenic Legionella bacteria. The detection of viable, clinically relevant Legionella strains indicates that garden soil is a potential source of Legionella bacteria, and future research should assess the public health implication of the presence of L. pneumophila in garden soil. PMID:27316958

  11. Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion.

    PubMed Central

    Cirillo, J D; Falkow, S; Tompkins, L S

    1994-01-01

    Legionella pneumophila is considered to be a facultative intracellular parasite. Therefore, the ability of these bacteria to enter, i.e., invade, eukaryotic cells is expected to be a key pathogenic determinant. We compared the invasive ability of bacteria grown under standard laboratory conditions with that of bacteria grown in Acanthamoeba castellanii, one of the protozoan species that serves as a natural host for L. pneumophila in the environment. Amoeba-grown L. pneumophila cells were found to be at least 100-fold more invasive for epithelial cells and 10-fold more invasive for macrophages and A. castellanii than were L. pneumophila cells grown on agar. Comparison of agar- and amoeba-grown L. pneumophila cells by light and electron microscopy demonstrated dramatic differences in the morphology and structure of the bacteria. Analyses of protein expression in the two strains of bacteria suggest that these phenotypic differences may be due to the expression of new proteins in amoeba-grown L. pneumophila cells. In addition, the amoeba-grown bacteria were found to enter macrophages via coiling phagocytosis at a higher frequency than agar-grown bacteria did. Replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease. Images PMID:8039895

  12. Population structure and minimum core genome typing of Legionella pneumophila

    PubMed Central

    Qin, Tian; Zhang, Wen; Liu, Wenbin; Zhou, Haijian; Ren, Hongyu; Shao, Zhujun; Lan, Ruiting; Xu, Jianguo

    2016-01-01

    Legionella pneumophila is an important human pathogen causing Legionnaires’ disease. In this study, whole genome sequencing (WGS) was used to study the characteristics and population structure of L. pneumophila strains. We sequenced and compared 53 isolates of L. pneumophila covering different serogroups and sequence-based typing (SBT) types (STs). We found that 1,896 single-copy orthologous genes were shared by all isolates and were defined as the minimum core genome (MCG) of L. pneumophila. A total of 323,224 single-nucleotide polymorphisms (SNPs) were identified among the 53 strains. After excluding 314,059 SNPs which were likely to be results of recombination, the remaining 9,165 SNPs were referred to as MCG SNPs. Population Structure analysis based on MCG divided the 53 L. pneumophila into nine MCG groups. The within-group distances were much smaller than the between-group distances, indicating considerable divergence between MCG groups. MCG groups were also supplied by phylogenetic analysis and may be considered as robust taxonomic units within L. pneumophila. Among the nine MCG groups, eight showed high intracellular growth ability while one showed low intracellular growth ability. Furthermore, MCG typing also showed high resolution in subtyping ST1 strains. The results obtained in this study provided significant insights into the evolution, population structure and pathogenicity of L. pneumophila. PMID:26888563

  13. Legionella pneumophila Seropositivity-Associated Factors in Latvian Blood Donors

    PubMed Central

    Valciņa, Olga; Pūle, Daina; Lucenko, Irina; Krastiņa, Dita; Šteingolde, Žanete; Krūmiņa, Angelika; Bērziņš, Aivars

    2015-01-01

    Continuous environmental exposure of humans to Legionella may induce immune responses and generation of antibodies. The aim of this study was to investigate the seroprevalence of Legionella pneumophila serogroups (SG) 1–6 in the general healthy population and identify the associated host-related and environmental risk factors. L. pneumophila SG 1–6 seroprevalence among a total of 2007 blood samples collected from healthy donors was 4.8%. Seroprevalence was higher in women (5.9%) than men (3.3%) and in areas with a larger number of inhabitants, ranging from 3.5% in rural regions to 6.8% in the capital, Riga. Blood samples from inhabitants of apartment buildings tested positive for L. pneumophila in more cases (5.8%) compared to those from inhabitants of single-family homes (2.7%). Residents of buildings with a municipal hot water supply system were more likely to be seropositive for L. pneumophila (OR = 3.16, 95% CI 1.26–7.91). Previous episodes of fever were additionally identified as a risk factor (OR = 2.42, 95% CI 1.43–4.1). In conclusion, centralized hot water supply, female gender and previous episodes of fever were determined as the main factors associated with L. pneumophila seropositivity in our study population. PMID:26703696

  14. Caenorhabditis elegans as an alternative model host for legionella pneumophila, and protective effects of Bifidobacterium infantis.

    PubMed

    Komura, Tomomi; Yasui, Chikako; Miyamoto, Hiroshi; Nishikawa, Yoshikazu

    2010-06-01

    The survival times of Caenorhabditis elegans worms infected with Legionella pneumophila from day 7.5 or later after hatching were shorter than those of uninfected worms. However, nematodes fed bifidobacteria prior to Legionella infection were resistant to Legionella. These nematodes may act as a unique alternative host for Legionella research.

  15. Incidence of Legionella pneumophila infections among Oklahoma pulmonary disease patients.

    PubMed Central

    Flournoy, D. J.; Guthrie, P. J.; Lawrence, C. H.; Silberg, S. L.; Beaver, S.

    1990-01-01

    Prior studies by the authors suggested high levels of Legionella pneumophila in the recreational and water supply reservoirs in central Oklahoma. This high exposure potential was supported by a relatively high prevalence of seropositive, asymptomatic infections among healthy blood donors in the area. In contrast, the present 9-month laboratory-based study confirmed only one clinical Legionella infection among 117 unidentified pulmonary disease patients admitted to the Oklahoma City Veterans Administration Medical Center. Comparison with the reports of others and with reported legionellosis in Oklahoma indicates that differences in cohort definition and variations in utilization and interpretation of clinical analyses leads to wide variations in the reported incidence of legionellosis. PMID:2304095

  16. Soil as a source of Legionella pneumophila sequence type 47.

    PubMed

    Schalk, Johanna A C; Euser, Sjoerd M; van Heijnsbergen, Eri; Bruin, Jacob P; den Boer, Jeroen W; de Roda Husman, Ana M

    2014-10-01

    Legionella pneumophila sequence type (ST) 47 was isolated from soil in a garden. We speculate that this strain was transmitted from soil to the whirlpool in the garden where it caused an outbreak of Legionnaires' disease and Pontiac fever. In the Netherlands, ST47 is frequently isolated from patients, but hardly ever from environmental sources. It is possible that human pathogenic Legionella strains, with ST47 as one of the predominant strains, are transmitted to humans from sources such as natural soil that are currently not targeted in outbreak investigations.

  17. Targeting single-nucleotide polymorphisms in the 16S rRNA gene to detect and differentiate Legionella pneumophila and non-Legionella pneumophila species.

    PubMed

    Zhan, Xiao-Yong; Hu, Chao-Hui; Zhu, Qing-Yi

    2016-08-01

    A PCR-based method targeting single-nucleotide polymorphisms (SNPs) in the 16S rRNA gene was developed for differential identification of Legionella pneumophila and non-Legionella pneumophila. Based on the bioinformatics analysis for 176 Legionella 16S rRNA gene fragments of 56 different Legionella species, a set of SNPs, A(628)C(629) was found to be highly specific to L. pneumophila strains. A multiplex assay was designed that was able to distinguish sites with limited sequence heterogeneity between L. pneumophila and non-L. pneumophila in the targeted 16S rRNA gene. The assay amplified a 261-bp amplicon for Legionella spp. and a set of 203- and 97-bp amplicons only specific to L. pneumophila species. Among 49 ATCC strains and 284 Legionella isolates from environmental water and clinical samples, 100 % of L. pneumophila and non-L. pneumophila strains were correctly identified and differentiated by this assay. The assay presents a more rapid, sensitive and alternative method to the currently available PCR-sequencing detection and differentiation method.

  18. Virulence Properties of the Legionella Pneumophila Cell Envelope

    PubMed Central

    Shevchuk, Olga; Jäger, Jens; Steinert, Michael

    2011-01-01

    The bacterial envelope plays a crucial role in the pathogenesis of infectious diseases. In this review, we summarize the current knowledge of the structure and molecular composition of the Legionella pneumophila cell envelope. We describe lipopolysaccharides biosynthesis and the biological activities of membrane and periplasmic proteins and discuss their decisive functions during the pathogen–host interaction. In addition to adherence, invasion, and intracellular survival of L. pneumophila, special emphasis is laid on iron acquisition, detoxification, key elicitors of the immune response and the diverse functions of outer membrane vesicles. The critical analysis of the literature reveals that the dynamics and phenotypic plasticity of the Legionella cell surface during the different metabolic stages require more attention in the future. PMID:21747794

  19. Legionella pneumophila decreases velocity of Acanthamoeba castellanii.

    PubMed

    Mengue, Luce; Richard, Freddie-Jeanne; Caubet, Yves; Rolland, Steven; Héchard, Yann; Samba-Louaka, Ascel

    2017-08-02

    Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus, A. castellanii is described as a Trojan horse able to harbor pathogenic bacteria. L. pneumophila is one of the amoeba-resisting bacteria able to avoid host degradation by phagocytosis and to multiply inside the amoeba. When infecting its host, L. pneumophila injects hundreds of effectors via a type IV secretion system that change physiology of the amoeba to its profit. In this study, we assess mobility of A. castellanii upon infection with L. pneumophila. Electron-microscopy analysis of amoebae revealed a reduction of acanthopodia on cells infected with L. pneumophila. Analysis of velocity showed that migration of A. castellanii infected with L. pneumophila was significantly impaired compare to uninfected cells. Taken together, infection with L. pneumophila could prevent formation of cytoplasmic extensions such as acanthopodia with consequences on the shape, adherence and mobility of A. castellanii. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Innate Immunity to Intracellular Pathogens: Lessons Learned from Legionella pneumophila.

    PubMed

    Shin, Sunny

    2012-01-01

    Intracellular bacterial pathogens have the remarkable ability to manipulate host cell processes in order to establish a replicative niche within the host cell. In response, the host can initiate immune defenses that lead to the eventual restriction and clearance of intracellular infection. The bacterial pathogen Legionella pneumophila has evolved elaborate virulence mechanisms that allow for its survival inside protozoa within a specialized membrane-bound organelle. These strategies also enable L. pneumophila to survive and replicate within alveolar macrophages, and can result in the severe pneumonia Legionnaires' disease. Essential to L. pneumophila's intracellular lifestyle is a specialized type IV secretion system, termed Dot/Icm, that translocates bacterial effector proteins into host cells. The ease with which L. pneumophila can be genetically manipulated has facilitated the comparison of host responses to virulent and isogenic avirulent mutants lacking a functional Dot/Icm system. This has made L. pneumophila an excellent model for understanding how the host discriminates between pathogenic and nonpathogenic bacteria and for systematically dissecting host defense mechanisms against intracellular pathogens. In this chapter, I discuss a few examples demonstrating how the study of immune responses triggered specifically by the L. pneumophila type IV secretion system has provided unique insight into our understanding of host immunity against intracellular bacterial pathogens.

  1. [Infection with Legionella pneumophila among workers of Polish sea drilling platforms].

    PubMed

    Lapiński, T W; Kruminis-Lozowski, J

    1997-01-01

    The epidemiology, clinic, diagnostic dates and treatment of Legionella pneumophila (L. pneumophila) were discussion. The our study were removal in 246 workers seas drill platforms. The antibody opposed of L. pneumophila were mean in 54 with 246 workers. Investigation were execution into conjunction of specifically job and life workers and frequently appearance of bronchitis. The antibody L. pneumophila were detection in 25% persons. These dates may suggest possibilities of L. pneumophila infections among workers of these professions.

  2. Isolation of Legionella pneumophila serogroup 14 from a human source.

    PubMed Central

    Pastoris, M. C.; Berchicci, C.; Pallonari, G.

    1992-01-01

    A strain of Legionella pneumophila serogroup 14 was isolated during a retrospective study, after death from the sputum of a patient who had had acute leukaemia and pneumonia. This is the third strain of that serogroup to be isolated from a human source. This event emphasises the importance of performing culture as well as serological tests, so as to detect cases of legionellosis caused by strains which rarely cause fatal clinical illness. PMID:1517467

  3. Absence of siderophore-like activity in Legionella pneumophila supernatants.

    PubMed Central

    Liles, M R; Cianciotto, N P

    1996-01-01

    Conflicting reports have been given as to the existence of a Legionella pneumophila siderophore. Hence, we rigorously examined the reported siderophore-like activity using the chrome azurol S indicator. Although chrome azurol S reactivity was detected in supernatants, control experiments indicate that it was due to cysteine in the media. When bacteria were cultured in media without cysteine, no siderophores were detected. PMID:8613408

  4. Bafilomycin A1 and intracellular multiplication of Legionella pneumophila.

    PubMed Central

    Cattani, L; Goldoni, P; Pastoris, M C; Sinibaldi, L; Orsi, N

    1997-01-01

    Multiplication of Legionella pneumophila in HeLa cells was found to be inhibited by noncytotoxic concentrations of bafilomycin A1, with blockage of bacterial growth at a concentration 15.6 nM. The inhibiting action was evident only when the antibiotic was present during the initial phase of intracellular multiplication, i.e., during the formation of the phagosome, whereas the addition of the drug did not affect microorganisms already actively multiplying within the phagosome. PMID:8980784

  5. Susceptibility of Legionella pneumophila to three cooling tower microbicides.

    PubMed

    Grace, R D; Dewar, N E; Barnes, W G; Hodges, G R

    1981-01-01

    Investigation of epidemic outbreaks of Legionnaires disease by Center for Disease Control personnel has resulted in the isolation of Legionella pneumophila from water in the air-conditioning cooling towers or evaporative condensers at the site of the outbreak. It is suspected that improperly maintained open, recirculating water systems may play a role in the growth and dissemination of this pathogen. The objective of this study was to determine the antimicrobial activity of three chemically different, commercially available, cooling tower microbicides against L. pneumophila. Using two in vitro test systems, a combination of N-alkyl dimethyl benzyl ammonium chloride and bis (tri-n-butyltin) oxide was found to kill L. pneumophila at a concentration 25 times less than the minimum recommended use concentration, whereas N-alkyl 1,3-propanediamine and methylene bis (thiocyanate) were active at concentrations equal to or greater than the concentrations recommended for use by the manufacturer.

  6. Susceptibility of Legionella pneumophila to three cooling tower microbicides.

    PubMed Central

    Grace, R D; Dewar, N E; Barnes, W G; Hodges, G R

    1981-01-01

    Investigation of epidemic outbreaks of Legionnaires disease by Center for Disease Control personnel has resulted in the isolation of Legionella pneumophila from water in the air-conditioning cooling towers or evaporative condensers at the site of the outbreak. It is suspected that improperly maintained open, recirculating water systems may play a role in the growth and dissemination of this pathogen. The objective of this study was to determine the antimicrobial activity of three chemically different, commercially available, cooling tower microbicides against L. pneumophila. Using two in vitro test systems, a combination of N-alkyl dimethyl benzyl ammonium chloride and bis (tri-n-butyltin) oxide was found to kill L. pneumophila at a concentration 25 times less than the minimum recommended use concentration, whereas N-alkyl 1,3-propanediamine and methylene bis (thiocyanate) were active at concentrations equal to or greater than the concentrations recommended for use by the manufacturer. PMID:7224623

  7. Susceptibility of Legionella pneumophila to three cooling tower microbicides

    SciTech Connect

    Grace, R.D.; Dewar, N.E.; Barnes, W.G.; Hodges, G.R.

    1981-01-01

    Investigation of epidemic outbreaks of Legionnaires disease by Center for Disease Control personnel has resulted in the isolation of Legionella pneumophila from water in the air-conditioning cooling towers or evaporative condensers at the site of the outbreak. It is suspected that improperly maintained open, recirculating water systems may play a role in the growth and dissemination of this pathogen. The objective of this study was to determine the antimicrobial activity of three chemically different, commercially available, cooling tower microbicides against L. pneumophila. Using two in vitro test systems, a combination of N-alkyl dimethyl benzyl ammonium chloride and bis (tri-n-butyltin) oxide was found to kill L. pneumophila at a concentration 25 times less than the minimum recommended use concentration, whereas N-alkyl 1,3-propanediamine and methylene bis(thiocyanate) were active at concentrations equal to or greater than the concentrations recommended for use by the manufacturer.

  8. Effects of three oxidizing biocides on Legionella pneumophila serogroup 1

    SciTech Connect

    Domingue, E.L.; Tyndall, R.L.; Mayberry, W.R.; Pancorbo, O.C.

    1988-03-01

    A study was conducted to determine the bactericidal effects of ozone and hydrogen peroxide relative to that of free chlorine on Legionella pneumophila serogroup 1. In laboratory batch-type experiments, organisms seeded at various densities were exposed to different concentrations of these biocides in demand-free buffers. Bactericidal effects were measured by determining the ability of L. pneumophila to grow on buffered charcoal-yeast extract agar supplemented with ..cap alpha..-ketoglutarate. Ozone was the most potent of the three biocide, with a greater than 99% kill of L. pneumophila occurring during a 5-min exposure to 0.10 to 0.30 ..mu..g of O/sub 3/ per ml. The bactericidal action of O/sub 3/ was not markedly affected by changes in pH or temperature. Concentrations of 0.30 and 0.40 /sup +/g of free chlorine per ml killed 99% of the L. pneumophila after 30- and 5-min exposures, respectively. A 30-min exposure to 1000 ..mu..g of H/sub 2/O/sub 2/ per ml was required to effect a 99% reduction of the viable L. pneumophila population. However, no viable L. pneumophila could be detected after a 24-h exposure to 100 or 300 ..mu..g of H/sub 2/O/sub 2/ per ml. Attempts were made to correlate the biocidal effects of O/sub 3/ and H/sub 2/O/sub 2/ with the oxidation of L. pneumophila fatty acids. These tests indicated that certain biocidal concentrations of O/sub 3/ and H/sub 2/O/sub 2/ resulted in a loss or severe reduction of L. pneumophila unsaturated fatty acids.

  9. Environmental surveillance of Legionella pneumophila in two Italian hospitals.

    PubMed

    Tesauro, Marina; Bianchi, Annalisa; Consonni, Michela; Pregliasco, Fabrizio; Galli, Maria Gabriella

    2010-01-01

    The aim of this study was to identify the most effective disinfection protocol to reduce the presence of Legionella pneumophila in the water system of two Italian hospitals. From 2004 to 2009, 271 samplings of hot water were carried out in 11 hospital units to detect the presence of L. pneumophila. Additionally, water samples collected from one boiler outlet and the hot water recirculation were tested. From 2004 to 2009, L. pneumophila was present in 37% of the samples. Of these, 68.3% and 18.8% were positive for serogroups 2-14 and 1, respectively. Furthermore, 12.9% of the samples were positive for both serogroups. Finally, a maximal count of 10(4) CFU/L was measured in the most distal sites. To reduce L. pneumophila colonization, a two-year long hyperchlorination (2004-2006) was carried out. Moreover, from June 2005 until now, continuous maintenance of boilers and tanks, substitution of the shower heads and increase of the boiler outlet temperature to 60 °C were performed. All these treatments led to a marked reduction of L. pneumophila colonization in the short but not in the medium-long term. Only the use of chlorine dioxide led, after four years, to a reduction of the loads of L. pneumophila to values below 100 CFU/L. However, in the distal sites a persistent degree of colonization (maximum value 700 CFU/L, average 600 CFU/L) was observed probably due to the presence of L. pneumophila in the stagnant water in dead legs. In conclusion, data show that long-term chlorination of hot water sources together with carefully aimed maintenance of water pipes can lead to an effective reduction of L. pneumophila concentration in hospital water systems.

  10. Plasmid Detection in Legionella pneumophila and Legionella-like Organisms.

    DTIC Science & Technology

    1980-04-30

    AD-ASM 700 ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FR--ETC F/6 6/5 PLASNIC DETECTION IN LEGIONELLA PNEUNOPHILA ND LESIONELLA-LIKE--ETe(U...in view of the narrow spectrum of antibiotics effective in the treatment of legionellosis and Legionella -like diseases , the acquisition of drug...pneumonia. Ann. Intern. Med. 91:831-834. 15. McDade, J. E., D. J. Brenner, and F. M. Bozeman. 1979. Legionnaires ’ disease bacterium isolated in 1947

  11. Legionella pneumophila lipopolysaccharide activates the classical complement pathway.

    PubMed Central

    Mintz, C S; Schultz, D R; Arnold, P I; Johnson, W

    1992-01-01

    Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes. PMID:1612744

  12. Association of Legionella pneumophila with the macrophage endoplasmic reticulum.

    PubMed Central

    Swanson, M S; Isberg, R R

    1995-01-01

    Legionella pneumophila replicates within a membrane-bounded compartment that is studded with ribosomes. In this study we investigated whether these ribosomes originate from the cytoplasmic pool or are associated with host endoplasmic reticulum (ER). Immunofluorescence and electron microscopic localization studies of ER proteins in macrophages infected with L. pneumophila indicated that the bacteria reside in a compartment surrounded by ER. An L. pneumophila mutant that grows slowly in macrophages was slow to associate with host ER, providing genetic evidence in support of the hypothesis that this specialized vacuole is required for intracellular bacterial growth. Ultrastructural studies, in which the ER luminal protein BiP was labeled by immunoperoxidase cytochemistry, revealed that L. pneumophila replication vacuoles resemble nascent autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagosomes. Furthermore, short-term amino acid starvation of macrophages, which stimulated host autophagy, increased association of the bacteria with the ER and enhanced bacterial growth. These results are compatible with the hypothesis that L. pneumophila exploits the autophagy machinery of macrophages to establish an intracellular niche favorable for replication. PMID:7642298

  13. Legionella pneumophila prevents proliferation of its natural host Acanthamoeba castellanii

    PubMed Central

    Mengue, Luce; Régnacq, Matthieu; Aucher, Willy; Portier, Emilie; Héchard, Yann; Samba-Louaka, Ascel

    2016-01-01

    Legionella pneumophila is a ubiquitous, pathogenic, Gram-negative bacterium responsible for legionellosis. Like many other amoeba-resistant microorganisms, L. pneumophila resists host clearance and multiplies inside the cell. Through its Dot/Icm type IV secretion system, the bacterium injects more than three hundred effectors that modulate host cell physiology in order to promote its own intracellular replication. Here we report that L. pneumophila prevents proliferation of its natural host Acanthamoeba castellanii. Infected amoebae could not undergo DNA replication and no cell division was observed. The Dot/Icm secretion system was necessary for L. pneumophila to prevent the eukaryotic proliferation. The absence of proliferation was associated with altered amoebal morphology and with a decrease of mRNA transcript levels of CDC2b, a putative regulator of the A. castellanii cell cycle. Complementation of CDC28-deficient Saccharomyces cerevisiae by the CDC2b cDNA was sufficient to restore proliferation of CDC28-deficient S. cerevisiae and suggests for the first time that CDC2b from A. castellanii could be functional and a bona fide cyclin-dependent kinase. Hence, our results reveal that L. pneumophila impairs proliferation of A. castellanii and this effect could involve the cell cycle protein CDC2b. PMID:27805070

  14. Severe Pneumonia Caused by Legionella pneumophila: Differential Diagnosis and Therapeutic Considerations.

    PubMed

    Chahin, Abdullah; Opal, Steven M

    2017-03-01

    Severe legionella pneumonia poses a diagnostic challenge and requires early intervention. Legionnaire's disease can have several presenting signs, symptoms, and laboratory abnormalities that suggest that Legionella pneumophila is the pathogen, but none of these are sufficient to distinguish L pneumophila pneumonia from other respiratory pathogens. L pneumophila is primarily an intracellular pathogen and needs treatment with antibiotics that efficiently enter the intracellular space. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Isolation of Legionella pneumophila from hospital cooling towers in Johor, Malaysia.

    PubMed

    Abdul Samad, B H; Suhaili, M R; Baba, N; Rajasekaran, G

    2004-08-01

    Water-based cooling towers and their water supply at two hospitals in Johor were surveyed for the presence Legionella pneumophila. L. pneumophila were grown from 19 (76%) out of 25 collected water samples. One hospital cooling tower was contaminated with L. pneumophila serogroup 1.

  16. A note on the survival of Legionella pneumophila in stagnant tap water.

    PubMed

    Schofield, G M

    1985-10-01

    Tap water, from an experimental hot water system, containing a known virulent strain of Legionella pneumophila was stored in screw-capped bottles for 14 months. Viable counts showed survival of L. pneumophila and at least three other bacterial species. This reinforces the view that L. pneumophila can survive in stagnant water for relatively long periods of time.

  17. Confirmation of Legionella pneumophila cultures with a fluorescein-labeled monoclonal antibody.

    PubMed Central

    Tenover, F C; Carlson, L; Goldstein, L; Sturge, J; Plorde, J J

    1985-01-01

    We compared a fluorescein-labeled monoclonal antibody directed against an outer membrane protein of Legionella pneumophila (Genetic Systems Corp. [GSC], Seattle, Wash.) with a similarly labeled polyclonal reagent (L. pneumophila serogroups 1 to 6, poly; BioDx, Inc., Denville, N.J.) for the confirmation of L. pneumophila isolates grown in culture. Duplicate suspensions of 52 organisms, including 21 L. pneumophila and 8 non-L. pneumophila species of legionella, were placed on individual glass slides, fixed, and stained with both reagents, and the results were compared. Both antisera correctly identified all L. pneumophila serogroups 1 to 6, but only the GSC reagent produced definitive staining of the L. pneumophila isolates of serogroups 7, 8, and 9. Additionally, the GSC reagent produced more uniform staining patterns around the legionella bacilli and displayed little background fluorescence when compared with the BioDx reagent. PMID:3891777

  18. Rapid quantification method for Legionella pneumophila in surface water.

    PubMed

    Wunderlich, Anika; Torggler, Carmen; Elsässer, Dennis; Lück, Christian; Niessner, Reinhard; Seidel, Michael

    2016-03-01

    World-wide legionellosis outbreaks caused by evaporative cooling systems have shown that there is a need for rapid screening methods for Legionella pneumophila in water. Antibody-based methods for the quantification of L. pneumophila are rapid, non-laborious, and relatively cheap but not sensitive enough for establishment as a screening method for surface and drinking water. Therefore, preconcentration methods have to be applied in advance to reach the needed sensitivity. In a basic test, monolithic adsorption filtration (MAF) was used as primary preconcentration method that adsorbs L. pneumophila with high efficiency. Ten-liter water samples were concentrated in 10 min and further reduced to 1 mL by centrifugal ultrafiltration (CeUF). The quantification of L. pneumophila strains belonging to the monoclonal subtype Bellingham was performed via flow-based chemiluminescence sandwich microarray immunoassays (CL-SMIA) in 36 min. The whole analysis process takes 90 min. A polyclonal antibody (pAb) against L. pneumophila serogroup 1-12 and a monoclonal antibody (mAb) against L. pneumophila SG 1 strain Bellingham were immobilized on a microarray chip. Without preconcentration, the detection limit was 4.0 × 10(3) and 2.8 × 10(3) CFU/mL determined by pAb and mAb 10/6, respectively. For samples processed by MAF-CeUF prior to SMIA detection, the limit of detection (LOD) could be decreased to 8.7 CFU/mL and 0.39 CFU/mL, respectively. A recovery of 99.8 ± 15.9% was achieved for concentrations between 1-1000 CFU/mL. The established combined analytical method is sensitive for rapid screening of surface and drinking water to allow fast hygiene control of L. pneumophila.

  19. Legionella pneumophila Arthritis: use of medium specific for Mycobacteria for isolation of L. pneumophila in culture of articular fluid specimens.

    PubMed

    Bemer, Pascale; Leautez, Sophie; Ninin, Emmanuelle; Jarraud, Sophie; Raffi, François; Drugeon, Henri

    2002-07-01

    We report the first case, to our knowledge, of acute purulent arthritis due to Legionella pneumophila in an immunosuppressed patient. L. pneumophila was isolated from samples of blood and articular fluid cultured with use of medium specific for mycobacteria (Bactec 13A medium).

  20. Virulence conversion of Legionella pneumophila: a one-way phenomenon.

    PubMed Central

    Catrenich, C E; Johnson, W

    1988-01-01

    Previous investigations have shown that Legionella pneumophila converts from virulence to avirulence after passage on supplemented Mueller-Hinton (SMH) agar and may convert back to virulence after passage in guinea pigs. However, there is no additional information concerning the apparent interconversion of virulent and avirulent derivatives of L. pneumophila cultures. We investigated the stability of a parental virulent culture and its avirulent derivatives and the growth and viability of these cultures on charcoal-yeast extract (CYE) and SMH agars. Avirulent derivatives of a highly virulent L. pneumophila culture were obtained by passage of the virulent parent culture on SMH agar. The only time a virulent L. pneumophila culture was recoverable from an avirulent culture was when the avirulent culture was derived from a saline suspension of a virulent culture which had been passaged only five times on SMH agar. When an avirulent culture was derived from a virulent culture passaged 25 times on SMH agar or from an isolated colony which grew on a SMH agar plate, we were unable to recover a virulent culture after successive passage through guinea pigs. These results suggest that the conversion process which occurs between virulent and avirulent forms of L. pneumophila is a one-way phenomenon from virulence to avirulence and that stable avirulent derivatives can be isolated. Furthermore, our findings suggest that SMH agar acts as a selective medium for the growth of avirulent L. pneumophila, and growth on SMH agar may be a phenotypic marker for avirulence. Virulent cells, although unable to grow on SMH agar, may remain viable for several passages on SMH agar and propagate when inoculated into guinea pigs. Images PMID:3182073

  1. A case of pneumonia caused by Legionella pneumophila serogroup 12 and treated successfully with imipenem.

    PubMed

    Nishizuka, Midori; Suzuki, Hiroki; Ara, Tomoka; Watanabe, Mari; Morita, Mami; Sato, Chisa; Tsuchida, Fumihiro; Seto, Junji; Amemura-Maekawa, Junko; Kura, Fumiaki; Takeda, Hiroaki

    2014-06-01

    The patient was an 83-year-old man hospitalized for Haemophilus influenzae pneumonia, who developed recurrent pneumonia after improvement of the initial episode. Legionella pneumophila serogroup 12 was isolated from the sputum, accompanied by increased serum antibody titers to L. pneumophila serogroup 12. Therefore, the patient was diagnosed as having Legionella pneumonia caused by L. pneumophila serogroup 12. Case reports of pneumonia caused by L. pneumophila serogroup 12 are rare, and the case described herein is the first report of clinical isolation of this organism in Japan. When the genotype was determined by the protocol of The European Working Group for Legionella Infections (Sequence-Based Typing [SBT] for epidemiological typing of L. pneumophila, Version 3.1), the sequence type was ST68. Imipenem/cilastatin therapy was found to be effective for the treatment of Legionella pneumonia in this patient.

  2. Characteristics of anti-Legionella antibodies in patients infected with Legionella pneumophila serogroups 1, 6, and 10.

    PubMed Central

    van Zwet, T L; Meenhorst, P L; Leijh, P C; Daha, M R; van Furth, R

    1988-01-01

    Nosocomial infections with Legionella pneumophila serogroups 1 and 10 in the Leiden University Hospital and infections with L. pneumophila serogroup 6 in neighboring hospitals gave us an opportunity to study the development of opsonizing antibodies against L. pneumophila serogroups 1, 6, and 10 in the serum of 13 patients. Seven of these patients were infected with L. pneumophila serogroup 1, two were infected with serogroup 6, and four were infected with serogroup 10. The opsonic cross-reactivity of antibodies against these serogroups of L. pneumophila and complement involvement in opsonization were also investigated. Convalescent-phase sera from patients infected with L. pneumophila serogroup 1 or 6 were able to promote ingestion of these serogroups by polymorphonuclear leukocytes, whereas ingestion of L. pneumophila serogroup 10 was enhanced only in the presence of convalescent-phase sera from patients infected with this serogroup. Opsonization of L. pneumophila serogroups 1, 6, and 10 was complement dependent. PMID:3235666

  3. Functional type 1 secretion system involved in Legionella pneumophila virulence.

    PubMed

    Fuche, Fabien; Vianney, Anne; Andrea, Claire; Doublet, Patricia; Gilbert, Christophe

    2015-02-01

    Legionella pneumophila is a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence of L. pneumophila is now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation of L. pneumophila, via its T1SS, in its internalization into host cells.

  4. Cell biology and immunology lessons taught by Legionella pneumophila.

    PubMed

    Zhu, Wenhan; Luo, Zhao-Qing

    2016-01-01

    Legionella pneumophila is a facultative intracellular pathogen capable of replicating within a broad range of hosts. One unique feature of this pathogen is the cohort of ca. 300 virulence factors (effectors) delivered into host cells via its Dot/Icm type IV secretion system. Study of these proteins has produced novel insights into the mechanisms of host function modulation by pathogens, the regulation of essential processes of eukaryotic cells and of immunosurveillance. In this review, we will briefly discuss the roles of some of these effectors in the creation of a niche permissive for bacterial replication in phagocytes and recent advancements in the dissection of the innate immune detection mechanisms by challenging immune cells with L. pneumophila.

  5. Legionella pneumophila pangenome reveals strain-specific virulence factors

    PubMed Central

    2010-01-01

    Background Legionella pneumophila subsp. pneumophila is a gram-negative γ-Proteobacterium and the causative agent of Legionnaires' disease, a form of epidemic pneumonia. It has a water-related life cycle. In industrialized cities L. pneumophila is commonly encountered in refrigeration towers and water pipes. Infection is always via infected aerosols to humans. Although many efforts have been made to eradicate Legionella from buildings, it still contaminates the water systems. The town of Alcoy (Valencian Region, Spain) has had recurrent outbreaks since 1999. The strain "Alcoy 2300/99" is a particularly persistent and recurrent strain that was isolated during one of the most significant outbreaks between the years 1999-2000. Results We have sequenced the genome of the particularly persistent L. pneumophila strain Alcoy 2300/99 and have compared it with four previously sequenced strains known as Philadelphia (USA), Lens (France), Paris (France) and Corby (England). Pangenome analysis facilitated the identification of strain-specific features, as well as some that are shared by two or more strains. We identified: (1) three islands related to anti-drug resistance systems; (2) a system for transport and secretion of heavy metals; (3) three systems related to DNA transfer; (4) two CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems, known to provide resistance against phage infections, one similar in the Lens and Alcoy strains, and another specific to the Paris strain; and (5) seven islands of phage-related proteins, five of which seem to be strain-specific and two shared. Conclusions The dispensable genome disclosed by the pangenomic analysis seems to be a reservoir of new traits that have mainly been acquired by horizontal gene transfer and could confer evolutionary advantages over strains lacking them. PMID:20236513

  6. Exploring the Legionella pneumophila positivity rate in hotel water samples from Antalya, Turkey.

    PubMed

    Sepin Özen, Nevgün; Tuğlu Ataman, Şenay; Emek, Mestan

    2017-05-01

    The genus Legionella is a fastidious Gram-negative bacteria widely distributed in natural waters and man made water supply systems. Legionella pneumophila is the aetiological agent of approximately 90% of reported Legionellosis cases, and serogroup 1 is the most frequent cause of infections. Legionnaires' disease is often associated with travel and continues to be a public health concern at present. The correct water management quality practices and rapid methods for analyzing Legionella species in environmental water is a key point for the prevention of Legionnaires' disease outbreaks. This study aimed to evaluate the positivity rates and serotyping of Legionella species from water samples in the region of Antalya, Turkey, which is an important tourism center. During January-December 2010, a total of 1403 samples of water that were collected from various hotels (n = 56) located in Antalya were investigated for Legionella pneumophila. All samples were screened for L. pneumophila by culture method according to "ISO 11731-2" criteria. The culture positive Legionella strains were serologically identified by latex agglutination test. A total of 142 Legionella pneumophila isolates were recovered from 21 (37.5%) of 56 hotels. The total frequency of L. pneumophila isolation from water samples was found as 10.1%. Serological typing of 142 Legionella isolates by latex agglutination test revealed that strains belonging to L. pneumophila serogroups 2-14 predominated in the examined samples (85%), while strains of L. pneumophila serogroup 1 were less numerous (15%). According to our knowledge, our study with the greatest number of water samples from Turkey demonstrates that L. pneumophila serogroups 2-14 is the most common isolate. Rapid isolation of L. pneumophila from environmental water samples is essential for the investigation of travel related outbreaks and the possible resources. Further studies are needed to have epidemiological data and to determine the types of L

  7. Legionella pneumophila serogroup 1 in a birthing pool.

    PubMed

    Teare, L; Millership, S

    2012-09-01

    This report describes a risk assessment and subsequent actions following isolation of Legionella pneumophila serogroup 1 in the water supply to a birthing pool during a planned maintenance programme. A literature search for cases of neonatal legionellosis identified 24 reports of cases among babies aged <2 months, two of which were associated with water births. On this basis, the pool was closed until Legionella spp. were undetectable. Control proved difficult as hyperchlorination failed, and a filter fitted to the thermostatic mixer tap supplying the pool slowed filling so much that additional taps were required to achieve a satisfactory flow rate. Copyright © 2012 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  8. Susceptibility of Legionella pneumophila to chlorine in tap water.

    PubMed

    Kuchta, J M; States, S J; McNamara, A M; Wadowsky, R M; Yee, R B

    1983-11-01

    A study was conducted to compare the susceptibility of legionellae and coliforms to disinfection by chlorine. The chlorine residuals used were similar to concentrations that might be found in the distribution systems of large public potable water supplies. The effects of various chlorine concentrations, temperatures, and pH levels were considered. A number of different Legionella strains, both environmental and clinical, were tested. The results indicate that legionellae are much more resistant to chlorine than are coliform bacteria. At 21 degrees C, pH 7.6, and 0.1 mg of free chlorine residual per liter, a 99% kill of L. pneumophila was achieved within 40 min, compared with less than 1 min for Escherichia coli. The observed resistance is enhanced as conditions for disinfection become less optimal. The required contact time for the removal of L. pneumophilia was twice as long at 4 degrees C than it was at 21 degrees C. These data suggest that legionellae can survive low levels of chlorine for relatively long periods of time.

  9. Legionella pneumophila 6S RNA optimizes intracellular multiplication

    PubMed Central

    Faucher, Sébastien P.; Friedlander, Gilgi; Livny, Jonathan; Margalit, Hanah; Shuman, Howard A.

    2010-01-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to identify sRNAs expressed by L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which 6 were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed in postexponential phase, and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of σ70-containing RNA polymerase. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA, as well as many genes involved in acquisition of nutrients and genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media. PMID:20368425

  10. Legionella pneumophila sequence type 1/Paris pulsotype subtyping by spoligotyping.

    PubMed

    Ginevra, Christophe; Jacotin, Nathalie; Diancourt, Laure; Guigon, Ghislaine; Arquilliere, Romain; Meugnier, Hélène; Descours, Ghislaine; Vandenesch, Francois; Etienne, Jerome; Lina, Gérard; Caro, Valérie; Jarraud, Sophie

    2012-03-01

    Endemic strains of Legionella pneumophila sequence type 1 (ST1), in particular the ST1/Paris pulsotype, are dispersed worldwide and represent about 10% of culture-proven clinical cases of Legionnaires' disease in France. The high rate of isolation of this strain from both clinical and environmental samples makes identification of the source of infection difficult during epidemiological investigations. The full-length genome sequence of this strain was recently determined, and it revealed the presence of a CRISPR/cas complex. The aim of this study was to develop and evaluate a spoligotyping tool based on the diversity of this CRISPR locus that would allow the accurate subtyping of the L. pneumophila serogroup 1 ST1/Paris pulsotype. The CRISPR loci of 28 L. pneumophila ST1/Paris pulsotype isolates were sequenced, and 42 different spacers regions were characterized. A membrane-based spoligotyping method was developed and used to determine the subtypes of 406 L. pneumophila isolates, including 233 with the ST1/Paris pulsotype profile that were collected in France from 2000 to 2011. A total of 46 different spoligotypes were detected, and 41 of these were specifically identified in the ST1/Paris pulsotype isolates. In 27 of 33 epidemiological investigations, the environmental source of contamination was confirmed by comparing spoligotypes of clinical isolates with those of environmental isolates. With an index of discrimination of 79.72% (95% confidence interval, 75.82 to 83.63), spoligotyping of the L. pneumophila ST1/Paris pulsotype has the potential to be a useful complementary genotyping tool for discriminating isolates with undistinguishable pulsed-field gel electrophoresis (PFGE) and ST genotypes, which could help to identify environmental sources of infection.

  11. Detection of Legionella pneumophila in environmental water samples using a fluorescein conjugated monoclonal antibody.

    PubMed Central

    Makin, T.; Hart, C. A.

    1989-01-01

    Sixty-three environmental water samples from various sources were examined for the presence of Legionella pneumophila with a commercially available direct fluorescent monoclonal antibody (GS), an indirect fluorescent antibody test (IFAT) and culture. GS detected L. pneumophila in 94% and 100% of environmental water samples which were culture and IFAT positive for L. pneumophila, respectively. IFAT detected 69% of L. pneumophila culture positive samples. Cultures of L. pneumophila serogroups 1 to 12, 14 and non-L. pneumophila bacteria which may be found in water, and bacteria containing non-specific binding proteins, were stained by GS and IFAT. GS identified all serogroups of L. pneumophila and did not cross react with any non-L. pneumophila bacteria. L. pneumophila in environmental samples was easy to detect against a clear dark background when stained with GS. Images Fig. 1 PMID:2673821

  12. Bus water storage tank as a reservoir of Legionella pneumophila.

    PubMed

    Jurčev-Savičević, Anamarija; Bradarić, Nikola; Paić, Vlado Ozic; Mulić, Rosanda; Puntarić, Dinko; Miše, Kornelija

    2014-09-01

    Health concerns associated with Legionnaires' disease have been identified as an area of the increasing public and professional interest. Any natural water or man-made water systems worldwide might be reservoirs of Legionellae. We presented a sporadic, community-acquired case of Legionnaires' disease caused by Legionellapneumophila serogroup 1 in a bus driver who used water for hand and face washing from a bus water storage tank. The history of any other usual place of exposure to Legionellae was negative. The water from the tank was dirty, filled with sediment and leaves, at the temperature of 22 degrees C. The water was heavily contaminated with Legionella pneumophila serogroup 1 isolated from each sample with the concentration of 66,000, 16,000, 42,000, 56,000 and 34,000 CFU/L. The disinfection of the bus water storage tank was made using hyperclorination with 50 mg/L of free residual chlorine. The control sampling one week after the disinfection yielded negative results. So far, there are no recommendations on regular management or disinfection of water in bus storage tanks, but it seems to be reasonable to assume that passengers as well as bus drivers may be exposed to Legionella and therefore at risk of acquiring the infection. These recommendations should include regular empting, rinsing and filling the tank with fresh tap water, at least once a week. Finally, we have to be aware that Legionella bacteria are ubiquitous and any potential mode of producing contaminated aerosol should not be overlooked during an epidemiological field investigation and proposed appropriate measures.

  13. Bus water storage tank as a reservoir of Legionella pneumophila.

    PubMed

    Jurčev-Savičević, Anamarija; Bradarić, Nikola; Paić, Vlado Ozic; Mulić, Rosanda; Puntarić, Dinko; Miše, Kornelija

    2014-09-01

    Health concerns associated with Legionnaires' disease have been identified as an area of the increasing public and professional interest. Any natural water or man-made water systems worldwide might be reservoirs of Legionellae. We presented a sporadic, community-acquired case of Legionnaires' disease caused by Legionellapneumophila serogroup 1 in a bus driver who used water for hand and face washing from a bus water storage tank. The history of any other usual place of exposure to Legionellae was negative. The water from the tank was dirty, filled with sediment and leaves, at the temperature of 22 degrees C. The water was heavily contaminated with Legionella pneumophila serogroup 1 isolated from each sample with the concentration of 66,000, 16,000, 42,000, 56,000 and 34,000 CFU/L. The disinfection of the bus water storage tank was made using hyperclorination with 50 mg/L of free residual chlorine. The control sampling one week after the disinfection yielded negative results. So far, there are no recommendations on regular management or disinfection of water in bus storage tanks, but it seems to be reasonable to assume that passengers as well as bus drivers may be exposed to Legionella and therefore at risk of acquiring the infection. These recommendations should include regular empting, rinsing and filling the tank with fresh tap water, at least once a week. Finally, we have to be aware that Legionella bacteria are ubiquitous and any potential mode of producing contaminated aerosol should not be overlooked during an epidemiological field investigation and proposed appropriate measures.

  14. Association of flagellum expression and intracellular growth of Legionella pneumophila.

    PubMed Central

    Pruckler, J M; Benson, R F; Moyenuddin, M; Martin, W T; Fields, B S

    1995-01-01

    We examined the role of the flagella of Legionella pneumophila in the infection of amoebae and human monocyte-like cells. Insertional mutants were constructed with mini-Tn10. Ten mutants (F-) which did not react with polyclonal L. pneumophila antiflagellar antisera were identified. Ten randomly selected mutants (F+) that did react with the polyclonal antiflagellar antiserum were also identified. The infectivity of these 20 mutants in Hartmannella vermiformis and human U937 cells was characterized. Seven of the 10 F- mutants were attenuated in their ability to multiply in the amoebae during the first 3 days of coincubation and failed to multiply in U937 cells. Three of the 10 F- mutants multiplied as well as the wild-type parent strain did in amoebae and to a limited degree in U937 cells. None of the 10 F+ mutants were attenuated in either the amoebae or U937 cells. While the flagellar structure is not essential for virulence, the ability of L. pneumophila to infect amoebae and human phagocytic cells appears to be linked to flagellar expression. We believe that the attenuated F- mutants contain insertions in genes critical to both flagellum expression and the infection process. PMID:7591159

  15. Occurrence of Opportunistic Pathogens Legionella pneumophila and non-tuberculous mycobacteria in hospital plumbing systems

    EPA Science Inventory

    Occurrence of Opportunistic Pathogens Legionella pneumophila and non-tuberculous mycobacteria in hospital plumbing systems Jill Hoelle, Michael Coughlin, Elizabeth Sotkiewicz, Jingrang Lu, Stacy Pfaller, Mark Rodgers, and Hodon Ryu U.S. Environmental Protection Agency, Cincinnati...

  16. Epidemiology and Ecology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa

    EPA Science Inventory

    BACKGROUND: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa are opportunistic premise plumbing pathogens (OPPPs) that persist and grow in household plumbing, habitats they share with humans. Infections caused by these OPPPs involve individuals with preexis...

  17. Occurrence of Opportunistic Pathogens Legionella pneumophila and non-tuberculous mycobacteria in hospital plumbing systems

    EPA Science Inventory

    Occurrence of Opportunistic Pathogens Legionella pneumophila and non-tuberculous mycobacteria in hospital plumbing systems Jill Hoelle, Michael Coughlin, Elizabeth Sotkiewicz, Jingrang Lu, Stacy Pfaller, Mark Rodgers, and Hodon Ryu U.S. Environmental Protection Agency, Cincinnati...

  18. Epidemiology and Ecology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa

    EPA Science Inventory

    BACKGROUND: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa are opportunistic premise plumbing pathogens (OPPPs) that persist and grow in household plumbing, habitats they share with humans. Infections caused by these OPPPs involve individuals with preexis...

  19. Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

    PubMed Central

    Dolezal, Pavel; Aili, Margareta; Tong, Janette; Jiang, Jhih-Hang; Marobbio, Carlo M.; Lee, Sau fung; Schuelein, Ralf; Belluzzo, Simon; Binova, Eva; Mousnier, Aurelie; Frankel, Gad; Giannuzzi, Giulia; Palmieri, Ferdinando; Gabriel, Kipros; Naderer, Thomas; Hartland, Elizabeth L.; Lithgow, Trevor

    2012-01-01

    The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms. PMID:22241989

  20. Reclamation of Ampicillin Sensitivity for the Genetic Manipulation of Legionella pneumophila

    PubMed Central

    Sutherland, Molly C.

    2012-01-01

    Research on Legionella pneumophila, the causative agent of Legionnaires' disease, has been hampered due to the lack of selectable markers for genetic manipulation. We report the construction of a mutant strain of L. pneumophila lacking loxA, a chromosomally encoded β-lactamase, that has enhanced sensitivity to ampicillin. Also described are a method for converting Legionella strains to ampicillin sensitivity and conditions for utilizing bla as a selectable marker. PMID:22635996

  1. Physiological and Antigenic Characteristics of Virulent and Attenuated Strains of Legionella pneumophila (Philadelphia 3)

    DTIC Science & Technology

    1981-07-01

    Shepard. 1979. Virulent to avirulent conversion of Legionnaires ’ disease bacterium ( Legionella pneumophila) - its effect on isolation techniques. J...8217 disease bacterium: Legionella pneumophila, genus novum, species nova, of the family Legionellaceae, familia nova. Ann. Intern, Med. 90:656-658. 5...J. C. Feeley. 1978. A microagglutination test for detecting antibodies, p. 163-168. In C. L. Jones and G. A. Hebert (ed.), " Legionnaires " the disease

  2. Spatial distribution of Legionella pneumophila MLVA-genotypes in a drinking water system.

    PubMed

    Rodríguez-Martínez, Sarah; Sharaby, Yehonatan; Pecellín, Marina; Brettar, Ingrid; Höfle, Manfred; Halpern, Malka

    2015-06-15

    Bacteria of the genus Legionella cause water-based infections, resulting in severe pneumonia. To improve our knowledge about Legionella spp. ecology, its prevalence and its relationships with environmental factors were studied. Seasonal samples were taken from both water and biofilm at seven sampling points of a small drinking water distribution system in Israel. Representative isolates were obtained from each sample and identified to the species level. Legionella pneumophila was further determined to the serotype and genotype level. High resolution genotyping of L. pneumophila isolates was achieved by Multiple-Locus Variable number of tandem repeat Analysis (MLVA). Within the studied water system, Legionella plate counts were higher in summer and highly variable even between adjacent sampling points. Legionella was present in six out of the seven selected sampling points, with counts ranging from 1.0 × 10(1) to 5.8 × 10(3) cfu/l. Water counts were significantly higher in points where Legionella was present in biofilms. The main fraction of the isolated Legionella was L. pneumophila serogroup 1. Serogroup 3 and Legionella sainthelensis were also isolated. Legionella counts were positively correlated with heterotrophic plate counts at 37 °C and negatively correlated with chlorine. Five MLVA-genotypes of L. pneumophila were identified at different buildings of the sampled area. The presence of a specific genotype, "MLVA-genotype 4", consistently co-occurred with high Legionella counts and seemed to "trigger" high Legionella counts in cold water. Our hypothesis is that both the presence of L. pneumophila in biofilm and the presence of specific genotypes, may indicate and/or even lead to high Legionella concentration in water. This observation deserves further studies in a broad range of drinking water systems to assess its potential for general use in drinking water monitoring and management.

  3. Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila

    PubMed Central

    Ginevra, Christophe; Jacotin, Nathalie; Forey, Françoise; Chastang, Joëlle; Kay, Elisabeth; Etienne, Jerome; Lina, Gérard; Doublet, Patricia; Jarraud, Sophie

    2017-01-01

    ABSTRACT Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila. Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings. PMID:28069647

  4. Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila.

    PubMed

    Descours, Ghislaine; Ginevra, Christophe; Jacotin, Nathalie; Forey, Françoise; Chastang, Joëlle; Kay, Elisabeth; Etienne, Jerome; Lina, Gérard; Doublet, Patricia; Jarraud, Sophie

    2017-03-01

    Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

  5. A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy

    PubMed Central

    Khweek, Arwa Abu; Caution, Kyle; Akhter, Anwari; Abdulrahman, Basant A.; Tazi, Mia; Hassan, Hoda; Majumdar, Neal; Doran, Andrew; Guirado, Evelyn; Schlesinger, Larry S.; Shuman, Howard; Amer, Amal O.

    2013-01-01

    Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant is the first L. pneumophila mutant to replicate in wild-type (WT) mice and their bone marrow derived macrophages (BMDMs). Less legA9 mutant- containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila -containing vacuole for autophagy uptake. PMID:23420491

  6. A comparison of assays measuring the viability of Legionella pneumophila after treatment with copper and silver ions

    EPA Science Inventory

    Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could p...

  7. Preferential colonization and release of Legionella pneumophila from mature drinking water biofilms grown on copper versus unplasticized polyvinylchloride coupons

    EPA Science Inventory

    Legionella persistence and amplification in premise drinking water systems is a known contributor to legionellosis outbreaks, especially in the presence of suitable eukaryotic hosts. Here we examined Legionella pneumophila behavior within drinking water biofilms grown on copper ...

  8. Preferential colonization and release of Legionella pneumophila from mature drinking water biofilms grown on copper versus unplasticized polyvinylchloride coupons

    EPA Science Inventory

    Legionella persistence and amplification in premise drinking water systems is a known contributor to legionellosis outbreaks, especially in the presence of suitable eukaryotic hosts. Here we examined Legionella pneumophila behavior within drinking water biofilms grown on copper ...

  9. A comparison of assays measuring the viability of Legionella pneumophila after treatment with copper and silver ions

    EPA Science Inventory

    Background: The relatively high prevalence of Legionella pneumophila in premise plumbing systems has been widely reported. Published reports indicate Legionella has a comparatively high resistance to chlorine and moreover has the ability to grow in phagocytic amoeba which could p...

  10. Temperature-dependent parasitic relationship between Legionella pneumophila and a free-living amoeba (Acanthamoeba castellanii).

    PubMed

    Ohno, Akira; Kato, Naoyuki; Sakamoto, Ryota; Kimura, Soichiro; Yamaguchi, Keizo

    2008-07-01

    We analyzed the effects of temperature on the interaction of Legionella pneumophila with Acanthamoeba castellanii. At <20 degrees C, overexpression of type 1 metacaspase, a stimulator of A. castellanii encystation, was associated with a reduced number of bacteria within amoeba. At low temperatures, A. castellanii seems to eliminate L. pneumophila by encystation and digestion.

  11. Widespread molecular detection of Legionella pneumophila Serogroup 1 in cold water taps across the United States

    EPA Science Inventory

    In the United States 3,522 cases of legionellosis were reported to the Center for Disease Control and Prevention in 2009. Of these reports, it is estimated that 84% are caused by the microorganism Legionella pneumophila Serogroup (Sg) 1. Legionella spp. have been isolated and r...

  12. Disseminated Legionella pneumophila infection in an immunocompromised patient treated with tigecycline.

    PubMed

    Valve, Kirsi; Vaalasti, Annikki; Anttila, Veli-Jukka; Vuento, Risto

    2010-01-01

    We describe an immunocompromised patient with disseminated Legionella pneumophila infection. A chronic leg ulcer was probably the port of entry for the infection. Treatment required several operations and prolonged antimicrobial treatment. To our knowledge, this is the first case report of Legionella soft tissue infection and pneumonia treated with tigecycline.

  13. Widespread molecular detection of Legionella pneumophila Serogroup 1 in cold water taps across the United States

    EPA Science Inventory

    In the United States 3,522 cases of legionellosis were reported to the Center for Disease Control and Prevention in 2009. Of these reports, it is estimated that 84% are caused by the microorganism Legionella pneumophila Serogroup (Sg) 1. Legionella spp. have been isolated and r...

  14. Draft Genome Sequences of Legionella pneumophila JR32 and Lp01 Laboratory Strains Domesticated in Japan

    PubMed Central

    Maita, Chinatsu; Matushita, Mizue; Okubo, Torahiko; Matsuo, Junji; Miyake, Masaki; Nagai, Hiroki

    2016-01-01

    We report here the draft genome sequences of two Legionella pneumophila variant strains (JR32 and Lp01_666) originally derived from a Philadelphia-1 clinical isolate, domesticated in Japan, with distinct susceptibility to amoebae. Detailed genomic analysis will allow us to better understand Legionella adaptation and survival mechanisms in host cells. PMID:27491976

  15. Identification of vacuoles containing extraintestinal differentiated forms of Legionella pneumophila in colonized Caenorhabditis elegans soil nematodes

    PubMed Central

    Hellinga, Jacqueline R; Garduño, Rafael A; Kormish, Jay D; Tanner, Jennifer R; Khan, Deirdre; Buchko, Kristyn; Jimenez, Celine; Pinette, Mathieu M; Brassinga, Ann Karen C

    2015-01-01

    Legionella pneumophila, a causative agent of Legionnaires’ disease, is a facultative intracellular parasite of freshwater protozoa. Legionella pneumophila features a unique developmental network that involves several developmental forms including the infectious cyst forms. Reservoirs of L. pneumophila include natural and man-made freshwater systems; however, recent studies have shown that isolates of L. pneumophila can also be obtained directly from garden potting soil suggesting the presence of an additional reservoir. A previous study employing the metazoan Caenorhabditis elegans, a member of the Rhabditidae family of free-living soil nematodes, demonstrated that the intestinal lumen can be colonized with L. pneumophila. While both replicative forms and differentiated forms were observed in C. elegans, these morphologically distinct forms were initially observed to be restricted to the intestinal lumen. Using live DIC imaging coupled with focused transmission electron microscopy analyses, we report here that L. pneumophila is able to invade and establish Legionella-containing vacuoles (LCVs) in the intestinal cells. In addition, LCVs containing replicative and differentiated cyst forms were observed in the pseudocoelomic cavity and gonadal tissue of nematodes colonized with L. pneumophila. Furthermore, establishment of LCVs in the gonadal tissue was Dot/Icm dependent and required the presence of the endocytic factor RME-1 to gain access to maturing oocytes. Our findings are novel as this is the first report, to our knowledge, of extraintestinal LCVs containing L. pneumophila cyst forms in C. elegans tissues, highlighting the potential of soil-dwelling nematodes as an alternate environmental reservoir for L. pneumophila. PMID:26131925

  16. Identification of vacuoles containing extraintestinal differentiated forms of Legionella pneumophila in colonized Caenorhabditis elegans soil nematodes.

    PubMed

    Hellinga, Jacqueline R; Garduño, Rafael A; Kormish, Jay D; Tanner, Jennifer R; Khan, Deirdre; Buchko, Kristyn; Jimenez, Celine; Pinette, Mathieu M; Brassinga, Ann Karen C

    2015-08-01

    Legionella pneumophila, a causative agent of Legionnaires' disease, is a facultative intracellular parasite of freshwater protozoa. Legionella pneumophila features a unique developmental network that involves several developmental forms including the infectious cyst forms. Reservoirs of L. pneumophila include natural and man-made freshwater systems; however, recent studies have shown that isolates of L. pneumophila can also be obtained directly from garden potting soil suggesting the presence of an additional reservoir. A previous study employing the metazoan Caenorhabditis elegans, a member of the Rhabditidae family of free-living soil nematodes, demonstrated that the intestinal lumen can be colonized with L. pneumophila. While both replicative forms and differentiated forms were observed in C. elegans, these morphologically distinct forms were initially observed to be restricted to the intestinal lumen. Using live DIC imaging coupled with focused transmission electron microscopy analyses, we report here that L. pneumophila is able to invade and establish Legionella-containing vacuoles (LCVs) in the intestinal cells. In addition, LCVs containing replicative and differentiated cyst forms were observed in the pseudocoelomic cavity and gonadal tissue of nematodes colonized with L. pneumophila. Furthermore, establishment of LCVs in the gonadal tissue was Dot/Icm dependent and required the presence of the endocytic factor RME-1 to gain access to maturing oocytes. Our findings are novel as this is the first report, to our knowledge, of extraintestinal LCVs containing L. pneumophila cyst forms in C. elegans tissues, highlighting the potential of soil-dwelling nematodes as an alternate environmental reservoir for L. pneumophila.

  17. Influence of aquatic microorganisms on Legionella pneumophila survival.

    PubMed

    Guerrieri, Elisa; Bondi, Moreno; Borella, Paola; Messi, Patrizia

    2007-07-01

    The ability of aquatic bacteria Pseudomonas fluorescens SSD (Ps-D) and Pseudomonas putida SSC (Ps-C) to support the persistence of Legionella pneumophila (Lp-1) in an artificial water microcosm was investigated for 42 day, at two different incubation temperatures. At 4 degrees C, individually suspended Lp-1 was no longer detectable just after 24 hours, while in co-cultures with Pseudomonas, Lp1 showed a better survival capability. At 30 degrees C, Lp-1 alone displayed high survival rates over the entire period of observation. When Lp-1 was inoculated with Ps-C and Ps-D, its count showed a marked decrease, followed by a gradual and costant decline.

  18. Isolation of Legionella pneumophila serogroup 3 from pericardial fluid in a case of pericarditis.

    PubMed

    Lück, P C; Helbig, J H; Wunderlich, E; Foelske, H; Selbitschka, M; Wenzel, D; Pätzold, L; Witzleb, W

    1989-01-01

    A 43-year-old woman was hospitalized for fulminant pericarditis. During diagnostic work-up, an as yet unknown bronchial carcinoma was detected. In the pericardial exudate Legionella pneumophila serogroup 3 was demonstrated by direct fluorescent antibody technique and by culture. In a lung biopsy L. pneumophila serogroup 3 was found, too. Using an antigen-ELISA for L. pneumophila serogroup 1, antigenuria was demonstrated. In cases of pericarditis negative for common bacterial pathogens, all diagnostic tests for legionellae, e.g. culture, antigen detection in pericardial, pleural effusion and urine and antibody detection should be included in the diagnostic programme.

  19. Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth ▿

    PubMed Central

    Nasrallah, Gheyath K.; Riveroll, Angela L.; Chong, Audrey; Murray, Lois E.; Lewis, P. Jeffrey; Garduño, Rafael A.

    2011-01-01

    The Gram-negative intracellular pathogen Legionella pneumophilareplicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophilaexpressing a translocation reporter consisting of the Bordetella pertussisadenylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophilamay require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophilareplication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophilaproliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila. PMID:21742865

  20. Difference in virulence of environmental isolates of Legionella pneumophila.

    PubMed Central

    Bollin, G E; Plouffe, J F; Para, M F; Prior, R B

    1985-01-01

    Endemic nosocomial Legionnaires disease has occurred at our medical center for several years. Two subtypes of Legionella pneumophila serogroup 1 (UH-1 and RH-1) have been isolated in approximately equal numbers from hospital potable water. However, almost all clinical isolates have been UH-1. To assess potential differences in virulence, 50% lethal doses (LD50) and 50% infective doses (ID50) of UH-1 and RH-1 were determined by intraperitoneal infection in guinea pigs. The UH-1 LD50 was 7.41 X 10(6) CFU, which was significantly lower than the RH-1 LD50 of 9.12 X 10(7) CFU (P = 0.0001). The mean time to death in UH-1-infected guinea pigs was also significantly shorter than in RH-1-infected animals (P = 0.0008). The UH-1 ID50 was 5.8 X 10(3) CFU, and although it was lower than the RH-1 ID50 of 1.4 X 10(4) CFU, this difference was not statistically significant (P = 0.21). This study demonstrates a difference in virulence between UH-1 and RH-1 in guinea pigs. Differences in strain virulence, as demonstrated between these two subtypes, may help to explain the widespread isolation of L. pneumophila from the environment contrasted with the limited occurrence of human Legionnaires disease. PMID:3998095

  1. Differentiate to thrive: lessons from the Legionella pneumophila life cycle.

    PubMed

    Molofsky, Ari B; Swanson, Michele S

    2004-07-01

    When confronted by disparate environments, microbes routinely alter their physiology to tolerate or exploit local conditions. But some circumstances require more drastic remodelling of the bacterial cell, as sporulation by the Bacillus and Streptomyces species of soil bacteria vividly illustrates. Cellular differentiation is also crucial for pathogens, the challenge for which is to colonize one host, then be transmitted to the next. Using the Gram-negative Legionella pneumophila as a model intracellular pathogen, we describe how biogenesis of the replication vacuole is determined by the developmental state of the bacterium. Subsequently, when replicating bacteria have exhausted the nutrient supply, the pathogens couple their conversion to stationary phase physiology with expression of traits that promote transmission to a new host. The cellular differentiation of L. pneumophila is co-ordinated by a regulatory circuit that integrates several elements that are broadly conserved in the microbial world. The alarmone (p)ppGpp promotes transcription directed by the alternative sigma factors RpoS, FliA and, probably, RpoN, and also post-transcriptional control mediated by a two-component regulatory system, LetA/S (GacA/S), and an mRNA-binding protein, CsrA (RsmA). By applying knowledge of microbial differentiation in combination with tools to screen the complete genomes of pathogens, experiments can be designed to identify two distinct classes of virulence traits: factors that promote replication and those dedicated to transmission.

  2. Chemical composition of a lipopolysaccharide from Legionella pneumophila.

    PubMed

    Sonesson, A; Jantzen, E; Bryn, K; Larsson, L; Eng, J

    1989-01-01

    Lipopolysaccharide isolated from Legionella pneumophila (Phil. 1) was examined for chemical composition. The polysaccharide split off by mild acid hydrolysis contained rhamnose, mannose, glucose, quinovosamine, glucosamine and 2-keto-3-deoxyoctonate, in molar proportions 1.6:1.8:1.0:1.5:4.1:2.7. Heptoses were absent and glucose was probably mainly phosphorylated. The carbohydrate backbone of the lipid A part consisted of glucosamine, quinovosamine and glycerol, in the molar ratios 3.9:1.0:3.4, with glycerol as a phosphorylated moiety. A complex fatty acid substitution pattern comprising eight O-ester-linked, exclusively nonhydroxylated acids, and nineteen amide-linked, exclusively 3-hydroxylated acids was revealed. Both straight- and branched (iso and anteiso) carbon chains occurred. The major hydroxy fatty acid was 3-hydroxy-12-methyltridecanoic acid and six others were of a chain-length above 20 carbon atoms, with 3-hydroxy-20-methyldocosanoic acid as the longest. Two dihydroxy fatty acids, 2,3-dihydroxy-12-methyltridecanoic and 2,3-dihydroxytetradecanoic acids, were also detected. These results suggest that L. pneumophila contains a rather complex and unusual lipopolysaccharide structure of considerable biological and chemotaxonomic interest.

  3. Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody.

    PubMed Central

    Barthe, C; Joly, J R; Ramsay, D; Boissinot, M; Benhamou, N

    1988-01-01

    Serogroup-specificity of Legionella pneumophila is related to lipopolysaccharide (LPS), and few cross-reactions between serogroups have been observed with rabbit or monkey antisera. C57BL/6 mice were sequentially immunized with crude outer membrane fractions of L. pneumophila serogroups 1, 5, and 7, Legionella bozemanii, and Legionella micdadei. Spleen cells from these mice were then fused with the Sp2-0/Ag14 mouse myeloma cell line. Outer membrane-rich fractions and LPS were prepared from L. pneumophila serogroups 1 to 8 and other Legionella and non-Legionella species. Immunoblots of these extracts were performed with monoclonal antibody obtained from these fusions. One of these monoclonal antibodies recognized an epitope common to all tested serogroups of L. pneumophila and attached to the major constituent of the outer membrane, LPS. This antibody did not react with other Legionella species and numerous gram-negative rods other than Pseudomonas fluorescens CDC93. This monoclonal antibody may be useful in preliminary identification of L. pneumophila as an alternative to direct fluorescent-antibody testing. Images PMID:2454935

  4. Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water.

    PubMed

    Cho, Min Seok; Ahn, Tae-Young; Joh, Kiseong; Lee, Eui Seok; Park, Dong Suk

    2015-11-01

    Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 264-bp product for only targeted six strains of L. pneumophila. The assay was also able to detect at least 1.39 × 10(3) copies/μl of cloned amplified target DNA using purified DNA or 7.4 × 10(0) colony-forming unit per reaction when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples.

  5. Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila.

    PubMed

    Dey, Rafik; Bodennec, Jacques; Mameri, Mouh Oulhadj; Pernin, Pierre

    2009-01-01

    Legionella pneumophila is known as a facultative intracellular parasite of free-living soil and freshwater amoebae, of which several species have been shown to support the growth of the pathogenic bacteria. We report for the first time the behaviour of two strains (c2c and Z503) of the amoeba Willaertia magna towards different strains of L. pneumophila serogroup 1 and compared it with Acanthamoeba castellanii and Hartmannella vermiformis, known to be L. pneumophila permissive. In contrast to the results seen with other amoebae, W. magna c2c inhibited the growth of one strain of Legionella (L. pneumophila, Paris), but not of others belonging to the same serogroup (L. pneumophila, Philadelphia and L. pneumophila, Lens). Also, the different L. pneumophila inhibited cell growth and induced cell death in A. castellanii, H. vermiformis and W. magna Z503 within 3-4 days while W. magna c2c strain remained unaffected even up to 7 days. Electron microscopy demonstrated that the formation of numerous replicative phagosomes observed within Acanthamoeba and Hartmannella is rarely seen in W. magna c2c cocultured with L. pneumophila. Moreover, the morphological differences were observed between L. pneumophila cultured either with Willaertia or other amoebae. These observations show that amoebae are not all equally permissive to L. pneumophila and highlight W. magna c2c as particularly resistant towards some strains of this bacterium.

  6. [Nosocomial Legionella pneumophila infection in a nephrology department].

    PubMed

    Gahrn-Hansen, B; Uldum, S A; Schmidt, J; Nielsen, B; Birkeland, S A; Jørgensen, K A

    1995-01-30

    During the autumn and winter of 1993-94 four cases of legionellosis were diagnosed in a Department of Nephrology. Three of the patients were kidney transplanted patients. Two of the patients died. The diagnosis was based on positive culture in two patients and by positive urinary antigen test in the other two patients. Serology was negative for all four patients. Legionella pneumophila was initially found in the cold and hot shower water, in ice-water from the ice machine, from the hot water tank and in the cold water inlet to the building. The isolate from patient no. one and isolates of serogroup 5 from the ice machine and the shower water had identical REA profiles, different from the profiles of the isolate from patient no. four. We concluded that at least one of the four patients was likely to have been infected from the water in the department, either by inhalation of contaminated aerosols from the shower or by aspiration of contaminated ice-water. Precautions were taken to reduce the number of Legionella in the shower- and ice-water. In addition, restrictions in the use of showers and ice-water from the ice machine were introduced.

  7. Diagnostic Utility of Splenial Lesions in a Case of Legionnaires' Disease due to Legionella pneumophila Serogroup 2.

    PubMed

    Tomizawa, Yuji; Hoshino, Yasunobu; Sasaki, Fuyuko; Kurita, Naohide; Kawajiri, Sumihiro; Noda, Kazuyuki; Hattori, Nobutaka; Amemura-Maekawa, Junko; Kura, Fumiaki; Okuma, Yasuyuki

    2015-01-01

    We herein report the case of a 49-year-old man with clinically mild encephalitis/encephalopathy with a reversible splenial lesion (MERS) associated with Legionnaires' disease due to Legionella pneumophila serogroup 2. Past reports suggest that Legionella infection is frequent in cases of MERS-associated pneumonia. Obtaining an early diagnosis of legionella infection is a challenge, especially if a Legionella pneumophila serogroup other than serogroup 1 contains the causative agent. In this case, the splenial lesion played an important role in recognizing the legionella infection. We suggest that legionella infection should be considered as a differential diagnosis in cases of splenial lesions associated with pneumonia.

  8. Commercial potting soils as an alternative infection source of Legionella pneumophila and other Legionella species in Switzerland.

    PubMed

    Casati, S; Gioria-Martinoni, A; Gaia, V

    2009-06-01

    Legionella spp. are pathogens that can cause Legionnaires' disease in humans through inhalation of contaminated aerosols. The principal reservoir for these microorganisms is water, but Legionella spp. have been isolated from composted vegetable and plant material, and from many potting mixes as well. In Australia, there have been several cases of Legionnaires' disease in which Legionella longbeachae has been isolated from potting soils. In Switzerland, the source of infection cannot always be identified as water or cooling towers: therefore, we have investigated 46 commercially available potting soils in Switzerland to determine the presence of Legionella spp. We were able to detect Legionella spp. in 45.7% (21/46) of the potting soil samples analysed by culture. Legionella pneumophila was present in 19.6% (9/46) of the samples and L. pneumophila serogroup 1 in 6.5% (3/46). Quantification by both culture and quantitative real-time PCR revealed high concentrations of legionellae in potting soils, ranging between 10(3) CFU/g and 10(5) CFU/g and 10(4) genomic units (GU)/g and 10(6) GU/g, respectively. Thus, potting soils may represent an alternative reservoir for Legionella spp. in Switzerland.

  9. Analysis of cell surface alterations in Legionella pneumophila cells treated with human apolipoprotein E.

    PubMed

    Palusinska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Cytryńska, Małgorzata; Wdowiak-Wróbel, Sylwia; Chmiel, Elżbieta; Gruszecki, Wiesław I

    2015-03-01

    Binding of human apolipoprotein E (apoE) to Legionella pneumophila lipopolysaccharide was analysed at the molecular level by Fourier-transform infrared spectroscopy, thereby providing biophysical evidence for apoE-L. pneumophila lipopolysaccharide interaction. Atomic force microscopy imaging of apoE-exposed L. pneumophila cells revealed alterations in the bacterial cell surface topography and nanomechanical properties in comparison with control bacteria. The changes induced by apoE binding to lipopolysaccharide on the surface of L. pneumophila cells may participate in: (1) impeding the penetration of host cells by the bacteria; (2) suppression of pathogen intracellular growth and eventually; and (3) inhibition of the development of infection.

  10. Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis.

    PubMed

    Reischl, Udo; Linde, Hans-Jörg; Lehn, Norbert; Landt, Olfert; Barratt, Kevin; Wellinghausen, Nele

    2002-10-01

    A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.

  11. [EFFECT OF TEMPERATURE ON THE VIABILITY OF PLANKTON CELLS AND MODEL LEGIONELLA PNEUMOPHILA BIOFILMS IN WATER].

    PubMed

    Tartakovsky, I S; Karpoval, T I; Gruzdeva, O A; Marinenko, O V; Dronina, Yu E

    2015-01-01

    Study the effect of water temperature from 40 to 70 degrees C on viability of plankton forms and model Legionella pneumophila under experimental conditions. Monospecies legionella biofilms, obtained in plates for enzyme immunoassay during 96 hours at 28 degrees C, and water suspension of BCYE agar cultivated cells of L. pneumophila at a concentration of 10(3) - 10(5) CFU per liter were used in the study for evaluation of bactericidal effect of temperature on various legionella forms. Analysis of effects of various temperature regimens on plankton forms and model legionella biofilms has shown that at a temperature range from 50 to 60 degrees C a significant reduction of quantity of viable legionella cells occurs. Model legionella biofilms have partially conserved viability at a temperature of 60 degrees C and only exposition to a temperature of 70 degrees C resulted in death of legionella biofilms and plankton forms of bacteria. A dependence of viability conservation of legionella from the initial concentration of the causative agent in water and duration of exposition at varying temperature was shown. Short-term heating at a temperature of at least 70 degrees C has the most pronounced bactericidal effect on plankton forms and model L. pneumophila biofilms under experimental conditions. Such temperature regimen could be used as one of the prophylaxis approaches during maintenance of especially dangerous water system and, fist of all, systems of hot water supply.

  12. The Legionella pneumophila kai operon is implicated in stress response and confers fitness in competitive environments

    PubMed Central

    Loza-Correa, Maria; Sahr, Tobias; Rolando, Monica; Daniels, Craig; Petit, Pierre; Skarina, Tania; Valero, Laura Gomez; Dervins-Ravault, Delphine; Honoré, Nadine; Savchenko, Aleksey; Buchrieser, Carmen

    2014-01-01

    Summary Legionella pneumophila uses aquatic protozoa as replication niche and protection from harsh environments. Although L. pneumophila is not known to have a circadian clock, it encodes homologues of the KaiBC proteins of Cyanobacteria that regulate circadian gene expression. We show that L. pneumophila kaiB, kaiC and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor RpoS. KaiC and KaiB of L. pneumophila do not interact as evidenced by yeast and bacterial two-hybrid analyses. Fusion of the C-terminal residues of cyanobacterial KaiB to Legionella KaiB restores their interaction. In contrast, KaiC of L. pneumophila conserved autophosphorylation activity, but KaiB does not trigger the dephosphorylation of KaiC like in Cyanobacteria. The crystal structure of L. pneumophila KaiB suggests that it is an oxidoreductase-like protein with a typical thioredoxin fold. Indeed, mutant analyses revealed that the kai operon-encoded proteins increase fitness of L. pneumophila in competitive environments, and confer higher resistance to oxidative and sodium stress. The phylogenetic analysis indicates that L. pneumophila KaiBC resemble Synechosystis KaiC2B2 and not circadian KaiB1C1. Thus, the L. pneumophila Kai proteins do not encode a circadian clock, but enhance stress resistance and adaption to changes in the environments. PMID:23957615

  13. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae.

    PubMed Central

    Rowbotham, T J

    1980-01-01

    Legionella pneumophila, the causative organism of Legionnaires' disease, is pathogenic for free living, ubiquitous, freshwater, and soil amoebae of the genera Acanthamoeba and Naegleria. Some species support the growth of strains from serogroups 1 to 6, others only strains from certain serogroups. Initial studies with seeded material indicate that amoebal enrichment could be utilised for the isolation of legionellae from clinical specimens and natural habitats. It is suggested that a vacuole, or amoeba, full of legionellae, rather than free legionellae, could be the infective particle for man. Images Fig. 1 Fig. 2 PMID:7451664

  14. Intrapulmonary Hartmannella vermiformis: a potential niche for Legionella pneumophila replication in a murine model of legionellosis.

    PubMed Central

    Brieland, J; McClain, M; LeGendre, M; Engleberg, C

    1997-01-01

    The potential role of inhaled protozoa as a niche for intrapulmonary replication of Legionella pneumophila was investigated in vivo with mutant strains of L. pneumophila which have reduced virulence for the amoeba Hartmannella vermiformis. L. pneumophila AA488 and AA502 were derived from wild-type strain AA100 after transposon mutagenesis. These mutants have reduced virulence for H. vermiformis but are fully virulent for mononuclear phagocytic cells. A/J mice, which are susceptible to replicative L. pneumophila lung infections, were inoculated intratracheally with L. pneumophila AA100, AA488, or AA502 (10[6] bacteria per mouse) or were coinoculated with one of the L. pneumophila strains (10[6] bacteria per mouse) and uninfected H. vermiformis (10[6] amoebae per mouse). The effect of coinoculation with H. vermiformis on intrapulmonary growth of each L. pneumophila strain was subsequently assessed. In agreement with our previous studies, coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of the parent L. pneumophila strain (AA100). In contrast, intrapulmonary growth of L. pneumophila AA488 or AA502 was not significantly enhanced by coinoculation of mice with H. vermiformis. These studies demonstrate that L. pneumophila virulence for amoebae is required for maximal intrapulmonary growth of the bacteria in mice coinoculated with H. vermiformis and support the hypothesis that inhaled amoebae may potentiate intrapulmonary growth of L. pneumophila by providing a niche for bacterial replication. PMID:9353084

  15. Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages.

    PubMed

    Abu Khweek, Arwa; Fernández Dávila, Natalia S; Caution, Kyle; Akhter, Anwari; Abdulrahman, Basant A; Tazi, Mia; Hassan, Hoda; Novotny, Laura A; Bakaletz, Lauren O; Amer, Amal O

    2013-01-01

    Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human.

  16. Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages

    PubMed Central

    Abu Khweek, Arwa; Fernández Dávila, Natalia S.; Caution, Kyle; Akhter, Anwari; Abdulrahman, Basant A.; Tazi, Mia; Hassan, Hoda; Novotny, Laura A.; Bakaletz, Lauren O.; Amer, Amal O.

    2013-01-01

    Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human. PMID:23750338

  17. Influence of growth temperature on virulence of Legionella pneumophila.

    PubMed Central

    Edelstein, P H; Beer, K B; DeBoynton, E D

    1987-01-01

    The effect of growth temperature on the virulence of a strain of broth-grown serogroup 1 Legionella pneumophila (Wadsworth F889) was examined by growing the bacterium at different temperatures and then infecting guinea pigs (by intratracheal injection) and guinea pig alveolar macrophages. The 50% lethal dose for guinea pigs infected with 25 degrees C-grown F889 was log10 5.0 CFU and that for 41 degrees C-grown F889 was log10 5.7 CFU, or a fivefold difference. Guinea pig alveolar macrophages were infected in quadruplicate with log10 3.8 CFU of F889 cells grown at either 25 or 41 degrees C. Counts of F889 in the alveolar macrophages infected with 25 degrees C-grown bacteria were 40% greater after 1 day of incubation (P = 2 X 10(-4)) than were counts in the alveolar macrophage suspensions inoculated with 41 degrees C-grown bacteria. However, the counts were not significantly different after 3 days of incubation. Examination of cover slip cultures of guinea pig alveolar macrophages infected with 25 degrees C-grown or 41 degrees C-grown bacteria showed that the bacteria grown at the lower temperature were twice as likely to be macrophage-associated after 1 h of incubation than were the bacteria grown at the higher temperature. Growth at the lower temperature was also associated with a change in reactivity with monoclonal antibodies, but not with a change in plasmid content. Thus, environmental temperature may play an important role in modulating the virulence of L. pneumophila, possibly by affecting bacterial adherence to host cells. PMID:3666960

  18. Mechanisms of Legionella pneumophila-induced interleukin-8 expression in human lung epithelial cells

    PubMed Central

    Teruya, Hiromitsu; Higa, Futoshi; Akamine, Morikazu; Ishikawa, Chie; Okudaira, Taeko; Tomimori, Koh; Mukaida, Naofumi; Tateyama, Masao; Heuner, Klaus; Fujita, Jiro; Mori, Naoki

    2007-01-01

    Background Legionella pneumophila is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of L. pneumophila on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines. Results Infection of L. pneumophila strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with L. pneumophila lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient Legionella. Activation of the IL-8 promoter by L. pneumophila infection occurred through the action of nuclear factor-κB (NF-κB). Transfection of dominant negative mutants of NF-κB-inducing kinase, IκB kinase and IκB inhibited L. pneumophila-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed L. pneumophila-induced IL-8 mRNA due to deactivation of NF-κB. Conclusion Collectively, these results suggest that L. pneumophila induces activation of NF-κB through an intracellular signaling pathway that involves NF-κB-inducing kinase and IκB kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in L. pneumophila-induced IL-8 expression, presumably contributing to immune response in L. pneumophila. The presence of flagellin and a type IV secretion system are critical for Legionella to induce IL-8 expression in lung epithelial cells. PMID:18034886

  19. Whole-Genome Mapping as a Novel High-Resolution Typing Tool for Legionella pneumophila.

    PubMed

    Bosch, Thijs; Euser, Sjoerd M; Landman, Fabian; Bruin, Jacob P; IJzerman, Ed P; den Boer, Jeroen W; Schouls, Leo M

    2015-10-01

    Legionella is the causative agent for Legionnaires' disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by Legionella pneumophila, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of L. pneumophila mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated L. pneumophila isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated L. pneumophila isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch L. pneumophila outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated L. pneumophila isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of L. pneumophila in real time since the turnaround time from culture to comparison of the L. pneumophila maps is less than 24 h.

  20. Micro- and macromethod assays for the ecological study of Legionella pneumophila.

    PubMed

    Guerrieri, Elisa; Bondi, Moreno; Ciancio, Claudia; Borella, Paola; Messi, Patrizia

    2005-11-01

    The survival of a strain of Legionella pneumophila (Lp-1) inoculated in artificial water microcosms was investigated with and without an amoebal host and varying environmental conditions, such as biofilm formation, amount of nutrients and incubation temperature. The results obtained using short (micromethod) and long (macromethod) term methods showed that L. pneumophila Lp-1 dies rapidly at 4 degrees C in the "macromethod" assay. When the same temperature (4 degrees C) was applied to the "micromethod" assay, L. pneumophila Lp-1 survived for three weeks, although it progressively decreased. At an incubation temperature of 30 degrees C, the aquatic environment was more favourable and better survival emerged in the "macromethod"; in contrast, this favourable temperature condition did not improve the survival of L. pneumophila Lp-1 cultured with the "micromethod". The role of the protozoa Acanthamoeba polyphaga proved to be indispensable for legionella survival only when environmental conditions become unfavourable.

  1. A case of community-acquired pneumonia due to Legionella pneumophila serogroup 9 in which initial treatment with single-dose oral azithromycin appeared useful.

    PubMed

    Ito, Akihiro; Ishida, Tadashi; Tachibana, Hiromasa; Ito, Yuhei; Takaiwa, Takuya; Fujii, Hiroyuki; Hashimoto, Toru; Nakajima, Hiroshi; Amemura-Maekawa, Junko

    2017-09-11

    Legionella species are important causative pathogens for severe community-acquired pneumonia (CAP). Most cases of Legionella pneumonia are due to Legionella pneumophila serogroup 1, and CAP due to Legionella pneumophila serogroup 9 is rare. A fourth case of CAP due to Legionella pneumophila serogroup 9 is reported, and initial treatment using single-dose oral azithromycin appeared useful. Azithromycin injection or fluoroquinolone injection is usually recommended for the treatment of Legionella pneumonia, and no previous reports have shown the effectiveness of single-dose oral azithromycin. This case report is therefore valuable from the perspective of possible treatment for mild to moderate Legionella pneumonia using single-dose oral azithromycin.

  2. Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages

    PubMed Central

    Jung, Anna Lena; Stoiber, Cornelia; Herkt, Christina E.; Schulz, Christine; Bertrams, Wilhelm; Schmeck, Bernd

    2016-01-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila), a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a’s targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host. PMID:27105429

  3. Hospital-acquired pneumonia and bacteremia caused by Legionella pneumophila in an immunocompromised patient.

    PubMed

    Lai, C-C; Tan, C-K; Chou, C-H; Hsu, H-L; Huang, Y-T; Liao, C-H; Hsueh, P-R

    2010-04-01

    The Legionella species is an important cause of communityand hospital-acquired pneumonia. Bacteremic pneumonia caused by L. pneumophila is rarely reported. We describe the first reported case of hospital-acquired pneumonia and bacteremia caused by L. pneumophila from Taiwan in a patient with idiopathic thrombocytopenic purpura who received steroid treatment. The patient was successfully treated with ceftazidime and clindamycin initially, followed by ciprofloxacin for 14 days. The blood isolate was further confirmed by 16S rDNA sequence analysis.

  4. Complete Genome Sequences of Three Outbreak-Associated Legionella pneumophila Isolates

    PubMed Central

    Morrison, Shatavia S.; Desai, Heta P.; Mercante, Jeffrey W.; Lapierre, Pascal; Raphael, Brian H.; Musser, Kimberlee

    2016-01-01

    We report here the complete genome sequences of three Legionella pneumophila isolates that are associated with a Legionnaires’ disease outbreak in New York in 2012. Two clinical isolates (D7630 and D7632) and one environmental isolate (D7631) were recovered from this outbreak. A single isolate-specific virulence gene was found in D7632. These isolates were included in a large study evaluating the genomic resolution of various bioinformatics approaches for L. pneumophila serogroup 1 isolates. PMID:27445383

  5. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila

    PubMed Central

    Chiaraviglio, Lucius

    2015-01-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  6. Soil as a source of Legionella pneumophila serogroup 1 (Lp1).

    PubMed

    Wallis, Lara; Robinson, Priscilla

    2005-12-01

    To investigate the potential source of a case of Legionnaires' disease caused by an unusual serotype of Legionella pneumophila serogroup 1 (Lp1) in regional Victoria in May 2001. Epidemiological and environmental investigation of the source of infection of a case of Legionnaires' disease in regional Victoria in May 2001. Extensive environmental investigations did not reveal any cooling water tower systems close to the residence or the shopping centre that the case visited prior to illness. The sputum culture and a soil sample from the field at the plant nursery where the case worked prior to illness were both positive for Legionella pneumophilia serogroup 1, MDU pulsovar 97:103. Legionella pneumophila has been found in soil and was further found to be associated with a case of Legionella pneumophila. Public health authorities should consider exposures to soil and potting mixes when investigating cases of Legionella pneumophila where the case has no apparent association with cooling towers. Safe gardening practices should be promoted among the community.

  7. The Legionella pneumophila Collagen-Like Protein Mediates Sedimentation, Autoaggregation, and Pathogen-Phagocyte Interactions

    PubMed Central

    Abdel-Nour, Mena; Duncan, Carla; Prashar, Akriti; Rao, Chitong; Ginevra, Christophe; Jarraud, Sophie; Low, Donald E.; Ensminger, Alexander W.; Terebiznik, Mauricio R.

    2014-01-01

    Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae. PMID:24334670

  8. The Legionella pneumophila collagen-like protein mediates sedimentation, autoaggregation, and pathogen-phagocyte interactions.

    PubMed

    Abdel-Nour, Mena; Duncan, Carla; Prashar, Akriti; Rao, Chitong; Ginevra, Christophe; Jarraud, Sophie; Low, Donald E; Ensminger, Alexander W; Terebiznik, Mauricio R; Guyard, Cyril

    2014-02-01

    Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae.

  9. Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

    PubMed Central

    Suter, T M; Viswanathan, V K; Cianciotto, N P

    1997-01-01

    A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae. PMID:9174205

  10. Preparation, Characterization, and Efficacy of Cell Wall and Ribosomal Vaccines from Legionella Pneumophila

    DTIC Science & Technology

    1981-05-01

    protection agaifist infection with Legionella pneumophila, the causative agent of Legionnaires ’ disease (15). Altheugh the guinea pig is the recomaended...response to Legionella uneuoph.ila ( Legionnaires ’ disease bacterium). J. Clin. Nicrobiol. 11:200-201. 29. Tsai. T. F., D. R. Finn, B. D. Plikavtis, W...preparations. _ _ _ __ _ __ _ __ _ __ _= ’I The epidemiology and clinical manifestations of Legionnaires ’ disease have been extensively defined (13, 22, 29, 30

  11. Necessity and Effect of Combating Legionella pneumophila in Municipal Shower Systems

    PubMed Central

    Wiik, Ragnhild; Krøvel, Anne Vatland

    2014-01-01

    The objective was to obtain research-based, holistic knowledge about necessity and effect of practiced measures against L. pneumophila in municipal shower systems in Stavanger, Norway. The effects of hot water treatment and membrane-filtering were investigated and compared to no intervention at all. The studies were done under real-world conditions. Additionally, a surveillance pilot study of municipal showers in Stavanger was performed. The validity of high total plate count (TPC) as an indication of L. pneumophila was evaluated. A simplified method, named “dripping method”, for detection and quantification of L. pneumophila was developed. The sensitivity of the dripping method is 5 colony-forming units of L. pneumophila/ml. The transference of L. pneumophila from shower water to aerosols was studied. Interviews and observational studies among the stakeholders were done in order to identify patterns of communication and behavior in a Legionella risk perspective. No substantial effects of the measures against L. pneumophila were demonstrated, except for a distally placed membrane filter. No significant positive correlation between TPC and L. pneumophila concentrations were found. L. pneumophila serogroup 2–14 was demonstrated in 21% of the 29 buildings tested in the surveillance pilot. Relatively few cells of L. pneumophila were transferred from shower water to aerosols. Anxiety appeared as the major driving force in the risk governance of Legionella. In conclusion, the risk of acquiring Legionnaires' disease from municipal shower systems is evaluated as low and uncertain. By eliminating ineffective approaches, targeted Legionella risk governance can be practiced. Risk management by surveillance is evaluated as appropriate. PMID:25490721

  12. Necessity and effect of combating Legionella pneumophila in municipal shower systems.

    PubMed

    Wiik, Ragnhild; Krøvel, Anne Vatland

    2014-01-01

    The objective was to obtain research-based, holistic knowledge about necessity and effect of practiced measures against L. pneumophila in municipal shower systems in Stavanger, Norway. The effects of hot water treatment and membrane-filtering were investigated and compared to no intervention at all. The studies were done under real-world conditions. Additionally, a surveillance pilot study of municipal showers in Stavanger was performed. The validity of high total plate count (TPC) as an indication of L. pneumophila was evaluated. A simplified method, named "dripping method", for detection and quantification of L. pneumophila was developed. The sensitivity of the dripping method is 5 colony-forming units of L. pneumophila/ml. The transference of L. pneumophila from shower water to aerosols was studied. Interviews and observational studies among the stakeholders were done in order to identify patterns of communication and behavior in a Legionella risk perspective. No substantial effects of the measures against L. pneumophila were demonstrated, except for a distally placed membrane filter. No significant positive correlation between TPC and L. pneumophila concentrations were found. L. pneumophila serogroup 2-14 was demonstrated in 21% of the 29 buildings tested in the surveillance pilot. Relatively few cells of L. pneumophila were transferred from shower water to aerosols. Anxiety appeared as the major driving force in the risk governance of Legionella. In conclusion, the risk of acquiring Legionnaires' disease from municipal shower systems is evaluated as low and uncertain. By eliminating ineffective approaches, targeted Legionella risk governance can be practiced. Risk management by surveillance is evaluated as appropriate.

  13. Endemicity of Legionella pneumophila serogroup 3 in a hospital water supply.

    PubMed

    Franzin, L; Castellani Pastoris, M; Gioannini, P; Villani, G

    1989-04-01

    A microbiological and epidemiological investigation at the Infectious Diseases Hospital in Turin, Italy, demonstrated Legionella pneumophila serogroup 3 at 10(2) to greater than 4 X 10(3) cfu l-1 from 24 of 32 hot water samples collected from hand-basins in six separate buildings. A sample taken from the public water supply, and a hot water sample (80 degrees C) collected from hot water tanks, did not yield legionellas. Legionella pneumophila serogroup 3 was found in samples taken at the first point of mixed hot and cold water (50 degrees C) at 3 X 10(2) cfu l-1. 12 of 26 samples from the shower-heads yielded 10(3) to 2.5 X 10(5) cfu l-1 and one of 12 water samples from oxygen bubble humidifiers tested yielded 1.6 X 10(4) cfu l-1. No other legionellas species or serogroups of Legionella pneumophila were isolated during the study. No cases of nosocomial pneumonia were detected among 3653 patients' records, nor was there serological evidence of Legionella infection in the 180 patients tested.

  14. Widespread molecular detection of Legionella pneumophila Serogroup 1 in cold water taps across the United States.

    PubMed

    Donohue, Maura J; O'Connell, Katharine; Vesper, Stephen J; Mistry, Jatin H; King, Dawn; Kostich, Mitch; Pfaller, Stacy

    2014-03-18

    In the United States, 6,868 cases of legionellosis were reported to the Center for Disease Control and Prevention in 2009-2010. Of these reports, it is estimated that 84% are caused by the microorganism Legionella pneumophila Serogroup (Sg) 1. Legionella spp. have been isolated and recovered from a variety of natural freshwater environments. Human exposure to L. pneumophila Sg1 may occur from aerosolization and subsequent inhalation of household and facility water. In this study, two primer/probe sets (one able to detect L. pneumophila and the other L. pneumophila Sg1) were determined to be highly sensitive and selective for their respective targets. Over 272 water samples, collected in 2009 and 2010 from 68 public and private water taps across the United States, were analyzed using the two qPCR assays to evaluate the incidence of L. pneumophila Sg1. Nearly half of the taps showed the presence of L. pneumophila Sg1 in one sampling event, and 16% of taps were positive in more than one sampling event. This study is the first United States survey to document the occurrence and colonization of L. pneumophila Sg1 in cold water delivered from point of use taps.

  15. Subtyping of Legionella pneumophila isolates by arbitrarily primed polymerase chain reaction.

    PubMed

    Ledesma, E; Camaró, M L; Carbonell, E; Sacristán, T; Martí, A; Pellicer, S; Llorca, J; Herrero, P; Dasí, M A

    1995-09-01

    Arbitrarily primed polymerase chain reaction (AP-PCR) was used to differentiate strains of Legionella pneumophila isolated from different water sources in a resort hotel in Benidorm, Alicante, Spain, where an outbreak of Legionnaires' disease occurred among a group of tourists between 65 and 80 years of age. All isolates were L. pneumophila serogroup 1, subtype Pontiac (Knoxville 1). Five different patterns (P1 to P5) were obtained by AP-PCR. The number of bands per pattern varied between 4 and 11. Patterns P1 and P2 represented 60 and 20% of L. pneumophila isolates, respectively. Since different subpopulations of L. pneumophila coexisted (up to three different AP-PCR patterns were identified in a single room), it was not possible to link an individual L. pneumophila strain to the occurrence of this outbreak.

  16. Antibacterial activities of gemifloxacin, levofloxacin, gatifloxacin, moxifloxacin and erythromycin against intracellular Legionella pneumophila and Legionella micdadei in human monocytes.

    PubMed

    Baltch, Aldona L; Bopp, Lawrence H; Smith, Raymond P; Michelsen, Phyllis B; Ritz, William J

    2005-07-01

    The antibacterial activity of a new fluoroquinolone, gemifloxacin, was tested against intracellular Legionella pneumophila and Legionella micdadei and was compared with the activities of levofloxacin, gatifloxacin, moxifloxacin and erythromycin. For intracellular assays, bacteria were used to infect human monocyte-derived macrophages prepared from heparinized blood of healthy volunteers. Antibiotics were added following phagocytosis. Numbers of viable bacteria were determined at 0, 24, 48, 72 and 96 h. The intracellular antibacterial activity of gemifloxacin was concentration- and time-dependent. All of the quinolones had similar activities against L. pneumophila and L. micdadei at 10 x MIC, but there were minor differences: at 24 h moxifloxacin was significantly more active than the other quinolones against L. pneumophila, while gemifloxacin was more active against L. micdadei (P < 0.01). All of the quinolones were markedly more active than erythromycin (P < 0.01). The antibacterial effect of gemifloxacin against L. pneumophila following drug removal at 24 h persisted for 72 h at 20 x MIC but not at 10 x MIC, while for L. micdadei the antibacterial effect persisted for 24 h at 10 x MIC. All of the quinolones had similar activities against intracellular L. pneumophila and L. micdadei and were markedly more effective than erythromycin.

  17. Isolation on Chocolate Agar Culture of Legionella pneumophila Isolates from Subcutaneous Abscesses in an Immunocompromised Patient

    PubMed Central

    Cavalie, Laurent; Daviller, Benjamin; Dubois, Damien; Mantion, Benoît; Delobel, Pierre; Debard, Alexa; Prere, Marie-Françoise; Marchou, Bruno; Martin-Blondel, Guillaume

    2015-01-01

    Cutaneous infections due to Legionella species have rarely been reported (L. J. Padrnos, J. E. Blair, S. Kusne, D. J. DiCaudo, and J. R. Mikhael, Transpl Infect Dis 16:307–314, 2014; P. W. Lowry, R. J. Blankenship, W. Gridley, N. J. Troup, and L. S. Tompkins, N Engl J Med 324:109–113, 1991; M. K. Waldor, B. Wilson, and M. Swartz, Clin Infect Dis 16:51–53, 1993). Here we report the identification of Legionella pneumophila isolates, from subcutaneous abscesses in an immunocompromised patient, that grew in an unusual medium for Legionella bacteria. PMID:26292305

  18. Isolation on Chocolate Agar Culture of Legionella pneumophila Isolates from Subcutaneous Abscesses in an Immunocompromised Patient.

    PubMed

    Barigou, Mohammed; Cavalie, Laurent; Daviller, Benjamin; Dubois, Damien; Mantion, Benoît; Delobel, Pierre; Debard, Alexa; Prere, Marie-Françoise; Marchou, Bruno; Martin-Blondel, Guillaume

    2015-11-01

    Cutaneous infections due to Legionella species have rarely been reported (L. J. Padrnos, J. E. Blair, S. Kusne, D. J. DiCaudo, and J. R. Mikhael, Transpl Infect Dis 16:307-314, 2014; P. W. Lowry, R. J. Blankenship, W. Gridley, N. J. Troup, and L. S. Tompkins, N Engl J Med 324:109-113, 1991; M. K. Waldor, B. Wilson, and M. Swartz, Clin Infect Dis 16:51-53, 1993). Here we report the identification of Legionella pneumophila isolates, from subcutaneous abscesses in an immunocompromised patient, that grew in an unusual medium for Legionella bacteria.

  19. Effects of culture conditions and biofilm formation on the iodine susceptibility of Legionella pneumophila

    NASA Technical Reports Server (NTRS)

    Cargill, K. L.; Pyle, B. H.; Sauer, R. L.; McFeters, G. A.

    1992-01-01

    The susceptibility of Legionella pneumophila to iodination was studied with cultures grown in well water, on rich agar media, and attached to stainless-steel surfaces. Legionella pneumophila grown in water cultures in association with other microorganisms were less sensitive to disinfection by chlorine and iodine than were agar-passaged cultures. Differences in sensitivity to disinfection between water-cultured and agar-grown legionellae were determined by comparing C x T values (concentration in milligrams per litre multiplied by time in minutes to achieve 99% decrease in viability) and CM x T values (concentration in molarity). Iodine (1500x) gave a greater difference in CM x T values than did chlorine (68x). Iodine was 50 times more effective than chlorine when used with agar-grown cultures but was only twice as effective when tested against water-grown Legionella cultures. C x T x S values (C x T multiplied by percent survivors), which take into consideration the percent surviving bacteria, were used to compare sensitivities in very resistant populations, such as those in biofilms. Water cultures of legionellae associated with stainless-steel surfaces were 135 times more resistant to iodination than were unattached legionellae, and they were 210,000 times more resistant than were agar-grown cultures. These results indicate that the conditions under which legionellae are grown can dramatically affect their susceptibility to some disinfectants and must be considered when evaluating the efficacy of a disinfecting agent.

  20. Effects of culture conditions and biofilm formation on the iodine susceptibility of Legionella pneumophila

    NASA Technical Reports Server (NTRS)

    Cargill, K. L.; Pyle, B. H.; Sauer, R. L.; McFeters, G. A.

    1992-01-01

    The susceptibility of Legionella pneumophila to iodination was studied with cultures grown in well water, on rich agar media, and attached to stainless-steel surfaces. Legionella pneumophila grown in water cultures in association with other microorganisms were less sensitive to disinfection by chlorine and iodine than were agar-passaged cultures. Differences in sensitivity to disinfection between water-cultured and agar-grown legionellae were determined by comparing C x T values (concentration in milligrams per litre multiplied by time in minutes to achieve 99% decrease in viability) and CM x T values (concentration in molarity). Iodine (1500x) gave a greater difference in CM x T values than did chlorine (68x). Iodine was 50 times more effective than chlorine when used with agar-grown cultures but was only twice as effective when tested against water-grown Legionella cultures. C x T x S values (C x T multiplied by percent survivors), which take into consideration the percent surviving bacteria, were used to compare sensitivities in very resistant populations, such as those in biofilms. Water cultures of legionellae associated with stainless-steel surfaces were 135 times more resistant to iodination than were unattached legionellae, and they were 210,000 times more resistant than were agar-grown cultures. These results indicate that the conditions under which legionellae are grown can dramatically affect their susceptibility to some disinfectants and must be considered when evaluating the efficacy of a disinfecting agent.

  1. Nosocomial transmission of Legionella pneumophila to a child from a hospital's cold-water supply.

    PubMed

    Johansson, P J Hugo; Andersson, Kaj; Wiebe, Thomas; Schalén, Claes; Bernander, Sverker

    2006-01-01

    Human Legionella infections mainly consist of community-acquired and nosocomial pneumonia and rarely affect children. We describe a nosocomial infection with Legionella pneumophila, serogroup 1, subgroup OLDA, in an immunocompromized 2-y-old girl at a paediatric clinic. L. pneumophila identical to that of the patient was found in the hospital's cold-water but not in the hot-water distribution system. Transmission of Legionella to the girl most probably occurred by Legionella-contaminated cold water mixed and heated by water from the hot-water system. Mixing of hot and cold water probably occurred through thermostatic water mixing valves connected to showers regulated by a handle at the shower head. Nosocomial Legionella infection might thus have occurred, although circulating hot water temperatures never dropped below 53 degrees C and cultures for surveillance of Legionella from central parts of the hot-water system have been consistently negative. Legionellae were successfully eliminated from the hospital's cold-water distribution system by hot water flushing at 73 degrees C for 1h.

  2. Legionnaires' disease caused by Legionella longbeachae and Legionella pneumophila: comparison of clinical features, host-related risk factors, and outcomes.

    PubMed

    Amodeo, M R; Murdoch, D R; Pithie, A D

    2010-09-01

    Legionnaires' disease remains an important cause of mortality and morbidity worldwide. Disease caused by Legionella pneumophila has been extensively studied, and its clinical characteristics have been well described. There is, however, little information on disease caused by Legionella longbeachae, despite its importance in some countries. We undertook a retrospective review of culture-positive cases of Legionnaires' disease in the Canterbury region of New Zealand over 10 years, in order to compare the clinical features and outcomes of Legionnaires' disease caused by these two species.

  3. Complete Genome Sequences of Legionella pneumophila subsp. fraseri Strains Detroit-1 and Dallas 1E.

    PubMed

    Raphael, Brian H; Kozak-Muiznieks, Natalia A; Morrison, Shatavia S; Mercante, Jeffrey W; Winchell, Jonas M

    2017-02-02

    We report here the complete genome sequences of two of the earliest known strains of Legionella pneumophila subsp. fraseri Detroit-1 is serogroup 1 and was isolated from a lung biopsy specimen in 1977. Dallas 1E is serogroup 5 and was isolated in 1978 from a cooling tower.

  4. Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.

    PubMed Central

    Steinert, M; Emödy, L; Amann, R; Hacker, J

    1997-01-01

    Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model. PMID:9143134

  5. Complete Genome Sequences of the Historical Legionella pneumophila Strains OLDA and Pontiac

    PubMed Central

    Mercante, Jeffrey W.; Morrison, Shatavia S.; Raphael, Brian H.

    2016-01-01

    Here, we report the complete genome sequences of Legionella pneumophila serogroup 1 strains OLDA and Pontiac, which predate the 1976 Philadelphia Legionnaires’ disease outbreak. Strain OLDA was isolated in 1947 from an apparent sporadic case, and strain Pontiac caused an explosive outbreak at a Michigan health department in 1968. PMID:27563044

  6. Complete Genome Sequences of Legionella pneumophila subsp. fraseri Strains Detroit-1 and Dallas 1E

    PubMed Central

    Raphael, Brian H.; Kozak-Muiznieks, Natalia A.; Morrison, Shatavia S.; Mercante, Jeffrey W.

    2017-01-01

    ABSTRACT We report here the complete genome sequences of two of the earliest known strains of Legionella pneumophila subsp. fraseri. Detroit-1 is serogroup 1 and was isolated from a lung biopsy specimen in 1977. Dallas 1E is serogroup 5 and was isolated in 1978 from a cooling tower. PMID:28153889

  7. The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire's disease.

    PubMed Central

    Brieland, J K; Fantone, J C; Remick, D G; LeGendre, M; McClain, M; Engleberg, N C

    1997-01-01

    Legionella pneumophila is a bacterial parasite of many species of freshwater protozoa and occasionally an intracellular pathogen of humans. While protozoa are known to play a key role in the persistence of L. pneumophila in the environment, there has been limited research addressing the potential role of L. pneumophila-infected protozoa in the pathogenesis of human infection. In this report, the potential role of an L. pneumophila-infected amoeba as an infectious particle in replicative L. pneumophila lung infection was investigated in vivo with the amoeba Hartmannella vermiformis, a natural reservoir of L. pneumophila in the environment. L. pneumophila-infected H. vermiformis organisms were prepared by coculture of the amoebae and virulent L. pneumophila cells in vitro. A/J mice, which are susceptible to replicative L. pneumophila lung infection, were subsequently inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms (10(6) amoebae containing 10(5) bacteria), and intrapulmonary growth of the bacteria was assessed. A/J mice inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms developed replicative L. pneumophila lung infections. Furthermore, L. pneumophila-infected H. vermiformis organisms were more pathogenic than an equivalent number of bacteria or a coinoculum of L. pneumophila cells and uninfected amoebae. These results demonstrate that L. pneumophila-infected amoebae are infectious particles in replicative L. pneumophila infections in vivo and support the hypothesis that inhaled protozoa may serve as cofactors in the pathogenesis of pulmonary disease induced by inhaled respiratory pathogens. PMID:9393834

  8. The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire's disease.

    PubMed

    Brieland, J K; Fantone, J C; Remick, D G; LeGendre, M; McClain, M; Engleberg, N C

    1997-12-01

    Legionella pneumophila is a bacterial parasite of many species of freshwater protozoa and occasionally an intracellular pathogen of humans. While protozoa are known to play a key role in the persistence of L. pneumophila in the environment, there has been limited research addressing the potential role of L. pneumophila-infected protozoa in the pathogenesis of human infection. In this report, the potential role of an L. pneumophila-infected amoeba as an infectious particle in replicative L. pneumophila lung infection was investigated in vivo with the amoeba Hartmannella vermiformis, a natural reservoir of L. pneumophila in the environment. L. pneumophila-infected H. vermiformis organisms were prepared by coculture of the amoebae and virulent L. pneumophila cells in vitro. A/J mice, which are susceptible to replicative L. pneumophila lung infection, were subsequently inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms (10(6) amoebae containing 10(5) bacteria), and intrapulmonary growth of the bacteria was assessed. A/J mice inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms developed replicative L. pneumophila lung infections. Furthermore, L. pneumophila-infected H. vermiformis organisms were more pathogenic than an equivalent number of bacteria or a coinoculum of L. pneumophila cells and uninfected amoebae. These results demonstrate that L. pneumophila-infected amoebae are infectious particles in replicative L. pneumophila infections in vivo and support the hypothesis that inhaled protozoa may serve as cofactors in the pathogenesis of pulmonary disease induced by inhaled respiratory pathogens.

  9. Travel-associated infections caused by unusual serogroups of Legionella pneumophila identified using Legionella BIOCHIP slides in Turkey and Iraq.

    PubMed

    Kocazeybek, Bekir S; Yuksel, Pelin; Keskin, Dilek; Sheikh, Suhail; Habip, Zafer; Yavuzer, Serap Sahin; Caliskan, Reyhan; Altun, Yagız Meric; Kuskucu, Mert; Cengiz, Mahir; Dinc, Harika Oyku; Karakullukcu, Asiye; Ergin, Sevgi; Saribas, Suat; Yilmaz, Nail; Tokman, Hrisi Bahar

    2016-01-01

    Although Legionella pneumophila serogroup 1 is the common disease causing serogroup, rare serogroups can also may cause legionellosis. A 54-year-old male patient (index case) reported that he had been on a religious trip (for visiting, tomb of Ali, which is important for Shias) to Iraq with a large group (50 shia pilgrims from Kars city of Turkey) two weeks prior to admission. Due to civil war, the hotel where the patient stayed in Iraq lacked proper hygiene. A large number of people in the travel group were experiencing the same symptoms. Other five cases were 2 males (ages; 50, 45) and 3 females including the wife of the index case (ages; 50, 28, 27). The detection of L. pneumophila IgG and IgM was performed by anti-L. pneumophila Indirect Immunofluorescent IgM, IgG kit. Legionella 1 biochip/verification BIOCHIP slides were used for serogrouping in Euroimmun AG, Leubeck, Germany. In index case, L. pneumophila IgM was positive with a titer of 1/32 titer. IgG was negative with a 1/100 titer. Another case (28 year old female), had clinical symptoms identical to the index case. L. pneumophila IgM and IgG were positive with titers of 1/64 and 1/100, respectively. These two cases were diagnosed with Legionnaires' disease caused by L. pneumophila serogroup 12 (index case) and female (28-year-old) by serogroup 11. The other 4 cases were diagnosed with possible Pontiac fever caused by L. pneumophila serogroups 14 (wife of the index case), 4, and 6 whereas the serogroup of L. pneumophila detected in 27 years old female case could not be identified. A major limitation of this work is the absence of genotyping and the serogroup difference between index case and his wife who shared the same hotel. We suggest that this serogroup difference may be caused by (for men and women) sitting separately in Islamic rules. On the other hand, the movement of people in the context of mutual visits between countries or neighboring countries for tourism-related (i.e., for religious events

  10. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets

    PubMed Central

    Supuran, Claudiu T.

    2016-01-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4–8.3) × 105 s−1 and kcat/KM values of (4.7–8.5) × 107 M−1·s−1. In vitro inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates, were also reported for the two β-CAs from this pathogen, LpCA1 and LpCA2. Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3–90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2–88.5 nM). Considering such preliminary results, the two bacterial CAs from this pathogen represent promising yet underexplored targets for obtaining antibacterials devoid of the resistance problems common to most of the clinically used antibiotics, but further studies are needed to validate them in vivo as drug targets. PMID:27322334

  11. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay

    PubMed Central

    Shen, Shu-Min; Chou, Ming-Yuan; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-01-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 102 and 3.3 × 105 cells/l in river water and 72.1–5.7 × 106 cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors. PMID:26184706

  12. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.

    PubMed

    Shen, Shu-Min; Chou, Ming-Yuan; Hsu, Bing-Mu; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-07-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

  13. WIN 57273 is bactericidal for Legionella pneumophila grown in alveolar macrophages.

    PubMed Central

    Edelstein, P H; Edelstein, M A

    1989-01-01

    The in vitro antimicrobial activity of WIN 57273, a new quinolone antimicrobial agent, was determined for 21 Legionella strains, using broth macrodilution and agar dilution testing methods; ciprofloxacin and erythromycin were tested as well. Three different buffered yeast extract media were used for the agar dilution studies, two of which were made with starch rather than charcoal. Broth macrodilution susceptibility testing was performed with buffered yeast extract broth and two Legionella pneumophila strains. Antimicrobial inhibition of L. pneumophila growth in guinea pig alveolar macrophages was also studied, using a method able to detect bacterial killing. The MICs for 90% of the 21 strains of Legionella spp. grown on buffered charcoal yeast extract medium were 0.125 microgram/ml for WIN 57273, 0.25 microgram/ml for ciprofloxacin, and 1.0 micrograms/ml for erythromycin. These MICs were falsely high, because of inhibition of drug activity by the medium used. Use of less drug-antagonistic, starch-containing media did not support good growth of the test strains. The broth macrodilution MICs for two strains of L. pneumophila serogroup 1 were less than or equal to 0.03 microgram/ml for WIN 57273 and ciprofloxacin and 0.125 microgram/ml for erythromycin. WIN 57273, ciprofloxacin, and erythromycin all inhibited growth of L. pneumophila in guinea pig alveolar macrophages at concentrations of 1 microgram/ml, but only WIN 57273 prevented regrowth or killed L. pneumophila after removal of extracellular antimicrobial agent. PMID:2619277

  14. Prevalence of Legionella pneumophila serogroup 1 in water distribution systems in Izmir province of Turkey.

    PubMed

    Uzel, Ataç; Uçar, Füsun; Hameş-Kocabaş, E Esin

    2005-10-01

    Legionella pneumophila serogroup 1 occurrence has been investigated in 168 hot water samples from 24 hotels, situated in 6 counties in Izmir province of Turkey, from 15 June to 30 September of the year 2000. Sampling was carried out at 15-day intervals and seven samples were taken from each of the hotels' hot water reservoirs and hot water networks. The samples were (1 L) concentrated using polycarbonate filters (mesh size 0.22 microm). Isolation was achieved using selective medium, GVPC agar. The samples were concentrated by membrane filtration, divided into three portions and cultured without pretreatment, after acid treatment, and after heat treatment, on GVPC agar. One hundred and ten isolates were identified as L.pneumophila sg 1 using the Legionella Latex Test (Oxoid). Arbitrarily primed PCR (AP PCR) was employed to assess the clonal relationship between Legionella pneumophila sg 1 isolates from the hot water samples of the hotels. Three genotypes of L. pneumophila sg 1 isolates were identified. With a high prevalence of type A, 22 hotels were found to be colonized with L. pneumophila serogroup 1, while only 2 were free from the bacteria.

  15. Detection limits of Legionella pneumophila in environmental samples after co-culture with Acanthamoeba polyphaga

    PubMed Central

    2013-01-01

    Background The efficiency of recovery and the detection limit of Legionella after co-culture with Acanthamoeba polyphaga are not known and so far no investigations have been carried out to determine the efficiency of the recovery of Legionella spp. by co-culture and compare it with that of conventional culturing methods. This study aimed to assess the detection limits of co-culture compared to culture for Legionella pneumophila in compost and air samples. Compost and air samples were spiked with known concentrations of L. pneumophila. Direct culturing and co-culture with amoebae were used in parallel to isolate L. pneumophila and recovery standard curves for both methods were produced for each sample. Results The co-culture proved to be more sensitive than the reference method, detecting 102-103 L. pneumophila cells in 1 g of spiked compost or 1 m3 of spiked air, as compared to 105-106 cells in 1 g of spiked compost and 1 m3 of spiked air. Conclusions Co-culture with amoebae is a useful, sensitive and reliable technique to enrich L. pneumophila in environmental samples that contain only low amounts of bacterial cells. PMID:23442526

  16. First Case of Legionnaire's Disease Caused by Legionella anisa in Spain and the Limitations on the Diagnosis of Legionella non-pneumophila Infections.

    PubMed

    Vaccaro, Lucianna; Izquierdo, Fernando; Magnet, Angela; Hurtado, Carolina; Salinas, Mireya A; Gomes, Thiago Santos; Angulo, Santiago; Salso, Santiago; Pelaez, Jesús; Tejeda, Maria Isabel; Alhambra, Almudena; Gómez, Carmen; Enríquez, Ana; Estirado, Eva; Fenoy, Soledad; Del Aguila, Carmen

    2016-01-01

    Legionnaires' disease is a severe form of pneumonia, with worldwide relevance, caused by Legionella spp. Approximately 90% of all cases of legionellosis are caused by Legionella pneumophila, but other species can also be responsible for this infection. These bacteria are transmitted by inhalation of aerosols or aspiration of contaminated water. In Spain, environmental studies have demonstrated the presence of Legionella non-pneumophila species in drinking water treatment plants and water distribution networks. Aware that this evidence indicates a risk factor and the lack of routine assays designed to detect simultaneously diverse Legionella species, we analyzed 210 urine samples from patients presenting clinical manifestations of pneumonia using a semi-nested PCR for partial amplification of the 16S rDNA gene of Legionella and a diagnostic method used in hospitals for Legionella antigen detection. In this study, we detected a total of 15 cases of legionellosis (7.1%) and the first case of Legionnaires' disease caused by L. anisa in Spain. While the conventional method used in hospitals could only detect four cases (1.9%) produced by L. pneumophila serogroup 1, using PCR, the following species were identified: Legionella spp. (10/15), L. pneumophila (4/15) and L. anisa (1/15). These results suggest the need to change hospital diagnostic strategies regarding the identification of Legionella species associated with this disease. Therefore, the detection of Legionella DNA by PCR in urine samples seems to be a suitable alternative method for a sensitive, accurate and rapid diagnosis of Legionella pneumonia, caused by L. pneumophila and also for L. non-pneumophila species.

  17. First Case of Legionnaire's Disease Caused by Legionella anisa in Spain and the Limitations on the Diagnosis of Legionella non-pneumophila Infections

    PubMed Central

    Vaccaro, Lucianna; Izquierdo, Fernando; Magnet, Angela; Hurtado, Carolina; Salinas, Mireya A.; Gomes, Thiago Santos; Angulo, Santiago; Salso, Santiago; Pelaez, Jesús; Tejeda, Maria Isabel; Alhambra, Almudena; Gómez, Carmen; Enríquez, Ana; Estirado, Eva; Fenoy, Soledad; del Aguila, Carmen

    2016-01-01

    Legionnaires’ disease is a severe form of pneumonia, with worldwide relevance, caused by Legionella spp. Approximately 90% of all cases of legionellosis are caused by Legionella pneumophila, but other species can also be responsible for this infection. These bacteria are transmitted by inhalation of aerosols or aspiration of contaminated water. In Spain, environmental studies have demonstrated the presence of Legionella non-pneumophila species in drinking water treatment plants and water distribution networks. Aware that this evidence indicates a risk factor and the lack of routine assays designed to detect simultaneously diverse Legionella species, we analyzed 210 urine samples from patients presenting clinical manifestations of pneumonia using a semi-nested PCR for partial amplification of the 16S rDNA gene of Legionella and a diagnostic method used in hospitals for Legionella antigen detection. In this study, we detected a total of 15 cases of legionellosis (7.1%) and the first case of Legionnaires’ disease caused by L. anisa in Spain. While the conventional method used in hospitals could only detect four cases (1.9%) produced by L. pneumophila serogroup 1, using PCR, the following species were identified: Legionella spp. (10/15), L. pneumophila (4/15) and L. anisa (1/15). These results suggest the need to change hospital diagnostic strategies regarding the identification of Legionella species associated with this disease. Therefore, the detection of Legionella DNA by PCR in urine samples seems to be a suitable alternative method for a sensitive, accurate and rapid diagnosis of Legionella pneumonia, caused by L. pneumophila and also for L. non-pneumophila species. PMID:27442238

  18. Ecological behaviour of three serogroups of Legionella pneumophila within a model plumbing system.

    PubMed

    Messi, P; Anacarso, I; Bargellini, A; Bondi, M; Marchesi, I; de Niederhäusern, S; Borella, P

    2011-02-01

    Three Legionella pneumophila strains isolated from water samples and belonging to serogroups (sgs) 1, 6 and 9 were analysed for their capacity to colonise an experimental model simulating a domestic hot water distribution system. Ecological factors that could influence the persistence of the sgs such as intracellular life within protozoan hosts and bacterial interference by the production of antagonistic compounds were also studied. Viable counts of L. pneumophila increased both in the planktonic and in the sessile phases. Sg 6 showed a marked prevalence during the whole experiment and exhibited the highest host infection efficiency. Sg 1 was significantly less represented, but showed the highest capacity to reproduce in the protozoan hosts. Sg 9 was poorly represented and less adapted to intracellular life. Among the 14 bacteria constantly isolated in the system, five (35.7%) produced antagonistic substances against Legionella, with differences according to the bacterial strain and L. pneumophila sgs.

  19. Role of biofilm in protection of the replicative form of Legionella pneumophila.

    PubMed

    Andreozzi, Elisa; Di Cesare, Andrea; Sabatini, Luigia; Chessa, Elisa; Sisti, Davide; Rocchi, Marco; Citterio, Barbara

    2014-12-01

    The dual nature of Legionella pneumophila enables its survival in free and intracellular environments and underpins its infection and spread mechanisms. Experiments using bacterial cultures and improved RTqPCR protocols were devised to gain fresh insights into the role of biofilm in protecting the replicative form of L. pneumophila. mip gene expression was used as a marker of virulence in sessile (biofilm-bound) and planktonic (free-floating) cells of L. pneumophila serotype 1 ATCC 33152. The ratio of mip gene expression to transcriptionally active Legionella cells increased both in sessile and free-floating cells demonstrating an up-regulation of mip gene under nutrient depletion. However, a different trend was observed between the two forms, in planktonic cells the mip gene expression/transcriptionally active Legionella cells increased until the end of the experiment, while in the biofilm such increase was observed at the end of the experiment. These findings suggest a possible association between the switch to the transmissive phase of Legionella and a mip up-regulation and a role for biofilm in preserving Legionella cells in replicative form. Moreover, it has been shown that improved RTqPCR protocols are valuable tools to explore bacterial virulence.

  20. Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR.

    PubMed

    Grúas, Cristina; Llambi, Silvia; Arruga, M Victoria

    2014-01-01

    Legionella pneumophila is the primary cause of the legionellosis diseases (90 %) (Yu et al. in J Infect Dis 186:127-128, 2002; Doleans et al. in J Clin Microbiol 42:458-460, 2004; Den Boer et al. in Clin Microbiol Infect 14:459-466, 2008). In this study, methodologies based on molecular biology were developed in order to provide a quick diagnosis of the bacterial presence in water samples of Spain. Multiplex real-time polymerase chain reaction assays were realized to target the 16S rRNA and macrophage infectivity potentiator (mip) genes of, respectively, Legionella spp. and L. pneumophila including in the design of an internal control. The results obtained by the culture and the gene amplification methods agreed in 94.44 % for the 16S rRNA gene, and a concordance of 66.67 % of the cases was obtained for the mip gene.

  1. Detection of Legionella Pneumophila in Urine and Serum Specimens of Neutropenic Febrile Patients with Haematological Malignancies

    PubMed Central

    Farzi, Nastaran; Abrehdari-Tafreshi, Zahra; Zarei, Omid; Chamani-Tabriz, Leili

    2017-01-01

    Background: Legionella pneumophila (L. pneumophila) is a gram-negative bacterium which causes ‎Legionnaires’ disease as well as Pontiac fever. The Legionella infections in patients suffering from ‎neutropenia- as a common complication of cancer chemotherapy- can distribute rapidly. We ‎aimed to detect of L. pneumophila in haematological malignancy suffering patients with ‎neutropenic fever by targeting the (macrophage infectivity potentiator) mip gene. Subjects and Methods: Serum and ‎urine specimens were obtained from 80 patients and presence of mip gene of L. pneumophila in ‎specimens was investigated by PCR. Results: The L. pneumophila infection was detected in 21 (26.2%) and 38 ‎‎(47.5%) of urine and serum specimens, respectively. Conclusion: Our findings indicated that the relative high ‎prevalence of L. pneumophila in the studied patients group which show the necessity of ‎considering this microorganism in future studies from detection and treatment point of view in ‎cancer patients. PMID:28286615

  2. Galleria mellonella apolipophorin III - an apolipoprotein with anti-Legionella pneumophila activity.

    PubMed

    Zdybicka-Barabas, Agnieszka; Palusińska-Szysz, Marta; Gruszecki, Wiesław I; Mak, Paweł; Cytryńska, Małgorzata

    2014-10-01

    The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III.

  3. The Legionella pneumophila GIG operon responds to gold and copper in planktonic and biofilm cultures

    PubMed Central

    Jwanoswki, Kathleen; Wells, Christina; Bruce, Terri; Rutt, Jennifer; Banks, Tabitha; McNealy, Tamara L.

    2017-01-01

    Legionella pneumophila contaminates man-made water systems and creates numerous exposure risks for Legionnaires’ Disease. Because copper/silver ionization is commonly used to control L. pneumophila, its mechanisms of metal response and detoxification are of significant interest. Here we describe an L. pneumophila operon with significant similarity to the GIG operon of Cupriavidus metallidurans. The Legionella GIG operon is present in a subset of strains and has been acquired as part of the ICE-βox 65-kB integrative conjugative element. We assessed GIG promoter activity following exposure of L. pneumophila to multiple concentrations of HAuCl4, CuSO4 and AgNO3. At 37°C, control stationary phase cultures exhibited GIG promoter activity. This activity increased significantly in response to 20 and 50uM HAuCl4 and CuSO4 but not in response to AgNO3. Conversely, at 26°C, cultures exhibited decreased promoter response to copper. GIG promoter activity was also induced by HAuCl4 or CuSO4 during early biofilm establishment at both temperatures. When an L. pneumophila GIG promoter construct was transformed into E. coli DH5α, cultures showed baseline expression levels that did not increase following metal addition. Analysis of L. pneumophila transcriptional regulatory mutants suggested that GIG up-regulation in the presence of metal ions may be influenced by the stationary phase sigma factor, RpoS. PMID:28463986

  4. The many forms of a pleomorphic bacterial pathogen—the developmental network of Legionella pneumophila

    PubMed Central

    Robertson, Peter; Abdelhady, Hany; Garduño, Rafael A.

    2014-01-01

    Legionella pneumophila is a natural intracellular bacterial parasite of free-living freshwater protozoa and an accidental human pathogen that causes Legionnaires' disease. L. pneumophila differentiates, and does it in style. Recent experimental data on L. pneumophila's differentiation point at the existence of a complex network that involves many developmental forms. We intend readers to: (i) understand the biological relevance of L. pneumophila's forms found in freshwater and their potential to transmit Legionnaires' disease, and (ii) learn that the common depiction of L. pneumophila's differentiation as a biphasic developmental cycle that alternates between a replicative and a transmissive form is but an oversimplification of the actual process. Our specific objectives are to provide updates on the molecular factors that regulate L. pneumophila's differentiation (Section The Differentiation Process and Its Regulation), and describe the developmental network of L. pneumophila (Section Dissecting Lp's Developmental Network), which for clarity's sake we have dissected into five separate developmental cycles. Finally, since each developmental form seems to contribute differently to the human pathogenic process and the transmission of Legionnaires' disease, readers are presented with a challenge to develop novel methods to detect the various L. pneumophila forms present in water (Section Practical Implications), as a means to improve our assessment of risk and more effectively prevent legionellosis outbreaks. PMID:25566200

  5. Influence of copper surfaces on biofilm formation by Legionella pneumophila in potable water.

    PubMed

    Gião, M S; Wilks, S A; Keevil, C W

    2015-04-01

    Legionella pneumophila is a waterborne pathogen that can cause Legionnaires' disease, a fatal pneumonia, or Pontiac fever, a mild form of disease. Copper is an antimicrobial material used for thousands of years. Its incorporation in several surface materials to control the transmission of pathogens has been gaining importance in the past decade. In this work, the ability of copper to control the survival of L. pneumophila in biofilms was studied. For that, the incorporation of L. pneumophila in polymicrobial drinking water biofilms formed on copper, PVC and PEX, and L. pneumophila mono-species biofilms formed on copper and uPVC were studied by comparing cultivable and total numbers (quantified by peptide nucleic acid (PNA) hybridisation). L. pneumophila was never recovered by culture from heterotrophic biofilms; however, PNA-positive numbers were slightly higher in biofilms formed on copper (5.9 × 10(5) cells cm(-2)) than on PVC (2.8 × 10(5) cells cm(-2)) and PEX (1.7 × 10(5) cells cm(-2)). L. pneumophila mono-species biofilms grown on copper gave 6.9 × 10(5) cells cm(-2) for PNA-positive cells and 4.8 × 10(5) CFU cm(-2) for cultivable numbers, showing that copper is not directly effective in killing L. pneumophila. Therefore previous published studies showing inactivation of L. pneumophila by copper surfaces in potable water polymicrobial species biofilms must be carefully interpreted.

  6. Iron Availability Modulates the Persistence of Legionella pneumophila in Complex Biofilms

    PubMed Central

    Portier, Emilie; Bertaux, Joanne; Labanowski, Jérôme; Hechard, Yann

    2016-01-01

    Legionella pneumophila is a pathogenic bacteria found in biofilms in freshwater. Iron is an essential nutrient for L. pneumophila growth. In this study, complex biofilms were developed using river water spiked with L. pneumophila, and the persistence of L. pneumophila in these complex biofilms was evaluated. In order to study the role of iron in the persistence of L. pneumophila, river water was supplied with either iron pyrophosphate or iron chelators (deferoxamine mesylate, DFX for ferric iron and dipyridyl, DIP for ferrous iron) to modulate iron availability. The addition of iron pyrophosphate and DFX did not markedly affect the persistence of L. pneumophila in the biofilms, whereas that of DIP had a beneficial effect. Since DIP specifically chelates ferrous iron, we hypothesized that DIP may protect L. pneumophila from the deleterious effects of ferrous iron. In conclusion, ferrous iron appears to be important for the persistence of L. pneumophila in complex biofilms. However, further studies are needed in order to obtain a better understanding of the role of ferrous iron in the behavior of this bacterium in the environment. PMID:27629106

  7. A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy.

    PubMed

    Khweek, Arwa A; Caution, Kyle; Akhter, Anwari; Abdulrahman, Basant A; Tazi, Mia; Hassan, Hoda; Majumdar, Neal; Doran, Andrew; Guirado, Evelyn; Schlesinger, Larry S; Shuman, Howard; Amer, Amal O

    2013-05-01

    Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow-derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow-derived macrophages other than the Fla mutant. Less legA9 mutant-containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila-containing vacuole for autophagy uptake.

  8. AUTOMATED DEAD-END ULTRAFILTRATION FOR ENHANCED SURVEILLANCE OF LEGIONELLA 2 PNEUMOPHILA AND LEGIONELLA SPP. IN COOLING TOWER WATERS

    SciTech Connect

    Brigmon, R.; Leskinen, S.; Kearns, E.; Jones, W.; Miller, R.; Betivas, C.; Kingsley, M.; Lim, D.

    2011-10-10

    Detection of Legionella pneumophila in cooling towers and domestic hot water systems involves concentration by centrifugation or membrane filtration prior to inoculation onto growth media or analysis using techniques such as PCR or immunoassays. The Portable Multi-use Automated Concentration System (PMACS) was designed for concentrating microorganisms from large volumes of water in the field and was assessed for enhancing surveillance of L. pneumophila at the Savannah River Site, SC. PMACS samples (100 L; n = 28) were collected from six towers between August 2010 and April 2011 with grab samples (500 ml; n = 56) being collected before and after each PMACS sample. All samples were analyzed for the presence of L. pneumophila by direct fluorescence immunoassay (DFA) using FITC-labeled monoclonal antibodies targeting serogroups 1, 2, 4 and 6. QPCR was utilized for detection of Legionella spp. in the same samples. Counts of L. pneumophila from DFA and of Legionella spp. from qPCR were normalized to cells/L tower water. Concentrations were similar between grab and PMACS samples collected throughout the study by DFA analysis (P = 0.4461; repeated measures ANOVA). The same trend was observed with qPCR. However, PMACS concentration proved advantageous over membrane filtration by providing larger volume, more representative samples of the cooling tower environment, which led to reduced variability among sampling events and increasing the probability of detection of low level targets. These data highlight the utility of the PMACS for enhanced surveillance of L. pneumophila by providing improved sampling of the cooling tower environment.

  9. Three Genome Sequences of Legionella pneumophila subsp. pascullei Associated with Colonization of a Health Care Facility

    PubMed Central

    Kozak-Muiznieks, Natalia A.; Morrison, Shatavia S.; Sammons, Scott; Rowe, Lori A.; Sheth, Mili; Frace, Michael; Lucas, Claressa E.; Loparev, Vladimir N.; Raphael, Brian H.

    2016-01-01

    Here, we report the complete genome sequences of three Legionella pneumophila subsp. pascullei strains (including both serogroup 1 and 5 strains) that were found in the same health care facility in 1982 and 2012. PMID:27151801

  10. Ruling out False-Positive Urinary Legionella pneumophila Serogroup 1 and Streptococcus pneumoniae Antigen Test Results by Heating Urine

    PubMed Central

    Pontoizeau, C.; Dangers, L.; Jarlier, V.; Luyt, C. E.; Guiller, E.; Fievet, M. H.; Lecsö-Bornet, M.; Aubry, A.

    2014-01-01

    We report here false-positive urinary Legionella pneumophila serogroup 1 and Streptococcus pneumoniae antigen test results due to rabbit antilymphocyte serum treatment and provide a simple and fast solution to rule them out by heating urine. PMID:25253788

  11. Effect of quinolones and other antimicrobial agents on cell-associated Legionella pneumophila.

    PubMed Central

    Havlichek, D; Saravolatz, L; Pohlod, D

    1987-01-01

    We evaluated the in vitro susceptibility of Legionella pneumophila ATCC 33152 (serogroup I) to 13 antibiotics alone and in combination with rifampin (0.1 mg/liter) by three methods. Extracellular susceptibility was determined by MIC determinations and time kill curves in buffered yeast extract broth, while intracellular susceptibility was determined by peripheral human monocytes in RPMI 1640 culture medium. Antibiotic concentrations equal to or greater than the broth dilution MIC inhibited or killed L. pneumophila by the time kill method, except this was not the case for trimethoprim-sulfamethoxazole. Antibiotic concentrations below the broth dilution MIC did not inhibit Legionella growth. The only antibiotic-rifampin combinations which produced improved killing of L. pneumophila by the time kill method were those in which the logarithmic growth of L. pneumophila occurred during the experiment (rosoxacin, amifloxacin, cinoxacin, trimethoprim-sulfamethoxazole, clindamycin, and doxycycline). Neither direct MICs nor time kill curve assays accurately predicted intracellular L. pneumophila susceptibility. Rifampin, erythromycin, ciprofloxacin, rosoxacin, enoxacin, amifloxacin, gentamicin, clindamycin, and doxycycline all inhibited intracellular L. pneumophila growth at readily achievable concentrations in serum. Cefoxitin and thienamycin showed no inhibition of growth, although they were present extracellularly at concentrations that were 20 to 1,000 times their broth dilution MICs. Clindamycin was the only antibiotic that was able to inhibit intracellular L. pneumophila growth at an extracellular concentration below its MIC. The gentamicin (5 mg/liter)-rifampin combination was the only antibiotic-rifampin combination which demonstrated decreased cell-associated Legionella survival in this model of in vitro susceptibility. PMID:3435101

  12. Comparison of Legionella longbeachae and Legionella pneumophila cases in Scotland; implications for diagnosis, treatment and public health response.

    PubMed

    Cameron, R L; Pollock, K G J; Lindsay, D S J; Anderson, E

    2016-02-01

    The reported incidence of Legionnaires' disease caused by Legionella longbeachae has increased since 2008 in Scotland. While microbiological and epidemiological studies have identified exposure to growing media as a risk factor for infection, little is known about the differences regarding disease risk factors, clinical features and outcomes of infection with L. longbeachae when compared with L. pneumophila. A nested case-case study was performed comparing 12 L. longbeachae cases with 25 confirmed L. pneumophila cases. Fewer L. longbeachae infected patients reported being smokers [27% (95% CI 2-52%) vs. 68% (95% CI 50-86%), P = 0.034] but more L. longbeachae patients experienced breathlessness [67% (95% CI 40-94%) vs. 28% (95% CI 10-46%), P = 0.036]. Significantly more L. longbeachae-infected patients received treatment in intensive care [50% (95% CI 22-78%) vs. 12% (95% CI 0-25%), P = 0.036]. However, the differences in diagnostic methods between the two groups may have led to only the most severe cases of L. longbeachae being captured by the surveillance system. No differences were observed in any of the other pre-hospital symptoms assessed. Our results highlight the similarity of Legionnaires' disease caused by L. pneumophila and L. longbeachae, and reinforce the importance of diagnostic tools other than the urinary antigen assays for the detection of non-L. pneumophila species. Unfortunately, cases of community-acquired pneumonia caused by Legionella species will continue to be underdiagnosed unless routine testing criteria changes.

  13. Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila

    SciTech Connect

    Barbaree, J.M.; Fields, B.S.; Feeley, J.C.; Gorman, G.W.; Martin, W.T.

    1986-02-01

    At the site of a legionellosis outbreak, amoebae and two ciliates, Tetrahymena sp. and Cyclidium sp., were isolated from cooling-tower water containing Legionella pneumophila. The Tetrahymena sp. and the amoebae repeatedly showed the ability to support intracellular multiplication of L. pneumophila. Both were isolated from cooling towers specifically implicated as the source for the spread of legionellosis. These protozoa may be reservoirs supporting the survival and multiplication of virulent legionellae in cooling-tower water.

  14. Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems

    PubMed Central

    Rogers, Julie; Dowsett, A. B.; Dennis, P. J.; Lee, J. V.; Keevil, C. W.

    1994-01-01

    A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of L. pneumophila. Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of L. pneumophila CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of L. pneumophila CFU. Images PMID:16349278

  15. [Microbial contamination of water by pipe and tubing material. 2. Growth of Legionella pneumophila].

    PubMed

    Schoenen, D; Schulze-Röbbecke, R; Schirdewahn, N

    1988-07-01

    In the 1st communication it was possible to show that some hoses and insufficiently cleaned high grade steel pipe can produce a microbial growth. The growth-promoting effect of materials in the water distribution system for Legionella pneumophila has been discussed before. In this investigation it was tested how L. pneumophila behaves in pipes and hoses with narrow diameter, at temperatures from 35 degrees C to 40 degrees C and over a time of half a year. L. pneumophila could be found in high numbers in the water from PVC, PE, PTFE, rubber and silicon hoses all over the time and regularly in low numbers or occasionally in glass, high-grade steel pipes and PA hose. L. pneumophila could be found only for the first 4 weeks in the copper pipe.

  16. Growth of Legionella pneumophila in association with blue-green algae (Cyanobacteria)

    SciTech Connect

    Tison, D.L.; Pope, D.H.; Cherry, W.B.; Fliermans, C.B.

    1980-02-01

    Legionella pneumophila (Legionnaires disease bacterium) of serogroup 1 was isolated from an algal-bacterial mat community growing at 45/sup 0/C in a man-made thermal effluent. This isolate was grown in mineral salts medium at 45/sup 0/C in association with the blue-green alga (cyanobacterium) Fischerella sp. over a pH range of 6.9 to 7.6. L. pneumophila was apparently using algal extracellular products as its carbon and energy sources. These observations indicate that the temperature, pH, and nutritional requirements of L. pneumophila are not as stringent as those previously observed when cultured on complex media. This association between L. pneumophila and certain blue-green algae suggests an explanation for the apparent widespread distribution of the bacterium in nature.

  17. Epidemiological and Environmental Investigations of Legionella pneumophila Infection in Cattle and Case Report of Fatal Pneumonia in a Calf

    PubMed Central

    Fabbi, Massimo; Pastoris, Maddalena Castellani; Scanziani, Eugenio; Magnino, Simone; Di Matteo, Luigi

    1998-01-01

    A fatal pneumonia due to Legionella pneumophila was diagnosed in a young calf reared in a dairy herd located in northern Italy. Clinical symptoms consisted of watery diarrhea, hyperthermia, anorexia, and severe dyspnea. The pathological and histological findings were very similar to those observed in human legionellosis. Legionella pneumophila serogroup 1 (SG1) and SG10 were isolated from the calf’s lung, and L. pneumophila SG1 was isolated from the calf’s liver. L. pneumophila SG1 was also demonstrated in the lung tissue by immunofluorescence and immunohistochemical examinations. Nine of 10 L. pneumophila SG1 isolates belonged to the Olda subtype, and 1 belonged to the Camperdown subtype. A very low prevalence of antibodies to Legionella was detected in cows and calves reared in the same herd. Cultures of aqueous sediment of an old electric water heater which supplied hot water for the feeding of the calves yielded L. pneumophila SG1. Four of the colonies tested belonged to the Olda subtype. Ten clinical and four environmental isolates were examined for the presence of plasmids. Nine of them were also examined by pulsed-field gel electrophoresis assay, and the same patterns were found for L. pneumophila SG1 Olda strains isolated from the calf and from the electric heater. This is the first report of a documented case of a naturally occurring Legionella pneumonia in an animal. Cattle probably act as accidental hosts for legionellae, much the same as humans. PMID:9650941

  18. [Evaluation of a new ELISA (Bartels) for detection of Legionella pneumophila antigen in urine].

    PubMed

    de Ory, Fernando

    2002-03-01

    Detection of Legionella pneumophila soluble antigens allows rapid diagnosis of pneumonia caused by these bacteria. A new ELISA (Bartels) for antigenuria detection has recently been commercialized. We compared the new ELISA with another well-established ELISA (Binax). To evaluate ELISA-Bartels (Legionella Urinary Antigen, Intracel, Issaquah, Washington, United States), urine samples previously characterized by ELISA Binax (Legionella Urinary Antigen Enzyme Immunoassay Kit, Binax, Portland, Maine, United States) were used. Samples came from Legionella outbreaks (n = 48), from sporadic legionellosis (n = 38), and from children with viral pneumonia (n = 21). Samples from the External Quality Control of Legionella of the European Working Group on Legionella Infections (n = 102) were also tested. Of the samples analyzed, 109 were positive in ELISA-Binax, 2 were equivocal and 98 were negative. Samples showing equivocal results were excluded from the analysis. The sensitivity of ELISA-Bartels in comparison with that of ELISA-Binax was 98.2% (107/109) and specificity was 82.7% (81/98). In the 17 samples that were positive in ELISA-Bartels and negative in ELISA-Binax, 10 were positive in ELISA-Binax after concentration by selective ultrafiltration and 6 further cases showed serology indicating or compatible with recent Legionella infection and were thus classified as true positives. ELISA-Bartels showed good sensitivity and specificity. Sensitivity was even higher than that of ELISA-Binax. Thus, we consider it to be an appropriate method for diagnosis of Legionella pneumonia.

  19. Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens.

    PubMed

    Horng, Yu-Tze; Soo, Po-Chi; Shen, Bin-Jon; Hung, Yu-Li; Lo, Kai-Yin; Su, Hsun-Pi; Wei, Jun-Rong; Hsieh, Shang-Chen; Hsueh, Po-Ren; Lai, Hsin-Chih

    2006-06-01

    A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.

  20. Role of biofilms in the survival of Legionella pneumophila in a model potable-water system.

    PubMed

    Murga, R; Forster, T S; Brown, E; Pruckler, J M; Fields, B S; Donlan, R M

    2001-11-01

    Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6.7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist.

  1. Regulation, Integrase-Dependent Excision, and Horizontal Transfer of Genomic Islands in Legionella pneumophila

    PubMed Central

    Lautner, Monika; Schunder, Eva; Herrmann, Vroni

    2013-01-01

    Legionella pneumophila is a Gram-negative freshwater agent which multiplies in specialized nutrient-rich vacuoles of amoebae. When replicating in human alveolar macrophages, Legionella can cause Legionnaires' disease. Recently, we identified a new type of conjugation/type IVA secretion system (T4ASS) in L. pneumophila Corby (named trb-tra). Analogous versions of trb-tra are localized on the genomic islands Trb-1 and Trb-2. Both can exist as an episomal circular form, and Trb-1 can be transferred horizontally to other Legionella strains by conjugation. In our current work, we discovered the importance of a site-specific integrase (Int-1, lpc2818) for the excision and conjugation process of Trb-1. Furthermore, we identified the genes lvrRABC (lpc2813 to lpc2816) to be involved in the regulation of Trb-1 excision. In addition, we demonstrated for the first time that a Legionella genomic island (LGI) of L. pneumophila Corby (LpcGI-2) encodes a functional type IV secretion system. The island can be transferred horizontally by conjugation and is integrated site specifically into the genome of the transconjugants. LpcGI-2 generates three different episomal forms. The predominant episomal form, form A, is generated integrase dependently (Lpc1833) and transferred by conjugation in a pilT-dependent manner. Therefore, the genomic islands Trb-1 and LpcGI-2 should be classified as integrative and conjugative elements (ICEs). Coculture studies of L. pneumophila wild-type and mutant strains revealed that the int-1 and lvrRABC genes (located on Trb-1) as well as lpc1833 and pilT (located on LpcGI-2) do not influence the in vivo fitness of L. pneumophila in Acanthamoeba castellanii. PMID:23354744

  2. Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax

    SciTech Connect

    Smith-Somerville, H.E.; Huryn, V.B.; Walker, C.; Winters, A.L. )

    1991-09-01

    The processing of phagosomes containing Legionella pneumophila and Escerichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.

  3. Asc-Dependent and Independent Mechanisms Contribute to Restriction of Legionella Pneumophila Infection in Murine Macrophages

    PubMed Central

    Abdelaziz, Dalia H. A.; Gavrilin, Mikhail A.; Akhter, Anwari; Caution, Kyle; Kotrange, Sheetal; Khweek, Arwa Abu; Abdulrahman, Basant A.; Hassan, Zeinab A.; El-Sharkawi, Fathia Z.; Bedi, Simranjit S.; Ladner, Katherine; Gonzalez-Mejia, M. Elba; Doseff, Andrea I.; Mostafa, Mahmoud; Kanneganti, Thirumala-Devi; Guttridge, Dennis; Marsh, Clay B.; Wewers, Mark D.; Amer, Amal O.

    2010-01-01

    The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase-1−/− and Nlrc4−/− allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc−/− macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation. PMID:21713115

  4. Role of stagnation and obstruction of water flow in isolation of Legionella pneumophila from hospital plumbing.

    PubMed Central

    Ciesielski, C A; Blaser, M J; Wang, W L

    1984-01-01

    The stagnation of water in two of four hospital hot-water storage tanks found to contain Legionella pneumophila was reduced by keeping the two tanks continually on-line for 1 year. L. pneumophila colony counts in these two tanks fell quickly to low levels, whereas the organisms persisted in the two tanks that were not in use. L. pneumophila continued to be isolated from 50 to 100% of the hospital showerheads which were sampled during this period. We also examined aerators and other hospital faucet fixtures which obstruct water flow. L. pneumophila was isolated from 22 of 30 faucet aerators and 2 of 16 vacuum breakers but not from 26 nonobstructed faucets or 6 backflow preventers. Over a 7-month period, after nine faucet aerators were sterilized, 10 of 60 surveillance cultures revealed L. pneumophila, despite the inability to isolate the organism from the potable-water tanks in use. These data suggest that prevention of stagnation in hot-water tanks may be effective in reducing L. pneumophila concentrations in potable-water systems serving high-risk populations. We have also shown that faucet aerators, by providing a surface for L. pneumophila to colonize, can become secondary reservoirs for the organism in hospital plumbing. PMID:6508313

  5. Effects of metals on Legionella pneumophila growth in drinking water plumbing systems.

    PubMed Central

    States, S J; Conley, L F; Ceraso, M; Stephenson, T E; Wolford, R S; Wadowsky, R M; McNamara, A M; Yee, R B

    1985-01-01

    An investigation of the chemical environment and growth of Legionella pneumophila in plumbing systems was conducted to gain a better understanding of its ecology in this habitat. Water samples were collected from hospital and institutional hot-water tanks known to have supported L. pneumophila and were analyzed for 23 chemical parameters. The chemical environment of these tanks was found to vary extensively, with the concentrations of certain metals reaching relatively high levels due to corrosion. The effect of various chemical conditions on L. pneumophila growth was then examined by observing its multiplication in the chemically analyzed hot-water tank samples after sterilization and reinoculation with L. pneumophila. L. pneumophila and associated microbiota used in these experiments were obtained from a hot-water tank. These stains were maintained in tap water and had never been passaged on agar. The results of the growth studies indicate that although elevated concentrations of a number of metals are toxic, lower levels of certain metals such as iron, zinc, and potassium enhance growth of naturally occurring L. pneumophila. Parallel observations on accompanying non-Legionellaceae bacteria failed to show the same relationship. These findings suggest that metal plumbing components and associated corrosion products are important factors in the survival and growth of L. pneumophila in plumbing systems and may also be important in related habitats such as cooling towers and air-conditioning systems. PMID:4091551

  6. Water characteristics associated with the occurrence of Legionella pneumophila in dental units.

    PubMed

    Zanetti, F; Stampi, S; De Luca, G; Fateh-Moghadam, P; Antonietta, M; Sabattini, B; Checchi, L

    2000-02-01

    This study evaluated the incidence of Legionella pneumophila in dental unit water samples and investigated how the occurrence of these bacteria may be related to some physical, chemical and bacteriological characteristics of the water. The samples were taken from the incoming tap water, oral rinsing cup, air-water syringe, ultrasonic scaler, and the turbine of 23 dental units of private and public institutions. Apart from L. pneumophila (serogroup 1 and 3) isolated in 22 out of the 101 (21.8%) water samples tested, two other species were found: L. bozemanii and L. dumoffii. The highest densities and frequency of L. pneumophila were observed in the water coming into the units and in the dental units of public institutions. A negative association between L. pneumophila and 36 degrees C and 22 degrees C heterotrophic total plate counts and other gram-negative bacteria was found. An inverse association between the concentration of L. pneumophila and water temperature was also observed. The values of pH and total hardness did not show any significant difference in the L. pneumophila-positive and -negative dental unit waters. Finally, the chemical oxygen demand (COD) and residual chlorine were found to correlate positively with L. pneumophila.

  7. Legionella pneumophila: From potable water to treated greywater; quantification and removal during treatment.

    PubMed

    Blanky, Marina; Rodríguez-Martínez, Sara; Halpern, Malka; Friedler, Eran

    2015-11-15

    Greywater is an alternative water source that can help alleviate stress on depleted water resources. The main options for greywater reuse are toilet flushing and garden irrigation, both producing aerosols. For that reason transmission of inhalable pathogens like Legionella present a potential risk. To improve the understanding about Legionella in greywater, we traced the pathogen seasonally from the potable water system to the final steps of the greywater treatment in four houses in northern Israel. Physicochemical and microbiological parameters were analyzed in order to assess background greywater quality and to establish possible associations with Legionella. The mean concentrations of Legionella pneumophila isolated from the potable water system were 6.4×10(2) and 5.9×10(3) cfu/l in cold and hot water respectively. By amending the ISO protocol for Legionella isolation from drinking water, we succeeded in quantifying Legionella in greywater. The mean Legionella concentrations that were found in raw, treated and treated chlorinated greywater were 1.2×10(5), 2.4×10(4) and 5.7×10(3) cfu/l respectively. While Legionella counts in potable water presented a seasonal pattern with high concentrations in summer, its counts in greywater presented an almost inversed pattern. Greywater treatment resulted in 95% decrease in Legionella counts. No significant difference was found between Legionella concentrations in potable water and the treated chlorinated greywater. These findings indicate that regarding Legionella, reusing treated chlorinated greywater would exhibit a risk that is very similar to the risk associated with using potable water for the same non-potable uses.

  8. Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

    PubMed Central

    Zheng, Huaixin; Chatfield, Christa H.; Liles, Mark R.

    2013-01-01

    Iron acquisition is critical to the growth and virulence of Legionella pneumophila. Previously, we found that L. pneumophila uses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted by L. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability of L. pneumophila and other species of Legionella to take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing of L. pneumophila culture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis of L. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin. PMID:23980114

  9. Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

    PubMed Central

    Aurell, Helena; Catala, Philippe; Farge, Pierre; Wallet, France; Le Brun, Matthieu; Helbig, Jürgen H.; Jarraud, Sophie; Lebaron, Philippe

    2004-01-01

    A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems. PMID:15006790

  10. Legionella pneumophila infection activates bystander cells differentially by bacterial and host cell vesicles.

    PubMed

    Jung, Anna Lena; Herkt, Christina Elena; Schulz, Christine; Bolte, Kathrin; Seidel, Kerstin; Scheller, Nicoletta; Sittka-Stark, Alexandra; Bertrams, Wilhelm; Schmeck, Bernd

    2017-07-24

    Extracellular vesicles from eukaryotic cells and outer membrane vesicles (OMVs) released from gram-negative bacteria have been described as mediators of pathogen-host interaction and intercellular communication. Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. The differential effect of bacterial and host cell vesicles in L. pneumophila infection is unknown so far. We infected THP-1-derived or primary human macrophages with L. pneumophila and isolated supernatant vesicles by differential centrifugation. We observed an increase of exosomes in the 100 k pellet by nanoparticle tracking analysis, electron microscopy, and protein markers. This fraction additionally contained Legionella LPS, indicating also the presence of OMVs. In contrast, vesicles in the 16 k pellet, representing microparticles, decreased during infection. The 100 k vesicle fraction activated uninfected primary human alveolar epithelial cells, A549 cells, and THP-1 cells. Epithelial cell activation was reduced by exosome depletion (anti-CD63, or GW4869), or blocking of IL-1β in the supernatant. In contrast, the response of THP-1 cells to vesicles was reduced by a TLR2-neutralizing antibody, UV-inactivation of bacteria, or - partially - RNase-treatment of vesicles. Taken together, we found that during L. pneumophila infection, neighbouring epithelial cells were predominantly activated by exosomes and cytokines, whereas myeloid cells were activated by bacterial OMVs.

  11. Impact of drinking water conditions and copper materials on downstream biofilm microbial communities and legionella pneumophila colonization

    EPA Science Inventory

    Legionella pneumophila, the medically important species within the genus Legionella, is a concern in engineered water systems. Its ability to amplify within free-living amoebae is well documented, but its interactions/ecology within the microbial community of drinking water biofi...

  12. Impact of drinking water conditions and copper materials on downstream biofilm microbial communities and legionella pneumophila colonization

    EPA Science Inventory

    Legionella pneumophila, the medically important species within the genus Legionella, is a concern in engineered water systems. Its ability to amplify within free-living amoebae is well documented, but its interactions/ecology within the microbial community of drinking water biofi...

  13. Molecular mimicry and original biochemical strategies for the biogenesis of a Legionella pneumophila replicative niche in phagocytic cells.

    PubMed

    Allombert, Julie; Fuche, Fabien; Michard, Céline; Doublet, Patricia

    2013-12-01

    Legionella pneumophila is a paradigm of highly adapted intravacuolar pathogens that acquired the rare ability to replicate within a phagocytic cell. Here, we review recent progress about the role of Type 4 secretion system effectors involved in the biogenesis of the replicative niche, the Legionella containing vacuole.

  14. Legionella pneumophila Persists within Biofilms Formed by Klebsiella pneumoniae, Flavobacterium sp., and Pseudomonas fluorescens under Dynamic Flow Conditions

    PubMed Central

    Stewart, Catherine R.; Muthye, Viraj; Cianciotto, Nicholas P.

    2012-01-01

    Legionella pneumophila, the agent of Legionnaires' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiella pneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4×104 CFU per cm2 of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species. PMID:23185637

  15. Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies.

    PubMed Central

    Joly, J R; McKinney, R M; Tobin, J O; Bibb, W F; Watkins, I D; Ramsay, D

    1986-01-01

    A panel of monoclonal antibodies to Legionella pneumophila serogroup 1 and a subclassification scheme were developed in a collaborative project among three laboratories. The seven most useful monoclonal antibodies were selected from three previously developed panels on the basis of indirect fluorescent antibody patterns with 83 strains of L. pneumophila serogroup 1 that were obtained from widely distributed geographic locations. The isolates were divided into 10 major subgroups on the basis of reactivity patterns that can be readily reproduced in any laboratory and are not subject to major inconsistencies of interpretation of staining intensity. A standard protocol for the indirect fluorescent antibody procedure was also developed. PMID:3517064

  16. The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis.

    PubMed Central

    Steinert, M; Engelhard, H; Flügel, M; Wintermeyer, E; Hacker, J

    1995-01-01

    The lly locus (legiolysin) mediates the browning of the culture medium of Legionella pneumophila in the late stationary growth phase, presumably as a result of synthesis of homogentisic acid. Mutagenesis of the lly gene of the L. pneumophila Philadelphia I derivative JR32 did not affect intracellular replication in the natural host Hartmannella vermiformis. The Lly-negative mutant, however, showed a markedly decreased resistance to ordinary light. The cloned lly gene conferred an increased resistance to light in recombinant L. pneumophila and Escherichia coli K-12, indicating a contribution of the Lly protein to ecological adaptation of Legionella species. PMID:7793965

  17. Severe Legionnaires' disease with pneumonia and biopsy-confirmed myocarditis most likely caused by Legionella pneumophila serogroup 6.

    PubMed

    Ishimaru, Naoto; Suzuki, Hiromichi; Tokuda, Yasuharu; Takano, Tomoko

    2012-01-01

    We herein describe the successful treatment of a patient with possible Legionella pneumophila serogroup 6 infection complicated by pneumonia and myocarditis. A 32-year-old man presented with a five-day history of cough, dyspnea and chest pain. Chest radiography revealed patchy opacities in both lungs suggestive of bilateral pneumonia, and a urinary antigen test for Legionella pneumophila was positive. After admission, the patient developed congestive heart failure due to pathologically confirmed myocarditis. He was successfully treated with minocycline, macrolide, steroids and noninvasive positive-pressure ventilation (NPPV). He eventually recovered with a normalized cardiac function. L. pneumophila serogroup 6 was isolated from the bathwater in the patient's home.

  18. Identification and functional characterization of K(+) transporters encoded by Legionella pneumophila kup genes.

    PubMed

    Hori, Juliana I; Pereira, Marcelo S F; Roy, Craig R; Nagai, Hiroki; Zamboni, Dario S

    2013-12-01

    Legionnaires' disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the Escherichia coli K(+) transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K(+) acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella-containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but itdid not influence the replication of wild-type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K(+) transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K(+) transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient-limited conditions.

  19. Identification and functional characterization of K+ transporters encoded by Legionella pneumophila kup genes

    PubMed Central

    Hori, Juliana I.; Pereira, Marcelo S.F.; Roy, Craig R.; Nagai, Hiroki; Zamboni, Dario S.

    2013-01-01

    Summary Legionnaires’ disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the E. coli K+ transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K+ acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella-containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but it did not influence the replication of wild-type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K+ transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K+ transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient-limited conditions. PMID:23848378

  20. Electrophoretic characterization of soluble protein extracts of Legionella pneumophila and other members of the family Legionellaceae.

    PubMed Central

    Lema, M; Brown, A

    1983-01-01

    The soluble peptides of strains of Legionella pneumophila, Tatlockia micdadei, Fluoribacter bozemanae, Fluoribacter dumoffii, and Fluoribacter gormanii were studied by polyacrylamide gel electrophoresis. Characteristic patterns were seen for Legionella and Tatlockia strains, whereas the patterns for the Fluoribacter strains were variable as would be expected for this genetically heterogeneous group. Grouping by peptide pattern was consistent with proposed taxons based on DNA-DNA homology. By using a new silver stain technique, the sensitivity and ease of pattern recognition were enhanced significantly. This technique is an easily applied general method for distinguishing between strains in epidemiological studies. Images PMID:6874904

  1. Clinical and environmental distribution of Legionella pneumophila in a university hospital in Italy: efficacy of ultraviolet disinfection.

    PubMed

    Triassi, M; Di Popolo, A; Ribera D'Alcalà, G; Albanese, Z; Cuccurullo, S; Montegrosso, S; Crispino, M; Borella, P; Zarrilli, R

    2006-04-01

    The molecular epidemiology of Legionella pneumophila in the 'V. Monaldi' University Hospital was studied. Seven cases of nosocomial Legionnaires' disease were diagnosed between 1999 and 2003. Two clinical legionella strains obtained from two patients in the adult cardiac surgery unit (CSU) and 30 environmental legionella strains from the paediatric and adult CSUs, neonatal intensive care unit (NICU) and the cardiorespiratory intensive care unit (CR-ICU) were serotyped and genotyped. L. pneumophila serogroup 1/Philadelphia with an identical pulsed-field gel electrophoresis (PFGE) profile A was isolated from two patients in the adult CSU, and from three and one water samples taken in the adult CSU and the paediatric CSU, respectively, from 2001 to 2002. Furthermore, L. pneumophila serogroup 3 with an identical PFGE profile B was identified in 20 environmental strains from all wards, L. pneumophila serogroup 3 with PFGE profile C was identified in a single environmental strain from the CR-ICU, and non-pneumophila Legionella with identical PFGE profile D was identified in five environmental strains from the adult CSU, paediatric CSU and NICU. Ultraviolet irradiation was effective in disinfection of the hospital water supplies in the adult and paediatric CSUs contaminated by L. pneumophila clone associated with nosocomial Legionnaires' disease. In conclusion, these data demonstrate that two cases of nosocomial legionellosis were caused by the persistence of a single clone of L. pneumophila serogroup 1/Philadelphia in the hospital environment, and that disinfection by ultraviolet irradiation may represent an effective measure to prevent nosocomial Legionnaires' disease.

  2. Immunochromatic kits Xpect Legionella and BinaxNOW Legionella for detection of Legionella pneumophila urinary antigen have low sensitivities for the diagnosis of Legionnaires' disease.

    PubMed

    Svarrer, Christina Wiid; Lück, Christian; Elverdal, Pernille Landsbo; Uldum, Søren A

    2012-02-01

    Urinary antigen tests are the most widely used methods for diagnosing Legionnaires' disease (LD). However, all available urinary antigen tests have the disadvantage that they have low or no sensitivity for serogroups (sgs) other than Legionella pneumophila sg 1. Recently, Oxoid introduced the Xpect Legionella test for detection of L. pneumophila sg 1 and sg 6. In this study, we have evaluated the Xpect kit together with the BinaxNOW kit and compared them with the BinaxEIA kit. One hundred and fifteen urine samples from 91 patients with laboratory-confirmed LD were examined. Ninety-three samples were from 69 culture-proven cases of which 27 samples were from 23 non-sg 1 cases. At the patient level, the overall sensitivities for the three Legionella urinary antigen kits were 79 % for the BinaxEIA, 47 % for the BinaxNOW and 32 % for the Xpect kit. None of the urine samples from the 10 L. pneumophila sg 6 cases were positive by the Xpect kit whereas samples from four of the patients were positive by the BinaxEIA. Overall, the sensitivities for both immunochromatic assays were poor and they should not be used as the sole method for the diagnosis of LD.

  3. Serological cross-reaction between Legionella pneumophila and campylobacter in the indirect fluorescent antibody test.

    PubMed Central

    Boswell, T. C.; Kudesia, G.

    1992-01-01

    Sera from 50 patients with culture-proven campylobacter gastroenteritis were examined for the presence of antibodies to Legionella pneumophila. Ten patients (20%) had a positive titre (> or = 16) as measured by indirect immunofluorescence. Antibodies were detected in only 1 of 36 acute sera but in 10 of 14 (71%) sera obtained more than 10 days after the onset of symptoms. All positive sera contained specific IgM antibodies but specific IgG or IgA could not be detected in any sample. No legionella antibodies could be detected in sera from 42 similar patients with salmonella gastroenteritis. These results were shown to be due to serological cross-reaction between L. pneumophila and campylobacter. PMID:1397117

  4. Protein expression by the protozoan Hartmannella vermiformis upon contact with its bacterial parasite Legionella pneumophila.

    PubMed Central

    abu Kwaik, Y; Fields, B S; Engleberg, N C

    1994-01-01

    Legionella pneumophila is ingested by both human macrophages and amoebae, and it multiplies within similar endocytic compartments in both eukaryotic species. Inhibitors of eukaryotic protein synthesis, such as cycloheximide and emetine, had no effect on the uptake of L. pneumophila by macrophages but completely abolished ingestion by the amoeba Hartmannella vermiformis. Therefore, host cell protein synthesis is required for the bacterium to infect the amoeba but not human macrophages. To identify proteins expressed by H. vermiformis upon contact with L. pneumophila, we radiolabeled amoebal proteins after contact with bacteria in bacteriostatic concentrations of tetracycline to inhibit bacterial protein synthesis. We analyzed protein expression by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found that 33 amoebal proteins were induced; 12 of these were not detected in resting amoebae. Eleven other amoebal proteins were repressed; four of them became undetectable. In contrast, no phenotypic changes were observed in H. vermiformis upon contact with Escherichia coli or heat-killed L. pneumophila. An isogenic, avirulent variant of L. pneumophila, incapable of infecting either macrophages or amoebae, induced a different pattern of protein expression upon contact with H. vermiformis. Our data showed that amoebae manifested a specific phenotypic response upon contact with virulent L. pneumophila. This phenotypic modulation may be necessary for uptake of the bacteria into an endocytic compartment that permits bacterial survival and multiplication. Images PMID:8168950

  5. DISTRIBUTION OF LEGIONELLA PNEUMOPHILA SEROGROUPS ISOLATED FROM WATER SYSTEMS OF PUBLIC FACILITIES IN BUSAN, SOUTH KOREA.

    PubMed

    Hwang, In-Yeong; Park, Eun-Hee; Park, Yon-Koung; Park, Sun-Hee; Sung, Gyung-Hye; Park, Hye-Young; Lee, Young-Choon

    2016-05-01

    Legionella pneumophila is the major causes of legionellosis worldwide. The distribution of L. pneumophila was investigated in water systems of public facilities in Busan, South Korea during 2007 and 2013-2014. L. pneumophila was isolated from 8.3% of 3,055 samples, of which the highest isolation rate (49%) was from ships and the lowest 4% from fountains. Serogroups of L. pneumophila isolated in 2007 were distributed among serogroups (sgs) 1-7 with the exception of sg 4, while those of isolates during 2013 and 2014 included also 11 sgs ( 1, 2, 3, 4, 5, 6, 7, 8, 12, 13, 15). L. pneumophila sg 1 was predominated among isolates from fountains (75%), hotels (60%), buildings (44%), hospitals (38%), and public baths (37%), whereas sg 3 and sg 7 was the most prevalent from ships (46%) and factories (40%), respectively. The predominated serogroup of L. pneumophila isolates from hot and cooling tower water was sg 1 (35% and 46%, respectively), while from cold water was sg 3 (29%). These results should be useful for epidemiological surveys to identify sources of outbreaks of legionellosis in Busan, South Korea.

  6. molecular typing of Legionella pneumophila strains isolated from environment in Morocco.

    PubMed

    Mekkour, M; Ben Driss, E; Tai, J; Squinazi, F; Forey, F; Jarraud, S; Cohen, N

    2012-06-30

    Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most legionellosis outbreaks are linked to contaminated hot water systems or cooling towers. The aim of this study was to determine the genetic diversity of (n= 55) environmental strains of L. pneumophila recovered from the hot water distribution systems of 16 establishments in seven Moroccan towns during the period 2009-2011. Thirteen chromosomal restriction patterns determined by Pulsed field gel electrophoresis were detected. The strains of L. pneumophila serogroup1 exhibited in 6/13 different PFGE patterns, while the strains of L. pneumophila serogroups 2-14 showed 7/13 PFGE patterns. The PFGE showed the existence of various patterns in Morocco, The pattern -XI- have tree similar profiles with the endemic L. pneumophila Paris's strain. This technique also allowed to conclude that the same pulsotype was found for many strains isolated from different establishments. Moreover, different pulsolypes were found for strains isolated from the same establishment. These results showed that PFGE analysis is a powerful tool to reveal the clonal nature and genetic differences among L. pneumophila strains.

  7. Permissiveness of freshly isolated environmental strains of amoebae for growth of Legionella pneumophila.

    PubMed

    Dupuy, Mathieu; Binet, Marie; Bouteleux, Celine; Herbelin, Pascaline; Soreau, Sylvie; Héchard, Yann

    2016-03-01

    Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater amoeba genera.

  8. Hybridoma-derived monoclonal immunoglobulin M antibodies to Legionella pneumophila serogroup 1 with diagnostic potential.

    PubMed Central

    Sethi, K K; Drüeke, V; Brandis, H

    1983-01-01

    Mouse hybridomas were isolated by fusing P3-X63-Ag 8.653 myeloma cells with spleen cells from mice that had been repeatedly immunized with Legionella pneumophila serogroup 1 organisms. In one fusion, three independent hybridoma cultures which secreted antibodies that reacted with the immunizing strain in the indirect immunofluorescent-antibody test were selected for cloning. Representative continuously growing clones, one of each hybridoma, which remained stable in producing high-titer antibodies were examined in detail. Extensive specificity tests revealed that these hybridoma-derived monoclonal antibodies were specifically directed against L. pneumophila serogroup 1 organisms and showed no cross-reactions in the indirect immunofluorescent-antibody test either with the other known serogroups of L. pneumophila or with other unrelated bacterial species. The three monoclonal antibodies F4/CB5/K18, F/4CB5/K104, and F4/JD3.8/K101 belonged to the immunoglobulin M class and were capable of agglutinating serogroup 1 organisms of L. pneumophila exquisitely. These monoclonal antibodies against L. pneumophila with defined fine specificity should enable purification and subsequent analysis of the corresponding antigenic determinant(s) and can also be used for the preparation of unlimited supplies of standard diagnostic reagents for the identification of L. pneumophila in the tissues and body fluids. PMID:6874913

  9. Characterization and resuscitation of ‘non-culturable’ cells of Legionella pneumophila

    PubMed Central

    2014-01-01

    Background Legionella pneumophila is a waterborne pathogen responsible for Legionnaires’ disease, an infection which can lead to potentially fatal pneumonia. After disinfection, L. pneumophila has been detected, like many other bacteria, in a “viable but non culturable” state (VBNC). The physiological significance of the VBNC state is unclear and controversial: it could be an adaptive response favoring long-term survival; or the consequence of cellular deterioration which, despite maintenance of certain features of viable cells, leads to death; or an injured state leading to an artificial loss of culturability during the plating procedure. VBNC cells have been found to be resuscitated by contact with amoebae. Results We used quantitative microscopic analysis, to investigate this “resuscitation” phenomenon in L. pneumophila in a model involving amending solid plating media with ROS scavengers (pyruvate or glutamate), and co-culture with amoebae. Our results suggest that the restoration observed in the presence of pyruvate and glutamate may be mostly due to the capacity of these molecules to help the injured cells to recover after a stress. We report evidence that this extracellular signal leads to a transition from a not-culturable form to a culturable form of L. pneumophila, providing a technique for recovering virulent and previously uncultivated forms of L. pneumophila. Conclusion These new media could be used to reduce the risk of underestimation of counts of virulent of L. pneumophila cells in environmental samples. PMID:24383402

  10. The Identification of a Legionella pneumophila Toxin with in vivo Lethality

    DTIC Science & Technology

    1980-01-01

    of the two peaks FIT PEAK SECOND PEAK COMPONENT (U4/mi) (tag/ml) Protein 50 325 Carbohydrate 7 23 KDO 0.8 0.6 endotoxicity of L. pneumophila LPS ...lipopolysaccharides ( LPS ) the effects caused by a toxin.’, that reacted positively with limulus This chapter reports the demon- lysate was not...unexpected. However, stration of a low molecular weight pro- these investigators demonstrated that tein toxin derived from a cell-free ex- Legionella LPS

  11. Localization of Legionella pneumophila in Tissue Using FITC-Conjugated Specific Antibody and a Background Stain

    DTIC Science & Technology

    1982-05-01

    Legionnaires ’ disease in tissue. N Engi J Med 1977; Manual of Clinical Microbiology. Third edition. Edited by EH 297:1218-1220 Lennette, A Balows, WJ Hausler...Pathologits P6 i U. S. A. Localization of Legionella pneumophila in Tissue Using FITC- Conjuga ted Specific Antibody and a Background Stain BARBARA S. LOWRY...kIstitute of Infectmau meth W.: Localization of Legieaelle jimewnophsila Ii, tissue Disease . Fort Detrick, Frederick, MAryland using FIT71C-coujugated

  12. Extensive recombination events and horizontal gene transfer shaped the Legionella pneumophila genomes

    PubMed Central

    2011-01-01

    Background Legionella pneumophila is an intracellular pathogen of environmental protozoa. When humans inhale contaminated aerosols this bacterium may cause a severe pneumonia called Legionnaires' disease. Despite the abundance of dozens of Legionella species in aquatic reservoirs, the vast majority of human disease is caused by a single serogroup (Sg) of a single species, namely L. pneumophila Sg1. To get further insights into genome dynamics and evolution of Sg1 strains, we sequenced strains Lorraine and HL 0604 1035 (Sg1) and compared them to the available sequences of Sg1 strains Paris, Lens, Corby and Philadelphia, resulting in a comprehensive multigenome analysis. Results We show that L. pneumophila Sg1 has a highly conserved and syntenic core genome that comprises the many eukaryotic like proteins and a conserved repertoire of over 200 Dot/Icm type IV secreted substrates. However, recombination events and horizontal gene transfer are frequent. In particular the analyses of the distribution of nucleotide polymorphisms suggests that large chromosomal fragments of over 200 kbs are exchanged between L. pneumophila strains and contribute to the genome dynamics in the natural population. The many secretion systems present might be implicated in exchange of these fragments by conjugal transfer. Plasmids also play a role in genome diversification and are exchanged among strains and circulate between different Legionella species. Conclusion Horizontal gene transfer among bacteria and from eukaryotes to L. pneumophila as well as recombination between strains allows different clones to evolve into predominant disease clones and others to replace them subsequently within relatively short periods of time. PMID:22044686

  13. Metabolism of myo-Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages

    PubMed Central

    Manske, Christian; Schell, Ursula

    2016-01-01

    ABSTRACT Legionella pneumophila is a natural parasite of environmental amoebae and the causative agent of a severe pneumonia termed Legionnaires' disease. The facultative intracellular pathogen employs a bipartite metabolism, where the amino acid serine serves as the major energy supply, while glycerol and glucose are mainly utilized for anabolic processes. The L. pneumophila genome harbors the cluster lpg1653 to lpg1649 putatively involved in the metabolism of the abundant carbohydrate myo-inositol (here termed inositol). To assess inositol metabolism by L. pneumophila, we constructed defined mutant strains lacking lpg1653 or lpg1652, which are predicted to encode the inositol transporter IolT or the inositol-2-dehydrogenase IolG, respectively. The mutant strains were not impaired for growth in complex or defined minimal media, and inositol did not promote extracellular growth. However, upon coinfection of Acanthamoeba castellanii, the mutants were outcompeted by the parental strain, indicating that the intracellular inositol metabolism confers a fitness advantage to the pathogen. Indeed, inositol added to L. pneumophila-infected amoebae or macrophages promoted intracellular growth of the parental strain, but not of the ΔiolT or ΔiolG mutant, and growth stimulation by inositol was restored by complementation of the mutant strains. The expression of the Piol promoter and bacterial uptake of inositol required the alternative sigma factor RpoS, a key virulence regulator of L. pneumophila. Finally, the parental strain and ΔiolG mutant bacteria but not the ΔiolT mutant strain accumulated [U-14C6]inositol, indicating that IolT indeed functions as an inositol transporter. Taken together, intracellular L. pneumophila metabolizes inositol through the iol gene products, thus promoting the growth and virulence of the pathogen. IMPORTANCE The environmental bacterium Legionella pneumophila is the causative agent of a severe pneumonia termed Legionnaires' disease. The

  14. Intra-Species and Inter-Kingdom Signaling of Legionella pneumophila

    PubMed Central

    Hochstrasser, Ramon; Hilbi, Hubert

    2017-01-01

    The ubiquitous Gram-negative bacterium Legionella pneumophila parasitizes environ mental amoebae and, upon inhalation, replicates in alveolar macrophages, thus causing a life-threatening pneumonia called “Legionnaires’ disease.” The opportunistic pathogen employs a bi-phasic life cycle, alternating between a replicative, non-virulent phase and a stationary, transmissive/virulent phase. L. pneumophila employs the Lqs (Legionella quorum sensing) system as a major regulator of the growth phase switch. The Lqs system comprises the autoinducer synthase LqsA, the homologous sensor kinases LqsS and LqsT, as well as a prototypic response regulator termed LqsR. These components produce, detect, and respond to the α-hydroxyketone signaling molecule LAI-1 (Legionella autoinducer-1, 3-hydroxypentadecane-4-one). LAI-1-mediated signal transduction through the sensor kinases converges on LqsR, which dimerizes upon phosphorylation. The Lqs system regulates the bacterial growth phase switch, pathogen-host cell interactions, motility, natural competence, filament production, and expression of a chromosomal “fitness island.” Yet, LAI-1 not only mediates bacterial intra-species signaling, but also modulates the motility of eukaryotic cells through the small GTPase Cdc42 and thus promotes inter-kingdom signaling. Taken together, the low molecular weight compound LAI-1 produced by L. pneumophila and sensed by the bacteria as well as by eukaryotic cells plays a major role in pathogen-host cell interactions. PMID:28217110

  15. Geographical and Temporal Structures of Legionella pneumophila Sequence Types in Comunitat Valenciana (Spain), 1998 to 2013.

    PubMed

    Sánchez-Busó, Leonor; Coscollà, Mireia; Palero, Ferran; Camaró, María Luisa; Gimeno, Ana; Moreno, Pilar; Escribano, Isabel; López Perezagua, María Mar; Colomina, Javier; Vanaclocha, Herme; González-Candelas, Fernando

    2015-10-01

    Legionella pneumophila is an accidental human pathogen associated with aerosol formation in water-related sources. High recombination rates make Legionella populations genetically diverse, and nearly 2,000 different sequence types (STs) have been described to date for this environmental pathogen. The spatial distribution of STs is extremely heterogeneous, with some variants being present worldwide and others being detected at only a local scale. Similarly, some STs have been associated with disease outbreaks, such as ST578 or ST23. Spain is among the European countries with the highest incidences of reported legionellosis cases, and specifically, Comunitat Valenciana (CV) is the second most affected area in the country. In this work, we aimed at studying the overall diversity of Legionella pneumophila populations found in the period from 1998 to 2013 in 79 localities encompassing 23 regions within CV. To do so, we performed sequence-based typing (SBT) on 1,088 L. pneumophila strains detected in the area from both environmental and clinical sources. A comparison with the genetic structuring detected in a global data set that included 20 European and 7 non-European countries was performed. Our results reveal a level of diversity in CV that can be considered representative of the diversity found in other countries worldwide. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Detection of Legionella, L. pneumophila and Mycobacterium Avium Complex (MAC) along Potable Water Distribution Pipelines

    PubMed Central

    Whiley, Harriet; Keegan, Alexandra; Fallowfield, Howard; Bentham, Richard

    2014-01-01

    Inhalation of potable water presents a potential route of exposure to opportunistic pathogens and hence warrants significant public health concern. This study used qPCR to detect opportunistic pathogens Legionella spp., L. pneumophila and MAC at multiple points along two potable water distribution pipelines. One used chlorine disinfection and the other chloramine disinfection. Samples were collected four times over the year to provide seasonal variation and the chlorine or chloramine residual was measured during collection. Legionella spp., L. pneumophila and MAC were detected in both distribution systems throughout the year and were all detected at a maximum concentration of 103 copies/mL in the chlorine disinfected system and 106, 103 and 104 copies/mL respectively in the chloramine disinfected system. The concentrations of these opportunistic pathogens were primarily controlled throughout the distribution network through the maintenance of disinfection residuals. At a dead-end and when the disinfection residual was not maintained significant (p < 0.05) increases in concentration were observed when compared to the concentration measured closest to the processing plant in the same pipeline and sampling period. Total coliforms were not present in any water sample collected. This study demonstrates the ability of Legionella spp., L. pneumophila and MAC to survive the potable water disinfection process and highlights the need for greater measures to control these organisms along the distribution pipeline and at point of use. PMID:25046636

  17. Detection of Legionella, L. pneumophila and Mycobacterium avium complex (MAC) along potable water distribution pipelines.

    PubMed

    Whiley, Harriet; Keegan, Alexandra; Fallowfield, Howard; Bentham, Richard

    2014-07-18

    Inhalation of potable water presents a potential route of exposure to opportunistic pathogens and hence warrants significant public health concern. This study used qPCR to detect opportunistic pathogens Legionella spp., L. pneumophila and MAC at multiple points along two potable water distribution pipelines. One used chlorine disinfection and the other chloramine disinfection. Samples were collected four times over the year to provide seasonal variation and the chlorine or chloramine residual was measured during collection. Legionella spp., L. pneumophila and MAC were detected in both distribution systems throughout the year and were all detected at a maximum concentration of 103 copies/mL in the chlorine disinfected system and 106, 103 and 104 copies/mL respectively in the chloramine disinfected system. The concentrations of these opportunistic pathogens were primarily controlled throughout the distribution network through the maintenance of disinfection residuals. At a dead-end and when the disinfection residual was not maintained significant (p < 0.05) increases in concentration were observed when compared to the concentration measured closest to the processing plant in the same pipeline and sampling period. Total coliforms were not present in any water sample collected. This study demonstrates the ability of Legionella spp., L. pneumophila and MAC to survive the potable water disinfection process and highlights the need for greater measures to control these organisms along the distribution pipeline and at point of use.

  18. Geographical and Temporal Structures of Legionella pneumophila Sequence Types in Comunitat Valenciana (Spain), 1998 to 2013

    PubMed Central

    Sánchez-Busó, Leonor; Coscollà, Mireia; Palero, Ferran; Camaró, María Luisa; Gimeno, Ana; Moreno, Pilar; Escribano, Isabel; López Perezagua, María Mar; Colomina, Javier; Vanaclocha, Herme

    2015-01-01

    Legionella pneumophila is an accidental human pathogen associated with aerosol formation in water-related sources. High recombination rates make Legionella populations genetically diverse, and nearly 2,000 different sequence types (STs) have been described to date for this environmental pathogen. The spatial distribution of STs is extremely heterogeneous, with some variants being present worldwide and others being detected at only a local scale. Similarly, some STs have been associated with disease outbreaks, such as ST578 or ST23. Spain is among the European countries with the highest incidences of reported legionellosis cases, and specifically, Comunitat Valenciana (CV) is the second most affected area in the country. In this work, we aimed at studying the overall diversity of Legionella pneumophila populations found in the period from 1998 to 2013 in 79 localities encompassing 23 regions within CV. To do so, we performed sequence-based typing (SBT) on 1,088 L. pneumophila strains detected in the area from both environmental and clinical sources. A comparison with the genetic structuring detected in a global data set that included 20 European and 7 non-European countries was performed. Our results reveal a level of diversity in CV that can be considered representative of the diversity found in other countries worldwide. PMID:26231651

  19. MTOR-Driven Metabolic Reprogramming Regulates Legionella pneumophila Intracellular Niche Homeostasis

    PubMed Central

    Abshire, Camille F.; Roy, Craig R.

    2016-01-01

    Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L

  20. Isolation and identification of Legionella pneumophila from drinking water in Basra governorate, Iraq.

    PubMed

    Al-Sulami, A A; Al-Taee, A M R; Yehyazarian, A A

    2013-11-01

    This study in Iraq investigated the occurrence of Legionella. pneumophila in different drinking-water sources in Basra governorate as well as the susceptibility of isolates to several antibiotics. A total of 222 water samples were collected in 2008-2009: 49 samples from water purification plants (at entry points, from precipitation tanks, from filtration tanks and at exit points), 127 samples of tap water; and 46 samples from tankers and plants supplying water by reverse osmosis. The findings confirmed the presence of L. pneumophila in sources of crude water, in general drinking water supplies and drinking water tankers. Of 258 isolates 77.1% were serotype 1 and 22.9% serotypes 2-15. All examined isolates displayed drug resistance, particularly to ampicillin, but were 100% susceptible to doxycycline. The prevalence of L. pneumophila, especially serogroup 1, is a strong indicator of unsuitability of drinking water and requires appropriate action.

  1. Real-time investigation of a Legionella pneumophila outbreak using whole genome sequencing.

    PubMed

    Graham, R M A; Doyle, C J; Jennison, A V

    2014-11-01

    Legionella pneumophila is the main pathogen responsible for outbreaks of Legionnaires' disease, which can be related to contaminated water supplies such as cooling towers or water pipes. We combined conventional molecular methods and whole genome sequence (WGS) analysis to investigate an outbreak of L. pneumophila in a large Australian hospital. Typing of these isolates using sequence-based typing and virulence gene profiling, was unable to discriminate between outbreak and non-outbreak isolates. WGS analysis was performed on isolates during the outbreak, as well as on unlinked isolates from the Public Health Microbiology reference collection. The more powerful resolution provided by analysis of whole genome sequences allowed outbreak isolates to be distinguished from isolates that were temporally and spatially unassociated with the outbreak, demonstrating that this technology can be used in real-time to investigate L. pneumophila outbreaks.

  2. Flow cytometric determination of endocytosis of viable labelled Legionella pneumophila by Acanthamoeba palestinensis.

    PubMed

    Harf, C; Goffinet, S; Meunier, O; Monteil, H; Colin, D A

    1997-03-01

    Endocytosis of fluorescently labelled cells of Legionella pneumophila (L. pneumophila) by free-living Acanthamoeba palestinensis (A. palestinensis) has been studied using flow cytometry. L. pneumophila cells were labelled with CM-DiI, a lipophilic fluorescent probe under conditions that did not modify viability. Coculturing the bacteria with amoebae was accompanied by rapid endocytosis; after 5 min, 90% of the amoebae had internalized bacteria. This percentage remained unchanged during further coculture, but the number of bacteria ingested per amoeba increased. Moreover, the number of ingested bacteria was found to be dependent on the size of the amoeba. The validity of the internalization analyzed by flow cytometry was confirmed by observation using epifluorescence and phase contrast microscopy. CM-DiI labelling associated with flow cytometry provides a very valuable technique for the determination of bacteria endocytosis by free-living amoeba.

  3. Effectiveness of bromicide against Legionella pneumophila in a cooling tower

    SciTech Connect

    Fliermans, C.B.; Harvey, R.S.

    1983-01-01

    Cooling towers are considered to be man-made amplifiers of Legionella. Thus the proper maintenance and choice of biocides is important. The only biocide that has thus far been shown to be effective in field tests is the judicious use of chlorination. Perturbation studies were conducted on an industrial cooling tower shown to contain Legionella, using 1-bromo-3-chloro-5,5-dimethylhydantoin (Bromicide, Great Lakes Chemical Corp.). At the manufacturer's recommended concentrations neither the density nor the activity of Legionella was affected. At concentrations greater than 2.0 ppM free residual, the Bromicide was not effective in reducing Legionella to source water concentrations, nor was it effective in reducing the INT activity of the bacterium in situ. The data indicate that at concentrations up to 2.0 ppM, Bromicide is not effective in these tower studies. 23 references, 3 tables.

  4. Contamination of Hospital Water Supplies in Gilan, Iran, with Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa

    PubMed Central

    Ahmadi Jalali Moghadam, Masoumeh; Asfaram Meshginshahr, Sajad

    2015-01-01

    This study is designed to determine the contamination degree of hospital water supplies with Pseudomonas aeruginosa, Legionella pneumophila, and E. coli in Gilan, Iran. Samples were collected directly into sterile containers and concentrated by centrifuge. Half part of any sample transferred to yeast extract broth and the second part transferred to Trypticase Soy Broth and incubated for 3 days. DNA was extracted by using commercial kit. Four rounds of PCR were performed as follows: multiplex PCR for detecting Pseudomonas aeruginosa, Integron 1, and Metallo-β-lactamases gene; PCR for detecting Legionella pneumophila and mip gene separately; PCR for detecting E. coli; and another PCR for detecting whole bacterial presence. Contamination rates of cold, warm, and incubator water samples with P. aeruginosa, were 16.6%, 37.5%, and 6.8% consequently. Degrees of contamination with L. pneumophila were 3.3%, 9.3%, and 10.9% and with E. coli were zero, 6.2%, and zero. Total bacterial contamination of cold, warm, and incubator water samples was 93.3%, 84.4%, and 89.0% consequently. Metallo-β-lactamases gene was found in 20.0% of all samples. Contamination degree with P. aeruginosa was considerable and with L. pneumophila was moderate. Metallo-β-lactamases gene was found frequently indicating widespread multiple drug resistance bacteria. We suggest using new decontamination method based on nanotechnology. PMID:26448745

  5. Side-polished fiber immunosensor based on surface plasmon resonance for detection of Legionella pneumophila

    NASA Astrophysics Data System (ADS)

    Tsao, Yu-Chia; Yang, Yi-Wen; Tsai, Woo-Hu; Yan, Tsong-Rong

    2008-02-01

    Side-polished fiber immunosensor based on surface plasmon resonance (SPR) onto self-assembled protein A layer was proposed for the detection of Legionella pneumophila. A self-assembled protein A layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and activated by N-Ethyl-N'-(3-dimethylaminopropyl) carbodiimide/ N-Hydroxysuccinimide (EDC/NHS). The formation of self-assembled protein A and gold layer on side-polished surface and the binding of antibody and antigen in series were confirmed by SPR response on spectrum. The binding protein A layer can improve the sensitivity, which indirectly supports the configurations that antibody layer is immobilized on the binding protein A layer with a well-ordered orientation. The surface morphology analyses of self-assembled protein A layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein A were demonstrated by SPR dip shifts on optical spectrum analyzer. The SPR fiber immunosensor for detection of L. pneumophila was developed and the detection limit was 10 CFU/ml with the SPR dip shift in wavelength from 1070 to 1105nm. The current fabrication technique of a SPR immunosensor using optical fiber for the detection of Legionella pneumophila could be applied to construct other biosensor.

  6. Contamination of Hospital Water Supplies in Gilan, Iran, with Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa.

    PubMed

    Ahmadi Jalali Moghadam, Masoumeh; Honarmand, Hamidreza; Asfaram Meshginshahr, Sajad

    2015-01-01

    This study is designed to determine the contamination degree of hospital water supplies with Pseudomonas aeruginosa, Legionella pneumophila, and E. coli in Gilan, Iran. Samples were collected directly into sterile containers and concentrated by centrifuge. Half part of any sample transferred to yeast extract broth and the second part transferred to Trypticase Soy Broth and incubated for 3 days. DNA was extracted by using commercial kit. Four rounds of PCR were performed as follows: multiplex PCR for detecting Pseudomonas aeruginosa, Integron 1, and Metallo-β-lactamases gene; PCR for detecting Legionella pneumophila and mip gene separately; PCR for detecting E. coli; and another PCR for detecting whole bacterial presence. Contamination rates of cold, warm, and incubator water samples with P. aeruginosa, were 16.6%, 37.5%, and 6.8% consequently. Degrees of contamination with L. pneumophila were 3.3%, 9.3%, and 10.9% and with E. coli were zero, 6.2%, and zero. Total bacterial contamination of cold, warm, and incubator water samples was 93.3%, 84.4%, and 89.0% consequently. Metallo-β-lactamases gene was found in 20.0% of all samples. Contamination degree with P. aeruginosa was considerable and with L. pneumophila was moderate. Metallo-β-lactamases gene was found frequently indicating widespread multiple drug resistance bacteria. We suggest using new decontamination method based on nanotechnology.

  7. Phylogenetic analysis of environmental Legionella pneumophila isolates from an endemic area (Alcoy, Spain).

    PubMed

    Sánchez-Busó, Leonor; Olmos, María Piedad; Camaró, María Luisa; Adrián, Francisco; Calafat, Juan Miguel; González-Candelas, Fernando

    2015-03-01

    Environmental surveillance of Legionella pneumophila is a key component of the control measures established in urban settlements to ensure water safety and quality, with the aim of minimizing and limiting opportunistic infections in humans. In this work, we present results on the detection and genetic characterization of these bacteria in the outbreak-recurrent region of Alcoy (Comunidad Valenciana, Spain) using water and biofilm samples. We were particularly interested in studying the presence and distribution of L. pneumophila in the absence of outbreak or sporadic cases of legionellosis and in comparing the efficacy of culturing from water samples with a biofilm-based detection procedure using molecular amplification. To this end, water samples were taken from 120 sites distributed all around the city and its surroundings, as well as 60 biofilm swabs from half of the sampling sites. L. pneumophila could be isolated from water in just 4 of the locations. Touchdown PCR was applied to DNA extracted from water and also biofilm swabs, as a rapid method for both routine and outbreak investigations. L. pneumophila was detected by this method in 14 of the sites in which both water and biofilms were taken, although 13 of them tested positive using only the biofilm samples. These results show a ten-fold increase in the success rate of Legionella detection over water samples. The application of this method to study the presence of L. pneumophila in the water-supply system and risk facilities of Alcoy revealed different strains distributed in different areas of the city. Sequence Type ST578, endemic in the area and responsible for most clinical cases, was detected in one of the sampling sites. The number of positive samples correlated with water temperature but not with chlorine levels. The direct analysis of biofilm swabs improves the detection rate and genetic characterization of L. pneumophila and can complement analyses based on bacterial culture. Copyright © 2014

  8. Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila.

    PubMed

    David, Sophia; Sánchez-Busó, Leonor; Harris, Simon R; Marttinen, Pekka; Rusniok, Christophe; Buchrieser, Carmen; Harrison, Timothy G; Parkhill, Julian

    2017-06-01

    Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires' disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic "hotspots" of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila

  9. Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes

    PubMed Central

    1987-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve

  10. Potential Virulence Role of the Legionella pneumophila ptsP Ortholog

    PubMed Central

    Higa, Futoshi; Edelstein, Paul H.

    2001-01-01

    We previously identified the Legionella pneumophila ptsP (phosphoenolpyruvate phosphotransferase) ortholog gene as a putative virulence factor in a study of signature-tagged mutagenesis using a guinea pig pneumonia model. In this study, we further defined the phenotypic properties of L. pneumophila ptsP and its complete sequence. The L. pneumophila ptsP was 2,295 bases in length. Its deduced amino acid sequence had high similarity with ptsP orthologs of Pseudomonas aeruginosa, Azotobacter vinelandii, and Escherichia coli, with nearly identical lengths. Here we show that while the mutant grew well in laboratory media, it was defective in both lung and spleen multiplication in guinea pigs. It grew slowly in guinea pig alveolar macrophages despite good uptake into the cells. Furthermore, there was minimal growth in a human alveolar epithelial cell line (A549). Transcomplementation of the L. pneumophila ptsP mutant almost completely rescued its growth in alveolar macrophages, in A549 cells, and in guinea pig lung and spleen. The L. pneumophila ptsP mutant was capable of evasion of phagosome-lysosome fusion and resided in ribosome-studded phagosomes. Pore formation activity of the mutant was normal. The L. pneumophila ptsP mutant expressed DotA and IcmX in apparently normal amounts, suggesting that the ptsP mutation did not affect dotA and icmX regulation. In addition, the mutant was resistant to serum and neutrophil killing. Taken together, these findings show that L. pneumophila ptsP is required for full in vivo virulence of L. pneumophila, most probably by affecting intracellular growth. PMID:11447151

  11. Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila

    PubMed Central

    Marttinen, Pekka; Rusniok, Christophe; Harrison, Timothy G.

    2017-01-01

    Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires’ disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic “hotspots” of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila

  12. Importance of Legionella pneumophila in the etiology of severe community-acquired pneumonia in Santiago, Chile.

    PubMed

    Arancibia, Francisco; Cortes, Claudia P; Valdés, Marcelo; Cerda, Javier; Hernández, Antonio; Soto, Luis; Torres, Antoni

    2014-02-01

    In US and European literature, Legionella pneumophila is reported as an important etiologic agent of severe community-acquired pneumonia (CAP), but in Chile this information is lacking. The aim of this study was to determine the incidence and identify predictors of severe CAP caused by L pneumophila in Santiago, Chile. A multicenter, prospective clinical study lasting 18 months was conducted; it included all adult patients with severe CAP admitted to the ICUs of four hospitals in Santiago. We excluded patients who were immunocompromised, had been hospitalized in the previous 4 weeks, or presented with another disease during their hospitalization. All data for the diagnosis of severe CAP were registered, and urinary antigens for L pneumophila serogroup 1 were determined. A total of 104 patients with severe CAP were included (mean ± SD age, 58.3 ± 19.3 years; men, 64.4%; APACHE (Acute Physiology and Chronic Health Evaluation) II score, 16.7 ± 6.3; Sepsis-related Organ Failure Assessment score, 6.1 ± 3.2; Pitt Bacteremia Score, 3.4 ± 2.5; Pao2/Fio2, 170.8 ± 87.1). An etiologic agent was identified in 62 patients (59.6%), with the most frequent being Streptococcus pneumoniae (27 patients [26%]) and L pneumophila (nine patients [8.6%]). Logistic regression analysis showed that a plasma sodium level of ≤ 130 mEq/L was an independent predictor for L pneumophila severe CAP (OR, 11.3; 95% CI, 2.5-50.5; P = .002). Global mortality was 26% and 33% for L pneumophila. The Pitt bacteremia score and pneumonia score index were the best predictors of mortality. We found that in Santiago, L pneumophila was second to S pneumoniae as the etiologic agent of severe CAP. Severe hyponatremia at admission appears to be an indicator for L pneumophila etiology in severe CAP.

  13. Community-acquired Legionnaires' disease caused by Legionella pneumophila serogroup 10 linked to the private home.

    PubMed

    Lück, Paul Christian; Schneider, Thomas; Wagner, Jutta; Walther, Ilona; Reif, Ursula; Weber, Stefan; Weist, Klaus

    2008-02-01

    We describe the case of a 66-year-old man with a culture-proven Legionella pneumonia after kidney transplantation. The patient developed the infection 15 days after discharge from a university hospital. Legionella pneumonia caused by Legionella pneumophila serogroup 5/10 was established by positive direct fluorescence assay, positive urinary-antigen detection and isolation of the causative agent. The infection was successfully treated by giving appropriate antibiotics, but the further course was complicated by invasive aspergillosis, cytomegalovirus pneumonia, failure of the transplanted kidney and development of septic anaemia. Four months after the diagnosis of Legionella pneumonia the patient died of multi-organ failure. The microbiological and epidemiological investigation revealed that strains from the water supply of the patient's private home were indistinguishable from the patient's isolate by amplified fragment length polymorphism analysis and sequence-based typing (SBT). Unrelated strains of serogroups 4, 5, 8 and 10 from the Dresden strain collection were of different SBT types. Thus, SBT is a very useful tool for epidemiological investigation of infections by L. pneumophila serogroups other than serogroup 1.

  14. [Legionella pneumophila pneumonia associated with the use of a home humidifier in an immunocompetent girl].

    PubMed

    Bonilla Escobar, Bertha Angélica; Montero Rubio, Juan Carlos; Martínez Juárez, Guadalupe

    2014-01-21

    Legionellosis is an uncommon cause of pneumonia in children and even more exceptionally in healthy children. The purpose of this study is to describe the procedure which led to the identification of an unusual source of legionella infection, in a healthy girl, and highlight the need to adopt preventive measures. A 4-year old girl without any risk factors was diagnosed of pneumonia caused by Legionella pneumophila serogroup 1. After investigating the environment, it was found that the source of the transmission was a home humidifier. Despite the fact that this equipment is classified with a low probability of spreading legionella, a lack of cleanliness and maintenance of these machines can increase the risk of transmission of the bacteria. Therefore, healthcare professionals must give adequate advice to their patients about the care of this equipment and how to use these machines properly. Copyright © 2012 Elsevier España, S.L. All rights reserved.

  15. Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region.

    PubMed

    Yang, G; Benson, R; Pelish, T; Brown, E; Winchell, J M; Fields, B

    2010-03-01

    Although the majority of cases of Legionnaires' disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.

  16. Relationship between Legionella pneumophila and Acanthamoeba polyphaga: Physiological status and susceptibility to chemical inactivation

    SciTech Connect

    Barker, J.; Farrell, I. ); Brown, M.R.W.; Collier, P.J.; Gilbert, P. )

    1992-08-01

    Survival studies were conducted on Legionella pneumophila cells that had been grown intracellulary in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 [times] MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure of PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.

  17. Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

    PubMed

    Barker, J; Brown, M R; Collier, P J; Farrell, I; Gilbert, P

    1992-08-01

    Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.

  18. Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

    PubMed Central

    Barker, J; Brown, M R; Collier, P J; Farrell, I; Gilbert, P

    1992-01-01

    Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila. PMID:1514790

  19. Genomic Resolution of Outbreak-Associated Legionella pneumophila Serogroup 1 Isolates from New York State

    PubMed Central

    Raphael, Brian H.; Baker, Deborah J.; Nazarian, Elizabeth; Lapierre, Pascal; Bopp, Dianna; Kozak-Muiznieks, Natalia A.; Morrison, Shatavia S.; Lucas, Claressa E.; Mercante, Jeffrey W.; Musser, Kimberlee A.

    2016-01-01

    ABSTRACT A total of 30 Legionella pneumophila serogroup 1 isolates representing 10 separate legionellosis laboratory investigations (“outbreaks”) that occurred in New York State between 2004 and 2012 were selected for evaluation of whole-genome sequencing (WGS) approaches for molecular subtyping of this organism. Clinical and environmental isolates were available for each outbreak and were initially examined by pulsed-field gel electrophoresis (PFGE). Sequence-based typing alleles were extracted from WGS data yielding complete sequence types (ST) for isolates representing 8 out of the 10 outbreaks evaluated in this study. Isolates from separate outbreaks sharing the same ST also contained the fewest differences in core genome single nucleotide polymorphisms (SNPs) and the greatest proportion of identical allele sequences in a whole-genome multilocus sequence typing (wgMLST) scheme. Both core SNP and wgMLST analyses distinguished isolates from separate outbreaks, including those from two outbreaks sharing indistinguishable PFGE profiles. Isolates from a hospital-associated outbreak spanning multiple years shared indistinguishable PFGE profiles but displayed differences in their genome sequences, suggesting the presence of multiple environmental sources. Finally, the rtx gene demonstrated differences in the repeat region sequence among ST1 isolates from different outbreaks, suggesting that variation in this gene may be useful for targeted molecular subtyping approaches for L. pneumophila. This study demonstrates the utility of various genome sequence analysis approaches for L. pneumophila for environmental source attribution studies while furthering the understanding of Legionella ecology. IMPORTANCE We demonstrate that whole-genome sequencing helps to improve resolution of Legionella pneumophila isolated during laboratory investigations of legionellosis compared to traditional subtyping methods. These data can be important in confirming the environmental sources

  20. Combination of Heat Shock and Enhanced Thermal Regime to Control the Growth of a Persistent Legionella pneumophila Strain

    PubMed Central

    Bédard, Emilie; Boppe, Inès; Kouamé, Serge; Martin, Philippe; Pinsonneault, Linda; Valiquette, Louis; Racine, Jules; Prévost, Michèle

    2016-01-01

    Following nosocomial cases of Legionella pneumophila, the investigation of a hot water system revealed that 81.5% of sampled taps were positive for L. pneumophila, despite the presence of protective levels of copper in the water. A significant reduction of L. pneumophila counts was observed by culture after heat shock disinfection. The following corrective measures were implemented to control L. pneumophila: increasing the hot water temperature (55 to 60 °C), flushing taps weekly with hot water, removing excess lengths of piping and maintaining a water temperature of 55 °C throughout the system. A gradual reduction in L. pneumophila counts was observed using the culture method and qPCR in the 18 months after implementation of the corrective measures. However, low level contamination was retained in areas with hydraulic deficiencies, highlighting the importance of maintaining a good thermal regime at all points within the system to control the population of L. pneumophila. PMID:27092528

  1. Activity of Six Essential Oils Extracted from Tunisian Plants against Legionella pneumophila.

    PubMed

    Chaftar, Naouel; Girardot, Marion; Quellard, Nathalie; Labanowski, Jérôme; Ghrairi, Tawfik; Hani, Khaled; Frère, Jacques; Imbert, Christine

    2015-10-01

    The aim of this study was to investigate the composition of six essential oils extracted from Tunisian plants, i.e., Artemisia herba-alba Asso, Citrus sinensis (L.) Osbeck, Juniperus phoenicea L., Rosmarinus officinalis L., Ruta graveolens L., and Thymus vulgaris L., and to evaluate their activity against Legionella pneumophila (microdilution assays). Eight Legionella pneumophila strains were studied, including the two well-known serogroup 1 Lens and Paris strains as controls and six environmental strains isolated from Tunisian spas belonging to serogroups 1, 4, 5, 6, and 8. The essential oils were generally active against L. pneumophila. The activities of the A. herba-alba, C. sinensis, and R. officinalis essential oils were strain-dependent, whereas those of the J. phoenicea and T. vulgaris oils, showing the highest anti-Legionella activities, with minimum inhibitory concentrations (MICs) lower than 0.03 and lower than or equal to 0.07 mg/ml, respectively, were independent of the strains' serogroup. Moreover, the microorganisms treated with T. vulgaris essential oil were shorter, swollen, and less electron-dense compared to the untreated controls. Isoborneol (20.91%), (1S)-α-pinene (18.30%) β-phellandrene (8.08%), α-campholenal (7.91%), and α-phellandrene (7.58%) were the major components isolated from the J. phoenicea oil, while carvacrol (88.50%) was the main compound of the T. vulgaris oil, followed by p-cymene (7.86%). This study highlighted the potential interest of some essential oils extracted from Tunisian plants as biocides to prevent the Legionella risk. Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

  2. Geostatistics - a tool applied to the distribution of Legionella pneumophila in a hospital water system.

    PubMed

    Laganà, Pasqualina; Moscato, Umberto; Poscia, Andrea; La Milia, Daniele Ignazio; Boccia, Stefania; Avventuroso, Emanuela; Delia, Santi

    2015-01-01

    Legionnaires' disease is normally acquired by inhalation of legionellae from a contaminated environmental source. Water systems of large buildings, such as hospitals, are often contaminated with legionellae and therefore represent a potential risk for the hospital population. The aim of this study was to evaluate the potential contamination of Legionella pneumophila (LP) in a large hospital in Italy through georeferential statistical analysis to assess the possible sources of dispersion and, consequently, the risk of exposure for both health care staff and patients. LP serogroups 1 and 2-14 distribution was considered in the wards housed on two consecutive floors of the hospital building. On the basis of information provided by 53 bacteriological analysis, a 'random' grid of points was chosen and spatial geostatistics or FAIk Kriging was applied and compared with the results of classical statistical analysis. Over 50% of the examined samples were positive for Legionella pneumophila. LP 1 was isolated in 69% of samples from the ground floor and in 60% of sample from the first floor; LP 2-14 in 36% of sample from the ground floor and 24% from the first. The iso-estimation maps show clearly the most contaminated pipe and the difference in the diffusion of the different L. pneumophila serogroups. Experimental work has demonstrated that geostatistical methods applied to the microbiological analysis of water matrices allows a better modeling of the phenomenon under study, a greater potential for risk management and a greater choice of methods of prevention and environmental recovery to be put in place with respect to the classical statistical analysis.

  3. Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila

    PubMed Central

    Zheng, Huaixin; VanRheenen, Susan M.; Ghosh, Soma; Cianciotto, Nicholas P.; Isberg, Ralph R.

    2016-01-01

    Legionella pneumophila is an intracellular bacterial pathogen that replicates in alveolar macrophages, causing a severe form of pneumonia. Intracellular growth of the bacterium depends on its ability to sequester iron from the host cell. In the L. pneumophila strain 130b, one mechanism used to acquire this essential nutrient is the siderophore legiobactin. Iron-bound legiobactin is imported by the transport protein LbtU. Here, we describe the role of LbtP, a paralog of LbtU, in iron acquisition in the L. pneumophila strain Philadelphia-1. Similar to LbtU, LbtP is a siderophore transport protein and is required for robust growth under iron-limiting conditions. Despite their similar functions, however, LbtU and LbtP do not contribute equally to iron acquisition. The Philadelphia-1 strain lacking LbtP is more sensitive to iron deprivation in vitro. Moreover, LbtP is important for L. pneumophila growth within macrophages while LbtU is dispensable. These results demonstrate that LbtP plays a dominant role over LbtU in iron acquisition. In contrast, loss of both LbtP and LbtU does not impair L. pneumophila growth in the amoebal host Acanthamoeba castellanii, demonstrating a host-specific requirement for the activities of these two transporters in iron acquisition. The growth defect of the ΔlbtP mutant in macrophages is not due to alterations in growth kinetics. Instead, the absence of LbtP limits L. pneumophila replication and causes bacteria to prematurely exit the host cell. These results demonstrate the existence of a preprogrammed exit strategy in response to iron limitation that allows L. pneumophila to abandon the host cell when nutrients are exhausted. PMID:27185787

  4. Legionella pneumophila serogroup Lansing 3 isolated from a patient with fatal pneumonia, and descriptions of L. pneumophila subsp. pneumophila subsp. nov., L. pneumophila subsp. fraseri subsp. nov., and L. pneumophila subsp. pascullei subsp. nov.

    PubMed Central

    Brenner, D J; Steigerwalt, A G; Epple, P; Bibb, W F; McKinney, R M; Starnes, R W; Colville, J M; Selander, R K; Edelstein, P H; Moss, C W

    1988-01-01

    Previous DNA relatedness and enzyme electrophoretic mobility studies indicated heterogeneity among strains of Legionella pneumophila serogroups 1, 4, 5, and Lansing 3 (a new, as yet unnumbered serogroup). In this study 60 L. pneumophila strains were studied by DNA hybridization (hydroxyapatite method) to assess their genomic relatedness. These strains were also studied biochemically and serologically to determine whether they formed one or more phenotypic groups. DNA relatedness studies identified three groups. DNA group 1 contained the type strain Philadelphia 1 and strains from serogroups 1 through 14 of L. pneumophila. The average relatedness of DNA group 1 strains was 88% at 60 degrees C with 1.1% divergence in related sequences and 85% at 75 degrees C. DNA group 2 contained strain Los Angeles 1, the reference strain of serogroup 4, and strains of serogroups 1, 4, 5, and Lansing 3, an unnumbered serogroup. Average relatedness of DNA group 2 strains was 84% at 60 degrees C with 0.7% divergence and 87% at 75 degrees C. Reciprocal relatedness of DNA groups 1 and 2 was approximately 67% at 60 degrees C with 6.0% divergence and 48% at 75 degrees C. DNA group 3 strains were in serogroup 5. They were 98% related at 60 degrees C with 0.5% divergence and 97% related at 75 degrees C. Reciprocal relatedness of DNA group 3 and DNA group 1 was approximately 74% at 60 degrees C with 5.3% divergence and 43% at 75 degrees C, and reciprocal relatedness of DNA groups 3 and 2 was 66% at 60 degrees C with 5.7% divergence and 55% at 75 degrees C. The DNA groups could not be separated biochemically or serologically or by cell wall fatty acid and isoprenoid quinone composition. Three subspecies of L. pneumophila are proposed to accommodate the three DNA groups: L. pneumophila subsp. pneumophila subsp. nov. for DNA group 1, L. pneumophila subsp. fraseri subsp. nov. for DNA group 2, and pneumophila subsp. pascullei subsp. nov. for DNA group 3. PMID:3053773

  5. Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila.

    PubMed

    Harrison, Christopher F; Chiriano, Gianpaolo; Finsel, Ivo; Manske, Christian; Hoffmann, Christine; Steiner, Bernhard; Kranjc, Agata; Patthey-Vuadens, Ophelie; Kicka, Sébastien; Trofimov, Valentin; Ouertatani-Sakouhi, Hajer; Soldati, Thierry; Scapozza, Leonardo; Hilbi, Hubert

    2015-07-10

    The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s).

  6. Validation of a Microbead-Based Format for Spoligotyping of Legionella pneumophila

    PubMed Central

    Gomgnimbou, Michel Kiréopori; Ginevra, Christophe; Peron-Cane, Caroline; Versapuech, Margaux; Refrégier, Guislaine; Jacotin, Nathalie; Jarraud, Sophie

    2014-01-01

    A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs. PMID:24759720

  7. [Cloning of major outer membrane protein gene of Legionella pneumophila and detection of its expression in prokaryotic cell].

    PubMed

    Zhang, Lei; Chen, Jianping; Wang, Tao; Zhang, Li; Tian, Yu

    2006-04-01

    In this study, the ompS gene, a major outer membrane protein gene of Legionella pneumophila, was obtained from the DNA of Legionella pneumophila by PCR. The gene was cloned into prokaryotic expressional plasmid pUC18 to construct recombinant plasmid. The recombinant plasmid was transformed into E. coli strain BL21. The identification was made by means of restriction enzyme analysis, polymerase chain reaction, DNA sequencing analysis, SDS--polyacrylamine gel electrophoresis analysis and Western blot. The results showed that the ompS gene of 914 bp was amplified from Legionella pneumophila DNA, the recombinant plasmid pLPompS was constructed and its expression in prokaryotic cell was detected successfully.

  8. Predictive parameters of Legionella pneumophila occurrence in hospital water: HPCs and plumbing system installation age.

    PubMed

    Ghanizadeh, Ghader; Mirmohamadlou, Ali; Esmaeli, Davoud

    2016-09-01

    Occurrence of Legionella pneumophila can be relevant to the installation age and the presence of heterotrophic plate counts (HPCs). This research illustrates L. pneumophila contamination of hospital water in accordance with the installation age and the presence of HPCs. One hundred and fifty samples were collected from hot and cold water systems and cultured on R2A and BCYE agar. L. pneumophila identification was done via specific biochemical tests. HPCs and L. pneumophila were detected in 96 and 37.3 % of the samples, respectively. The mean of HPCs density was 947 ± 998 CFU/ml; therefore, 52 % of the samples had higher densities than 500 CFU/ml. High densities of HPCs (>500 CFU/ml) led to colonization of L. pneumophila (≥1000 CFU/ml), mainly observed in cooling systems, gynecological, sonography, and NICU wards. Chi(2) test demonstrated that higher densities (>500 CFU/ml) of HPCs and L. pneumophila contamination in cold water were more frequent than warm water (OR: 2.3 and 1.49, respectively). Univariate regressions implied a significant difference between HPCs density and installation age in positive and negative tests of L. pneumophila (OR = 1.1, p < 0.001, OR = 1.2, p < 0.001). Mann-Whitney U test implied the significant effects of HPCs and installation age on L. pneumophila occurrences (p < 0.001). Spearman correlation and multivariate linear regression revealed significant differences between L. pneumophila and HPCs densities (r s  = 0.33, p < 0.001 and ß = 0.11, p = 0.02), but nonsignificant difference with installation age (r s  = 0.33, p < 0.001 and ß = 0.0, p = 0.91). The occurrence of L. pneumophila, HPCs, and installation age are relevant; so, plumbing system renovation with appropriate materials and promotion of the effective efforts for hospital's water quality assurance is highly recommended.

  9. An alkaline approach to treating cooling towers for control of Legionella pneumophila.

    PubMed Central

    States, S J; Conley, L F; Towner, S G; Wolford, R S; Stephenson, T E; McNamara, A M; Wadowsky, R M; Yee, R B

    1987-01-01

    Earlier field and laboratory studies have shown that Legionella species survive and multiply in the pH range 5.5 to 9.2. Additionally, the technical feasibility of operating cooling towers at elevated alkalinities and pH has previously been documented by published guidelines. The guidelines indicate that these conditions facilitate corrosion control and favor chlorine persistence which enhances the effectiveness of continuous chlorination in biofouling control. This information suggests that control of Legionella species in cooling towers can be accomplished by operating the towers under alkaline conditions. To test this possibility, we collected water samples over a period of months from a hospital cooling tower. The samples were analyzed for a variety of chemical parameters. Subsamples were pasteurized and inoculated with non-agar-passaged Legionella pneumophila which had been maintained in tap water. Correlation of subsequent Legionella growth with corresponding pH and alkalinity values revealed statistically significant inverse associations. These data support the hypothesis that operating cooling towers outside of the optimal conditions for Legionella growth (e.g., at elevated alkalinities and a pH greater than 9) may be a useful approach to controlling growth in this habitat. PMID:3662515

  10. Caspase-11 stimulates rapid flagellin-independent pyroptosis in response to Legionella pneumophila.

    PubMed

    Case, Christopher L; Kohler, Lara J; Lima, Jonilson B; Strowig, Till; de Zoete, Marcel R; Flavell, Richard A; Zamboni, Dario S; Roy, Craig R

    2013-01-29

    A flagellin-independent caspase-1 activation pathway that does not require NAIP5 or NRLC4 is induced by the intracellular pathogen Legionella pneumophila. Here we demonstrate that this pathway requires caspase-11. Treatment of macrophages with LPS up-regulated the host components required for this caspase-11 activation pathway. Activation by Legionella differed from caspase-11 activation using previously described agonists in that Legionella caspase-11 activation was rapid and required bacteria with a functional type IV secretion system called Dot/Icm. Legionella activation of caspase-11 induced pyroptosis by a mechanism independent of the NAIP/NLRC4 and caspase-1 axis. Legionella activation of caspase-11 stimulated activation of caspase-1 through NLRP3 and ASC. Induction of caspase-11-dependent responses occurred in macrophages deficient in the adapter proteins TRIF or MyD88 but not in macrophages deficient in both signaling factors. Although caspase-11 was produced in macrophages deficient in the type-I IFN receptor, there was a severe defect in caspase-11-dependent pyroptosis in these cells. These data indicate that macrophages respond to microbial signatures to produce proteins that mediate a capsase-11 response and that the caspase-11 system provides an alternative pathway for rapid detection of an intracellular pathogen capable of evading the canonical caspase-1 activation system that responds to bacterial flagellin.

  11. Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems.

    PubMed

    Touron-Bodilis, A; Pougnard, C; Frenkiel-Lebossé, H; Hallier-Soulier, S

    2011-08-01

    This study was designed to evaluate the usefulness of quantification by real-time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90-431). Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10(5) GU l(-1) ) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57-100% of the samples. These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real-time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. This study shows the possibility of using real-time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology. No claim to French Government works.

  12. Overexpresssion of a Legionella pneumophila homologue of the E. coli regulator csrA affects cell size, flagellation, and pigmentation.

    PubMed

    Fettes, P S; Forsbach-Birk, V; Lynch, D; Marre, R

    2001-11-01

    Legionella pneumophila is an inhabitant of the aquatic environment and the causative agent of a bacterial pneumonia. We identified the presence of an L. pneumophila homologue of csrA of E. coli and rsmA of Erwinia carotovora, genes which regulate gene expression by destabilising mRNA and which have been shown to relate to environmental fitness and pathogenicity. The Legionella csrA was able to complement a csrA-negative mutant of E. coli. Overproduction of csrA in L. pneumophila lead to a reduction of flagellation and pigmentation and an increase in bacterial cell size. csrA overproduction was associated with a reduction of fliA and flaA transcripts. This suggests that similar to E. coli and Erwinia, L. pneumophila csrA is a regulator of gene expression and may contribute to the capability of the pathogen to rapidly adapt to changing environments.

  13. Dendrimers and Polyamino-Phenolic Ligands: Activity of New Molecules Against Legionella pneumophila Biofilms

    PubMed Central

    Andreozzi, Elisa; Barbieri, Federica; Ottaviani, Maria F.; Giorgi, Luca; Bruscolini, Francesca; Manti, Anita; Battistelli, Michela; Sabatini, Luigia; Pianetti, Anna

    2016-01-01

    Legionnaires’ disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems

  14. Legionella pneumophila Catalase-Peroxidases Are Required for Proper Trafficking and Growth in Primary Macrophages

    PubMed Central

    Bandyopadhyay, Purnima; Byrne, Brenda; Chan, Yolande; Swanson, Michele S.; Steinman, Howard M.

    2003-01-01

    Legionella pneumophila, a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of Legionella-containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in katA and katB, encoding the two Legionella catalase-peroxidases, were analyzed to evaluate the hypothesis that L. pneumophila must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing katA or katB mutant Legionella colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in dotA and dotB, encoding components of the Type IV secretion system. Quantitative similarity of the katA/B phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H2O2 compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that dotA and dotB mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba Acanthamoeba castellanii, indicating that certain dotA/B functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent Legionella phagosomes in macrophages. PMID:12874332

  15. Assessment of fluorescent amplified fragment length polymorphism analysis for epidemiological genotyping of Legionella pneumophila serogroup 1.

    PubMed

    Fry, N K; Afshar, B; Visca, P; Jonas, D; Duncan, J; Nebuloso, E; Underwood, A; Harrison, T G

    2005-09-01

    This study assessed the reproducibility and epidemiological concordance of double-enzyme fluorescent amplified fragment length polymorphism (fAFLP) analysis for genotyping of Legionella pneumophila serogroup (sg) 1. fAFLP fragment analysis was performed on three different sequencing platforms (one gel- and two capillary-based) in different laboratories with a well-characterised set of 50 strains of L. pneumophila sg 1. fAFLP data were analysed with the Pearson correlation similarity coefficient, using a range of parameters, and dendrogram outputs were converted to arbitrary types after selection of a specified percentage similarity threshold. The results obtained were compared with those obtained by the standard non-fluorescent AFLP method and were found to be broadly concordant. Using optimised settings for each fAFLP method to analyse the panel of 50 strains, epidemiological concordance (E) and reproducibility (R) values of 1.00 were obtained, and the number of types ranged from nine to 15, compared with E=1.00 and R=1.00, with 16 types, for the non-fluorescent AFLP protocol. The study demonstrated the potential of fAFLP for typing strains of L. pneumophila sg 1 on all three platforms; however, inter-platform comparison of fAFLP data was not achieved. fAFLP analysis may have a role in the fingerprinting of multiple isolates during Legionella outbreak investigations, but further work is required before type designations and identification libraries can be developed.

  16. A field study of the survival of Legionella pneumophila in a hospital hot-water system.

    PubMed Central

    Farrell, I. D.; Barker, J. E.; Miles, E. P.; Hutchison, J. G.

    1990-01-01

    The colonization, survival and control of Legionella pneumophila in a hospital hot-water system was examined. The organism was consistently isolated from calorifier drain-water samples at temperatures of 50 degrees C or below, despite previous chlorination of the system. When the temperature of one of two linked calorifiers was raised to 60 degrees C, by closing off the cold-water feed, the legionella count decreased from c. 10(4) c.f.u./l to an undetectable level. However, 10 min after turning on the cold-water feed which produced a fall in calorifier temperature, the count in the calorifier drain water returned to its original level. Investigations revealed that the cold-water supply was continually feeding the calorifiers with L. pneumophila. Simple modifications in the design of the system were made so that the cold-water feed no longer exceeds 20 degrees C; these measures have considerably reduced the number of L. pneumophila reaching the calorifiers. PMID:2189741

  17. Isolation and characterization of auxotrophic mutants of Legionella pneumophila that fail to multiply in human monocytes.

    PubMed Central

    Mintz, C S; Chen, J X; Shuman, H A

    1988-01-01

    Attempts to isolate auxotrophic mutants of Legionella pneumophila have been hampered by the complex nutritional composition of the media used to cultivate this organism. We developed a semidefined medium, designated CAA, to facilitate the isolation and characterization of Legionella auxotrophs. Unlike previously described chemically defined media for this organism, L. pneumophila formed colonies on CAA agar. Using this medium, we isolated several independent tryptophan auxotrophs of strain Philadelphia-1 after ethyl methanesulfonate mutagenesis and penicillin enrichment. Trimethoprim selection was used to isolate several independent thymidine-requiring mutants of the same strain. The thymidine auxotrophs exhibited a marked decrease in viability when they were deprived of thymidine. The results of monocyte infection experiments with both the tryptophan and thymidine auxotrophs indicated that the thymidine auxotrophs were incapable of intracellular survival or multiplication. In contrast, the tryptophan auxotrophs grew well in monocyte cultures. The isolation of additional auxotrophic mutants will facilitate the study of the nutritional requirements of L. pneumophila for growth in human mononuclear phagocytes. PMID:3372016

  18. Influence of Legionella pneumophila and other water bacteria on the survival and growth of Acanthamoeba polyphaga.

    PubMed

    Anacarso, I; Guerrieri, E; Bondi, M; de Niederhäusern, S; Iseppi, R; Sabia, C; Contri, M; Borella, P; Messi, P

    2010-10-01

    We investigated in solid medium, in water microcosm co-cultures and by light and transmission electron microscopy the influence of Legionella pneumophila Lp-1, Pseudomonas aeruginosa ATCC 27853, Burkholderia cepacia ATCC 25416 and Pseudomonas fluorescens SSD35 on the growth and survival of Acanthamoeba polyphaga. The infection with L. pneumophila was microscopically characterized by the presence of few bacteria inside protozoa at 4th h, and by the beginning of disruptive effects in late phase of trial. In water microcosm studies, performed at different temperature, the more significant interactions were observed at 30°C. In these conditions, L. pneumophila caused a marked reduction in trophozoite and cyst counts from the 4th day until the end of incubation (11 days). B. cepacia showed, by microscopic observation, few and generally single rods within protozoan phagosomes and caused a light reduction of trophozoite viability and cyst formation in co-cultures. A more invasive type of endocytosis, characterized by an early invasion with the presence of a high bacteria number inside amoebae, was observed for Pseudomonas strains. P. fluorescens produced a violent lysis of the host, whereas P. aeruginosa did not cause lysis or suffering. These results underline that water bacteria other than legionella are capable of intracellular survival in Acanthamoeba, influencing the protozoa viable cycle.

  19. Host FIH-Mediated Asparaginyl Hydroxylation of Translocated Legionella pneumophila Effectors

    PubMed Central

    Price, Christopher; Merchant, Michael; Jones, Snake; Best, Ashley; Von Dwingelo, Juanita; Lawrenz, Matthew B.; Alam, Nawsad; Schueler-Furman, Ora; Kwaik, Yousef A.

    2017-01-01

    FIH-mediated post-translational modification through asparaginyl hydroxylation of eukaryotic proteins impacts regulation of protein-protein interaction. We have identified the FIH recognition motif in 11 Legionella pneumophila translocated effectors, YopM of Yersinia, IpaH4.5 of Shigella and an ankyrin protein of Rickettsia. Mass spectrometry analyses of the AnkB and AnkH effectors of L. pneumophila confirm their asparaginyl hydroxylation. Consistent with localization of the AnkB effector to the Legionella-containing vacuole (LCV) membrane and its modification by FIH, our data show that FIH and its two interacting proteins, Mint3 and MT1-MMP are acquired by the LCV in a Dot/Icm type IV secretion-dependent manner. Chemical inhibition or RNAi-mediated knockdown of FIH promotes LCV-lysosomes fusion, diminishes decoration of the LCV with polyubiquitinated proteins, and abolishes intra-vacuolar replication of L. pneumophila. These data show acquisition of the host FIH by a pathogen-containing vacuole and that asparaginyl-hydroxylation of translocated effectors is indispensable for their function. PMID:28321389

  20. Legionella Pneumophila and TATLOCK Bacterium: Simple, Effective Giemsa Stain.

    DTIC Science & Technology

    1980-11-07

    presumptive diagnosis of Legionnaires ’ disease and Pittsburgh pneumonia. i9 ! I __ _ _ _ _ _ _ _ _ _ _ THE ORGANISMS which cause so called Legionnaires ...demonstrating L. pneumophila in impression smears and scrapings of fresh or formalin-fixed lung tissue from a patient who died of Legionnaires ’ disease , and...erium- (3) in t;iem .k stai:ned impression :;mcars of Lullg tissue from suspected cases of Legionnaires ’ Disease and Pittsburgh pneumonia may be i

  1. Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires.

    PubMed

    Wood, Rebecca E; Newton, Patrice; Latomanski, Eleanor A; Newton, Hayley J

    2015-10-01

    Legionella organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed the Legionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of Legionella pneumophila. Here, we have characterized the Legionella longbeachae replicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of L. pneumophila (pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deleting dotB and demonstrated the inability of this mutant to replicate inside THP-1 cells. L. longbeachae does not enter THP-1 cells as efficiently as L. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO (where RalFLLO is the L. longbeachae homologue of RalF) into THP-1 cells by the L. longbeachae Dot/Icm system is less efficient than that by L. pneumophila. This difference is negated in A549 cells where L. longbeachae and L. pneumophila infect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the Rab-like effector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the longbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the longbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the L. longbeachae replicative vacuole, highlighting phenotypic similarities to the vacuole of L. pneumophila as well as unique aspects of LCV biology.

  2. Phase-variable Expression of Lipopolysaccharide Contributes to the Virulence of Legionella pneumophila

    PubMed Central

    Lüneberg, Edeltraud; Zähringer, Ulrich; Knirel, Yuriy A.; Steinmann, Dorothee; Hartmann, Maike; Steinmetz, Ivo; Rohde, Manfred; Köhl, Jörg; Frosch, Matthias

    1998-01-01

    With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625–positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement components, whereas the parental strain RC1 was almost serum resistant. Moreover, mutant 811 was not able to replicate intracellularly in macrophage-like cell line HL-60. In the guinea pig animal model, mutant 811 exhibited significantly reduced ability to replicate. Among recovered bacteria, mAb 2625–positive revertants were increased by fourfold. The relevance of LPS phase switch for pathogenesis of Legionella infection was further corroborated by the observation that 5% of the bacteria recovered from the lungs of guinea pigs infected with the wild-type strain RC1 were negative for mAb 2625 binding. These findings strongly indicate that under in vivo conditions switching between two LPS phenotypes occurs and may promote adaptation and replication of L. pneumophila. This is the first description of phase-variable expression of Legionella LPS. PMID:9653083

  3. Growth-related Metabolism of the Carbon Storage Poly-3-hydroxybutyrate in Legionella pneumophila.

    PubMed

    Gillmaier, Nadine; Schunder, Eva; Kutzner, Erika; Tlapák, Hana; Rydzewski, Kerstin; Herrmann, Vroni; Stämmler, Maren; Lasch, Peter; Eisenreich, Wolfgang; Heuner, Klaus

    2016-03-18

    Legionella pneumophila, the causative agent of Legionnaires disease, has a biphasic life cycle with a switch from a replicative to a transmissive phenotype. During the replicative phase, the bacteria grow within host cells in Legionella-containing vacuoles. During the transmissive phenotype and the postexponential (PE) growth phase, the pathogens express virulence factors, become flagellated, and leave the Legionella-containing vacuoles. Using (13)C labeling experiments, we now show that, under in vitro conditions, serine is mainly metabolized during the replicative phase for the biosynthesis of some amino acids and for energy generation. During the PE phase, these carbon fluxes are reduced, and glucose also serves as an additional carbon substrate to feed the biosynthesis of poly-3-hydroxybuyrate (PHB), an essential carbon source for transmissive L. pneumophila. Whole-cell FTIR analysis and comparative isotopologue profiling further reveal that a putative 3-ketothiolase (Lpp1788) and a PHB polymerase (Lpp0650), but not enzymes of the crotonyl-CoA pathway (Lpp0931-0933) are involved in PHB metabolism during the PE phase. However, the data also reflect that additional bypassing reactions for PHB synthesis exist in agreement with in vivo competition assays using Acanthamoeba castellannii or human macrophage-like U937 cells as host cells. The data suggest that substrate usage and PHB metabolism are coordinated during the life cycle of the pathogen.

  4. Genetic diversity and evolutionary relationships among Legionella pneumophila clinical isolates, Portugal, 1987 to 2012.

    PubMed

    Chasqueira, M J; Rodrigues, L; Nascimento, M; Ramos, M; Marques, T

    2014-11-20

    The genetic diversity of 89 clinical Legionella isolates, collected between 1987 and 2012, in 22 hospitals from the five regions of Portugal, was analysed in this study using monoclonal antibodies (MAbs) of the Dresden panel and the sequence-based typing (SBT) protocol. The eBURST algorithm was used to infer levels of relatedness between isolates. All isolates collected were Legionella pneumophila, which were further characterised into four subgroups by MAbs, and 30 sequence types (STs) by SBT. Twelve of the STs were unique to Portugal; one of them (ST100) was represented by 32 epidemiologically related isolates. The ST44 was the profile with the highest number of epidemiologically unrelated isolates. The eBURST analyses indicate that, within the group formed by the 30 STs identified in this study, 17 STs were genetically close to at least another ST in the group. The comparison between the eBURST diagrams obtained with the STs from this study and the entire SBT database of the European Working Group for Legionella, showed that 24 (seven of them unique to Portugal) of our 30 STs were related with STs identified in others countries. These results suggest that the population of L. pneumophila clinical strains in Portugal includes both worldwide and local strains.

  5. [Comparison of five commercial assays for the detection of Legionella pneumophila antigens in urine].

    PubMed

    de Ory, Fernando; Minguito, Teodora

    2009-02-01

    Antigenuria detection is the main approach for diagnosing Legionella infections. The aim of this study was to compare 5 commercially available methods for detecting Legionella pneumophila soluble antigens in urine. Seventy-one urine samples were tested, 62 from patients with bacterial infection and 9 from patients with respiratory syncytial virus infection. All samples were assayed for the presence of L. pneumophila by immunoenzymatic (ELISA) (Binax and Bartels), and immunochromatographic (IC) (Binax, SAS and Uni-Gold) methods. Identical results (35 positive and 17 negative) were obtained by the 5 assays in 52 samples (73.2%). Samples showing discrepant results were classified by the majority criterion, and/or other laboratory results (serology), and/or epidemiological findings. On this basis, 51 samples were ultimately classified as positive, and 20 as negative. Sensitivity values of ELISA-Binax, ELISA-Bartels, IC-Binax, IC-SAS and IC-Uni-Gold were 80.4, 100, 82.4, 86.3, and 70.6%, respectively. Corresponding values for specificity were 90, 95, 100, 95 and 100%. The results indicate that the methods compared are all adequate for diagnosing Legionella infection, although some have certain limitations regarding sensitivity.

  6. A simple method for the eradication of Legionella pneumophila from potable water systems.

    PubMed

    Moreno, C; de Blas, I; Miralles, F; Apraiz, D; Catalan, V

    1997-12-01

    In this paper we describe a simple method, noncorrosive to pipes, for the eradication of Legionella pneumophila from potable water systems. This method is based on the systematic purging of the pipe networks with cold water containing 1-1.5 mg residual chlorine/L. In the hot water system, a new pipe bypassing the water heater was installed, whereas in the air conditioning system, the circuit is purged with water from the tap water system. The feasibility of this method was studied in two hotels in which the presence of Legionella was detected despite treatment of the water by the hyperchlorination method. The evolution of the presence of Legionella was studied by culture and polymerase chain reaction. Eighty samples from hotel A and sixty-seven samples from hotel B were analyzed during the time that the eradication method was applied. Our results showed that this method permitted the effective elimination of L. pneumophila after 5 months in hotel A and 7 months in hotel B.

  7. Comparison of arbitrarily primed polymerase chain reaction, ribotyping, and monoclonal antibody analysis for subtyping Legionella pneumophila serogroup 1.

    PubMed Central

    Gomez-Lus, P; Fields, B S; Benson, R F; Martin, W T; O'Connor, S P; Black, C M

    1993-01-01

    Arbitrarily primed polymerase chain reaction (AP-PCR) was used to characterize Legionella pneumophila serogroup 1. Cells from a single colony could be subtyped by AP-PCR within a few hours. The discrimination between strains of L. pneumophila serogroup 1 by AP-PCR was equivalent to that by monoclonal antibody analysis and ribotyping. Four strains representing the monoclonal antibody pattern most frequently associated with outbreaks all yielded unique amplicon patterns by AP-PCR. Images PMID:8394380

  8. Survival and multiplication of Legionella pneumophila in municipal drinking water systems.

    PubMed Central

    States, S J; Conley, L F; Kuchta, J M; Oleck, B M; Lipovich, M J; Wolford, R S; Wadowsky, R M; McNamara, A M; Sykora, J L; Keleti, G

    1987-01-01

    Studies were conducted to investigate the survival and multiplication of Legionella spp. in public drinking water supplies. An attempt was made, over a period of several years, to isolate legionellae from a municipal system. Sampling sites included the river water supply, treatment plant, finished water reservoir system, mains, and distribution taps. Despite the use of several isolation techniques, Legionella spp. could not be detected in any of the samples other than those collected from the river. It was hypothesized that this was due to the maintenance of a chlorine residual throughout the system. To investigate the potential for Legionella growth, additional water samples, collected from throughout the system, were dechlorinated, pasteurized, and inoculated with Legionella pneumophila. Subsequent growth indicated that many of these samples, especially those collected from areas affected by an accumulation of algal materials, exhibited a much greater ability to support Legionella multiplication than did river water prior to treatment. Chemical analyses were also performed on these samples. Correlation of chemical data and experimental growth results indicated that the chemical environment significantly affects the ability of the water to support multiplication, with turbidity, organic carbon, and certain metals being of particular importance. These studies indicate that the potential exists for Legionella growth within municipal systems and support the hypothesis that public water supplies may contaminate the plumbing systems of hospitals and other large buildings. The results also suggest that useful methods to control this contamination include adequate treatment plant filtration, maintenance of a chlorine residual throughout the treatment and distribution network, and effective covering of open reservoirs. PMID:3606101

  9. Rapid on-site monitoring of Legionella pneumophila in cooling tower water using a portable microfluidic system.

    PubMed

    Yamaguchi, Nobuyasu; Tokunaga, Yusuke; Goto, Satoko; Fujii, Yudai; Banno, Fumiya; Edagawa, Akiko

    2017-06-08

    Legionnaires' disease, predominantly caused by the bacterium Legionella pneumophila, has increased in prevalence worldwide. The most common mode of transmission of Legionella is inhalation of contaminated aerosols, such as those generated by cooling towers. Simple, rapid and accurate methods to enumerate L. pneumophila are required to prevent the spread of this organism. Here, we applied a microfluidic device for on-chip fluorescent staining and semi-automated counting of L. pneumophila in cooling tower water. We also constructed a portable system for rapid on-site monitoring and used it to enumerate target bacterial cells rapidly flowing in the microchannel. A fluorescently-labelled polyclonal antibody was used for the selective detection of L. pneumophila serogroup 1 in the samples. The counts of L. pneumophila in cooling tower water obtained using the system and fluorescence microscopy were similar. The detection limit of the system was 10(4) cells/ml, but lower numbers of L. pneumophila cells (10(1) to 10(3) cells/ml) could be detected following concentration of 0.5-3 L of the water sample by filtration. Our technique is rapid to perform (1.5 h), semi-automated (on-chip staining and counting), and portable for on-site measurement, and it may therefore be effective in the initial screening of Legionella contamination in freshwater.

  10. Lactoferrin inhibits or promotes Legionella pneumophila intracellular multiplication in nonactivated and interferon gamma-activated human monocytes depending upon its degree of iron saturation. Iron-lactoferrin and nonphysiologic iron chelates reverse monocyte activation against Legionella pneumophila.

    PubMed Central

    Byrd, T F; Horwitz, M A

    1991-01-01

    We have been exploring the role of iron in the pathogenesis of the intracellular bacterial pathogen Legionella pneumophila. In previous studies, we have demonstrated that L. pneumophila intracellular multiplication in human monocytes is iron dependent and that IFN gamma-activated monocytes inhibit L. pneumophila intracellular multiplication by limiting the availability of iron. In this study, we have investigated the effect on L. pneumophila intracellular multiplication of lactoferrin, an iron-binding protein which is internalized via specific receptors on monocytes, and of nonphysiologic iron chelates which enter monocytes by a receptor-independent route. Apolactoferrin completely inhibited L. pneumophila multiplication in nonactivated monocytes, and enhanced the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication. In contrast, iron-saturated lactoferrin had no effect on the already rapid rate of L. pneumophila multiplication in nonactivated monocytes. Moreover, it reversed the capacity of activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron from the lactoferrin-lactoferrin receptor pathway. The capacity of iron-lactoferrin to reverse monocyte activation was dependent upon its percent iron saturation and not just its total iron content. Similarly, the nonphysiologic iron chelates ferric nitrilotriacetate and ferric ammonium citrate completely reverse and ferric pyrophosphate partially reversed the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron derived from nonphysiologic iron chelates internalized by monocytes independently of the transferrin and lactoferrin endocytic pathways. This study suggests that at sites of inflammation, lactoferrin may inhibit or promote L. pneumophila intracellular multiplication in mononuclear phagocytes depending upon

  11. Mixed infection by Legionella pneumophila in outbreak patients.

    PubMed

    Coscollá, Mireia; Fernández, Carmen; Colomina, Javier; Sánchez-Busó, Leonor; González-Candelas, Fernando

    2014-05-01

    During the molecular epidemiological study of a legionellosis outbreak, we obtained sequence based typing (SBT) profiles from uncultured respiratory samples of 15 affected patients. We detected several distinct allelic profiles some of which were a mixture of alleles present in the more common profiles. Chromatograms from the sequences of one patient with mixed profile showed polymorphisms in several positions, which could result from the simultaneous presence of different Legionella variants in the sample. In order to test this possibility, we cloned PCR amplification products from six loci for two patients with a mixed profile and a patient with a pure profile. After obtaining around 20 sequences for each locus of three patients, we detected several variants in two of them and two variants in the third one. In summary, the three analyzed patients showed evidence of more than one Legionella variant during the acute infection. These results indicate that probably some patients were infected by more than one strain, which could be due to co-infection from the same environmental source or, alternatively, to independent infections in a very short period of time. Although our data cannot discriminate between these hypotheses, these results suggest that Legionella infection patterns can be more complex than previously assumed. None of the environmental samples analyzed during this outbreak was even similar to any of the clinical ones.

  12. Integration of Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms grown on domestic plumbing materials.

    PubMed

    Moritz, Miriam M; Flemming, Hans-Curt; Wingender, Jost

    2010-06-01

    Drinking water biofilms were grown on coupons of plumbing materials, including ethylene-propylene-diene-monomer (EPDM) rubber, silane cross-linked polyethylene (PE-X b), electron-ray cross-linked PE (PE-X c) and copper under constant flow-through of cold tap water. After 14 days, the biofilms were spiked with Pseudomonas aeruginosa, Legionella pneumophila and Enterobacter nimipressuralis (10(6) cells/mL each). The test bacteria were environmental isolates from contamination events in drinking water systems. After static incubation for 24 h, water flow was resumed and continued for 4 weeks. Total cell count and heterotrophic plate count (HPC) of biofilms were monitored, and P. aeruginosa, L. pneumophila and E. nimipressuralis were quantified, using standard culture-based methods or culture-independent fluorescence in situ hybridization (FISH). After 14 days total cell counts and HPC values were highest on EPDM followed by the plastic materials and copper. P. aeruginosa and L. pneumophila became incorporated into drinking water biofilms and were capable to persist in biofilms on EPDM and PE-X materials for several weeks, while copper biofilms were colonized only by L. pneumophila in low culturable numbers. E. nimipressuralis was not detected in any of the biofilms. Application of the FISH method often yielded orders of magnitude higher levels of P. aeruginosa and L. pneumophila than culture methods. These observations indicate that drinking water biofilms grown under cold water conditions on domestic plumbing materials, especially EPDM and PE-X in the present study, can be a reservoir for P. aeruginosa and L. pneumophila that persist in these habitats mostly in a viable but non-culturable state.

  13. The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

    PubMed Central

    Hales, Laura M.; Shuman, Howard A.

    1999-01-01

    To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS. PMID:10438758

  14. A single Legionella pneumophila genotype in the freshwater system in a ship experiencing three separate outbreaks of legionellosis in 6 years

    PubMed Central

    Ahlen, Catrine; Aas, Marianne; Krusnell, Jadwiga; Iversen, Ole-Jan

    2016-01-01

    Background Recurrent legionella outbreaks at one and the same location are common. We have identified a single Legionella pneumophila genotype associated with recurrent Legionella outbreaks over 6 years. Methods Field emergency surveys following Legionella outbreaks were performed on a vessel in 2008, 2009 and 2013. Water samples from both the distribution and technical parts of the potable water system were analyzed with respect to L. pneumophila [Real-Time PCR, cultivation, serotyping and genotyping (PFGE)] and free-living amoebae, (FLA). Results Legionella pneumophila serogroup 1 was present in the ship's potable water system during every outbreak. Genotyping of the 2008 survey material showed two separate PFGE genotypes while those in 2009 and 2013 demonstrated the presence of only one of the two genotypes. FLA with intracellular L. pneumophila of the same genotype were also detected. Analyses of the freshwater system on a ship following three separate Legionella outbreaks, for L. pneumophila and FLAs, revealed a single L. pneumophila genotype and FLA (Hartmanella). Conclusions It is reasonable to assume that the L. pneumophila genotype detected in the freshwater system was the causal agent in the outbreaks onboard. Persistence of an apparently low-pathogenic L. pneumophila genotype and FLA in a potable water system represent a potential risk for recurrent outbreaks. PMID:27515183

  15. Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila.

    PubMed Central

    Elliott, J A; Winn, W C

    1986-01-01

    Legionella pneumophila multiplied rapidly in guinea pig and rat alveolar macrophages but failed to grow when phagocytic activity was inhibited by pretreatment with 0.5 or 1.0 microgram of cytochalasin D per ml. Attachment was not inhibited by cytochalasin D. No extracellular multiplication occurred when L. pneumophila were in close proximity to viable functional macrophages or even when the bacteria were attached to plasma membranes of the macrophages. Nonopsonized L. pneumophila were avidly phagocytized by alveolar macrophages. When bacteria were centrifuged onto a cell pellet, more than 85% of the phagocytes contained one or more bacteria within 15 min. In contrast, under the same conditions only approximately 15% of the macrophages contained nonopsonized Escherichia coli or Staphylococcus aureus. Phagocytosis of L. pneumophila by untreated guinea pig macrophages occurred by extension of pseudopodia around the bacteria in a classical manner. The failure of the bacteria to actively penetrate the phagocyte suggests that their intracellular survival must not depend on avoidance of a phagosome but rather on an inhibition of or resistance to subsequent microbicidal functions of the macrophage. Images PMID:3941000

  16. Passage through Tetrahymena tropicalis enhances the resistance to stress and the infectivity of Legionella pneumophila.

    PubMed

    Koubar, Mohamad; Rodier, Marie-Hélène; Garduño, Rafael A; Frère, Jacques

    2011-12-01

    Legionella pneumophila is a gram-negative bacterium prevalent in fresh water which accidentally infects humans and is responsible for the disease called legionellosis. Intracellular growth of L. pneumophila in Tetrahymena is inconsistent; in the species Tetrahymena tropicalis stationary-phase forms (SPFs) of L. pneumophila differentiate into mature intracellular forms (MIFs) without apparent bacterial replication and are expelled from the ciliate as pellets containing numerous MIFS. In the present work, we tested the impact of L. pneumophila passage through T. tropicalis. We observed that MIFs released from T. tropicalis are more resistant to various stresses than SPFs. Under our conditions, MIFs harboured a higher gentamicin resistance, maintained even after 3 months as pellets. Long-term survival essays revealed that MIFs survived better in a nutrient-poor environment than SFPs, as a reduction of only about 3 logs was observed after 4 months in the MIF population, whereas no cultivable SPFs were detected after 3 months in the same medium, corresponding to a loss of about 7 logs. We have also observed that MIFs are significantly more infectious in human pneumocyte cells compared with SPFs. These results strongly suggest a potential role of ciliates in increasing the risk of legionellosis.

  17. Sensitivity and resistance of Legionella pneumophila to some antibiotics and combinations of antibiotics.

    PubMed

    Moffie, B G; Mouton, R P

    1988-10-01

    For the treatment of Legionella pneumophila infections erythromycin and rifampicin are the antibiotics of choice. In view of reported therapy failures other antibiotics, e.g. the quinolones, are currently under investigation. The sensitivity of L. pneumophila to four antibiotics and to combinations of antibiotics was investigated and the rate of mutations was calculated. For 20 L. pneumophila strains we determined the MIC of rifampicin (0.002-0.004 mg/l), erythromycin (0.063-0.125 mg/l), norfloxacin (0.125 mg/l) and ciprofloxacin (0.016-0.032 mg/l). Mutation rates ranged from 1 x 10(-8) for ciprofloxacin to greater than 1 x 10(-7) for erythromycin, resulting in high-level resistance to rifampicin in most strains and erythromycin resistance in one strain, but not in resistance to the quinolones. The combination of erythromycin and rifampicin was synergistic (FIC index less than 0.5) against four of the L. pneumophila strains and showed indifference (FIC index 0.5-2.0) for the remainder (mean FIC index 0.79). Combinations of ciprofloxacin and erythromycin and of rifampicin and ciprofloxacin showed only indifference (mean FIC index respectively 1.05 and 1.21). Combining rifampicin with ciprofloxacin was not effective in reducing the number of mutants for either of these antibiotics, whereas the other combinations did prevent this.

  18. Factors affecting the recovery of Legionella pneumophila serogroup 1 from cooling tower water systems.

    PubMed

    Lu, H F; Tsou, M F; Huang, S Y; Tsai, W C; Chung, J G; Cheng, K S

    2001-09-01

    A total of 20 water samples collected from the cooling towers at 20 different sites were analyzed under various conditions for the presence of Legionella pneumophila serogroup 1. A comparative assessment was performed to evaluate methods of sample collection (spray drops, beneath water at 20- to 40-cm depth, and water outlet), concentration (filtration and centrifugation), acid buffer treatment (no treatment, treatment for 3, 5, and 15 min), and CO2 incubation or candle jar incubation. The reduction in viable colonies and false negative rate were compared for the different factors. No quantitative differences in isolation of L. pneumophila serogroup 1 was found among samples collected from water at a depth of 20 to 40 cm, from water outlet, and from spray drops. Treatment in an acid buffer for 15 min significantly reduced the recovery rate, with a reduction in bacterial counts of about 40%, compared with a 3-min (12%) or a 5-min (25%) treatment. Acid buffer treatment for 3 or 5 min reduced the overgrowth of commensal flora. This treatment improved the selectivity but not the sensitivity for L. pneumophila serogroup 1. Colonies on plates incubated at 37 degrees C in a candle jar with a humidified atmosphere grew better than those incubated at 35 degrees C with 5% CO2. These results demonstrate that methods of sample collection, concentration, and incubation, but not collection site, can affect the isolation rate for L. pneumophila serogroup 1.

  19. Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans

    PubMed Central

    Shadoud, Lubana; Almahmoud, Iyad; Jarraud, Sophie; Etienne, Jérôme; Larrat, Sylvie; Schwebel, Carole; Timsit, Jean-François; Schneider, Dominique; Maurin, Max

    2015-01-01

    Background Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila. Methods We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples. Findings We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment. Interpretation In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics. PMID:26501115

  20. Cytopathogenicity and molecular subtyping of Legionella pneumophila environmental isolates from 17 hospitals.

    PubMed

    Garcia-Nuñez, M; Pedro-Botet, M L; Ragull, S; Sopena, N; Morera, J; Rey-Joly, C; Sabria, M

    2009-02-01

    The cytopathogenicity of 22 Legionella pneumophila isolates from 17 hospitals was determined by assessing the dose of bacteria necessary to produce 50% cytopathic effect (CPED50) in U937 human-derived macrophages. All isolates were able to infect and grow in macrophage-like cells (range log10 CPED50: 2.67-6.73 c.f.u./ml). Five groups were established and related to the serogroup, the number of PFGE patterns coexisting in the same hospital water distribution system, and the possible reporting of hospital-acquired Legionnaires' disease cases. L. pneumophila serogroup 1 isolates had the highest cytopathogenicity (P=0.003). Moreover, a trend to more cytopathogenic groups (groups 1-3) in hospitals with more than one PFGE pattern of L. pneumophila in the water distribution system (60% vs. 17%) and in hospitals reporting cases of hospital-acquired Legionnaires' disease (36.3% vs. 16.6%) was observed. We conclude that the cytopathogenicty of environmental L. pneumophila should be taken into account in evaluating the risk of a contaminated water reservoir in a hospital and hospital acquisition of Legionnaires' disease.

  1. Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans.

    PubMed

    Shadoud, Lubana; Almahmoud, Iyad; Jarraud, Sophie; Etienne, Jérôme; Larrat, Sylvie; Schwebel, Carole; Timsit, Jean-François; Schneider, Dominique; Maurin, Max

    2015-09-01

    Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila. We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples. We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment. In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

  2. Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication.

    PubMed

    Molofsky, Ari B; Swanson, Michele S

    2003-10-01

    Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.

  3. Legionella pneumophila couples fatty acid flux to microbial differentiation and virulence.

    PubMed

    Edwards, Rachel L; Dalebroux, Zachary D; Swanson, Michele S

    2009-03-01

    During its life cycle, Legionella pneumophila alternates between at least two phenotypes: a resilient, infectious form equipped for transmission and a replicative cell type that grows in amoebae and macrophages. Considering its versatility, we postulated that multiple cues regulate L. pneumophila differentiation. Beginning with a Biolog Phenotype MicroArray screen, we demonstrate that excess short-chain fatty acids (SCFAs) trigger replicative cells to cease growth and activate their panel of transmissive traits. To co-ordinate their response to SCFAs, L. pneumophila utilizes the LetA/LetS two-component system, but not phosphotransacetylase or acetyl kinase, two enzymes that generate high-energy phosphate intermediates. Instead, the stringent response enzyme SpoT appears to monitor fatty acid biosynthesis to govern transmission trait expression, as an altered distribution of acylated acyl carrier proteins correlated with the SpoT-dependent differentiation of cells treated with either excess SCFAs or the fatty acid biosynthesis inhibitors cerulenin and 5-(tetradecyloxy)-2-furoic acid. We postulate that, by exploiting the stringent response pathway to couple cellular differentiation to its metabolic state, L. pneumophila swiftly acclimates to stresses encountered in its host or the environment, thereby enhancing its overall fitness.

  4. Cutting edge: pulmonary Legionella pneumophila is controlled by plasmacytoid dendritic cells but not type I IFN.

    PubMed

    Ang, Desmond K Y; Oates, Clare V L; Schuelein, Ralf; Kelly, Michelle; Sansom, Fiona M; Bourges, Dorothée; Boon, Louis; Hertzog, Paul J; Hartland, Elizabeth L; van Driel, Ian R

    2010-05-15

    Plasmacytoid dendritic cells (pDCs) are well known as the major cell type that secretes type I IFN in response to viral infections. Their role in combating other classes of infectious organisms, including bacteria, and their mechanisms of action are poorly understood. We have found that pDCs play a significant role in the acute response to the intracellular bacterial pathogen Legionella pneumophila. pDCs were rapidly recruited to the lungs of L. pneumophila-infected mice, and depletion of pDCs resulted in increased bacterial load. The ability of pDCs to combat infection did not require type I IFN. This study points to an unappreciated role for pDCs in combating bacterial infections and indicates a novel mechanism of action for this cell type.

  5. Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

    PubMed

    Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge

    2015-04-01

    The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but could no longer be detected after exposure to 6 mg/L of ClO2, although viable but not culturable (VBNC) cells were found after exposure to 4-5 mg/L of ClO2. When testing whether these VBNC were infective, two of the strains were resuscitated after co-culture with Acanthamoeba polyphaga, but neither of them could infect macrophage-like cells.

  6. Legionella pneumophila, armed to the hilt: justifying the largest arsenal of effectors in the bacterial world.

    PubMed

    Ensminger, Alexander W

    2016-02-01

    Many bacterial pathogens use dedicated translocation systems to deliver arsenals of effector proteins to their hosts. Once inside the host cytosol, these effectors modulate eukaryotic cell biology to acquire nutrients, block microbial degradation, subvert host defenses, and enable pathogen transmission to other hosts. Among all bacterial pathogens studied to date, the gram-negative pathogen, Legionella pneumophila, maintains the largest arsenal of effectors, with over 330 effector proteins translocated by the Dot/Icm type IVB translocation system. In this review, I will discuss some of the recent work on understanding the consequences of this large arsenal. I will also present several models that seek to explain how L. pneumophila has acquired and subsequently maintained so many more effectors than its peers.

  7. [Identification of chemical structure of antibacterial components against Legionella pneumophila in a coffee beverage].

    PubMed

    Dogasaki, Chikaku; Shindo, Tetsuya; Furuhata, Katsunori; Fukuyama, Masafumi

    2002-07-01

    We previously reported that certain constituents in brewed coffee exhibited antibacterial activities against a strain of Legionella pneumophila. The constituents showing antibacterial activities were included only in extracts cold with water or hot water. To determine the antibacterial substances in coffee extract, the extract was fractionated by HPLC using a UV/photodiode array detector. The optimum HPLC conditions for analysis were UV wavelength of 250 nm and eluents of methanol/acetic acid (10/90), pH 3.0. When several fractions separated by HPLC were investigated for antibacterial activities against L. pneumophila, it was found that three peak fractions exhibited strong antibacterial activities. Each product from these fractions was analyzed by NMR and LC-mass spectrometry, and the chemical structure of each was determined. It was shown that the antibacterial substances was were protocatechuic acid (3,4-dihydroxy benzoic acid), chlorogenic acid, and caffeic acid.

  8. Discovery of a Specific Inhibitor of Pyomelanin Synthesis in Legionella pneumophila.

    PubMed

    Aubi, Oscar; Flydal, Marte I; Zheng, Huaixin; Skjærven, Lars; Rekand, Illimar; Leiros, Hanna-Kirsti S; Haug, Bengt Erik; Cianciotto, Nicholas P; Martinez, Aurora; Underhaug, Jarl

    2015-11-12

    Phenylalanine hydroxylase catalyzes the first step in the synthesis of pyomelanin, a pigment that aids in the acquisition of essential iron in certain bacteria. In this work, we present the development and application of a drug discovery protocol by targeting this enzyme in Legionella pneumophila, the major causative agent of Legionnaires' disease. We employ a combination of high-throughput screening to identify small-molecule binders, enzymatic activity measurements to identify inhibitors in vitro, and the verification of the inhibitory effect in vivo. The most potent inhibitor shows an IC50 value in the low micromolar range and successfully abolishes the synthesis of pyomelanin in L. pneumophila cultures at 10 μM. Thus, this compound represents a novel and effective tool for investigating the role of pyomelanin in the biology and pathogenicity of this organism. Altogether, the results demonstrate a successful pathway for drug development focusing on binding specificity in the initial high-throughput screening steps.

  9. Major outer membrane protein of Legionella pneumophila carries a species-specific epitope.

    PubMed Central

    Nolte, F S; Conlin, C A

    1986-01-01

    A monoclonal antibody (LP3IIG2) directed against a species-specific epitope of Legionella pneumophila is available from Genetic Systems Corp., Seattle, Wash., for use as a diagnostic reagent. Outer membrane protein-rich fractions were prepared from L. pneumophila serogroups 1 to 8 by treatment of cell envelopes with 2% Triton X-100. Immunoblots of sodium dodecyl sulfate-polyacrylamide gels demonstrated that each membrane fraction contained two bands that reacted with LP3IIG2. The monoclonal antibody bound preferentially to a 26,000-molecular-weight band that appears to result from modification of the 29,000-molecular-weight major outer membrane protein. Images PMID:2420824

  10. The Legionella pneumophila replication vacuole: making a cosy niche inside host cells.

    PubMed

    Isberg, Ralph R; O'Connor, Tamara J; Heidtman, Matthew

    2009-01-01

    The pathogenesis of Legionella pneumophila is derived from its growth within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this bacterium stems from its ability to manipulate host cell vesicular-trafficking pathways and establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This Review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted at host cells.

  11. The Legionella pneumophila replication vacuole: making a cozy niche inside host cells

    PubMed Central

    Isberg, Ralph R.; O'Connor, Tamara; Heidtman, Matthew

    2008-01-01

    The pathogenesis of Legionella pneumophila results from growth of the bacterium within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this microorganism stems from its ability to manipulate host cell vesicular trafficking pathways and to establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why the microorganism has accumulated an unprecedented number of translocated substrates targeted at host cells. PMID:19011659

  12. Genomic Characterization of a Large Outbreak of Legionella pneumophila Serogroup 1 Strains in Quebec City, 2012

    PubMed Central

    Mendis, Nilmini; Cantin, Philippe; Marchand, Geneviève; Charest, Hugues; Raymond, Frédéric; Huot, Caroline; Goupil-Sormany, Isabelle; Desbiens, François; Faucher, Sébastien P.; Corbeil, Jacques; Tremblay, Cécile

    2014-01-01

    During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaire's disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source. PMID:25105285

  13. Evaluation of a most probable number method for the enumeration of Legionella pneumophila from potable and related water samples.

    PubMed

    Sartory, D P; Spies, K; Lange, B; Schneider, S; Langer, B

    2017-04-01

    This study compared the performance of a novel MPN method (Legiolert/Quanti-Tray) with the ISO 11731-2 membrane filtration method for the enumeration of Legionella pneumophila from 100 ml potable water and related samples. Data from a multi-laboratory study analysed according to ISO 17994 showed that Legiolert™/Quanti-Tray® yielded on average higher counts of L. pneumophila. The Legiolert medium had a high specificity of 96·4%. The new method represents a significant improvement in the enumeration of L. pneumophila from drinking water-related samples.

  14. Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

    PubMed

    Schroeder, Gunnar N; Aurass, Philipp; Oates, Clare V; Tate, Edward W; Hartland, Elizabeth L; Flieger, Antje; Frankel, Gad

    2015-10-01

    Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.

  15. Crystallization and preliminary crystallographic analysis of an aminoglycoside kinase from Legionella pneumophila

    SciTech Connect

    Lemke, Christopher T.; Hwang, Jiyoung; Xiong, Bing; Cianciotto, Nicholas P.; Berghuis, Albert M.

    2005-06-01

    Two crystal forms of the antibiotic resistance enzyme APH(9)-Ia from L. pneumophila are reported. 9-Aminoglycoside phosphotransferase type Ia [APH(9)-Ia] is a resistance factor in Legionella pneuemophila, the causative agent of legionnaires’ disease. It is responsible for providing intrinsic resistance to the antibiotic spectinomycin. APH(9)-Ia phosphorylates one of the hydroxyl moieties of spectinomycin in an ATP-dependent manner, abolishing the antibiotic properties of this drug. Here, the crystallization and preliminary X-ray studies of this enzyme in two crystal forms is reported. One of the these crystal forms provides diffraction data to a resolution of 1.7 Å.

  16. In Vitro Response of Guinea Pig Peritoneal Macrophages to Legionella pneumophila

    DTIC Science & Technology

    1981-03-01

    causative agent of I strains. were cultured onl Mueller-Hinton agar supt)I- Legionnaires disease , have niot heeni well defined. niented with 2...In Vitro Responlse of Guinea Pig Peritoneal Macrophages to Legionella pneumophila It. A. KISIIIMi~O~’ .1. Ii.,W11ITE, F. G. SIREY, V. (U.1 Mc(GANN, R...obtained from the Centers for two washes of Hlanks balanced salt solution. Bacteria. Disease Control. Atlanta, Ga. The virulent P1hiladel- suspended in Earle

  17. Comparison of Binax Legionella Urinary Antigen EIA kit with Binax RIA Urinary Antigen kit for detection of Legionella pneumophila serogroup 1 antigen.

    PubMed Central

    Hackman, B A; Plouffe, J F; Benson, R F; Fields, B S; Breiman, R F

    1996-01-01

    The Legionella Urinary Antigen EIA kit (Binax, Portland, Maine) was compared with the EQUATE RIA Legionella Urinary Antigen kit (Binax) for its ability to detect the presence of urinary antigens to Legionella pneumophila serogroup 1. Urine specimens from patients without Legionnaires' disease (n = 33) were negative by both methods (specificity, 100%). Twenty (77%) of 26 urine specimens from patients with Legionnaires' disease positive by the radioimmunoassay kit were also positive by the enzyme immunoassay (EIA) kit. If the cutoff for a positive EIA result were lowered to a ration of > or = 2.5, 23 of 26 (88%) urine specimens would have been positive by EIA and the specificity would remain 100%. Use of the EIA kit is an acceptable method for detecting L. pneumophila serogroup 1 urinary antigens by laboratories that do not want to handle radioactive materials. PMID:8735125

  18. Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila.

    PubMed Central

    Barker, J; Lambert, P A; Brown, M R

    1993-01-01

    The surface properties of Legionella pneumophila were examined by analyzing outer membrane (OM) proteins, lipopolysaccharides (LPS), and cellular fatty acids after growth within Acanthamoeba polyphaga and in vitro under various nutrient-depleted conditions. Intra-amoeba-grown legionellae were found to differ in several respects from cells grown in vitro; most notably, they contained a 15-kDa OM protein and a monounsaturated straight-chain fatty acid (18:1(9)). These compounds were also found in abundant quantities in the host amoeba. Immunoblot analysis of intra-amoeba-grown legionellae with antiacanthamoebic serum revealed that both the bacterial whole cells and Sarkosyl-extracted OMs contained amoebic antigens. The findings suggest that the 15-kDa OM protein is likely to be of amoebic origin and associates with the OM of the bacterium. It is proposed that disruption of amoebic membranes, as a result of intra-amoebic infection, may liberate macromolecules, including a 15-kDa polypeptide, a major constituent of the amoebic membrane, which adhere to the surface of the legionellae. Growth under specific nutrient depletions also had a significant effect on the surface composition of L. pneumophila. Cells grown under phosphate depletion were markedly sensitive to protease K digestion and contained lower levels of LPS, as observed by silver staining of the digests on polyacrylamide gels. Intra-amoeba-grown cells contained more bands than the in vitro-grown organisms, reflecting further differences in the nature of the LPS. The whole-cell fatty acids of the phosphate-depleted cells were appreciably different from those of cells grown under other nutritional conditions. We found no evidence for expression of iron-regulated OM proteins under iron depletion. Images PMID:8335382

  19. [Investigation of Legionella pneumophila seropositivity in the professional long distance drivers as a risky occupation].

    PubMed

    Polat, Yusuf; Ergin, Cağri; Kaleli, Ilknur; Pinar, Ahmet

    2007-04-01

    Contaminated water sources, reservoirs and systems such as evaporative condensers of air-conditioners are known to be the main transmission routes of Legionella spp. which are ubiquitous aquatic bacteria. By virtue of this point the aim of this study was to investigate the rate of Legionella pneumophila seropositivity in a profession considered as risky due to the direct and prolonged exposure to air-conditioning and air-circulating systems. For this purpose, in the period of February-August 2004 a total of 79 male subjects (63 were bus drivers and 16 were driver assistants) who were continously travelling to two different route (South part as hot climate and Middle/North parts as cold climate of Turkey) from Denizli province coach station (a province located in internal Aegian where accepted as crossroads), were included to the study. The mean age and mean working duration of bus drivers were 43.0 +/- 1.1 years and 20.0 +/- 1.1 years, respectively, while these values were 22.5 +/- 0.9 years and 4.0 +/- 0.6 years, respectively, for the drivers' assistants. The serum samples collected from the subjects were screened by a commercial indirect immunofluorescence method (Euroimmun, Germany) using L. pneumophila serogrup 1-14 antigens for the presence of specific antibodies. Additionally, air-conditioners' moisture exhaust samples of the busses in which seropositive subjects travelling with have been examined by culture and 5S rRNA gene targeted polymerase chain reaction (PCR) methods, for the presence of Legionella spp. The overall L. pneumophila seropositivity rate was detected as 15.2% (12/79). This rate was 19% (12/63) for bus drivers while all of the drivers' assistants were found seronegative. The seropositivity rate was found statistically higher in the personnel who were travelling to the hot climates (10/36, 27.8%) than those who travel to cold climates (2/43, 4.6%), (p < 0.05). The culture and PCR yielded negative results for Legionella spp. in the exhaust

  20. Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

    PubMed Central

    Copenhaver, Alan M.; Casson, Cierra N.; Nguyen, Hieu T.; Fung, Thomas C.; Duda, Matthew M.; Roy, Craig R.

    2014-01-01

    Legionella pneumophila, an intracellular pathogen responsible for the severe pneumonia Legionnaires' disease, uses its dot/icm-encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol, L. pneumophila also activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate the control of infection. Thus, the pulmonary cell types that L. pneumophila infects not only may act as an intracellular niche that facilitates its pathogenesis but also may contribute to the immune response against L. pneumophila. The identity of these host cells remains poorly understood. Here, we developed a strain of L. pneumophila producing a fusion protein consisting of β-lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils both are the primary recipients of T4SS-translocated effectors and harbor viable L. pneumophila during pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced tumor necrosis factor and interleukin-1α in response to T4SS-sufficient, but not T4SS-deficient, L. pneumophila. Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir for L. pneumophila and a source of proinflammatory cytokines that contribute to the host immune response against L. pneumophila during pulmonary infection. PMID:25092908

  1. Preferential colonization and release of Legionella pneumophila from mature drinking water biofilms grown on copper versus unplasticized polyvinylchloride coupons.

    PubMed

    Buse, Helen Y; Lu, Jingrang; Struewing, Ian T; Ashbolt, Nicholas J

    2014-03-01

    Legionella occurrence in premise drinking water (DW) systems contributes to legionellosis outbreaks, especially in the presence of suitable protozoan hosts. This study examined L. pneumophila behavior within DW biofilms grown on copper (Cu) and unplasticized polyvinylchloride (uPVC) surfaces in the presence of Acanthamoeba polyphaga. One year-old DW biofilms were established within six CDC biofilm reactors: three each containing Cu or uPVC coupons. Biofilms were then inoculated with L. pneumophila (uPVC-Lp and Cu-Lp), or L. pneumophila and A. polyphaga (uPVC-Lp/Ap and Cu-Lp/Ap) and compared to sterile water inoculated controls (uPVC- and Cu-Control) over a 4 month period. L. pneumophila appeared more persistent by qPCR within Cu biofilms in the presence of A. polyphaga compared to uPVC biofilms with or without A. polyphaga, but maintained their cultivability in uPVC biofilms compared to Cu biofilms. Also, persistent shedding of L. pneumophila cells (assayed by qPCR) in the effluent water implied colonization of L. pneumophila within Cu-coupon reactors compared to no detection from uPVC-coupon reactor effluent 14 days after inoculation. Hence, L. pneumophila appeared to colonize Cu surfaces more effectively and may be shed from the biofilms at a greater frequency and duration compared to L. pneumophila colonized uPVC surfaces with host amoebae playing a role in L. pneumophila persistence within Cu biofilms. Published by Elsevier GmbH.

  2. Effect of Disinfectant Exposure on Legionella pneumophila Associated with Simulated Drinking Water Biofilms: Release, Inactivation, and Infectivity.

    PubMed

    Shen, Yun; Huang, Conghui; Lin, Jie; Wu, Wenjing; Ashbolt, Nicholas J; Liu, Wen-Tso; Nguyen, Thanh H

    2017-02-21

    Legionella pneumophila, the most commonly identified causative agent in drinking water associated with disease outbreaks, can be harbored by and released from drinking water biofilms. In this study, the release of biofilm-associated L. pneumophila under simulated drinking water flow containing a disinfectant residual was examined. Meanwhile, the inactivation and infectivity (to amoebae) of the released L. pneumophila were studied. To simulate drinking water system conditions, biofilms were prepared under either disinfectant exposure (predisinfected biofilms) or disinfectant-free (untreated biofilms) conditions, respectively. For experiments with water flow containing a disinfectant to release the biofilm-associated L. pneumophila from these two types of biofilms, the L. pneumophila release kinetics values from predisinfected and untreated biofilms under flow condition were not statistically different (one-way ANOVA, p > 0.05). However, inactivation of the L. pneumophila released from predisinfected biofilms was 1-2 times higher and amoeba infectivity was 2-29 times lower than that from untreated biofilms. The higher disinfectant resistance of L. pneumophila released from untreated biofilms was presumably influenced by the detachment of a larger amount of biofilm material (determined by 16S rRNA qPCR) surrounding the released L. pneumophila. This study highlights the interaction among disinfectant residual, biofilms, and L. pneumophila, which provides guidelines to assess and control pathogen risk.

  3. A STUDY ON LEGIONELLA PNEUMOPHILA, WATER CHEMISTRY, AND ATMOSPHERIC CONDITIONS IN COOLING TOWERS AT THE SAVANNAH RIVER SITE

    SciTech Connect

    Smith, C.; Brigmon, R.

    2009-10-20

    Legionnaires disease is a pneumonia caused by the inhalation of the bacterium Legionella pneumophila. The majority of illnesses have been associated with cooling towers since these devices can harbor and disseminate the bacterium in the aerosolized mist generated by these systems. Historically, Savannah River Site (SRS) cooling towers have had occurrences of elevated levels of Legionella in all seasons of the year and in patterns that are difficult to predict. Since elevated Legionella in cooling tower water are a potential health concern a question has been raised as to the best control methodology. In this work we analyze available chemical, biological, and atmospheric data to determine the best method or key parameter for control. The SRS 4Q Industrial Hygiene Manual, 4Q-1203, 1 - G Cooling Tower Operation and the SRNL Legionella Sampling Program, states that 'Participation in the SRNL Legionella Sampling Program is MANDATORY for all operating cooling towers'. The resulting reports include L. pneumophila concentration information in cells/L. L. pneumophila concentrations >10{sup 7} cells/L are considered elevated and unsafe so action must be taken to reduce these densities. These remedial actions typically include increase biocide addition or 'shocking'. Sometimes additional actions are required if the problem persists including increase tower maintenance (e.g. cleaning). Evaluation of 14 SRS cooling towers, seven water quality parameters, and five Legionella serogroups over a three-plus year time frame demonstrated that cooling tower water Legionella densities varied widely though out this time period. In fact there was no one common consistent significant variable across all towers. The significant factors that did show up most frequently were related to suspended particulates, conductivity, pH, and dissolved oxygen, not chlorine or bromine as might be expected. Analyses of atmospheric data showed that there were more frequent significant elevated Legionella

  4. Retrospective evaluation of the Du Pont radioimmunoassay kit for detection of Legionella pneumophila serogroup 1 antigenuria in humans.

    PubMed Central

    Aguero-Rosenfeld, M E; Edelstein, P H

    1988-01-01

    We used the Du Pont radioimmunoassay kit for soluble Legionella pneumophila serogroup 1 antigenuria (Du Pont Co., Wilmington, Del.) to test 422 urine samples from patients with and without Legionnaires disease (LD). The urine specimens were collected from 23 patients with culture-proven LD and from 346 patients without LD. L. pneumophila serogroup 1 was isolated from 14 patients with culture-proven LD, and other L. pneumophila serogroups or other Legionella species were isolated from 9 patients; 58 urine specimens were tested from these 23 patients. The non-LD group was composed of 75 bacteremic patients (35 gram-negative and 40 gram-positive bacteremias), 7 patients with candidemia, 48 patients with non-LD pneumonia, 90 patients with gram-negative bacteriuria (greater than 10(5) CFU/ml), 23 patients with gram-positive bacteriuria (greater than 10(5) CFU/ml), 14 patients with candiduria (greater than 10(5) CFU/ml), and 89 outpatients with negative urine cultures. All tests were performed in duplicate, including positive and negative controls. Sample results with values greater than or equal to 3.0 times those of the negative controls were considered positive for L. pneumophila serogroup 1 antigenuria. The average sample-to-negative ratios were 19.1 for the L. pneumophila serogroup 1 specimens, and 1.0 for both the non-serogroup 1 legionella group and the non-LD specimens. All but one of the patients who were culture positive for L. pneumophila serogroup 1 had at least one specimen positive for serogroup 1 antigenuria; none of the non-L. pneumophila serogroup 1 patients had a positive urine test. The test was highly specific (100%) and sensitive (93%) for the detection of L. pneumophila serogroup 1 antigenuria. Concentrations of urine by vacuum evaporation increased test sensitivity without apparently affecting specificity. PMID:3183024

  5. Retrospective evaluation of the Du Pont radioimmunoassay kit for detection of Legionella pneumophila serogroup 1 antigenuria in humans.

    PubMed

    Aguero-Rosenfeld, M E; Edelstein, P H

    1988-09-01

    We used the Du Pont radioimmunoassay kit for soluble Legionella pneumophila serogroup 1 antigenuria (Du Pont Co., Wilmington, Del.) to test 422 urine samples from patients with and without Legionnaires disease (LD). The urine specimens were collected from 23 patients with culture-proven LD and from 346 patients without LD. L. pneumophila serogroup 1 was isolated from 14 patients with culture-proven LD, and other L. pneumophila serogroups or other Legionella species were isolated from 9 patients; 58 urine specimens were tested from these 23 patients. The non-LD group was composed of 75 bacteremic patients (35 gram-negative and 40 gram-positive bacteremias), 7 patients with candidemia, 48 patients with non-LD pneumonia, 90 patients with gram-negative bacteriuria (greater than 10(5) CFU/ml), 23 patients with gram-positive bacteriuria (greater than 10(5) CFU/ml), 14 patients with candiduria (greater than 10(5) CFU/ml), and 89 outpatients with negative urine cultures. All tests were performed in duplicate, including positive and negative controls. Sample results with values greater than or equal to 3.0 times those of the negative controls were considered positive for L. pneumophila serogroup 1 antigenuria. The average sample-to-negative ratios were 19.1 for the L. pneumophila serogroup 1 specimens, and 1.0 for both the non-serogroup 1 legionella group and the non-LD specimens. All but one of the patients who were culture positive for L. pneumophila serogroup 1 had at least one specimen positive for serogroup 1 antigenuria; none of the non-L. pneumophila serogroup 1 patients had a positive urine test. The test was highly specific (100%) and sensitive (93%) for the detection of L. pneumophila serogroup 1 antigenuria. Concentrations of urine by vacuum evaporation increased test sensitivity without apparently affecting specificity.

  6. Nuclease activity of Legionella pneumophila Cas2 promotes intracellular infection of amoebal host cells.

    PubMed

    Gunderson, Felizza F; Mallama, Celeste A; Fairbairn, Stephanie G; Cianciotto, Nicholas P

    2015-03-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts.

  7. Interaction of legionella pneumophila and helicobacter pylori with bacterial species isolated from drinking water biofilms

    PubMed Central

    2011-01-01

    Background It is well established that Legionella pneumophila is a waterborne pathogen; by contrast, the mode of Helicobacter pylori transmission remains unknown but water seems to play an important role. This work aims to study the influence of five microorganisms isolated from drinking water biofilms on the survival and integration of both of these pathogens into biofilms. Results Firstly, both pathogens were studied for auto- and co-aggregation with the species isolated from drinking water; subsequently the formation of mono and dual-species biofilms by L. pneumophila or H. pylori with the same microorganisms was investigated. Neither auto- nor co-aggregation was observed between the microorganisms tested. For biofilm studies, sessile cells were quantified in terms of total cells by SYTO 9 staining, viable L. pneumophila or H. pylori cells were quantified using 16 S rRNA-specific peptide nucleic acid (PNA) probes and cultivable cells by standard culture techniques. Acidovorax sp. and Sphingomonas sp. appeared to have an antagonistic effect on L. pneumophila cultivability but not on the viability (as assessed by rRNA content using the PNA probe), possibly leading to the formation of viable but noncultivable (VBNC) cells, whereas Mycobacterium chelonae increased the cultivability of this pathogen. The results obtained for H. pylori showed that M. chelonae and Sphingomonas sp. help this pathogen to maintain cultivability for at least 24 hours. Conclusions It appears that M. chelonae may have an important role in the survival of both pathogens in drinking water. This work also suggests that the presence of some microorganisms can decrease the cultivability of L. pneumophila but not the viability which indicates that the presence of autochthonous microorganisms can lead to misleading results when the safety of water is assessed by cultivable methods alone. PMID:21418578

  8. Identification of Legionella pneumophila genes required for growth within and killing of human macrophages.

    PubMed Central

    Sadosky, A B; Wiater, L A; Shuman, H A

    1993-01-01

    Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl. Images PMID:8225610

  9. Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

    PubMed Central

    Gunderson, Felizza F.; Mallama, Celeste A.; Fairbairn, Stephanie G.

    2014-01-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. PMID:25547789

  10. Genome-Scale Identification of Legionella pneumophila Effectors Using a Machine Learning Approach

    PubMed Central

    Burstein, David; Zusman, Tal; Degtyar, Elena; Viner, Ram; Segal, Gil; Pupko, Tal

    2009-01-01

    A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF) was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine learning algorithms for

  11. Interaction of Legionella pneumophila and Helicobacter pylori with bacterial species isolated from drinking water biofilms.

    PubMed

    Gião, Maria S; Azevedo, Nuno F; Wilks, Sandra A; Vieira, Maria J; Keevil, Charles W

    2011-03-18

    It is well established that Legionella pneumophila is a waterborne pathogen; by contrast, the mode of Helicobacter pylori transmission remains unknown but water seems to play an important role. This work aims to study the influence of five microorganisms isolated from drinking water biofilms on the survival and integration of both of these pathogens into biofilms. Firstly, both pathogens were studied for auto- and co-aggregation with the species isolated from drinking water; subsequently the formation of mono and dual-species biofilms by L. pneumophila or H. pylori with the same microorganisms was investigated. Neither auto- nor co-aggregation was observed between the microorganisms tested. For biofilm studies, sessile cells were quantified in terms of total cells by SYTO 9 staining, viable L. pneumophila or H. pylori cells were quantified using 16 S rRNA-specific peptide nucleic acid (PNA) probes and cultivable cells by standard culture techniques. Acidovorax sp. and Sphingomonas sp. appeared to have an antagonistic effect on L. pneumophila cultivability but not on the viability (as assessed by rRNA content using the PNA probe), possibly leading to the formation of viable but noncultivable (VBNC) cells, whereas Mycobacterium chelonae increased the cultivability of this pathogen. The results obtained for H. pylori showed that M. chelonae and Sphingomonas sp. help this pathogen to maintain cultivability for at least 24 hours. It appears that M. chelonae may have an important role in the survival of both pathogens in drinking water. This work also suggests that the presence of some microorganisms can decrease the cultivability of L. pneumophila but not the viability which indicates that the presence of autochthonous microorganisms can lead to misleading results when the safety of water is assessed by cultivable methods alone.

  12. Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones.

    PubMed

    Finsel, Ivo; Hilbi, Hubert

    2015-07-01

    Legionella species are ubiquitous, waterborne bacteria that thrive in numerous ecological niches. Yet, in contrast to many other environmental bacteria, Legionella spp. are also able to grow intracellularly in predatory protozoa. This feature mainly accounts for the pathogenicity of Legionella pneumophila, which causes the majority of clinical cases of a severe pneumonia termed Legionnaires' disease. The pathomechanism underlying L. pneumophila infection is based on macrophage resistance, which in turn is largely defined by the opportunistic pathogen's resistance towards amoebae. L. pneumophila replicates in macrophages or amoebae in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system and involves a plethora of translocated effector proteins, which subvert pivotal processes in the host cell. Of the ca. 300 different experimentally validated Icm/Dot substrates, about 50 have been studied and attributed a cellular function to date. The versatility and ingenuity of these effectors' mode of actions is striking. In this review, we summarize insight into the cellular functions and biochemical activities of well-characterized L. pneumophila effector proteins and the host pathways they target. Recent studies not only substantially increased our knowledge about pathogen-host interactions, but also shed light on novel biological mechanisms.

  13. In vivo regulation of replicative Legionella pneumophila lung infection by endogenous tumor necrosis factor alpha and nitric oxide.

    PubMed Central

    Brieland, J K; Remick, D G; Freeman, P T; Hurley, M C; Fantone, J C; Engleberg, N C

    1995-01-01

    The in vivo role of endogenous tumor necrosis factor alpha (TNF-alpha) and reactive nitrogen intermediates (RNIs) in modulation of growth of Legionella pneumophila in the lung was assessed using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of mice with L. pneumophila resulted in induction of endogenous TNF-alpha, which preceded clearance of L. pneumophila from the lung. Inhibition of endogenous TNF-alpha activity, via in vivo administration of TNF-alpha neutralizing antibody, or inhibition of endogenous RNIs, via administration of the nitric oxide (NO) synthetase inhibitor N-monomethyl-L-arginine (NMMA), resulted in enhanced growth of L. pneumophila in the lung at > or = 3 days postinfection (when compared with untreated L. pneumophila-infected mice). Because of the similar kinetics of enhanced pulmonary growth of L. pneumophila in mice treated in vivo with either anti-TNF-alpha antibody or NMMA, the immunomodulatory effect of NO on endogenous TNF-alpha activity in the lung was assessed. Administration of NMMA to L. pneumophila-infected mice resulted in a significant decrease in endogenous TNF-alpha activity in the lung during replicative L. pneumophila infections in vivo. However, administration of exogenous TNF-alpha to NMMA-treated mice failed to significantly enhance clearance of L. pneumophila from the lung. Results of these studies indicate that both endogenous NO and TNF-alpha facilitate resolution of replicative L. pneumophila lung infections and that regulation of L. pneumophila replication by TNF-alpha is mediated, at least in part, by NO. PMID:7642253

  14. Molecular typing of Legionella pneumophila from air-conditioning cooling waters using mip gene, SBT, and FAFLP methods.

    PubMed

    Gong, Xiangli; Li, Juntao; Zhang, Ying; Hou, Shuiping; Qu, Pinghua; Yang, Zhicong; Chen, Shouyi

    2017-04-07

    Legionella spp. are important waterborne pathogens. Molecular typing has become an important method for outbreaks investigations and source tracking of Legionnaires. In a survey program conducted by the Guangzhou Center for Disease Control and Prevention, multiple serotypes Legionella pneumophila (L. pneumophila) were isolated from waters in air-conditioning cooling towers in urban Guangzhou region, China between 2008 and 2011. Three genotyping methods, mip (macrophage infectivity potentiator) genotyping, SBT (sequence-based typing), and FAFLP (fluorescent amplified fragment length polymorphism analysis) were used to type these waterborne L. pneumophila isolates. The three methods were capable of typing all the 134 isolates and a reference strain of L. pneumophila (ATCC33153), with discriminatory indices of 0.7034, 0.9218, and 0.9376, for the mip, SBT, and FAFLP methods respectively. Among the 9 serotypes of the 134 isolates, 10, 50, and 34 molecular types were detected by the mip, SBT, and FAFLP methods respectively. The mip genotyping and SBT typing are more feasible for inter-laboratory results sharing and comparison of different types of L. pneumophila. The SBT and FAFLP typing methods were rapid with higher discriminatory abilities. Combinations of two or more of the typing methods enables more accurate typing of Legionella isolates for outbreak investigations and source tracking of Legionnaires.

  15. Severe Legionella Pneumophila Infection in an Immunocompetent Patient: A Success Story 300 Kilometers Away

    PubMed Central

    Jacob, Miguel; Ramos, Helena C

    2016-01-01

    The most significant outbreak of Legionella pneumophila, or Legionnaires’ Disease, ever registered in Portugal occurred in 2014, and was considered one of the largest in European history. This relatively rare infection has a dire prognosis if not timely identified and correctly treated, presenting with a high lethality rate. We describe a case of infection by Legionella pneumophila in a previously healthy individual during an outbreak that originated 300 kilometers away from our hospital. The patient presented to the Emergency Department and after an initial assessment, was admitted to the Intensive Care Unit (ICU). He underwent supportive treatment with invasive mechanical ventilation and antibiotic therapy, having been discharged with functional improvement 21 days after admission. During follow-up, the patient presented well without residual clinical or radiological findings. Prompt management following established guidelines allowed for the appropriate treatment and a favorable prognosis. This case serves as a reminder that early management is important, healthy individuals without known risk factors may present with severe infection, and there is the possibility for individual cases of Legionellosis to present far from the outbreak source. PMID:28123918

  16. Severe Legionella Pneumophila Infection in an Immunocompetent Patient: A Success Story 300 Kilometers Away.

    PubMed

    Jacob, Miguel; Ramos, Helena C; Morgado, Bruno

    2016-12-21

    The most significant outbreak of Legionella pneumophila, or Legionnaires' Disease, ever registered in Portugal occurred in 2014, and was considered one of the largest in European history. This relatively rare infection has a dire prognosis if not timely identified and correctly treated, presenting with a high lethality rate. We describe a case of infection by Legionella pneumophila in a previously healthy individual during an outbreak that originated 300 kilometers away from our hospital. The patient presented to the Emergency Department and after an initial assessment, was admitted to the Intensive Care Unit (ICU). He underwent supportive treatment with invasive mechanical ventilation and antibiotic therapy, having been discharged with functional improvement 21 days after admission. During follow-up, the patient presented well without residual clinical or radiological findings. Prompt management following established guidelines allowed for the appropriate treatment and a favorable prognosis. This case serves as a reminder that early management is important, healthy individuals without known risk factors may present with severe infection, and there is the possibility for individual cases of Legionellosis to present far from the outbreak source.

  17. Variable genetic element typing: a quick method for epidemiological subtyping of Legionella pneumophila.

    PubMed

    Pannier, K; Heuner, K; Lück, C

    2010-04-01

    A total of 57 isolates of Legionella pneumophila were randomly selected from the German National Legionella strain collection and typed by monoclonal antibody subgrouping, seven-gene locus sequence-based typing (SBT) scheme and a newly developed variable element typing (VET) system based on the presence or absence of ten variable genetic elements. These elements were detected while screening a genomic library of strain Corby, as well as being taken from published data for PAI-1 (pathogenicity island) from strain Philadelphia. Specific primers were designed and used in gel-based polymerase chain reaction (PCR) assays. PCR amplification of the mip gene served as a control. The end-point was the presence/absence of a PCR product on an ethidium bromide-strained gel. In the present study, the index of discrimination was somewhat lower than that of the SBT (0.87 versus 0.97). Nevertheless, the results obtained showed as a 'proof of principle' that this simple and quick typing assay might be useful for the epidemiological characterisation of L. pneumophila strains.

  18. Broad-host-range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome.

    PubMed Central

    Keen, M G; Street, E D; Hoffman, P S

    1985-01-01

    Transposon Tn5 was introduced into Legionella pneumophila on plasmid pRK340, which is temperature sensitive for plasmid maintenance. The presence of plasmid DNA was confirmed by agarose gel electrophoresis and by conjugal transfer of the plasmid to Escherichia coli. Tn5 insertions were obtained by culturing L. pneumophila at the nonpermissive temperature (43 degrees C) on buffered charcoal-yeast extract agar containing kanamycin. Of the 260 kanamycin-resistant colonies picked, 220 failed to conjugate pRK340 to E. coli. Plasmid DNA was not visualized from eight randomly picked Tn5-containing strains, and Southern hybridizations indicated that Tn5, but not pRK340, inserted into multiple sites in the Legionella chromosome. In addition, the streptomycin resistance determinant on Tn5 was expressed in L. pneumophila. Images PMID:2987191

  19. Legionella pneumophila Philadelphia-1 tatB and tatC affect intracellular replication and biofilm formation.

    PubMed

    De Buck, Emmy; Maes, Liesbeth; Meyen, Eef; Van Mellaert, Lieve; Geukens, Nick; Anné, Jozef; Lammertyn, Elke

    2005-06-17

    Legionella pneumophila is a facultative intracellular human pathogen and an important cause of Legionnaires' disease, a severe form of pneumonia. Recently, we showed the presence of a putative twin-arginine translocation (Tat) pathway in L. pneumophila Philadelphia-1. This secretion pathway is used to transport completely folded proteins across the cytoplasmic membrane. The importance of the Tat pathway in L. pneumophila was investigated by constructing a tatB and a tatC mutant. Functionality of the Tat pathway was shown using a proven heterologous Tat substrate. It was shown that tatB and tatC are involved in intracellular replication in Acanthamoeba castellanii and differentiated U937 cells, and in biofilm forming ability. A putative Legionella Tat substrate was identified via 2D gel electrophoresis.

  20. Birc1e/Naip5 rapidly antagonizes modulation of phagosome maturation by Legionella pneumophila.

    PubMed

    Fortier, Anne; de Chastellier, Chantal; Balor, Stéphanie; Gros, Philippe

    2007-04-01

    Legionella survives intracellularly by preventing fusion with lysosomes, due to phagosome escape from the endocytic pathway at an early stage of phagosome maturation, and by creating a replicative organelle that acquires endoplasmic reticulum (ER) characteristics through sustained interactions and fusion with the ER. Intracellular replication of Legionella pneumophila in mouse macrophages is controlled by the Lgn1 locus. Functional complementation in vivo has identified the Birc1e/Naip5 gene as being responsible for the Lgn1 effect. To understand the function and temporal site of action of Birc1e/Naip5 in susceptibility to L. pneumophila, we examined the biogenesis of Legionella-containing vacuoles (LCVs) formed in permissive A/J macrophages and in their Birc1e/Naip5 transgenic non-permissive counterpart. Birc1e/Naip5 effects on acquisition of lysosomal and ER markers were evident within 1-2 h following infection. A significantly higher proportion of LCVs formed in Birc1e/Naip5 transgenic macrophages had acquired the lysosomal markers cathepsin D and Lamp1 by 2 h post infection, whereas a significantly higher proportion of LCVs formed in permissive macrophages were positively stained for the ER markers BAP31 and calnexin, 6 h post infection. Likewise, studies by electron microscopy showed acquisition of lysosomal contents (horseradish peroxidase), within the first hour following phagocytic uptake, by LCVs formed in Birc1e/Naip5 transgenic macrophages and delivery of the ER marker glucose 6-phosphatase (G6Pase) only to the lumen of LCVs formed in A/J macrophages. Finally, a larger proportion of LCVs formed in A/J macrophages were studded with ribosomes 24 h post infection, compared with LCVs formed in Birc1e/Naip5 transgenic macrophages. These results suggest that sensing of L. pneumophila products by Birc1e/Naip5 in macrophages occurs rapidly following phagocytosis, a process that antagonizes the ability of L. pneumophila to remodel its phagosome into a specialized

  1. Influence of temperature, chlorine residual and heavy metals on the presence of Legionella pneumophila in hot water distribution systems.

    PubMed

    Rakić, Anita; Perić, Jelena; Foglar, Lucija

    2012-01-01

    The microbiological colonisation of buildings and man-made structures often occurs on the walls of plumbing systems; therefore, monitoring of opportunistic pathogens such as Legionella pneumophila (L. pneumophila), both in water distribution mains and in consumers' plumbing systems, is an important issue according to the international and national guidelines that regulate the quality of drinking water. This paper investigates the presence of L. pneumophila in the Dalmatian County of Croatia and the relationship between L. pneumophila presence and heavy metals concentrations, free residual chlorine and water temperature in hot water distribution systems (WDS). Investigations were performed on a large number of hot water samples taken from taps in kitchens and bathrooms in hotels and homes for the elderly and disabled in the Split region. Of the 127 hot water samples examined, 12 (9.4%) were positive for Legionella spp. with median values concentration of 450 cfu × L(-1). Among positive isolates, 10 (83.3%) were L. pneumophila sg 1, and two of them (16.6%) belonged to the genera L. pneumophila sg 2-14. The positive correlation between the water temperature, iron and manganese concentrations, and L. pneumophila contamination was proved by statistical analysis of the experimental data. On the contrary, zinc and free residual chlorine had no observed influence on the presence of L. pneumophila. The presence of heavy metals in water samples confirms the corrosion of distribution system pipes and fittings, and suggests that metal plumbing components and associated corrosion products are important factors in the survival and growth of L. pneumophila in WDS.

  2. [Intra-amoebal development of Legionella pneumophila and the potential role of amoebae in the transmission of Legionnaires' disease].

    PubMed

    Philippe, C; Blech, M F; Hartemann, P

    2006-04-01

    Legionnaires' disease is one of the major infectious risks related to hospital water systems. It is commonly accepted, that the disease is transmitted to man mostly by inhalation of water aerosols contaminated by Legionella pneumophila. The ability of L. pneumophila to multiply intracellularly within some amoebae better explains the ecology, the pathogenicity, and the virulence of this bacterium against human alveolar macrophages. The presence of these amoebae in water systems located where cases of Legionnaire's disease broke out, partly explains the difficulty in eradicating Legionella. Some studies also show that amoebae can play a major role in the transmission of the disease to man. Some other studies point out that inhaled amoebae could be involved in the pathogenesis of Legionnaire's disease. Future strategies to prevent the transmission of Legionella will probably have to include efficient treatments against amoebae.

  3. Diverse populations of Legionella pneumophila present in the water of geographically clustered institutions served by the same water reservoir.

    PubMed Central

    Bezanson, G; Burbridge, S; Haldane, D; Yoell, C; Marrie, T

    1992-01-01

    We cultured potable water from seven institutions (six hospitals and one medical school) every 2 weeks for 6 months for Legionella pneumophila. All of the institutions were located close to each other and received water from the same freshwater source. Two institutions (the medical school and hospital F, a maternity hospital) never had L. pneumophila isolated from their potable water. The remaining five had 17 to 72% of their water samples positive for L. pneumophila. Most of the isolates were serogroup 1; however, in hospital B serogroup 5 accounted for 56% of the isolates. Oxford and OLDA monoclonal antibody subtypes of L. pneumophila serogroup 1 coexisted in four of the five institutions, while subtype France only was found in one institution. All 10 isolates from this institution lacked plasmids. The other four institutions had Legionella populations with plasmid profiles II, III, and VI. Two of these institutions also had isolates with no plasmids. The distribution of the plasmid types was significantly different for all institutions except C and D. The distribution of monoclonal antibody subtypes was significantly different for L. pneumophila isolates recovered from institutions C and D. There were no characteristics that distinguished the culture-positive institutions from the culture-negative areas. We conclude that diverse populations of L. pneumophila exist within these institutions despite their geographic proximity and identical potable water source. PMID:1551972

  4. Apoptosis in Macrophages and Alveolar Epithelial Cells during Early Stages of Infection by Legionella pneumophila and Its Role in Cytopathogenicity

    PubMed Central

    Gao, Lian-Yong; Abu Kwaik, Yousef

    1999-01-01

    The hallmark of Legionnaires’ disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellular L. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophila correlates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection. PMID:9916101

  5. Role of biofilm roughness and hydrodynamic conditions in Legionella pneumophila adhesion to and detachment from simulated drinking water biofilms.

    PubMed

    Shen, Yun; Monroy, Guillermo L; Derlon, Nicolas; Janjaroen, Dao; Huang, Conghui; Morgenroth, Eberhard; Boppart, Stephen A; Ashbolt, Nicholas J; Liu, Wen-Tso; Nguyen, Thanh H

    2015-04-07

    Biofilms in drinking water distribution systems (DWDS) could exacerbate the persistence and associated risks of pathogenic Legionella pneumophila (L. pneumophila), thus raising human health concerns. However, mechanisms controlling adhesion and subsequent detachment of L. pneumophila associated with biofilms remain unclear. We determined the connection between L. pneumophila adhesion and subsequent detachment with biofilm physical structure characterization using optical coherence tomography (OCT) imaging technique. Analysis of the OCT images of multispecies biofilms grown under low nutrient condition up to 34 weeks revealed the lack of biofilm deformation even when these biofilms were exposed to flow velocity of 0.7 m/s, typical flow for DWDS. L. pneumophila adhesion on these biofilm under low flow velocity (0.007 m/s) positively correlated with biofilm roughness due to enlarged biofilm surface area and local flow conditions created by roughness asperities. The preadhered L. pneumophila on selected rough and smooth biofilms were found to detach when these biofilms were subjected to higher flow velocity. At the flow velocity of 0.1 and 0.3 m/s, the ratio of detached cell from the smooth biofilm surface was from 1.3 to 1.4 times higher than that from the rough biofilm surface, presumably because of the low shear stress zones near roughness asperities. This study determined that physical structure and local hydrodynamics control L. pneumophila adhesion to and detachment from simulated drinking water biofilm, thus it is the first step toward reducing the risk of L. pneumophila exposure and subsequent infections.

  6. The Surfactant of Legionella pneumophila Is Secreted in a TolC-Dependent Manner and Is Antagonistic toward Other Legionella Species ▿†

    PubMed Central

    Stewart, Catherine R.; Burnside, Denise M.; Cianciotto, Nicholas P.

    2011-01-01

    When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent “sliding” motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment. PMID:21890700

  7. Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

    SciTech Connect

    Butler, C.A.; Hoffman, P.S. )

    1990-05-01

    A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled ((35S)cysteine or (35S)methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid per mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.

  8. Resistance of Legionella pneumophila serotype 1 biofilms to chlorine-based disinfection.

    PubMed

    Cooper, I R; Hanlon, G W

    2010-02-01

    The presence of Legionella spp. in potable water systems is a major concern to municipal water providers and consumers alike. Despite the inclusion of chlorine in potable supplies and frequent chlorination cycles, the bacterium is a recalcitrant human pathogen capable of causing incidents of Legionnaires' disease, Pontiac fever and community-acquired pneumonia in humans. Using two materials routinely employed for the delivery of potable water as a substratum, copper and stainless steel, the development of Legionella pneumophila biofilms and their response to chlorination was monitored over a three-day and a three-month period, respectively. Preliminary in vitro studies using broth and sterile tap water as culture media indicated that the bacterium was capable of surviving in low numbers for 28 days in the presence of chlorine. Subsequently, biofilms were grown for three days, one month and two months, respectively, on stainless steel and copper sections, which are widely used for the conveyance of potable water. Immediately after exposure to 50mg/L chlorine for 1h, the biofilms yielded no recoverable colonies, but colonies did reappear in low numbers over the following days. Despite chlorination at 50mg/L for 1h, both one- and two-month-old L. pneumophila biofilms were able to survive this treatment and to continue to grow, ultimately exceeding 1x10(6)cfu per disc. This research provides an insight into the resistance afforded to L. pneumophila against high levels of chlorine by the formation of biofilms and has implications for the delivery of potable water.

  9. Genetic Characterization of Legionella pneumophila Isolated from a Common Watershed in Comunidad Valenciana, Spain.

    PubMed

    Sánchez-Busó, Leonor; Coscollá, Mireia; Pinto-Carbó, Marta; Catalán, Vicente; González-Candelas, Fernando

    2013-01-01

    Legionella pneumophila infects humans to produce legionellosis and Pontiac fever only from environmental sources. In order to establish control measures and study the sources of outbreaks it is essential to know extent and distribution of strain variants of this bacterium in the environment. Sporadic and outbreak-related cases of legionellosis have been historically frequent in the Comunidad Valenciana region (CV, Spain), with a high prevalence in its Southeastern-most part (BV). Environmental investigations for the detection of Legionella pneumophila are performed in this area routinely. We present a population genetics study of 87 L. pneumophila strains isolated in 13 different localities of the BV area irrigated from the same watershed and compare them to a dataset of 46 strains isolated in different points of the whole CV. Our goal was to compare environmental genetic variation at two different geographic scales, at county and regional levels. Genetic diversity, recombination and population structure were analyzed with Sequence-Based Typing data and three intergenic regions. The results obtained reveal a low, but detectable, level of genetic differentiation between both datasets, mainly, but not only, attributed to the occurrence of unusual variants of the neuA locus present in the BV populations. This differentiation is still detectable when the 10 loci considered are analyzed independently, despite the relatively high incidence of the most common genetic variant in this species, sequence type 1 (ST-1). However, when the genetic data are considered without their associated geographic information, four major groups could be inferred at the genetic level which did not show any correlation with sampling locations. The overall results indicate that the population structure of these environmental samples results from the joint action of a global, widespread ST-1 along with genetic differentiation at shorter geographic distances, which in this case are related to

  10. Genetic Characterization of Legionella pneumophila Isolated from a Common Watershed in Comunidad Valenciana, Spain

    PubMed Central

    Sánchez-Busó, Leonor; Coscollá, Mireia; Pinto-Carbó, Marta; Catalán, Vicente; González-Candelas, Fernando

    2013-01-01

    Legionella pneumophila infects humans to produce legionellosis and Pontiac fever only from environmental sources. In order to establish control measures and study the sources of outbreaks it is essential to know extent and distribution of strain variants of this bacterium in the environment. Sporadic and outbreak-related cases of legionellosis have been historically frequent in the Comunidad Valenciana region (CV, Spain), with a high prevalence in its Southeastern-most part (BV). Environmental investigations for the detection of Legionella pneumophila are performed in this area routinely. We present a population genetics study of 87 L. pneumophila strains isolated in 13 different localities of the BV area irrigated from the same watershed and compare them to a dataset of 46 strains isolated in different points of the whole CV. Our goal was to compare environmental genetic variation at two different geographic scales, at county and regional levels. Genetic diversity, recombination and population structure were analyzed with Sequence-Based Typing data and three intergenic regions. The results obtained reveal a low, but detectable, level of genetic differentiation between both datasets, mainly, but not only, attributed to the occurrence of unusual variants of the neuA locus present in the BV populations. This differentiation is still detectable when the 10 loci considered are analyzed independently, despite the relatively high incidence of the most common genetic variant in this species, sequence type 1 (ST-1). However, when the genetic data are considered without their associated geographic information, four major groups could be inferred at the genetic level which did not show any correlation with sampling locations. The overall results indicate that the population structure of these environmental samples results from the joint action of a global, widespread ST-1 along with genetic differentiation at shorter geographic distances, which in this case are related to

  11. Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells.

    PubMed Central

    Wintermeyer, E; Ludwig, B; Steinert, M; Schmidt, B; Fischer, G; Hacker, J

    1995-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive intracellularly in eukaryotic cells such as monocytes, macrophages, and protozoan organisms. The Mip (macrophage infectivity potentiator) protein represents a factor of L. pneumophila necessary for optimal intracellular survival. Interestingly, Mip belongs to the substance class of FK 506-binding proteins and exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. In order to identify amino acids most likely to be involved in the enzymatic activity of Mip, site-directed mutagenized Mip proteins were constructed and characterized. It was shown that an Asp-142 to Leu-142 mutation and a Tyr-185 to Ala-185 substitution resulted in strongly reduced PPIase activity of the recombinant Mip proteins (5.3 and 0.6% of the activity of the wild-type Mip, respectively). Genes coding for the wild-type and for site-directed-mutagenized Mip proteins were used to complement three different Mip-negative mutants of the L. pneumophila Corby, Philadelphia I, and Wadsworth. While Mip protein expression could be restored in the corresponding complementants, significant Mip-specific PPIase activity could be detected only in Mip mutants complemented with wild-type mip genes. To investigate the influence of the PPIase activity of Mip on intracellular survival of L. pneumophila, invasion assays were performed using the macrophage-like cell line U937, human blood monocytes, and Acanthamoeba castellanii. The Mip-negative mutants were approximately 50- to 100-fold less infective for A. castellanii and for human mononuclear phagocytes in vitro compared with their isogenic Mip-positive parental strains. The wild-type invasion rate could be restored by introducing an intact copy of the mip gene into Mip-negative strains. In addition, no differences in intracellular survival were observed between the wild-type isolates and the Legionella strains

  12. Methodologies for quantifying culturable, viable, and total Legionella pneumophila in indoor air.

    PubMed

    Chang, C-W; Chou, F-C

    2011-08-01

    Legionella pneumophila, aerosolized from numerous indoor facilities (e.g., shower heads, hot tubs, spas), may cause Pontiac fever (PF) and lethal pneumonia named Legionnaires' disease (LD) in humans. Reliable methods on quantitative exposure assessment of this bioaerosol are essential for the prevention of PF and LD. Coupled with culture, ethidium monoazide with qPCR, and qPCR assays, the collection efficiency for culturable, viable, and total L. pneumophila was assessed by means of filtration sampling (IOM with gelatin filter and cassette with polycarbonate filter) and liquid-based sampling methods (BioSampler, AGI-30, MAS-100 sampler with Tween mixture and deionized water (DW)). Results show IOM/gelatin filter was comparable to cassette/polycarbonate filter (P = 0.33) and performed greater than all of tested liquid-based methods for total cell collection. On the other hand, IOM/gelatin filter obtained greater efficiencies than cassette/polycarbonate filter by a factor of 3.8-8.6 for viable cells (P = 0.0006) and two orders of magnitude for culturable cells (P = 0.00002). Further comparison between liquid impingement and filtration methods indicates the sampling by IOM/gelatin filter, AGI-30, and BioSampler with DW were the most appropriate for viable cells, while culturable cells were collected most efficiently by BioSampler/DW with periodical replenishment during the sampling. This study recommends the most suitable methodologies for quantifying culturable, viable, and total Legionella pneumophila in indoor air. By using appropriate sampling and analytical methods, the residents and building owners are able to obtain the reliable data and further characterize the exposure risk and/or intervention efficacy against L. pneumophila. Moreover, the adoption of suitable monitoring methods also assists the investigators to explore the sources linked to PF and LD during the outbreaks. Considering reliable microbial monitoring is fundamental for epidemiological survey and

  13. Roles of iron in the survival, growth, and pathogenesis of Legionella pneumophila

    SciTech Connect

    Quinn, F.D.

    1985-01-01

    The essentially of iron for living cells has long been recognized, and the availability of host-iron has been proposed as a contributing factor to virulence in bacterial, fungal, and protozoan infections. The mechanism by which legionella pneumophila causes disease is unknown. Growth of fresh clinical or environmental isolates in pure culture requires 20 times more iron than is needed for most other bacteria. Thus, increased plasma iron levels may be needed for multiplication within human hosts. It was observed that: (1) this organism can be more readily deprived of iron by iron binding agents than all other bacteria studied, and this inhibition can be reversed by the addition of iron; (2) normal human blood serum kills L. pneumophila and the bactericidal action is decreased when complement is inactivated or enough iron to saturate serum transferrin is added to the system; (3) in assays with a radioactive isotope of iron (/sup 55/Fe), no specific iron sequestering system was detected; (4) in analysis of outer membrane proteins with /sup 55/Fe, SDS-polyacrylamide gel electrophoresis, and autoradiography, no specific outer membrane proteins responsible for iron acquisition were observed; and (5) in assays for protease, iron does not stimulate production of extracellular proteases. These observations indicate that L. pneumophila has no specific iron uptake mechanism, but instead relies on passive diffusion and/or non-specific mechanisms to obtain its iron.

  14. Sequence determination and mutational analysis of the lly locus of Legionella pneumophila.

    PubMed Central

    Wintermeyer, E; Flügel, M; Ott, M; Steinert, M; Rdest, U; Mann, K H; Hacker, J

    1994-01-01

    The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus. Images PMID:8112844

  15. Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

    PubMed Central

    Roy, C R; Isberg, R R

    1997-01-01

    The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 and 73 amino acids and a carboxyl-terminal cytoplasmic domain of 122 amino acids. Protein fractionation studies were consistent with DotA residing in the inner membrane. An alkaline phosphatase fusion located 9 amino acids upstream from the C terminus of DotA still retained function and was able to restore intracellular growth when harbored by two L. pneumophila dotA mutants. A hybrid protein from which the carboxyl-terminal 48 amino acids of DotA were deleted was unable to complement the intracellular growth defect in the dotA mutants, indicating that this cytoplasmic region is required for function. PMID:9009315

  16. Virulence factor rtx in Legionella pneumophila, evidence suggesting it is a modular multifunctional protein

    PubMed Central

    D'Auria, Giuseppe; Jiménez, Núria; Peris-Bondia, Francesc; Pelaz, Carmen; Latorre, Amparo; Moya, Andrés

    2008-01-01

    Background The repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity. Results The comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity. Conclusion In contrast to the extracellular bacteria Vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen L. pneumophila shows a rapid evolution. Changes in the rtx could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences. PMID:18194518

  17. VBNC Legionella pneumophila cells are still able to produce virulence proteins.

    PubMed

    Alleron, Laëtitia; Khemiri, Arbia; Koubar, Mohamad; Lacombe, Christian; Coquet, Laurent; Cosette, Pascal; Jouenne, Thierry; Frere, Jacques

    2013-11-01

    Legionella pneumophila is the agent responsible for legionellosis. Numerous bacteria, including L. pneumophila, can enter into a viable but not culturable (VBNC) state under unfavorable environmental conditions. In this state, cells are unable to form colonies on standard medium but are still alive. Here we show that VBNC L. pneumophila cells, obtained by monochloramine treatment, were still able to synthesize proteins, some of which are involved in virulence. Protein synthesis was measured using (35)S-labeling and the proteomes of VBNC and culturable cells then compared. This analysis allowed the identification of nine proteins that were accumulated in the VBNC state. Among them, four were involved in virulence, i.e., the macrophage infectivity potentiator protein, the hypothetical protein lpl2247, the ClpP protease proteolytic subunit and the 27 kDa outer membrane protein. Others, i.e., the enoyl reductase, the electron transfer flavoprotein (alpha and beta subunits), the 50S ribosomal proteins (L1 and L25) are involved in metabolic and energy production pathways. However, resuscitation experiments performed with Acanthamoeba castellanii failed, suggesting that the accumulation of virulence factors by VBNC cells is not sufficient to maintain their virulence. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Genotyping of Legionella pneumophila serogroup 1 strains isolated in Northern Sicily, Italy.

    PubMed

    Chiarini, Alfredo; Bonura, Celestino; Ferraro, Donatella; Barbaro, Roberta; Calà, Cinzia; Distefano, Salvatore; Casuccio, Nicolò; Belfiore, Santina; Giammanco, Anna

    2008-04-01

    During a three-year period, from April 2002 to May 2005, one hundred-forty-seven samples, taken from technical systems of water distribution at point of use, were repeatedly collected at six different sites in Northern Sicily and assayed for the presence of Legionella pneumophila serogroup 1 and serogroups 2 to 14. At the first samplings, the water distribution systems of all the sites were heavily contaminated, and disinfection treatments by the superheat and flush method were therefore performed. Treatments were always successful against L. pneumophila sg.1, but only in a few cases against all other serogroups. Eighty-six strains of L. pneumophila sg. 1, isolated from 26 of these samples, were characterized by amplified fragment length polymorphism (AFLP) analysis and sequence-based typing (SBT) procedure. Perfectly overlapping results were obtained by both the procedures and four genotypes were identified, accounting for all the isolates. The easy transferability of the SBT data through a web-based database made it possible to identify the presence in Northern Sicily of the two SBT types most commonly circulating in Europe.

  19. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy.

    PubMed

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Bedia, Carmen; Daniels, Craig; Abraham, Gilu; Stogios, Peter J; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Ong, Sze Ying; Hartland, Elizabeth L; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-02-16

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

  20. Protozoa and human macrophages infection by Legionella pneumophila environmental strains belonging to different serogroups.

    PubMed

    Messi, Patrizia; Patrizia, Messi; Bargellini, Annalisa; Annalisa, Bargellini; Anacarso, Immacolata; Immacolata, Anacarso; Marchesi, Isabella; Isabella, Marchesi; de Niederhäusern, Simona; Bondi, Moreno; Moreno, Bondi

    2013-02-01

    Three Legionella pneumophila strains isolated from municipal hot tap water during a multicentric Italian survey and belonging to serogroups 1, 6, 9 and the reference strain Philadelphia-1 were studied to determine the intracellular replication capability and the cytopathogenicity in human monocyte cell line U937 and in an Acanthamoeba polyphaga strain. Our results show that both serogroups 1 and Philadelphia-1 were able to multiply into macrophages inducing cytopathogenicity, while serogroup 6 and ever more serogroup 9 were less efficient in leading to death of the infected macrophages. Both serogroups 1 and 6 displayed a quite good capability of intracellular replication in A. polyphaga, although serogroup 1 was less cytopathogenic than serogroup 6. Serogroup 9, like Philadelphia-1 strain, showed a reduced efficiency of infection and replication and a low cytopathogenicity towards the protozoan. Our study suggests that bacterial pathogenesis is linked to the difference in the virulence expression of L. pneumophila serogroups in both hosts, as demonstrated by the fact that only L. pneumophila serogroup 1 shows the contextual expression of the two virulence traits. Serogroup 6 proves to be a good candidate as pathogen since it shows a good capacity for intracellular replication in protozoan.

  1. The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages.

    PubMed

    Sauer, John-Demian; Bachman, Michael A; Swanson, Michele S

    2005-07-12

    Differentiation in response to environmental cues is integral to the success of many intracellular pathogens. By characterizing a Legionella pneumophila mutant defective for differentiation in broth and replication in macrophages, we identified a subfamily of major facilitator superfamily transporters, here named Pht (phagosomal transporter), that also is conserved in two other vacuolar pathogens, Coxiella burnetii and Francisella tularensis. Biolog phenotype microarray analysis indicated that PhtA transports threonine, an essential amino acid. Either excess threonine or threonine peptides bypass phtA function. In minimal medium, phtA mutants do not replicate; in rich broth, the bacteria prematurely differentiate to the transmissive phase, as judged by the kinetics of flaA-gfp expression, heat resistance, and sodium sensitivity. PhtA is dispensable for transmissive L. pneumophila to establish and persist within a replication vacuole but is essential for their differentiation to the replicative phase, based on phenotypic and RT-PCR analysis. Accordingly, we propose that the Pht transporter family equips transmissive L. pneumophila, C. burnetii, and F. tularensis to assess their phagosomal nutrient supply before committing to reenter the cell cycle.

  2. Legionella pneumophila S1P-lyase targets host sphingolipid metabolism and restrains autophagy

    PubMed Central

    Rolando, Monica; Escoll, Pedro; Nora, Tamara; Botti, Joëlle; Boitez, Valérie; Daniels, Craig; Abraham, Gilu; Stogios, Peter J.; Skarina, Tatiana; Christophe, Charlotte; Dervins-Ravault, Delphine; Cazalet, Christel; Hilbi, Hubert; Rupasinghe, Thusitha W. T.; Tull, Dedreia; McConville, Malcolm J.; Ong, Sze Ying; Hartland, Elizabeth L.; Codogno, Patrice; Levade, Thierry; Naderer, Thomas; Savchenko, Alexei; Buchrieser, Carmen

    2016-01-01

    Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen’s Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis. PMID:26831115

  3. Phosphatidylcholine synthesis is required for optimal function of Legionella pneumophila virulence determinants

    PubMed Central

    Conover, Gloria M; Martinez-Morales, Fernando; Heidtman, Matthew I.; Luo, Zhao-Qing; Tang, May; Chen, Cui; Geiger, Otto; Isberg, Ralph R.

    2009-01-01

    The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N-methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Secondly, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion. PMID:17979985

  4. Phosphatidylcholine synthesis is required for optimal function of Legionella pneumophila virulence determinants.

    PubMed

    Conover, Gloria M; Martinez-Morales, Fernando; Heidtman, Matthew I; Luo, Zhao-Qing; Tang, May; Chen, Cui; Geiger, Otto; Isberg, Ralph R

    2008-02-01

    The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N-methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Second, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady-state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion.

  5. Icm/Dot-Independent Entry of Legionella pneumophila into Amoeba and Macrophage Hosts

    PubMed Central

    Bandyopadhyay, Purnima; Xiao, Huifang; Coleman, Hope A.; Price-Whelan, Alexa; Steinman, Howard M.

    2004-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, expresses a type IVB secretion apparatus that translocates bacterial proteins into amoeba and macrophage hosts. When stationary-phase cultures are used to infect hosts, the type IVB apparatus encoded by the icm/dot genes is required for entry, delay of phagosome-lysosome fusion, and intracellular multiplication within host cells. Null mutants with mutations in icm/dot genes are defective in these phenotypes. Here a new model is described in which hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.5 buffer. This model is called Ers treatment because it enhances the resistance to acid, hydrogen peroxide, and antibiotic stress beyond that of stationary-phase cultures. Following Ers treatment entry into amoeba and macrophage hosts does not require dotA, which is essential for Legionella virulence phenotypes when hosts are infected with stationary-phase cultures, dotB, icmF, icmV, or icmX. Defective host entry is also suppressed for null mutants with mutations in the KatA and KatB catalase-peroxidase enzymes, which are required for proper intracellular growth in amoeba and macrophage hosts. Ers treatment-induced suppression of defective entry is not associated with increased bacterial adhesion to host cells or with morphological changes in the bacterial envelope but is dependent on protein expression during Ers treatment. By using proteomic analysis, Ers treatment was shown to induce a protein predicted to contain eight tetratricopeptide repeats, a motif previously implicated in enhanced entry of L. pneumophila. Characterization of Ers treatment-dependent changes in expression is proposed as an avenue for identifying icm/dot-independent factors that function in the entry of Legionella into amoeba and macrophage hosts. PMID:15271914

  6. Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires' disease.

    PubMed Central

    Brieland, J; McClain, M; Heath, L; Chrisp, C; Huffnagle, G; LeGendre, M; Hurley, M; Fantone, J; Engleberg, C

    1996-01-01

    The effect of inhaled amoebae on the pathogenesis of Legionnaires' disease was investigated in vivo. A/J mice, which are susceptible to replicative Legionella pneumophila infections, were inoculated intratracheally with L. pneumophila (10(6) bacteria per mouse) or were coinoculated with L. pneumophila (10(6) bacteria per mouse) and Hartmannella vermiformis (10(6) amoebae per mouse). The effect of coinoculation with H. vermiformis on bacterial clearance, histopathology, cellular recruitment into the lung, and intrapulmonary levels of cytokines including gamma interferon and tumor necrosis factor alpha was subsequently assessed. Coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of L. pneumophila in A/J mice. Histopathologic and flow cytometric analysis of lung tissue demonstrated that while A/J mice inoculated with L. pneumophila alone develop multifocal pneumonitis which resolves with minimal mortality, mice coinoculated with H. vermiformis develop diffuse pneumonitis which is associated with diminished intrapulmonary recruitment of lymphocytes and mononuclear phagocytic cells and significant mortality. Furthermore, coinoculation of mice with H. vermiformis resulted in a fourfold enhancement in intrapulmonary levels of gamma interferon and tumor necrosis factor alpha compared with mice infected with L. pneumophila alone. The effect of H. vermiformis on intrapulmonary growth of L. pneumophila in a resistant host (i.e., BALB/c mice) was subsequently evaluated. While BALB/c mice do not develop replicative L. pneumophila infections following inoculation with L. pneumophila alone, there was an eightfold increase in intrapulmonary L. pneumophila in BALB/c mice coinoculated with H. vermiformis. These studies, demonstrating that intrapulmonary amoebae potentiate replicative L. pneumophila lung infection in both a susceptible and a resistant host, have significant implications with regard to the potential role of protozoa in the pathogenesis of

  7. Effect of Prior Influenza Virus Infection on Susceptibility of AKR/J Mice and Squirrel Monkeys to Respiratory Challenge with Legionella pneumophila.

    DTIC Science & Technology

    1980-07-30

    influenza virus and Legionella pneumophila than to either agent alone. b 3 F As knowledge of Legionnaires ’ disease has accumulated, the evidence...suggests that many infections occur in individuals with underlying disease . Since Legionella pneumophila appears to spread by the airborne route (5, 6, 8, 9...The sequence of influenza followed by Legionnaires ’ disease may be relatively unco mon in nature because of the differing seasonal patterns of the

  8. Isolation of Legionella pneumophila from cooling towers, public baths, hospitals, and fountains in Seoul, Korea, from 2010 to 2012.

    PubMed

    Kim, Changkyu; Jeon, Sujin; Jung, Jihun; Oh, Younghee; Kim, Yeonsun; Lee, Jaein; Choi, Sungmin; Chae, Youngzoo; Lee, Young-Ki

    2015-01-01

    Legionnaire's disease is associated with a high mortality rate. The authors collected 3,495 water samples in Seoul, Korea, between 2010 and 2012 from public facilities (cooling towers, public baths, hospitals, and decorative fountains), which are considered the major habitats of Legionella pneumophila. In all, 527 (15.1%) isolates of L. pneumophila were obtained by microbial culture and polymerase chain reaction. Serological diagnosis and pulsed-field gel electrophoresis (PFGE) analysis were performed for the samples. The authors categorized the samples into four groups (A-D) on the basis of PFGE results. The analysis revealed that cooling towers containing the most samples with L. pneumophila serogroup 1 constituted the highest proportion of isolate. Samples from public facilities and serogroups could be distinctively classified by PFGE patterns. Thus, it is expected that source-specific features revealed through PFGE and serological analyses could serve as the basis for effectively coping with future outbreaks of L. pneumophila.

  9. An update on iron acquisition by Legionella pneumophila: new pathways for siderophore uptake and ferric iron reduction.

    PubMed

    Cianciotto, Nicholas P

    2015-01-01

    Iron acquisition is critical for the growth and pathogenesis of Legionella pneumophila, the causative agent of Legionnaires' disease. L. pneumophila utilizes two main modes of iron assimilation, namely ferrous iron uptake via the FeoB system and ferric iron acquisition through the action of the siderophore legiobactin. This review highlights recent studies concerning the mechanism of legiobactin assimilation, the impact of c-type cytochromes on siderophore production, the importance of legiobactin in lung infection and a newfound role for a bacterial pyomelanin in iron acquisition. These data demonstrate that key aspects of L. pneumophila iron acquisition are significantly distinct from those of long-studied, 'model' organisms. Indeed, L. pneumophila may represent a new paradigm for a variety of other intracellular parasites, pathogens and under-studied bacteria.

  10. An update on iron acquisition by Legionella pneumophila: new pathways for siderophore uptake and ferric iron reduction

    PubMed Central

    Cianciotto, Nicholas P

    2015-01-01

    Iron acquisition is critical for the growth and pathogenesis of Legionella pneumophila, the causative agent of Legionnaires’ disease. L. pneumophila utilizes two main modes of iron assimilation, namely ferrous iron uptake via the FeoB system and ferric iron acquisition through the action of the siderophore legiobactin. This review highlights recent studies concerning the mechanism of legiobactin assimilation, the impact of c-type cytochromes on siderophore production, the importance of legiobactin in lung infection and a newfound role for a bacterial pyomelanin in iron acquisition. These data demonstrate that key aspects of L. pneumophila iron acquisition are significantly distinct from those of long-studied, ‘model’ organisms. Indeed, L. pneumophila may represent a new paradigm for a variety of other intracellular parasites, pathogens and under-studied bacteria. PMID:26000653

  11. Human Lung Tissue Explants Reveal Novel Interactions during Legionella pneumophila Infections

    PubMed Central

    Jäger, Jens; Marwitz, Sebastian; Tiefenau, Jana; Rasch, Janine; Shevchuk, Olga; Kugler, Christian

    2014-01-01

    Histological and clinical investigations describe late stages of Legionnaires' disease but cannot characterize early events of human infection. Cellular or rodent infection models lack the complexity of tissue or have nonhuman backgrounds. Therefore, we developed and applied a novel model for Legionella pneumophila infection comprising living human lung tissue. We stimulated lung explants with L. pneumophila strains and outer membrane vesicles (OMVs) to analyze tissue damage, bacterial replication, and localization as well as the transcriptional response of infected tissue. Interestingly, we found that extracellular adhesion of L. pneumophila to the entire alveolar lining precedes bacterial invasion and replication in recruited macrophages. In contrast, OMVs predominantly bound to alveolar macrophages. Specific damage to septa and epithelia increased over 48 h and was stronger in wild-type-infected and OMV-treated samples than in samples infected with the replication-deficient, type IVB secretion-deficient DotA− strain. Transcriptome analysis of lung tissue explants revealed a differential regulation of 2,499 genes after infection. The transcriptional response included the upregulation of uteroglobin and the downregulation of the macrophage receptor with collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context of L. pneumophila infections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires' disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations. PMID:24166955

  12. Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing

    PubMed Central

    Rhoads, William J.; Pruden, Amy; Edwards, Marc A.

    2016-01-01

    Legionella pneumophila is known to proliferate in hot water plumbing systems, but little is known about the specific physicochemical factors that contribute to its regrowth. Here, L. pneumophila trends were examined in controlled, replicated pilot-scale hot water systems with continuous recirculation lines subject to two water heater settings (40 °C and 58 °C) and three distal tap water use frequencies (high, medium, and low) with two pipe configurations (oriented upward to promote convective mixing with the recirculating line and downward to prevent it). Water heater temperature setting determined where L. pneumophila regrowth occurred in each system, with an increase of up to 4.4 log gene copies/mL in the 40 °C system tank and recirculating line relative to influent water compared to only 2.5 log gene copies/mL regrowth in the 58 °C system. Distal pipes without convective mixing cooled to room temperature (23–24 °C) during periods of no water use, but pipes with convective mixing equilibrated to 30.5 °C in the 40 °C system and 38.8 °C in the 58 °C system. Corresponding with known temperature effects on L. pneumophila growth and enhanced delivery of nutrients, distal pipes with convective mixing had on average 0.2 log more gene copies/mL in the 40 °C system and 0.8 log more gene copies/mL in the 58 °C system. Importantly, this work demonstrated the potential for thermal control strategies to be undermined by distal taps in general, and convective mixing in particular. PMID:26985908

  13. Exposure to Synthetic Gray Water Inhibits Amoeba Encystation and Alters Expression of Legionella pneumophila Virulence Genes

    PubMed Central

    Lu, Jingrang; Ashbolt, Nicholas J.

    2014-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems. PMID:25381242

  14. Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

    PubMed Central

    Zusman, Tal; Speiser, Yariv

    2014-01-01

    Legionella pneumophila is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. For most of these effectors, there is no information regarding their regulation. Therefore, the aim of this study was to examine the involvement of the three L. pneumophila Fis homologs in the regulation of effector-encoding genes. Deletion mutants constructed in the genes encoding the three Fis regulators revealed that Fis1 (lpg0542 gene) and Fis3 (lpg1743) but not Fis2 (lpg1370) are partially required for intracellular growth of L. pneumophila in Acanthamoeba castellanii. To identify pathogenesis-related genes directly regulated by Fis, we established a novel in vivo system which resulted in the discovery of numerous effector-encoding genes directly regulated by Fis. Further examination of these genes revealed that Fis1 and Fis3 repress the level of expression of effector-encoding genes during exponential phase. Three groups of effector-encoding genes were identified: (i) effectors regulated mainly by Fis1, (ii) effectors regulated mainly by Fis3, and (iii) effectors regulated by both Fis1 and Fis3. Examination of the upstream regulatory region of all of these effector-encoding genes revealed multiple putative Fis regulatory elements, and site-directed mutagenesis confirmed that a few of these sites constitute part of a repressor binding element. Furthermore, gel mobility shift assays demonstrated the direct relation between the Fis1 and Fis3 regulators and these regulatory elements. Collectively, our results demonstrate for the first time that two of the three L. pneumophila Fis regulators directly repress the expression of Icm/Dot effector-encoding genes. PMID:25225276

  15. Exposure to synthetic gray water inhibits amoeba encystation and alters expression of Legionella pneumophila virulence genes.

    PubMed

    Buse, Helen Y; Lu, Jingrang; Ashbolt, Nicholas J

    2015-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems.

  16. Convective Mixing in Distal Pipes Exacerbates Legionella pneumophila Growth in Hot Water Plumbing.

    PubMed

    Rhoads, William J; Pruden, Amy; Edwards, Marc A

    2016-03-12

    Legionella pneumophila is known to proliferate in hot water plumbing systems, but little is known about the specific physicochemical factors that contribute to its regrowth. Here, L. pneumophila trends were examined in controlled, replicated pilot-scale hot water systems with continuous recirculation lines subject to two water heater settings (40 °C and 58 °C) and three distal tap water use frequencies (high, medium, and low) with two pipe configurations (oriented upward to promote convective mixing with the recirculating line and downward to prevent it). Water heater temperature setting determined where L. pneumophila regrowth occurred in each system, with an increase of up to 4.4 log gene copies/mL in the 40 °C system tank and recirculating line relative to influent water compared to only 2.5 log gene copies/mL regrowth in the 58 °C system. Distal pipes without convective mixing cooled to room temperature (23-24 °C) during periods of no water use, but pipes with convective mixing equilibrated to 30.5 °C in the 40 °C system and 38.8 °C in the 58 °C system. Corresponding with known temperature effects on L. pneumophila growth and enhanced delivery of nutrients, distal pipes with convective mixing had on average 0.2 log more gene copies/mL in the 40 °C system and 0.8 log more gene copies/mL in the 58 °C system. Importantly, this work demonstrated the potential for thermal control strategies to be undermined by distal taps in general, and convective mixing in particular.

  17. Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin.

    PubMed Central

    Horwitz, M A; Silverstein, S C

    1983-01-01

    We have previously reported that virulent egg yolk-grown Legionella pneumophila, Philadelphia 1 strain, multiplies intracellularly in human blood monocytes and only intracellularly under tissue culture conditions. In this paper, we have investigated the effect of erythromycin and rifampin on L. pneumophila-monocyte interaction in vitro; erythromycin and rifampin are currently the drugs of choice for the treatment of Legionnaires' disease. The intracellular multiplication of L. pneumophila is inhibited by erythromycin and rifampin, as measured by colony-forming units, whether the antibiotics are added just before or just after infection of monocytes with L. pneumophila, or 2 d after infection when L. pneumophila is in the logarithmic phase of growth in monocytes. Intracellular multiplication of L. pneumophila is inhibited by 1.25 microgram/ml but not less than or equal to 0.125 microgram/ml erythromycin and 0.01 microgram/ml but not less than or equal to 0.001 microgram/ml rifampin. These concentrations of antibiotics are comparable to those that inhibit extracellular multiplication of L. pneumophila under cell-free conditions in artificial medium; the minimal inhibitory concentration is 0.37 microgram/ml for erythromycin and 0.002 microgram/ml for rifampin. Multiplication of L. pneumophila in the logarithmic phase of growth in monocytes is inhibited within 1 h of the addition of antibiotics. Intracellular bacteria inhibited from multiplying by antibiotics are not killed. By electron microscopy, the bacteria appear intact within membrane-bound vacuoles, studded with ribosomelike structures. L. pneumophila multiplying extracellularly on artificial medium is killed readily by relatively low concentrations of erythromycin and rifampin; the minimal bactericidal concentration is 1 microgram/ml for erythromycin and 0.009 microgram/ml for rifampin. In contrast, L. pneumophila multiplying intracellularly is resistant to killing by these concentrations of erythromycin and

  18. Environmental surveillance and molecular epidemiology of waterborne pathogen Legionella pneumophila in health-care facilities of Northeastern Greece: a 4-year survey.

    PubMed

    Alexandropoulou, Ioanna G; Ntougias, Spyridon; Konstantinidis, Theocharis G; Parasidis, Theodoros A; Panopoulou, Maria; Constantinidis, Theodoros C

    2015-05-01

    A 4-year proactive environmental surveillance of Legionella spp. in the water distribution and cooling systems of five health-care facilities was carried out as part of the strategy for the prevention of hospital-acquired Legionnaires' disease in Northeastern Greece. Legionella spp. were detected in 71 out of 458 collected samples. The majority of strains belonged to Legionella pneumophila serogroups 2-15 (75.0%), while all L. pneumophila serogroup 1 strains (23.6%) were isolated from a single hospital. The highest percentage of positive samples was found in distal sites (19.4%), while no Legionella strains were detected in cooling systems. Each hospital was colonized at least once with L. pneumophila, while remedial actions resulted in significant reduction of Legionella concentration. The molecular epidemiology of environmental L. pneumophila strains was also investigated using random amplified polymorphic DNA (RAPD) and multi-gene sequence-based analysis. Based on RAPD patterns, L. pneumophila serogroups 2-15 and serogroup 1 strains were classified into 24 and 9 operational taxonomic units (OTUs), respectively. Sequencing of housekeeping and diversifying pressure-related genes recommended by European Working Group for Legionella Infections (EWGLI) revealed not only a high intraspecies variability but also the circulation and persistence of one specific genotyping profile in the majority of hospitals. This study highlights the necessity for diachronic surveillance of Legionella in health-care facilities by adopting both cultural and molecular methods.

  19. Colonization of hospital water systems by Legionella pneumophila, Pseudomonas aeroginosa, and Acinetobacter in ICU wards of Tehran hospitals.

    PubMed

    Yaslianifard, Somayeh; Mobarez, Ashraf Mohabati; Fatolahzadeh, Bahram; Feizabadi, Mohammad Mehdi

    2012-01-01

    Nosocomial infection caused by non-Enterobacteriaceae gram negative bacteria (GNB-NE) is increasing in intensive care units (ICU). The objective of this study was to determine whether potable water in ICU wards at Tehran hospitals is contaminated with L. pneomophila, P. aeroginosa and Acinetobacter spp. A total of 52 water samples from shower bath and taps water in seven hospitals of Tehran were collected. The water sample concentrated by filtering through millipore cellulose filters and cultured on BCYE agar and tryptic soya agar media. The presence of Legionella pneumophila was confirmed by real time PCR assay using primers-probe designed for the mip gene. Legionella pneumophila, Pseudomonas aeroginosa and Acinetobacter were isolated from 5 (9.6%), 6 (11.4%) and 1 (1.8%) of the hospital water systems, respectively. This study demonstrated the presence of Legionella, Pseudomonas and Acinetobacter in water system in ICU wards of different hospitals in Tehran. Hot water from shower heads could be a potential source of infection for Legionella pneumophila. Water was also proved to contain Pseudomonas aeruginonsa, the main GNB-NE causing nosocomila pneumonia at Tehran hospitals. Care should be taken concerning cleanliness and decontamination of water supplies at ICUs for pathogenic organisms.

  20. Fast immunosensing technique to detect Legionella pneumophila in different natural and anthropogenic environments: comparative and collaborative trials

    PubMed Central

    2013-01-01

    Background Legionellosis is an uncommon form of pneumonia. After a clinical encounter, the necessary antibiotic treatment is available if the diagnosis is made early in the illness. Before the clinical encounter, early detection of the main pathogen involved, Legionella pneumophila, in hazardous environments is important in preventing infectious levels of this bacterium. In this study a qualitative test based on combined magnetic immunocapture and enzyme-immunoassay for the fast detection of Legionella pneumophila in water samples was compared with the standard method, in both comparative and collaborative trials. The test was based on the use of anti-Legionella pneumophila antibodies immobilized on magnetic microspheres. The final protocol included concentration by filtration, resuspension and immunomagnetic capture. The whole assay took less than 1 hour to complete. Results A comparative trial was performed against the standard culture method (ISO 11731) on both artificially and naturally contaminated water samples, for two matrices: chlorinated tap water and cooling tower water. Performance characteristics of the test used as screening with culture confirmation resulted in sensitivity, specificity, false positive, false negative, and efficiency of 96.6%, 100%, 0%, 3.4%, and 97.8%, respectively. The detection limit at the level under which the false negative rate increases to 50% (LOD50) was 93 colony forming units (CFU) in the volume examined for both tested matrices. The collaborative trial included twelve laboratories. Water samples spiked with certified reference materials were tested. In this study the coincidence level between the two methods was 95.8%. Conclusion Results demonstrate the applicability of this immunosensing technique to the rapid, simple, and efficient detection of Legionella pneumophila in water samples. This test is not based on microbial growth, so it could be used as a rapid screening technique for the detection of L. pneumophila in

  1. Effect of temperature, pH, and oxygen level on the multiplication of naturally occurring Legionella pneumophila in potable water.

    PubMed Central

    Wadowsky, R M; Wolford, R; McNamara, A M; Yee, R B

    1985-01-01

    A water culture containing naturally occurring Legionella pneumophila and associated microbiota was maintained in the laboratory by serially transferring the culture in tap water which had been sterilized by membrane filtration. Successful maintenance of the water culture depended upon transferring the culture when the growth of L. pneumophila was in the late-exponential to early-stationary phase. The water culture was used as a source of naturally occurring bacteria to determine some of the parameters which affect the multiplication of L. pneumophila in tap water. Naturally occurring L. pneumophila multiplied at a temperature between 25 and 37 degrees C, at pH levels of 5.5 to 9.2, and at concentrations of dissolved oxygen of 6.0 to 6.7 mg/liter. Multiplication did not occur in tap water which contained less than 2.2 mg of dissolved oxygen per liter. An association was observed between the multiplication of L. pneumophila and the non-Legionellaceae bacteria which were also present in the water culture. The method of preserving naturally occurring L. pneumophila and associated microbiota may facilitate studies on the symbiosis of L. pneumophila with other microorganisms. PMID:4004233

  2. Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

    PubMed

    Nasrallah, Gheyath K; Abdelhady, Hany; Tompkins, Nicholas P; Carson, Kaitlyn R; Garduño, Rafael A

    2014-07-01

    L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted.

  3. Role of Biofilm Roughness and Hydrodynamic Conditions in Legionella pneumophila Adhesion to and Detachment from Simulated Drinking Water Biofilms

    PubMed Central

    Shen, Yun; Monroy, Guillermo L.; Derlon, Nicolas; Janjaroen, Dao; Huang, Conghui; Morgenroth, Eberhard; Boppart, Stephen A.; Ashbolt, Nicholas J.; Liu, Wen-Tso; Nguyen, Thanh H.

    2015-01-01

    Biofilms in drinking water distribution systems (DWDS) could exacerbate the persistence and associated risks of pathogenic Legionella pneumophila (L. pneumophila), thus raising human health concerns. However, mechanisms controlling adhesion and subsequent detachment of L. pneumophila associated with biofilms remain unclear. We determined the connection between L. pneumophila adhesion and subsequent detachment with biofilm physical structure characterization using optical coherence tomography (OCT) imaging technique. Analysis of the OCT images of multispecies biofilms grown under low nutrient condition up to 34 weeks revealed the lack of biofilm deformation even when these biofilms were exposed to flow velocity of 0.7 m/s, typical flow for DWDS. L. pneumophila adhesion on these biofilm under low flow velocity (0.007 m/s) positively correlated with biofilm roughness due to enlarged biofilm surface area and local flow conditions created by roughness asperities. The preadhered L. pneumophila on selected rough and smooth biofilms were found to detach when these biofilms were subjected to higher flow velocity. At the flow velocity of 0.1 and 0.3 m/s, the ratio of detached cell from the smooth biofilm surface was from 1.3 to 1.4 times higher than that from the rough biofilm surface, presumably because of the low shear stress zones near roughness asperities. This study determined that physical structure and local hydrodynamics control adhesion and detachment from simulated drinking water biofilm, thus it is the first step toward reducing the risk of L. pneumophila exposure and subsequent infections. PMID:25699403

  4. Pathway analysis using (13) C-glycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila.

    PubMed

    Häuslein, Ina; Manske, Christian; Goebel, Werner; Eisenreich, Wolfgang; Hilbi, Hubert

    2016-04-01

    Amino acids represent the prime carbon and energy source for Legionella pneumophila, a facultative intracellular pathogen, which can cause a life-threatening pneumonia termed Legionnaires' disease. Genome, transcriptome and proteome studies indicate that L. pneumophila also utilizes carbon substrates other than amino acids. We show here that glycerol promotes intracellular replication of L. pneumophila in amoeba or macrophages (but not extracellular growth) dependent on glycerol-3-phosphate dehydrogenase, GlpD. An L. pneumophila mutant strain lacking glpD was outcompeted by wild-type bacteria upon co-infection of amoeba, indicating an important role of glycerol during infection. Isotopologue profiling studies using (13) C-labelled substrates were performed in a novel minimal defined medium, MDM, comprising essential amino acids, proline and phenylalanine. In MDM, L. pneumophila utilized (13) C-labelled glycerol or glucose predominantly for gluconeogenesis and the pentose phosphate pathway, while the amino acid serine was used for energy generation via the citrate cycle. Similar results were obtained for L. pneumophila growing intracellularly in amoeba fed with (13) C-labelled glycerol, glucose or serine. Collectively, these results reveal a bipartite metabolism of L. pneumophila, where glycerol and carbohydrates like glucose are mainly fed into anabolic processes, while serine serves as major energy supply.

  5. First report of Legionella pneumophila in car cabin air filters. Are these a potential exposure pathway for professional drivers?

    PubMed

    Alexandropoulou, Ioanna G; Konstantinidis, Theocharis G; Parasidis, Theodoros A; Nikolaidis, Christos; Panopoulou, Maria; Constantinidis, Theodoros C

    2013-12-01

    Recent findings have identified professional drivers as being at an increased risk of Legionnaires' disease. Our hypothesis was that used car cabin air filters represent a reservoir of Legionella bacteria, and thus a potential pathway for contamination. We analysed used cabin air filters from various types of car. The filters were analysed by culture and by molecular methods. Our findings indicated that almost a third of air filters were colonized with Legionella pneumophila. Here, we present the first finding of Legionella spp. in used car cabin air filters. Further investigations are needed in order to confirm this exposure pathway. The presence of Legionella bacteria in used cabin air filters may have been an unknown source of infection until now.

  6. [Comparative study of the biological properties of various nutrient media used for the isolation of Legionella pneumophila].

    PubMed

    Shelokhovich, A I; Kharabadzhakhian, G D; Karbyshev, G L; Terent'ev, A N; Savel'eva, I K; Savel'eva, E P; Sokirkina, O G

    2009-10-01

    Three diagnostic selective media used for the isolation of Legionella pneumophila were compared. These included BCYEAalpha (Oxoid, a reference medium), BCYEAalpha (Hi Media), and elective legionellosis medium (ELM) developed at the Rostov-on-Don Research Institute for Plague Control. The virulent L. pneumophila strain Philadelphia-1 (LD50 was 10(5) CFU for guinea-pigs) was used a test culture. The susceptibility of the media was determined, by culturing 10(-6) and 10(-7) dilutions of the suspension of the macerated Legionella-infected guinea-pig spleen, as well as the suspension of a culture of this strain (100 CFU) and 6 L. pneumophila cultures freshly isolated from water. The BCYEAalpha (Oxoid) and ELM media demonstrated the similar growth characteristics (chi2 < 0.7; p = 0.05) while the BCYEAalpha (Hi Media) medium showed a low susceptibility. The BCYEAalpha (Oxoid) and ELM media were first found to be successful in detecting Legionella in viable, but nonculturable state, induced by the following factors: 1) starvation in distilled water; 2) exposure to hydroquinone (oxidative shock, and 3) elevated temperature (56 degrees C). The BCYEAalpha (Oxoid) and EML media did not differ either in their ability to suppress extraneous microflora and to maintain stable initial pH under the conditions of incubation of culture plates, as well as in their Na+ concentration (15-19 mmol/l). However, the BCYEAalpha (Oxoid) medium exceeded the ELM one in the growth rate and diameter of Legionella colonies. Two L. pneumophila cultures were isolated in the BCYEAalpha (Oxoid) and EML media used in the field experiment studying 15 water samples from different hot water supply systems. Thus, the conclusion can be drawn that the EML medium is comparable with the reference BCYEAalpha (Oxoid) medium in its susceptibility and ability to detect Legionella in both vegetative and viable, but nonculturable states and is suitable for practical application.

  7. Characterization of the selective inhibition of growth of virulent Legionella pneumophila by supplemented Mueller-Hinton medium.

    PubMed Central

    Catrenich, C. E.; Johnson, W.

    1989-01-01

    The phenotypic difference between virulent and avirulent Legionella pneumophila with regard to growth on supplemented Mueller-Hinton (SMH) agar was investigated. Defined populations of virulent and avirulent L. pneumophila were inoculated onto hybrid growth media containing the combination of SMH agar components with potassium phosphate-buffered charcoal-yeast extract agar. The casein acid hydrolysate component of SMH agar was found to be inhibitory to the growth of virulent but not avirulent cells. The selectively inhibitory component of the casein acid hydrolysate was identified as NaCl. Images PMID:2722245

  8. Distinct difference of flaA genotypes of Legionella pneumophila between isolates from bath water and cooling tower water.

    PubMed

    Amemura-Maekawa, Junko; Kura, Fumiaki; Chang, Bin; Suzuki-Hashimoto, Atsuko; Ichinose, Masayuki; Endo, Takuro; Watanabe, Haruo

    2008-09-01

    To investigate the genetic difference of Legionella pneumophila in human-made environments, we collected isolates of L. pneumophila from bath water (n = 167) and cooling tower water (n = 128) primarily in the Kanto region in 2001 and 2005. The environmental isolates were serogrouped and sequenced for a target region of flaA. A total of 14 types of flaA genotypes were found: 10 from cooling tower water and nine from bath water. The flaA genotypes of isolates from cooling tower water were quite different from those of bath water.

  9. Legionella pneumophila serogroup 1 subgrouping by monoclonal antibodies--an epidemiological tool.

    PubMed Central

    Watkins, I. D.; Tobin, J. O.; Dennis, P. J.; Brown, W.; Newnham, R.; Kurtz, J. B.

    1985-01-01

    A panel of 10 monoclonal antibodies was used to subgroup 326 strains of Legionella pneumophila serogroup 1. All but two strains could be classified into three major subgroups named after their representative strains Pontiac 1, Olda and Bellingham 1. Of the 50 isolates from patients, 44 representing 32 separate incidents were of the Pontiac subgroup. This subgroup was also found in 16 of 18 buildings epidemiologically associated with Legionnaires' Disease. In contrast, strains of the Olda subgroup predominated in buildings where no infections had occurred. In 9 of the 11 incidents where isolates were available from at least one patient as well as from the suspected environmental source, the monoclonal antibody reaction patterns of strains from patients were identical to those of one or more of their environmental counterparts. PMID:3905954

  10. Genetic variability in environmental isolates of Legionella pneumophila from Comunidad Valenciana (Spain).

    PubMed

    Coscollá, Mireia; Gosalbes, María José; Catalán, Vicente; González-Candelas, Fernando

    2006-06-01

    Legionella pneumophila is associated to recurrent outbreaks in several Comunidad Valenciana (Spain) localities, especially in Alcoi, where social and climatic conditions seem to provide an excellent environment for bacterial growth. We have analysed the nucleotide sequences of three loci from 25 environmental isolates from Alcoi and nearby locations sampled over 3 years. The analysis of these isolates has revealed a substantial level of genetic variation, with consistent patterns of variability across loci, and comparable to that found in a large, European-wide sampling of clinical isolates. Among the tree loci studied, fliC showed the highest level of nucleotide diversity. The analysis of isolates sampled in different years revealed a clear differentiation, with samples from 2001 being significantly distinct from those obtained in 2002 and 2003. Furthermore, although linkage disequilibrium measures indicate a clonal nature for population structure in this sample, the presence of some recombination events cannot be ruled out.

  11. SPR based immunosensor for detection of Legionella pneumophila in water samples

    NASA Astrophysics Data System (ADS)

    Enrico, De Lorenzis; Manera, Maria G.; Montagna, Giovanni; Cimaglia, Fabio; Chiesa, Maurizio; Poltronieri, Palmiro; Santino, Angelo; Rella, Roberto

    2013-05-01

    Detection of legionellae by water sampling is an important factor in epidemiological investigations of Legionnaires' disease and its prevention. To avoid labor-intensive problems with conventional methods, an alternative, highly sensitive and simple method is proposed for detecting L. pneumophila in aqueous samples. A compact Surface Plasmon Resonance (SPR) instrumentation prototype, provided with proper microfluidics tools, is built. The developed immunosensor is capable of dynamically following the binding between antigens and the corresponding antibody molecules immobilized on the SPR sensor surface. A proper immobilization strategy is used in this work that makes use of an important efficient step aimed at the orientation of antibodies onto the sensor surface. The feasibility of the integration of SPR-based biosensing setups with microfluidic technologies, resulting in a low-cost and portable biosensor is demonstrated.

  12. Passage through Tetrahymena tropicalis triggers a rapid morphological differentiation in Legionella pneumophila.

    PubMed

    Faulkner, Gary; Berk, Sharon G; Garduño, Elizabeth; Ortiz-Jiménez, Marco A; Garduño, Rafael A

    2008-12-01

    The intracellular bacterial pathogen Legionella pneumophila follows a developmental cycle in which replicative forms (RFs) differentiate into infectious stationary-phase forms (SPFs) in vitro and in vivo into highly infectious mature intracellular forms (MIFs). The potential relationships between SPFs and MIFs remain uncharacterized. Previously we determined that L. pneumophila survives, but does not replicate, while it transiently resides (for 1 to 2 h) in food vacuoles of the freshwater ciliate Tetrahymena tropicalis before being expelled as legionellae-laden pellets. We report here that SPFs have the ability to rapidly (<1 h) and directly (in the absence of bacterial replication) differentiate into MIFs while in transit through T. tropicalis, indicating that SPFs and MIFs constitute a differentiation continuum. Mutant RFs lacking the sigma factor gene rpoS, or the response regulator gene letA, were unable to produce normal SPFs in vitro and did not fully differentiate into MIFs in vivo, further supporting the existence of a common mechanism of differentiation shared by SPFs and MIFs. Mutants with a defective Dot/Icm system morphologically differentiated into MIFs while in transit through T. tropicalis. Therefore, T. tropicalis has allowed us to unequivocally conclude that SPFs can directly differentiate into MIFs and that the Dot/Icm system is not required for differentiation, two events that could not be experimentally addressed before. The Tetrahymena model can now be exploited to study the signals that trigger MIF development in vivo and is the only replication-independent model reported to date that allows the differentiation of Dot/Icm mutants into MIFs.

  13. Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila.

    PubMed

    Aragon, V; Kurtz, S; Flieger, A; Neumeister, B; Cianciotto, N P

    2000-04-01

    Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

  14. Growth-supporting activity for Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors.

    PubMed Central

    Wadowsky, R M; Butler, L J; Cook, M K; Verma, S M; Paul, M A; Fields, B S; Keleti, G; Sykora, J L; Yee, R B

    1988-01-01

    Photosynthetic cyanobacteria, heterotrophic bacteria, free-living amoebae, and ciliated protozoa may support growth of Legionella pneumophila. Studies were done with two tap water cultures (WS1 and WS2) containing L. pneumophila and associated microbiota to characterize growth-supporting activity and assess the relative importance of the microbiota in supporting multiplication of L. pneumophila. The water cultures were incubated in the dark at 35 degrees C. The growth-supporting factor(s) was separated from each culture by filtration through 1-micron-pore-size membrane filters. The retentate was then suspended in sterile tap water. Multiplication of L. pneumophila occurred when both the retentate suspension and the filtrate from either culture were inoculated into sterile tap water. L. pneumophila did not multiply in tap water inoculated with only the filtrate, even though filtration did not reduce the concentration of L. pneumophila or heterotrophic bacteria in either culture. Growth-supporting activity of the retentate suspension from WS1 was inactivated at 60 degrees C but unaffected at 0, 25, and 45 degrees C after 30-min incubations. Filtration experiments indicated that the growth-supporting factor(s) in WS1 was 2 to 5 micron in diameter. Ciliated protozoa were not detected in either culture. Hartmannellid amoebae were conclusively demonstrated in WS2 but not in WS1. L. pneumophila multiplied in tap water inoculated with the amoebae (10(3)/ml) and the 1-micron filtrate of WS2. No multiplication occurred in tap water inoculated with the filtrate only. Growth-supporting activity for L. pneumophila may be present in plumbing systems; hartmannellid amoebae appear to be important determinants of multiplication of L. pneumophila in some tap water cultures. PMID:3214153

  15. Alveolar macrophages and neutrophils are the primary reservoirs for Legionella pneumophila and mediate cytosolic surveillance of type IV secretion.

    PubMed

    Copenhaver, Alan M; Casson, Cierra N; Nguyen, Hieu T; Fung, Thomas C; Duda, Matthew M; Roy, Craig R; Shin, Sunny

    2014-10-01

    Legionella pneumophila, an intracellular pathogen responsible for the severe pneumonia Legionnaires' disease, uses its dot/icm-encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol, L. pneumophila also activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate the control of infection. Thus, the pulmonary cell types that L. pneumophila infects not only may act as an intracellular niche that facilitates its pathogenesis but also may contribute to the immune response against L. pneumophila. The identity of these host cells remains poorly understood. Here, we developed a strain of L. pneumophila producing a fusion protein consisting of β-lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils both are the primary recipients of T4SS-translocated effectors and harbor viable L. pneumophila during pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced tumor necrosis factor and interleukin-1α in response to T4SS-sufficient, but not T4SS-deficient, L. pneumophila. Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir for L. pneumophila and a source of proinflammatory cytokines that contribute to the host immune response against L. pneumophila during pulmonary infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  16. Heterogeneity in the attachment and uptake mechanisms of the Legionnaires' disease bacterium, Legionella pneumophila, by protozoan hosts.

    PubMed

    Harb, O S; Venkataraman, C; Haack, B J; Gao, L Y; Kwaik, Y A

    1998-01-01

    Invasion and intracellular replication of Legionella pneumophila within protozoa in the environment plays a major role in the transmission of Legionnaires' disease. Intracellular replication of L. pneumophila within protozoa occurs in a rough endoplasmic reticulum (RER)-surrounded phagosome (Y. Abu Kwaik, Appl. Environ. Microbiol. 62:2022-2028, 1996). Since the subsequent fate of many intracellular pathogens is determined by the route of entry, we compared the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoa Hartmannella vermiformis and Acanthamoeba polyphaga. Our data provide biochemical and genetic evidence that the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoan hosts are, in part, different. First, uptake of L. pneumophila by H. vermiformis is completely blocked by the monovalent sugars galactose and N-acetyl-D-galactosamine, but these sugars partially blocked A. polyphaga. Second, attachment of L. pneumophila to H. vermiformis is associated with a time-dependent and reversible tyrosine dephosphorylation of multiple host proteins. In contrast, only a slight dephosphorylation of a 170-kDa protein of A. polyphaga is detected upon infection. Third, synthesis of H. vermiformis proteins but not of A. polyphaga proteins is required for uptake of L. pneumophila. Fourth, we have identified L. pneumophila mutants that are severely defective in attachment to A. polyphaga but which exhibit minor reductions in attachment to H. vermiformis and, thus, provide a genetic basis for the difference in mechanisms of attachment to both protozoa. The data indicate a remarkable adaptation of L. pneumophila to attach and invade different protozoan hosts by different mechanisms, yet invasion is followed by a remarkably similar intracellular replication within a RER-surrounded phagosome and subsequent killing of the host cell.

  17. Multiple major disease-associated clones of Legionella pneumophila have emerged recently and independently

    PubMed Central

    David, Sophia; Rusniok, Christophe; Mentasti, Massimo; Gomez-Valero, Laura; Harris, Simon R.; Lechat, Pierre; Lees, John; Ginevra, Christophe; Glaser, Philippe; Ma, Laurence; Bouchier, Christiane; Underwood, Anthony; Jarraud, Sophie; Harrison, Timothy G.; Parkhill, Julian; Buchrieser, Carmen

    2016-01-01

    Legionella pneumophila is an environmental bacterium and the leading cause of Legionnaires’ disease. Just five sequence types (ST), from more than 2000 currently described, cause nearly half of disease cases in northwest Europe. Here, we report the sequence and analyses of 364 L. pneumophila genomes, including 337 from the five disease-associated STs and 27 representative of the species diversity. Phylogenetic analyses revealed that the five STs have independent origins within a highly diverse species. The number of de novo mutations is extremely low with maximum pairwise single-nucleotide polymorphisms (SNPs) ranging from 19 (ST47) to 127 (ST1), which suggests emergences within the last century. Isolates sampled geographically far apart differ by only a few SNPs, demonstrating rapid dissemination. These five STs have been recombining recently, leading to a shared pool of allelic variants potentially contributing to their increased disease propensity. The oldest clone, ST1, has spread globally; between 1940 and 2000, four new clones have emerged in Europe, which show long-distance, rapid dispersal. That a large proportion of clinical cases is caused by recently emerged and internationally dispersed clones, linked by convergent evolution, is surprising for an environmental bacterium traditionally considered to be an opportunistic pathogen. To simultaneously explain recent emergence, rapid spread and increased disease association, we hypothesize that these STs have adapted to new man-made environmental niches, which may be linked by human infection and transmission. PMID:27662900

  18. Nosocomial legionnaires' disease: epidemiologic demonstration of cooling towers as a source. [Legionella pneumophila

    SciTech Connect

    Garbe, P.L.; Davis, B.J.; Weisfeld, J.S.; Markowitz, L.; Miner, P. Garrity, F.; Barbaree, J.M.; Reingold, A.L.

    1985-07-26

    Investigation of a recent outbreak of nosocomial legionnaires' disease - initially thought to be due to the documented presence of Legionella pneumophila in the hospital potable water - showed that aerosols from one or more cooling towers were the actual source of infection. From June 27 to Aug 25, 1983, nosocomial legionnaires' disease developed in 15 persons at a hospital in Rhode Island. Twelve (80%) of 15 case-patients occupied rooms in building 1, unit B, compared with eight (28%) of 29 control patients (odds ratio = 10.8; 95% confidence interval = 1.4 to 85.6). Subsequent investigation demonstrated that water in a cooling tower located 100 ft upwind of unit B was heavily contaminated with L. pneumophila, serogroup 1, subgroup 1, 2, 4, 5. The same strain was isolated from nine of the patients and from the make-up water for the tower. Active surveillance during the ten months following decontamination of the cooling tower identified no additional cases of nosocomial legionnaires' disease, although the hospital potable water had not been treated. While recommendations have been made for controlling nosocomial legionnaires' disease by heating or hyperchlorination of hospital potable water, this outbreak demonstrates the importance of an adequate epidemiologic-environmental investigation in choosing the appropriate control strategy.

  19. Effects of alpha-amylase on in vitro growth of Legionella pneumophila.

    PubMed Central

    Bortner, C A; Miller, R D; Arnold, R R

    1983-01-01

    Sterile parotid saliva inhibited growth of Legionella pneumophila on solid media, and the salivary component involved in this inhibition has been shown to be amylase. Disk diffusion and well plate assays were used to study possible mechanisms for this effect. The amylolytic activity of saliva copurified with inhibitory activity, and both activities were sensitive to proteinase K digestion and heat treatment. In addition, purified alpha-amylase from several sources (bacteria, fungi, porcine pancreas, and human saliva) exhibited similar activity. Incorporation of charcoal or bovine serum albumin into media blocked inhibition by amylase. Replacement of Bacto-Agar with Noble agar (both from Difco Laboratories) prevented growth inhibition in the absence of starch. However, when corn starch was present with Noble agar, amylase-induced growth inhibition occurred. Purification of starch by washing with methanol eliminated some toxic component. The toxic component from starch could be recovered from the methanol wash and inhibited growth of L. pneumophila in the absence of amylase activity. The results suggest that toxic substances exist in media components which may be unmasked during salivary amylase digestion of starch. This effect may explain, in part, the difficulty in recovery of the organism from clinical specimens containing amylase. PMID:6190756

  20. Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation

    PubMed Central

    Dalebroux, Zachary D.; Yagi, Brian F.; Sahr, Tobias; Buchrieser, Carmen; Swanson, Michele S.

    2010-01-01

    SUMMARY To transit between hosts, intracellular Legionella pneumophila transform into a motile, infectious, transmissive state. Here we exploit the pathogen’s life cycle to examine how guanosine tetraphosphate (ppGpp) and DksA cooperate to govern bacterial differentiation. Transcriptional profiling revealed that during transmission alarmone accumulation increases the mRNA for flagellar and Type IV-secretion components, secreted host effectors, and regulators, and decreases transcripts for translation, membrane modification and ATP synthesis machinery. DksA is critical for differentiation, since mutants are defective for stationary phase survival, flagellar gene activation, lysosome avoidance, and macrophage cytotoxicity. The roles of ppGpp and DksA depend on the context. For macrophage transmission, ppGpp is essential, whereas DksA is dispensable, indicating ppGpp can act autonomously. In broth, DksA promotes differentiation when ppGpp levels increase, or during fatty acid stress, as judged by flaA expression and evasion of degradation by macrophages. For flagella morphogenesis, DksA is required for basal fliA (σ28) promoter activity. When alarmone levels increase, DksA cooperates with ppGpp to generate a pulse of Class II rod RNA or to amplify the Class III sigma factor and Class IV flagellin RNAs. Thus, DksA responds to the level of ppGpp and other stress signals to coordinate L. pneumophila differentiation. PMID:20199605

  1. Multiple major disease-associated clones of Legionella pneumophila have emerged recently and independently.

    PubMed

    David, Sophia; Rusniok, Christophe; Mentasti, Massimo; Gomez-Valero, Laura; Harris, Simon R; Lechat, Pierre; Lees, John; Ginevra, Christophe; Glaser, Philippe; Ma, Laurence; Bouchier, Christiane; Underwood, Anthony; Jarraud, Sophie; Harrison, Timothy G; Parkhill, Julian; Buchrieser, Carmen

    2016-11-01

    Legionella pneumophila is an environmental bacterium and the leading cause of Legionnaires' disease. Just five sequence types (ST), from more than 2000 currently described, cause nearly half of disease cases in northwest Europe. Here, we report the sequence and analyses of 364 L. pneumophila genomes, including 337 from the five disease-associated STs and 27 representative of the species diversity. Phylogenetic analyses revealed that the five STs have independent origins within a highly diverse species. The number of de novo mutations is extremely low with maximum pairwise single-nucleotide polymorphisms (SNPs) ranging from 19 (ST47) to 127 (ST1), which suggests emergences within the last century. Isolates sampled geographically far apart differ by only a few SNPs, demonstrating rapid dissemination. These five STs have been recombining recently, leading to a shared pool of allelic variants potentially contributing to their increased disease propensity. The oldest clone, ST1, has spread globally; between 1940 and 2000, four new clones have emerged in Europe, which show long-distance, rapid dispersal. That a large proportion of clinical cases is caused by recently emerged and internationally dispersed clones, linked by convergent evolution, is surprising for an environmental bacterium traditionally considered to be an opportunistic pathogen. To simultaneously explain recent emergence, rapid spread and increased disease association, we hypothesize that these STs have adapted to new man-made environmental niches, which may be linked by human infection and transmission.

  2. Trans-translation is essential in the human pathogen Legionella pneumophila.

    PubMed

    Brunel, Romain; Charpentier, Xavier

    2016-11-28

    Trans-translation is a ubiquitous bacterial mechanism for ribosome rescue in the event of translation stalling. Although trans-translation is not essential in several bacterial species, it has been found essential for viability or virulence in a wide range of pathogens. We describe here that trans-translation is essential in the human pathogen Legionella pneumophila, the etiologic agent of Legionnaire's disease (LD), a severe form of nosocomial and community-acquired pneumonia. The ssrA gene coding for tmRNA, the key component of trans-translation, could not be deleted in L. pneumophila. To circumvent this and analyse the consequences of impaired trans-translation, we placed ssrA under the control of a chemical inducer. Phenotypes associated with the inhibition of ssrA expression include growth arrest in rich medium, hampered cell division, and hindered ability to infect eukaryotic cells (amoebae and human macrophages). LD is often associated with failure of antibiotic treatment and death (>10% of clinical cases). Decreasing tmRNA levels led to significantly higher sensitivity to ribosome-targeting antibiotics, including to erythromycin. We also detected a higher sensitivity to the transcription inhibitor rifampicin. Both antibiotics are recommended treatments for LD. Thus, interfering with trans-translation may not only halt the infection, but could also potentiate the recommended therapeutic treatments of LD.

  3. Trans-translation is essential in the human pathogen Legionella pneumophila

    PubMed Central

    Brunel, Romain; Charpentier, Xavier

    2016-01-01

    Trans-translation is a ubiquitous bacterial mechanism for ribosome rescue in the event of translation stalling. Although trans-translation is not essential in several bacterial species, it has been found essential for viability or virulence in a wide range of pathogens. We describe here that trans-translation is essential in the human pathogen Legionella pneumophila, the etiologic agent of Legionnaire’s disease (LD), a severe form of nosocomial and community-acquired pneumonia. The ssrA gene coding for tmRNA, the key component of trans-translation, could not be deleted in L. pneumophila. To circumvent this and analyse the consequences of impaired trans-translation, we placed ssrA under the control of a chemical inducer. Phenotypes associated with the inhibition of ssrA expression include growth arrest in rich medium, hampered cell division, and hindered ability to infect eukaryotic cells (amoebae and human macrophages). LD is often associated with failure of antibiotic treatment and death (>10% of clinical cases). Decreasing tmRNA levels led to significantly higher sensitivity to ribosome-targeting antibiotics, including to erythromycin. We also detected a higher sensitivity to the transcription inhibitor rifampicin. Both antibiotics are recommended treatments for LD. Thus, interfering with trans-translation may not only halt the infection, but could also potentiate the recommended therapeutic treatments of LD. PMID:27892503

  4. Legionella pneumophila Transcriptional Response following Exposure to CuO Nanoparticles

    PubMed Central

    Struewing, Ian; Buse, Helen Y.; Kou, Jiahui; Shuman, Howard A.; Faucher, Sébastien P.; Ashbolt, Nicholas J.

    2013-01-01

    Copper ions are an effective antimicrobial agent used to control Legionnaires' disease and Pontiac fever arising from institutional drinking water systems. Here, we present data on an alternative bactericidal agent, copper oxide nanoparticles (CuO-NPs), and its efficacy on Legionella pneumophila. In broth cultures, the CuO-NPs caused growth inhibition, which appeared to be concentration and exposure time dependent. The transcriptomic response of L. pneumophila to CuO-NP exposure was investigated by using a whole-genome microarray. The expression of genes involved in metabolism, transcription, translation, DNA replication and repair, and unknown/hypothetical proteins was significantly affected by exposure to CuO-NPs. In addition, expression of 21 virulence genes was also affected by exposure to CuO-NP and further evaluated by quantitative reverse transcription-PCR (qRT-PCR). Some virulence gene responses occurred immediately and transiently after addition of CuO-NPs to the cells and faded rapidly (icmV, icmW, lepA), while expression of other genes increased within 6 h (ceg29, legLC8, legP, lem19, lem24, lpg1689, and rtxA), 12 h (cegC1, dotA, enhC, htpX, icmE, pvcA, and sidF), and 24 h (legP, lem19, and ceg19), but for most of the genes tested, expression was reduced after 24 h of exposure. Genes like ceg29 and rtxA appeared to be the most responsive to CuO-NP exposures and along with other genes identified in this study may prove useful to monitor and manage the impact of drinking water disinfection on L. pneumophila. PMID:23416998

  5. Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

    PubMed Central

    Amyot, Whitney M.; deJesus, Dennise

    2013-01-01

    Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system. PMID:23798536

  6. Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

    SciTech Connect

    Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.; Morar, Mariya; Rao, Chitong; Di Leo, Rosa; Evdokimova, Elena; Lam, Mandy; Oatway, Christina; Cuff, Marianne E.; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw P.; Taipale, Mikko; Savchenko, Alexei; Ensminger, Alexander W.

    2016-12-16

    Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.

  7. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response.

    PubMed

    Hempstead, Andrew D; Isberg, Ralph R

    2015-12-08

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.

  8. The Legionella pneumophila Siderophore Legiobactin Is a Polycarboxylate That Is Identical in Structure to Rhizoferrin.

    PubMed

    Burnside, Denise M; Wu, Yuyang; Shafaie, Saman; Cianciotto, Nicholas P

    2015-10-01

    Legionella pneumophila, the agent of Legionnaires' disease, secretes a siderophore (legiobactin) that promotes bacterial infection of the lung. In past work, we determined that cytoplasmic LbtA (from Legiobactin gene A) promotes synthesis of legiobactin, inner membrane LbtB aids in export of the siderophore, and outer membrane LbtU and inner membrane LbtC help mediate ferrilegiobactin uptake and assimilation. However, the past studies examined legiobactin contained within bacterial culture supernatants. By utilizing high-pressure liquid chromatography that incorporates hydrophilic interaction-based chemistry, we have now purified legiobactin from supernatants of virulent strain 130b that is suitable for detailed chemical analysis. High-resolution mass spectrometry (MS) revealed that the molecular mass of (protonated) legiobactin is 437.140 Da. On the basis of the results obtained from both MS analysis and various forms of nuclear magnetic resonance, we found that legiobactin is composed of two citric acid residues linked by a putrescine bridge and thus is identical in structure to rhizoferrin, a polycarboxylate-type siderophore made by many fungi and several unrelated bacteria. Both purified legiobactin and rhizoferrin obtained from the fungus Cunninghamella elegans were able to promote Fe(3+) uptake by wild-type L. pneumophila as well as enhance growth of iron-starved bacteria. These results did not occur with 130b mutants lacking lbtU or lbtC, indicating that both endogenously made legiobactin and exogenously derived rhizoferrin are assimilated by L. pneumophila in an LbtU- and LbtC-dependent manner.

  9. Sequence types diversity of Legionella pneumophila isolates from environmental water sources in Guangzhou and Jiangmen, China.

    PubMed

    Guo, Jingyu; Liang, Ting; Hu, Chaohui; Lv, Ruichen; Yang, Xianwei; Cui, Yujun; Song, Youtao; Yang, Ruifu; Zhu, Qingyi; Song, Yajun

    2015-01-01

    In this study, 159 Legionella pneumophila strains isolated from various natural and artificial water sources in Guangzhou and Jiangmen, China, were subjected to genotyping by the sequence-based typing (SBT) scheme. These isolates were assigned into 53 sequence types (STs) (50 STs with seven loci data and three unidentified STs with incomplete loci profiles) with ST1 as the dominant one (14.5%), and the index of diversity (IOD) was 0.950. Eight new alleles and 34 new STs were reported here. Notably, most of the newly identified STs with seven loci data (24/34) contained no new allele, implying frequent recombination events in L. pneumophila. Five intragenic recombination events were identified in the concatenated sequences of seven loci. The diversity of STs in natural environmental isolates (41 STs, IOD=0.956) is higher than that of artificial environmental ones (17 STs, IOD=0.824). The ST patterns varied in isolates from these two sources: the most common STs from artificial water sources, ST1 and ST752 (39.2% and 13.7%), were only occasionally isolated from natural water sources (2.9% and 3.8%, respectively); while the predominant STs from natural water sources, ST1048, ST739 and ST1267 (15.2%, 6.7% and 6.7%), were less frequently seen in artificial environments (2.0%, 0% and 0%, respectively). We also found out that Legionnaires' disease associated STs might be more frequently isolated in artificial environments than in natural ones. Our data revealed remarkable genetic diversity of L. pneumophila isolates from environmental water systems of Guangzhou and Jiangmen, and the different ST distribution patterns between natural water and artificial water sources as well.

  10. Zinc Metalloproteinase ProA Directly Activates Legionella pneumophila PlaC Glycerophospholipid:cholesterol Acyltransferase*

    PubMed Central

    Lang, Christina; Rastew, Elena; Hermes, Björn; Siegbrecht, Enrico; Ahrends, Robert; Banerji, Sangeeta; Flieger, Antje

    2012-01-01

    Enzymes secreted by Legionella pneumophila, such as phospholipases A (PLAs) and glycerophospholipid:cholesterol acyltransferases (GCATs), may target host cell lipids and therefore contribute to the establishment of Legionnaires disease. L. pneumophila possesses three proteins, PlaA, PlaC, and PlaD, belonging to the GDSL family of lipases/acyltransferases. We have shown previously that PlaC is the major GCAT secreted by L. pneumophila and that the zinc metalloproteinase ProA is essential for GCAT activity. Here we characterized the mode of PlaC GCAT activation and determined that ProA directly processes PlaC. We further found that not only cholesterol but also ergosterol present in protozoa was palmitoylated by PlaC. Such ester formations were not induced by either PlaA or PlaD. PlaD was shown here to possess lysophospholipase A activity, and interestingly, all three GDSL enzymes transferred short chain fatty acids to sterols. The three single putative catalytic amino acids (Ser-37, Asp-398, and His-401) proved essential for all PlaC-associated PLA, lysophospholipase A, and GCAT activities. A further four cysteine residues are important for the PLA/GCAT activities as well as their oxidized state, and we therefore conclude that PlaC likely forms at least one disulfide loop. Analysis of cleavage site and loop deletion mutants suggested that for GCAT activation deletion of several amino acids within the loop is necessary rather than cleavage at a single site. Our data therefore suggest a novel enzyme inhibition/activation mechanism where a disulfide loop inhibits PlaC GCAT activity until the protein is exported to the external space where it is ProA-activated. PMID:22582391

  11. Factors Influencing Survival of Legionella pneumophila Serotype 1 in Hot Spring Water and Tap Water

    PubMed Central

    Ohno, Akira; Kato, Naoyuki; Yamada, Koji; Yamaguchi, Keizo

    2003-01-01

    The factors involved in the survival of Legionella pneumophila in the microcosms of both hot spring water and tap water were studied by examining cultivability and metabolic activity. L. pneumophila could survive by maintaining metabolic activity but was noncultivable in all microcosms at 42°C, except for one microcosm with a pH of <2.0. Lower temperatures supported survival without loss of cultivability. The cultivability declined with increasing temperature, although metabolic activity was observed at temperatures of up to 45°C. The optimal range of pH for survival was between 6.0 and 8. The metabolic activity could be maintained for long periods even in microcosms with high concentrations of salt. The cultivability of organisms in the post-exponential phase in a tap water microcosm with a low inoculum size was more rapidly reduced than that of organisms in the exponential phase. In contrast, the loss of cultivability in microcosms of a high inoculum size was significant in the exponential phase. Random(ly) amplified polymorphic DNA analysis of microcosms where cultivability was lost but metabolic activity was retained showed no change compared to cells grown freshly, although an effect on the amplified DNA band pattern by production of stress proteins was expected. Resuscitation by the addition of Acanthamoeba castellanii to the microcosm in which cultivability was completely lost but metabolic activity was maintained was observed only in part of the cell population. Our results suggest that L. pneumophila cell populations can potentially survive as free organisms for long periods by maintaining metabolic activity but temporarily losing cultivability under strict environments and requiring resuscitation by ingestion by amoebas. PMID:12732519

  12. Structural insights into a unique Legionella pneumophila effector LidA recognizing both GDP and GTP bound Rab1 in their active state.

    PubMed

    Cheng, Wei; Yin, Kun; Lu, Defen; Li, Bingqing; Zhu, Deyu; Chen, Yuzhen; Zhang, Hao; Xu, Sujuan; Chai, Jijie; Gu, Lichuan

    2012-01-01

    The intracellular pathogen Legionella pneumophila hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated Legionella-containing vacuole (LCV) required for bacterial replication. Maturation of the LCV involved acquisition of Rab1, which is mediated by the bacterial effector protein SidM/DrrA. SidM/DrrA is a bifunctional enzyme having the activity of both Rab1-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). LidA, another Rab1-interacting bacterial effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rab1 to the LCV as well. Here we report the crystal structures of LidA complexes with GDP- and GTP-bound Rab1 respectively. Structural comparison revealed that GDP-Rab1 bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rab1 and render it persistently active. As with GTP, LidA maintains GDP-Rab1 in the active conformation through interaction with its two conserved switch regions. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP- and GTP-Rab1 equally well with an affinity approximately 7.5 nM. We propose that the tight interaction with Rab1 allows LidA to facilitate SidM/DrrA-catalyzed release of Rab1 from GDIs. Taken together, our results support a unique mechanism by which a bacterial effector protein regulates Rab1 recycling.

  13. Structural Insights into a Unique Legionella pneumophila Effector LidA Recognizing Both GDP and GTP Bound Rab1 in Their Active State

    PubMed Central

    Lu, Defen; Li, Bingqing; Zhu, Deyu; Chen, Yuzhen; Zhang, Hao; Xu, Sujuan; Chai, Jijie; Gu, Lichuan

    2012-01-01

    The intracellular pathogen Legionella pneumophila hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated Legionella-containing vacuole (LCV) required for bacterial replication. Maturation of the LCV involved acquisition of Rab1, which is mediated by the bacterial effector protein SidM/DrrA. SidM/DrrA is a bifunctional enzyme having the activity of both Rab1-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). LidA, another Rab1-interacting bacterial effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rab1 to the LCV as well. Here we report the crystal structures of LidA complexes with GDP- and GTP-bound Rab1 respectively. Structural comparison revealed that GDP-Rab1 bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rab1 and render it persistently active. As with GTP, LidA maintains GDP-Rab1 in the active conformation through interaction with its two conserved switch regions. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP- and GTP-Rab1 equally well with an affinity approximately 7.5 nM. We propose that the tight interaction with Rab1 allows LidA to facilitate SidM/DrrA-catalyzed release of Rab1 from GDIs. Taken together, our results support a unique mechanism by which a bacterial effector protein regulates Rab1 recycling. PMID:22416225

  14. Inhibition of Legionella pneumophila multiplication within human macrophages by antimicrobial agents.

    PubMed Central

    Vildé, J L; Dournon, E; Rajagopalan, P

    1986-01-01

    The activity of serial concentrations of different antimicrobial agents on the multiplication of Legionella pneumophila within human monocyte-derived macrophages was studied. The results led to the definition of a minimal extracellular concentration inhibiting intracellular multiplication (MIEC). According to the MIECs, the antimicrobial agents tested were classified in three groups: very active (MIEC less than or equal to 0.06 microgram/ml), such as erythromycin, rifampin, and pefloxacin; active (1 microgram/ml greater than or equal to MIEC greater than or equal to 0.1 microgram/ml), such as sulfamethoxazole-trimethoprim or doxycycline; and ineffective, such as cefoxitin, which was not active within macrophages at as high as 64 micrograms/ml despite a low MIC (0.2 microgram/ml) on bacterial charcoal-yeast extract agar. The activity of netilmicin was difficult to assess because of its effect on extracellular legionellae. Combinations of erythromycin with rifampin and pefloxacin with erythromycin, rifampin, doxycycline, or netilmicin showed an additive effect and no antagonism. These results obtained in a cellular model are in agreement with the efficacy of antimicrobial agents in experimental infections and in Legionnaires disease. They sustain clinical interest in the new quinolones, such as pefloxacin, and in combinations of antimicrobial agents for the treatment of Legionnaires disease. PMID:3492176

  15. Crystal structure and tartrate inhibition of Legionella pneumophila histidine acid phosphatase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Reilly, Thomas J; Tanner, John J

    2015-11-01

    Histidine acid phosphatases (HAPs) utilize a nucleophilic histidine residue to catalyze the transfer of a phosphoryl group from phosphomonoesters to water. HAPs function as protein phosphatases and pain suppressors in mammals, are essential for Giardia lamblia excystation, and contribute to virulence of the category A pathogen Francisella tularensis. Herein we report the first crystal structure and steady-state kinetics measurements of the HAP from Legionella pneumophila (LpHAP), also known as Legionella major acid phosphatase. The structure of LpHAP complexed with the inhibitor l(+)-tartrate was determined at 2.0 Å resolution. Kinetics assays show that l(+)-tartrate is a 50-fold more potent inhibitor of LpHAP than of other HAPs. Electrostatic potential calculations provide insight into the basis for the enhanced tartrate potency: the tartrate pocket of LpHAP is more positive than other HAPs because of the absence of an ion pair partner for the second Arg of the conserved RHGXRXP HAP signature sequence. The structure also reveals that LpHAP has an atypically expansive active site entrance and lacks the nucleotide substrate base clamp found in other HAPs. These features imply that nucleoside monophosphates may not be preferred substrates. Kinetics measurements confirm that AMP is a relatively inefficient in vitro substrate of LpHAP.

  16. Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non-culturable cells.

    PubMed

    Al-Bana, Badii H; Haddad, Moreen T; Garduño, Rafael A

    2014-02-01

    Legionella pneumophila is an intracellular bacterial parasite of freshwater protozoa and an accidental waterborne human pathogen. L. pneumophila is highly pleomorphic showing several forms that differentiate within its developmental cycle. In water, L. pneumophila produces viable but non-culturable cells (VBNCCs), which remain largely uncharacterized. We produced VBNCCs from two developmental forms of L. pneumophila [stationary phase forms (SPFs) and mature infectious forms (MIFs)] in two water microcosms [double-deionized (dd) and tap water] at 45°C. In contrast with SPFs, MIFs upheld a robust ultrastructure and high viability in the two water microcosms. In dd-water, MIFs and SPFs lost their culturability faster than in tap water and did not consume their poly-β-hydroxybutyrate inclusions. Resuscitation in Acanthamoeba castellani was only possible for VBNCCs produced from SPFs in tap water. Addition of salts to dd-water prolonged L. pneumophila culturability to tap water levels, suggesting that L. pneumophila requires ions to maintain its readiness to resume growth. VBNCCs resisted detergent lysis and digestion in the ciliate Tetrahymena, except for VBNCCs produced from SPFs in dd-water. L. pneumophila VBNCCs thus show distinct traits according to its originating developmental form and the surrounding water microcosm.

  17. IroT/mavN, a new iron-regulated gene involved in Legionella pneumophila virulence against amoebae and macrophages.

    PubMed

    Portier, Emilie; Zheng, Huaixin; Sahr, Tobias; Burnside, Denise M; Mallama, Celeste; Buchrieser, Carmen; Cianciotto, Nicholas P; Héchard, Yann

    2015-04-01

    Legionella pneumophila is a pathogenic bacterium commonly found in water. Eventually, it could be transmitted to humans via inhalation of contaminated aerosols. Iron is known as a key requirement for the growth of L. pneumophila in the environment and within its hosts. Many studies were performed to understand iron utilization by L. pneumophila but no global approaches were conducted. In this study, transcriptomic analyses were performed, comparing gene expression in L. pneumophila in standard versus iron restricted conditions. Among the regulated genes, a newly described one, lpp_2867, was highly induced in iron-restricted conditions. Mutants lacking this gene in L. pneumophila were not affected in siderophore synthesis or utilization. On the contrary, they were defective for growth on iron-depleted solid media and for ferrous iron uptake. A sequence analysis predicts that Lpp_2867 is a membrane protein, suggesting that it is involved in ferrous iron transport. We thus named it IroT, for iron transporter. Infection assays showed that the mutants are highly impaired in intracellular growth within their environmental host Acanthamoeba castellanii and human macrophages. Taken together, our results show that IroT is involved, directly or indirectly, in ferrous iron transport and is a key virulence factor for L. pneumophila.

  18. IroT/mavN, a new iron-regulated gene involved in Legionella pneumophila virulence against amoebae and macrophages

    PubMed Central

    PORTIER, Emilie; ZHENG, Huaixin; SAHR, Tobias; BURNSIDE, Denise M.; MALLAMA, Celeste; BUCHRIESER, Carmen; CIANCIOTTO, Nicholas P.; HÉCHARD, Yann

    2015-01-01

    Summary Legionella pneumophila is a pathogenic bacterium commonly found in water. Eventually, it could be transmitted to humans via inhalation of contaminated aerosols. Iron is known as a key requirement for the growth of L. pneumophila in the environment and within its hosts. Many studies were performed to understand iron utilization by L. pneumophila but no global approaches were conducted. In this study, transcriptomic analyses were performed, comparing gene expression in L. pneumophila in standard vs. iron restricted conditions. Among the regulated genes, a newly described one, lpp_2867, was highly induced in iron restricted conditions. Mutants lacking this gene in L. pneumophila were not affected in siderophore synthesis or utilization. On the contrary, they were defective for growth on iron depleted solid media and for ferrous iron uptake. A sequence analysis predicts that Lpp_2867 is a membrane protein, suggesting that it is involved in ferrous iron transport. We thus named it IroT, for iron transporter. Infection assays showed that the mutants are highly impaired in intracellular growth within their environmental host Acanthamoeba castellanii and human macrophages. Taken together, our results show that IroT is involved, directly or indirectly, in ferrous iron transport and is a key virulence factor for L. pneumophila. PMID:25141909

  19. Distribution of Legionella species from environmental water sources of public facilities and genetic diversity of L. pneumophila serogroup 1 in South Korea.

    PubMed

    Lee, Hae Kyung; Shim, Jung Im; Kim, Hye Eun; Yu, Jae Yon; Kang, Yeon Ho

    2010-10-01

    A total of 560 Legionella species were isolated from environmental water sources from public facilities from June to September 2008 throughout South Korea. The distribution of Legionella isolates was investigated according to geographical region, facility type, and sample type. The genetic diversity of 104 isolates of Legionella pneumophila serogroup 1 (sg 1) was analyzed by sequence-based typing (SBT). L. pneumophila was distributed broadly throughout Korea, accounting for 85.0% of the isolates, and L. pneumophila sg 1 predominated in all of the public facilities except for the springs. Legionella anisa and Legionella bozemanii predominated among non-L. pneumophila species (48.1% and 21.0%, respectively). The second most dominant strain differed depending on the facility type: L. anisa was the second most dominant strain in the buildings (10.8%), L. pneumophila sg 5 in public baths (21.6%), L. pneumophila sg 6 in factories (12.0%), and L. pneumophila sg 7 in hospitals (13.1%). In the SBT analysis, 104 L. pneumophila sg 1 isolates were differentiated into 26 sequence types (STs) and categorized into 3 clonal groups (CGs) and 10 singleton STs via the eBURST V3 program. ST1, a potential founder of major CG1, was commonly distributed (48.1%). The dominant ST in hot water was ST-K1 (7, 12, 17, 3, 35, 11, 11), which was designated in this study (36.1%). The second most dominant strain differed depending on the type of facility from which the samples were obtained. The unique allelic profile of ST-K1, obtained from hot water, was not found in the European Working Group for Legionella Infections (EWGLI) SBT database.

  20. Improved facility and sensitivity in the use of guinea pigs for the isolation of Legionella pneumophila from cooling tower water

    SciTech Connect

    Leinbach, E.D.; Winkler, H.H.; Wood, D.O.; Coggin, J.H. Jr.

    1983-03-01

    The established criteria for the determination of the optimum time for the sacrifice of guinea pigs inoculated with samples of cooling tower water were found to be inadequate for the detection of low levels of Legionella pneumophila. By ignoring the requirement for fever and by sequentially sacrificing the infected guinea pigs on days 3 through 5 postinoculation, we simplified the procedure, and the sensitivity of detection was improved a great deal.

  1. Common epitope on urinary antigen derived from different Legionella pneumophila serogroup 1 strains recognized by a monoclonal antibody.

    PubMed

    Helbig, J H; Lück, P C; Pilz, C; Witzleb, W

    1990-09-01

    Guinea pigs were infected intraperitoneally with 4 subgroup reference strains (Knoxville 1, Philadelphia 1, Bellingham 1, OLDA) and 2 clinical isolates of Legionella pneumophila serogroup 1. Antigenuria was demonstrated by the enzyme-linked immunosorbent assay using polyclonal antibodies and monoclonal antibody (mab) F8/5. Mab F8/5 recognizes a hitherto undetected common epitope on urinary antigen of the investigated strains.

  2. Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR.

    PubMed

    Yáñez, M Adela; Nocker, Andreas; Soria-Soria, Elena; Múrtula, Raquel; Martínez, Lorena; Catalán, Vicente

    2011-05-01

    One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.

  3. Molecular typing of Legionella pneumophila serogroup 1 clinical strains isolated in Italy.

    PubMed

    Fontana, Stefano; Scaturro, Maria; Rota, Maria Cristina; Caporali, Maria Grazia; Ricci, Maria Luisa

    2014-07-01

    Molecular typing methods for discriminating different bacterial isolates are essential epidemiological tools in prevention and control of Legionella infections and outbreaks. A selection of 56 out of 184 Legionella pneumophila serogroup 1 (Lp1) clinical isolates, collected from different Italian regions between 1987 and 2012, and stored at the National Reference Laboratory for Legionella, were typed by monoclonal antibody (MAb) subgrouping, amplified fragment length polymorphism (AFLP) and sequence based typing (SBT). These strains were isolated from 39 community (69.6%), 14 nosocomial (25%) and 3 travel associated (5.4%) Legionnaires'disease cases. MAb typing results showed a prevalence of MAb 3/1 positive isolates (75%) with the Philadelphia subgroup representing 35.7%, followed by Knoxville (23.2%), Benidorm (12.5%), Allentown/France (1.8%), Allentown/France-Philadelphia (1.8%). The remaining 25% were MAb 3/1 negative, namely 11 Olda (19.6%), 2 Oxford (3.6%) and 1 Bellingham (1.8%) subgroups. AFLP analysis detected 20 different genomic profiles. SBT analysis revealed 32 different sequence types (STs) with high diversity of STs (IODSTs=0.952) 12 of which were never described before. ST1 and ST23 were most frequently isolated as observed worldwide. A helpful analysis of data from SBT, MAb subgrouping and AFLP is provided, as well as a comparison to the Lp1 types investigated from other countries. This study describes the first Italian Lp1 strains database, providing molecular epidemiology data useful for future epidemiological investigations, especially of travel associated Legionnaires' diseases (TALD) cases, Italy being the country associated with the highest number of clusters.

  4. The Membrane Protein LasM Promotes the Culturability of Legionella pneumophila in Water

    PubMed Central

    Li, Laam; Faucher, Sébastien P.

    2016-01-01

    The water-borne pathogen Legionella pneumophila (Lp) strongly expresses the lpg1659 gene in water. This gene encodes a hypothetical protein predicted to be a membrane protein using in silico analysis. While no conserved domains were identified in Lpg1659, similar proteins are found in many Legionella species and other aquatic bacteria. RT-qPCR showed that lpg1659 is positively regulated by the alternative sigma factor RpoS, which is essential for Lp to survive in water. These observations suggest an important role of this novel protein in the survival of Lp in water. Deletion of lpg1659 did not affect cell morphology, membrane integrity or tolerance to high temperature. Moreover, lpg1659 was dispensable for growth of Lp in rich medium, and during infection of the amoeba Acanthamoeba castellanii and of THP-1 human macrophages. However, deletion of lpg1659 resulted in an early loss of culturability in water, while over-expression of this gene promoted the culturability of Lp. Therefore, these results suggest that lpg1659 is required for Lp to maintain culturability, and possibly long-term survival, in water. Since the loss of culturability observed in the absence of Lpg1659 was complemented by the addition of trace metals into water, this membrane protein is likely a transporter for acquiring essential trace metal for maintaining culturability in water and potentially in other metal-deprived conditions. Given its role in the survival of Lp in water, Lpg1659 was named LasM for Legionella aquatic survival membrane protein. PMID:27734007

  5. Intragenic Recombination Has a Critical Role on the Evolution of Legionella pneumophila Virulence-Related Effector sidJ

    PubMed Central

    Costa, Joana; Teixeira, Paulo Gonçalves; d'Avó, Ana Filipa; Júnior, Célio Santos; Veríssimo, António

    2014-01-01

    SidJ is a Dot/Icm effector involved in the trafficking or retention of ER-derived vesicles to Legionella pneumophila vacuoles whose mutation causes an observable growth defect, both in macrophage and amoeba hosts. Given the crucial role of this effector in L. pneumophila virulence we investigated the mechanisms shaping its molecular evolution. The alignment of SidJ sequences revealed several alleles with amino acid variations that may influence the protein properties. The identification of HGT events and the detection of balancing selection operating on sidJ evolution emerge as a clear result. Evidence suggests that intragenic recombination is an important strategy in the evolutionary adaptive process playing an active role on sidJ genetic plasticity. This pattern of evolution is in accordance with the life style of L. pneumophila as a broad host-range pathogen by preventing host-specialization and contributing to the resilience of the species. PMID:25299187

  6. Proteomic regulation during Legionella pneumophila biofilm development: decrease of virulence factors and enhancement of response to oxidative stress.

    PubMed

    Khemiri, Arbia; Lecheheb, Sandra Ahmed; Chi Song, Philippe Chan; Jouenne, Thierry; Cosette, Pascal

    2014-06-01

    Legionella pneumophila (L. pneumophila) is a Gram-negative bacterium, which can be found worldwide in aquatic environments. It tends to persist because it is often protected within biofilms or amoebae. L. pneumophila biofilms have a major impact on water systems, making the understanding of the bacterial physiological adaptation in biofilms a fundamental step towards their eradication. In this study, we report for the first time the influence of the biofilm mode of growth on the proteome of L. pneumophila. We compared the protein patterns of microorganisms grown as suspensions, cultured as colonies on agar plates or recovered with biofilms formed on stainless steel coupons. Statistical analyses of the protein expression data set confirmed the biofilm phenotype specificity which had been previously observed. It also identified dozens of proteins whose abundance was modified in biofilms. Proteins corresponding to virulence factors (macrophage infectivity potentiator protein, secreted proteases) were largely repressed in adherent cells. In contrast, a peptidoglycan-associated lipoprotein (Lpg2043) and a peroxynitrite reductase (Lpg2965) were accumulated by biofilm cells. Remarkably, hypothetical proteins, that appear to be unique to the Legionella genus (Lpg0563, Lpg1111 and Lpg1809), were over-expressed by sessile bacteria.

  7. Temperature diagnostic to identify high risk areas and optimize Legionella pneumophila surveillance in hot water distribution systems.

    PubMed

    Bédard, Emilie; Fey, Stéphanie; Charron, Dominique; Lalancette, Cindy; Cantin, Philippe; Dolcé, Patrick; Laferrière, Céline; Déziel, Eric; Prévost, Michèle

    2015-03-15

    Legionella pneumophila is frequently detected in hot water distribution systems and thermal control is a common measure implemented by health care facilities. A risk assessment based on water temperature profiling and temperature distribution within the network is proposed, to guide effective monitoring strategies and allow the identification of high risk areas. Temperature and heat loss at control points (water heater, recirculation, representative points-of-use) were monitored in various sections of five health care facilities hot water distribution systems and results used to develop a temperature-based risk assessment tool. Detailed investigations show that defective return valves in faucets can cause widespread temperature losses because of hot and cold water mixing. Systems in which water temperature coming out of the water heaters was kept consistently above 60 °C and maintained above 55 °C across the network were negative for Legionella by culture or qPCR. For systems not meeting these temperature criteria, risk areas for L. pneumophila were identified using temperature profiling and system's characterization; higher risk was confirmed by more frequent microbiological detection by culture and qPCR. Results confirmed that maintaining sufficiently high temperatures within hot water distribution systems suppressed L. pneumophila culturability. However, the risk remains as shown by the persistence of L. pneumophila by qPCR. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Legionella pneumophila serogroup 3 pneumonia in a patient with low-grade 4 non-Hodgkin lymphoma: a case report

    PubMed Central

    2011-01-01

    Introduction Nosocomial legionellosis has generally been described in immunodepressed patients, but Legionella pneumophila serogroup 3 has rarely been identified as the causative agent. Case presentation We report the case of nosocomial L. pneumophila serogroup 3 pneumonia in a 70-year-old Caucasian man with non-Hodgkin lymphoma. Diagnosis was carried out by culture and real-time polymerase chain reaction of bronchoalveolar lavage fluid. The results of a urinary antigen test were negative. A hospital environmental investigation revealed that the hospital water system was highly colonized by L. pneumophila serogroups 3, 4, and 8. The hospital team involved in the prevention of infections was informed, long-term control measures to reduce the environmental bacterial load were adopted, and clinical monitoring of legionellosis occurrence in high-risk patients was performed. No further cases of Legionella pneumonia have been observed so far. Conclusions In this report, we describe a case of legionellosis caused by L. pneumophila serogroup 3, which is not usually a causative agent of nosocomial infection. Our research confirms the importance of carrying out cultures of respiratory secretions to diagnose legionellosis and highlights the limited value of the urinary antigen test for hospital infections, especially in immunocompromised patients. It also indicates that, to reduce the bacterial load and prevent nosocomial legionellosis, appropriate control measures should be implemented with systematic monitoring of hospital water systems. PMID:21849075

  9. A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila--characterization, molecular cloning and overexpression.

    PubMed

    Schmidt, B; Tradler, T; Rahfeld, J U; Ludwig, B; Jain, B; Mann, K; Rücknagel, K P; Janowski, B; Schierhorn, A; Küllertz, G; Hacker, J; Fischer, G

    1996-09-01

    Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.

  10. Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria.

    PubMed

    Shadrach, Winlet Sheba; Rydzewski, Kerstin; Laube, Ulrike; Holland, Gudrun; Ozel, Muhsin; Kiderlen, Albrecht F; Flieger, Antje

    2005-05-01

    Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.

  11. Identification of Two Legionella pneumophila Effectors that Manipulate Host Phospholipids Biosynthesis

    PubMed Central

    Viner, Ram; Chetrit, David; Ehrlich, Marcelo; Segal, Gil

    2012-01-01

    The intracellular pathogen Legionella pneumophila translocates a large number of effector proteins into host cells via the Icm/Dot type-IVB secretion system. Some of these effectors were shown to cause lethal effect on yeast growth. Here we characterized one such effector (LecE) and identified yeast suppressors that reduced its lethal effect. The LecE lethal effect was found to be suppressed by the over expression of the yeast protein Dgk1 a diacylglycerol (DAG) kinase enzyme and by a deletion of the gene encoding for Pah1 a phosphatidic acid (PA) phosphatase that counteracts the activity of Dgk1. Genetic analysis using yeast deletion mutants, strains expressing relevant yeast genes and point mutations constructed in the Dgk1 and Pah1 conserved domains indicated that LecE functions similarly to the Nem1-Spo7 phosphatase complex that activates Pah1 in yeast. In addition, by using relevant yeast genetic backgrounds we examined several L. pneumophila effectors expected to be involved in phospholipids biosynthesis and identified an effector (LpdA) that contains a phospholipase-D (PLD) domain which caused lethal effect only in a dgk1 deletion mutant of yeast. Additionally, LpdA was found to enhance the lethal effect of LecE in yeast cells, a phenomenon which was found to be dependent on its PLD activity. Furthermore, to determine whether LecE and LpdA affect the levels or distribution of DAG and PA in-vivo in mammalian cells, we utilized fluorescent DAG and PA biosensors and validated the notion that LecE and LpdA affect the in-vivo levels and distribution of DAG and PA, respectively. Finally, we examined the intracellular localization of both LecE and LpdA in human macrophages during L. pneumophila infection and found that both effectors are localized to the bacterial phagosome. Our results suggest that L. pneumophila utilize at least two effectors to manipulate important steps in phospholipids biosynthesis. PMID:23133385

  12. Inactivation of Legionella pneumophila and Pseudomonas aeruginosa: evaluation of the bactericidal ability of silver cations.

    PubMed

    Hwang, Myoung Goo; Katayama, Hiroyuki; Ohgaki, Shinichiro

    2007-10-01

    In this study, silver cations dissolved as silver nitrate at various concentrations were exposed to Legionella pneumophila, Pseudomonas aeruginosa, and Escherichia coli to quantitatively estimate the bactericidal ability of silver. Observed data were analyzed using a newly developed model (Cs x T) that introduced a specific amount of chemisorbed silver onto a bacterial cell (Cs), which represented the chemisorption properties of silver on the bacterial cell body. Silver cations were rapidly chemisorbed onto bacterial cells after injection into samples, and Cs values (initial concentration of silver was 0.1 mg Ag/l) were calculated as 1.810 x 10(-6) (L. pneumophila), 1.102 x 10(-6) (P. aeruginosa), and 1.638 x 10(-6) microg Ag/cell(i) (E. coli) after incubation for 8 h. During that time, the three tested bacteria were completely inactivated under the detection limit (>7.2 log reduction). Based on the calculated Cs values, bacterial tolerance against silver was estimated by using the equation (Cs x T) multiplying the Cs values with exposure time (T). The Cs x T values well represented the bactericidal abilities of silver against the tested bacteria. The demanded Cs x T values to accomplish a 1 log inactivation (90% reduction) of L. pneumophila, P. aeruginosa, and E. coli (the initial numbers of bacteria were 1.5 x 10(7) CFU/ml, approximately) were estimated as 2.44 x 10(-6), 0.63 x 10(-6), and 0.46 x 10(-6) microgh/cell(i) of silver. The values were significantly reduced to 1.54 x 10(-6), 0.31 x 10(-6), and 0.25 x 10(-6) microgh/cell(i), respectively, with simultaneous injection of silver and copper. This study shows the successful quantitative estimation of the bactericidal ability of silver by applying the newly developed model (Cs x T). Among the tested bacteria, L. pneumophila showed the strongest tolerance to exposure of the same concentration of silver.

  13. Legionella pneumophila: The Paradox of a Highly Sensitive Opportunistic Waterborne Pathogen Able to Persist in the Environment

    PubMed Central

    Berjeaud, Jean-Marc; Chevalier, Sylvie; Schlusselhuber, Margot; Portier, Emilie; Loiseau, Clémence; Aucher, Willy; Lesouhaitier, Olivier; Verdon, Julien

    2016-01-01

    Legionella pneumophila, the major causative agent of Legionnaires’ disease, is found in freshwater environments in close association with free-living amoebae and multispecies biofilms, leading to persistence, spread, biocide resistance, and elevated virulence of the bacterium. Indeed, legionellosis outbreaks are mainly due to the ability of this bacterium to colonize and persist in water facilities, despite harsh physical and chemical treatments. However, these treatments are not totally efficient and, after a lag period, L. pneumophila may be able to quickly re-colonize these systems. Several natural compounds (biosurfactants, antimicrobial peptides…) with anti-Legionella properties have recently been described in the literature, highlighting their specific activities against this pathogen. In this review, we first consider this hallmark of Legionella to resist killing, in regard to its biofilm or host-associated life style. Then, we focus more accurately on natural anti-Legionella molecules described so far, which could provide new eco-friendly and alternative ways to struggle against this important pathogen in plumbing. PMID:27092135

  14. Legionella pneumophila: The Paradox of a Highly Sensitive Opportunistic Waterborne Pathogen Able to Persist in the Environment.

    PubMed

    Berjeaud, Jean-Marc; Chevalier, Sylvie; Schlusselhuber, Margot; Portier, Emilie; Loiseau, Clémence; Aucher, Willy; Lesouhaitier, Olivier; Verdon, Julien

    2016-01-01

    Legionella pneumophila, the major causative agent of Legionnaires' disease, is found in freshwater environments in close association with free-living amoebae and multispecies biofilms, leading to persistence, spread, biocide resistance, and elevated virulence of the bacterium. Indeed, legionellosis outbreaks are mainly due to the ability of this bacterium to colonize and persist in water facilities, despite harsh physical and chemical treatments. However, these treatments are not totally efficient and, after a lag period, L. pneumophila may be able to quickly re-colonize these systems. Several natural compounds (biosurfactants, antimicrobial peptides…) with anti-Legionella properties have recently been described in the literature, highlighting their specific activities against this pathogen. In this review, we first consider this hallmark of Legionella to resist killing, in regard to its biofilm or host-associated life style. Then, we focus more accurately on natural anti-Legionella molecules described so far, which could provide new eco-friendly and alternative ways to struggle against this important pathogen in plumbing.

  15. Ultra-structure and localisation of formazan formed by human neutrophils and amoebae phagocytosing virulent and avirulent Legionella pneumophila.

    PubMed

    Halablab, M A; Bazin, M; Richards, L; Pacy, J

    1990-12-01

    Legionella pneumophila (LP) strains of differing virulence were incubated with a solution of nitroblue-tetrazolium (NBT) at a concentration of 1 mg.ml-1 in the presence of Acanthamoeba polyphaga or human polymorphonuclear neutrophils (PMN). Reduction of NBT to formazan occurred at a faster rate in the presence of virulent strains. Reduction appeared to be temperature dependent; at 37 degrees C the reaction rate was higher than at 20 degrees C. On microscopic examination, deposits of formazan around Legionella cells were observed inside amoebae similar to those deposited in human neutrophils. Electron microscopy revealed electron-dense particles surrounding virulent legionellae, which appeared to be associated with formazan formation. Formazan formation inside amoebae may suggest the presence of a respiratory burst against LP, which is more intense with virulent strains.

  16. Lpg0393 of Legionella pneumophila is a guanine-nucleotide exchange factor for Rab5, Rab21 and Rab22.

    PubMed

    Sohn, Young-Sik; Shin, Ho-Chul; Park, Wei Sun; Ge, Jianning; Kim, Chan-Hee; Lee, Bok Luel; Heo, Won Do; Jung, Jae U; Rigden, Daniel John; Oh, Byung-Ha

    2015-01-01

    Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella.

  17. Interactive effects of temperature, organic carbon, and pipe material on microbiota composition and Legionella pneumophila in hot water plumbing systems.

    PubMed

    Proctor, Caitlin R; Dai, Dongjuan; Edwards, Marc A; Pruden, Amy

    2017-10-04

    Several biotic and abiotic factors have been reported to influence the proliferation of microbes, including Legionella pneumophila, in hot water premise plumbing systems, but their combined effects have not been systematically evaluated. Here, we utilize simulated household water heaters to examine the effects of stepwise increases in temperature (32-53 °C), pipe material (copper vs. cross-linked polyethylene (PEX)), and influent assimilable organic carbon (0-700 μg/L) on opportunistic pathogen gene copy numbers and the microbiota composition, as determined by quantitative polymerase chain reaction and 16S rRNA gene amplicon sequencing. Temperature had an overarching influence on both the microbiota composition and L. pneumophila numbers. L. pneumophila peaked at 41 °C in the presence of PEX (1.58 × 10(5) gene copies/mL). At 53 °C, L. pneumophila was not detected. Several operational taxonomic units (OTUs) persisted across all conditions, accounting for 50% of the microbiota composition from 32 to 49 °C and 20% at 53 °C. Pipe material most strongly influenced microbiota composition at lower temperatures, driven by five to six OTUs enriched with each material. Copper pipes supported less L. pneumophila than PEX pipes (mean 2.5 log10 lower) at temperatures ≤ 41 °C, but showed no difference in total bacterial numbers. Differences between pipe materials diminished with elevated temperature, probably resulting from decreased release of copper ions. At temperatures ≤ 45 °C, influent assimilable organic carbon correlated well with total bacterial numbers, but not with L. pneumophila numbers. At 53 °C, PEX pipes leached organic carbon, reducing the importance of dosed organic carbon. L. pneumophila numbers correlated with a Legionella OTU and a Methylophilus OTU identified by amplicon sequencing. Temperature was the most effective factor for the control of L. pneumophila, while microbiota composition shifted with each stepwise temperature

  18. Water heater temperature set point and water use patterns influence Legionella pneumophila and associated microorganisms at the tap.

    PubMed

    Rhoads, William J; Ji, Pan; Pruden, Amy; Edwards, Marc A

    2015-12-01

    Lowering water heater temperature set points and using less drinking water are common approaches to conserving water and energy; yet, there are discrepancies in past literature regarding the effects of water heater temperature and water use patterns on the occurrence of opportunistic pathogens, in particular Legionella pneumophila. Our objective was to conduct a controlled, replicated pilot-scale investigation to address this knowledge gap using continuously recirculating water heaters to examine five water heater set points (39-58 °C) under three water use conditions. We hypothesized that L. pneumophila levels at the tap depend on the collective influence of water heater temperature, flow frequency, and the resident plumbing ecology. We confirmed temperature setting to be a critical factor in suppressing L. pneumophila growth both in continuously recirculating hot water lines and at distal taps. For example, at 51 °C, planktonic L. pneumophila in recirculating lines was reduced by a factor of 28.7 compared to 39 °C and was prevented from re-colonizing biofilm. However, L. pneumophila still persisted up to 58 °C, with evidence that it was growing under the conditions of this study. Further, exposure to 51 °C water in a low-use tap appeared to optimally select for L. pneumophila (e.g., 125 times greater numbers than in high-use taps). We subsequently explored relationships among L. pneumophila and other ecologically relevant microbes, noting that elevated temperature did not have a general disinfecting effect in terms of total bacterial numbers. We documented the relationship between L. pneumophila and Legionella spp., and noted several instances of correlations with Vermamoeba vermiformis, and generally found that there is a dynamic relationship with this amoeba host over the range of temperatures and water use frequencies examined. Our study provides a new window of understanding into the microbial ecology of potable hot water systems and helps to resolve

  19. Legionella pneumophila Secretes an Endoglucanase That Belongs to the Family-5 of Glycosyl Hydrolases and Is Dependent upon Type II Secretion

    PubMed Central

    Pearce, Meghan M.; Cianciotto, Nicholas P.

    2009-01-01

    Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. L. pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium’s type II protein secretion system. Inactivation of celA, encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli. Thus, CelA is the major secreted endoglucanase of L. pneumophila. Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila. The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophila and the seventeenth type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the type II secretion output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella. PMID:19817866

  20. The occurrence of Legionella species other than Legionella pneumophila in clinical and environmental samples in Denmark identified by mip gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

    PubMed

    Svarrer, C W; Uldum, S A

    2012-10-01

    In Denmark, several laboratories use PCR as a routine diagnostic method for Legionnaires' disease, and almost all PCR-positive samples are investigated by culture. From 1993 to 2010, isolates of Legionella species other than Legionella pneumophila were obtained from respiratory samples from 33 patients, and from 1997 to 2010, 42 isolates of Legionella non-pneumophila species were obtained and saved from water samples from 39 different sites in Denmark. Macrophage infectivity potentiator gene (mip) sequencing was used as a reference method to identify the Legionella non-pneumophila species. Only one of the 75 isolates did not meet the acceptance criterion of a similarity of ≥98% to sequences in the database. The species distribution between clinical and environmental isolates varied. For the former, four species were detected, with Legionella bozemanae and Legionella micdadei predominating (both 44%). For the latter, eight species were detected, with Legionella anisa predominating (52%). The distribution among the Danish clinical isolates was different from the general distribution both in Europe and outside Europe, where L. bozemanae and Legionella longbeachae are the most commonly found clinical Legionella non-pneumophila species. The 75 isolates were also investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): 64 were correctly identified, with a score of ≥2.0; eight had a score of <2.0, but only two of these were wrongly identified; and three gave no results with MALDI-TOF MS. Both mip sequencing and MALDI-TOF MS are robust methods for Legionella species identification.

  1. Identification and characterization of a new conjugation/type IVA secretion system (trb/tra) of Legionella pneumophila Corby localized on two mobile genomic islands.

    PubMed

    Glöckner, Gernot; Albert-Weissenberger, Christiane; Weinmann, Erik; Jacobi, Sebastian; Schunder, Eva; Steinert, Michael; Hacker, Jörg; Heuner, Klaus

    2008-07-01

    Horizontal gene transfer probably contributes to evolution of Legionella pneumophila and its adaptation to different environments. Although horizontal gene transfer was observed in Legionella, the mechanism is still not specified. In this study we identified and analysed a new type of conjugation/type IVA secretion system (trb/tra) of L. pneumophila Corby, a virulent human isolate. Two similar versions of this conjugation system were identified, localized on two different genomic islands (Trb-1, 42,710 bp and Trb-2, 34,434 bp). Trb-1 and Trb-2 are integrated within the tRNA(Pro) gene (lpc2778) and the tmRNA gene (lpc0164), respectively. Both islands exhibit an oriT region and both can be excised from the chromosome forming episomal circles. Trb-1 was analysed in more detail. It is active and can be horizontally transferred to other Legionella strains by conjugation and then integrated into the genome in a site-specific manner within the tRNA(Pro) gene. We characterized the sequence of the excision and integration sites of Trb-1 in three different L. pneumophila strains. Here we demonstrate that L. pneumophila exhibits a functional oriT region and that genomic islands in Legionella can be mobilized and conjugated to other species of Legionella. Thus, we describe for the first time a mechanism that may explain the observed horizontal transfer of chromosomal DNA in Legionella.

  2. Legionella pneumophila Catalase-Peroxidases: Cloning of the katB Gene and Studies of KatB Function

    PubMed Central

    Bandyopadhyay, Purnima; Steinman, Howard M.

    1998-01-01

    Legionella pneumophila, the causative organism of Legionnaires’ pneumonia, is spread by aerosolization from man-made reservoirs, e.g., water cooling towers and air conditioning ducts, whose nutrient-poor conditions are conducive to entrance into stationary phase. Exposure to starvation conditions is known to induce several virulence traits in L. pneumophila. Since catalase-peroxidases have been extremely useful markers of the stationary-phase response in many bacterial species and may be an avenue for identifying virulence genes in L. pneumophila, an investigation of these enzymes was initiated. L. pneumophila was shown to contain two bifunctional catalase-peroxidases and to lack monofunctional catalase and peroxidase. The gene encoding the KatB catalase-peroxidase was cloned and sequenced, and lacZ fusion and null mutant strains were constructed. Null mutants in katB are delayed in the infection and lysis of cultured macrophage-like cell lines. KatB is similar to the KatG catalase-peroxidase of Escherichia coli in its 20-fold induction during exponential growth and in playing a role in resistance to hydrogen peroxide. Analysis of the changes in katB expression and in the total catalase and peroxidase activity during growth indicates that the 8- to 10-fold induction of peroxidase activity that occurs in stationary phase is attributable to KatA, the second L. pneumophila catalase-peroxidase. PMID:9765568

  3. The impact of monochloramine on the diversity and dynamics of Legionella pneumophila subpopulations in a nuclear power plant cooling circuit.

    PubMed

    Jakubek, Delphine; Le Brun, Matthieu; Leblon, Gerard; DuBow, Michael; Binet, Marie

    2013-08-01

    Members of the pathogenic Legionella genus encounter suitable growth conditions in nuclear power plant cooling circuits. To limit its proliferation and ensure that levels remain below regulatory thresholds, chemical treatment with monochloramine can be used in continuous or sequential conditions. The aim of this study was to determine the impact of monochloramine on L. pneumophila subpopulations in the cooling circuits of a nuclear power plant. The chosen procedure involved monitoring the diversity and dynamics of L. pneumophila subpopulations every month over the course of a year in a nuclear power plant cooling circuit, which was treated for 2 months during the period under study. This study confirmed the effectiveness of monochloramine to limit L. pneumophila concentrations in cooling circuits. The culturable L. pneumophila community was strongly affected by the injection of monochloramine. Several subpopulations persisted during treatment at low concentrations (below the detection limit of standard methods), suggesting that the susceptibility of L. pneumophila is strain dependent. Although the composition of the subpopulations was not similar, the resilience of the community structure was observed. Indeed, the community eventually returned to its initial structure and presented a similar pattern of richness, diversity and uniformity to that seen before treatment.

  4. Different growth rates in amoeba of genotypically related environmental and clinical Legionella pneumophila strains isolated from a thermal spa.

    PubMed

    Molmeret, M; Jarraud, S; Mori, J P; Pernin, P; Forey, F; Reyrolle, M; Vandenesch, F; Etienne, J; Farge, P

    2001-04-01

    Two cases of legionellosis occurring 3 years apart were acquired in the same French thermal spa and were apparently due to the same strain of Legionella pneumophila serogroup 1, as shown by genomic macrorestriction analysis. Minor differences between the two isolates were found by random amplification PCR profiling which showed an additional band with one of the isolates. Analysis of 107 L. pneumophila strains isolated from the spa waters by genome macrorestriction failed to identify the infective strain, but a closely related L. pneumophila serogroup 3 strain differing from the clinical isolates by only one band was found. To determine if the clinical L. pneumophila serogroup 1 isolates was better adapted for intracellular multiplication than related serogroup 3 environmental isolates, the growth kinetics of six isolates were determined in co-culture with Acanthamoeba lenticulata. One clinical isolate failed to grow within amoeba, while the other clinical isolate yielded the highest increase in bacterial cell count per amoeba (1,200%) and the environmental isolates gave intermediate values. Genetic analysis of L. pneumophila isolates by DNA macrorestriction does not therefore appear to reflect their growth kinetics within amoeba, and is not sufficiently discriminatory to identify potentially virulent strains.

  5. Different growth rates in amoeba of genotypically related environmental and clinical Legionella pneumophila strains isolated from a thermal spa.

    PubMed Central

    Molmeret, M.; Jarraud, S.; Mori, J. P.; Pernin, P.; Forey, F.; Reyrolle, M.; Vandenesch, F.; Etienne, J.; Farge, P.

    2001-01-01

    Two cases of legionellosis occurring 3 years apart were acquired in the same French thermal spa and were apparently due to the same strain of Legionella pneumophila serogroup 1, as shown by genomic macrorestriction analysis. Minor differences between the two isolates were found by random amplification PCR profiling which showed an additional band with one of the isolates. Analysis of 107 L. pneumophila strains isolated from the spa waters by genome macrorestriction failed to identify the infective strain, but a closely related L. pneumophila serogroup 3 strain differing from the clinical isolates by only one band was found. To determine if the clinical L. pneumophila serogroup 1 isolates was better adapted for intracellular multiplication than related serogroup 3 environmental isolates, the growth kinetics of six isolates were determined in co-culture with Acanthamoeba lenticulata. One clinical isolate failed to grow within amoeba, while the other clinical isolate yielded the highest increase in bacterial cell count per amoeba (1,200%) and the environmental isolates gave intermediate values. Genetic analysis of L. pneumophila isolates by DNA macrorestriction does not therefore appear to reflect their growth kinetics within amoeba, and is not sufficiently discriminatory to identify potentially virulent strains. PMID:11349974

  6. Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress.

    PubMed

    Gião, M S; Wilks, S; Azevedo, N F; Vieira, M J; Keevil, C W

    2009-01-01

    Legionella pneumophila is a waterborne pathogen that has been isolated sporadically from drinking water distribution systems (DWDS). Resistance to disinfectants is mainly attributed to the association of cells with amoebae, but biofilms are also thought to provide some degree of protection. In the present work, a two-stage chemostat was used to form heterotrophic biofilms from drinking water to study the influence of chlorine on the presence of naturally occurring L. pneumophila. The pathogen was tracked in planktonic and sessile biofilm phases using standard culture recovery techniques for cultivable cells and a peptide nucleic acid fluorescence in situ hybridisation assay for total cells. The results showed that the total number of L. pneumophila cells in biofilms was not affected by the concentrations of chlorine tested, and the presence of L. pneumophila could not be detected by culturing. To restrict the outbreaks of disease caused by this bacterium, efforts need to be concentrated on preventing L. pneumophila from re-entering an infectious state by maintaining residual disinfectant levels through the entire DWDS network so that the resuscitation of cells via contact with amoebae is prevented.

  7. Epidemiologic investigation by macrorestriction analysis and by using monoclonal antibodies of nosocomial pneumonia caused by Legionella pneumophila serogroup 10.

    PubMed

    Lück, P C; Helbig, J H; Günter, U; Assmann, M; Blau, R; Koch, H; Klepp, M

    1994-11-01

    A 67-year-old woman was hospitalized with an acute pneumonia of the left lower lobe. Legionella pneumophila serogroup 10 was cultured from two sputum specimens taken on days 18 and 20 and was also detected by direct immunofluorescence assay by using a commercially available species-specific monoclonal antibody as well as serogroup 10-specific monoclonal antibodies. Antigenuria was detected in enzyme-linked immunosorbent assays by using serogroup 10-specific polyclonal and monoclonal antibodies. In the indirect immunofluorescence test rising antibody titers against serogroups 1, 4, 5, 8, 9, 10, 14, and 15 were found in serum, with the highest titers found against serogroups 8, 9, and 10. L. pneumophila serogroups 10 and 6 and a strain that reacted with serogroup 4 and 14 antisera were cultured from both central and peripheral hot water systems of the hospital. Macrorestriction analyses of the genomic DNAs by pulsed-field gel electrophoresis showed that the isolate from the patient was identical to the serogroup 10 strains from the hospital hot water system. In contrast, the genomic DNAs of 16 unrelated L. pneumophila serogroup 10 strains showed 12 different restriction patterns. Monoclonal antibody subtyping revealed only minor differences in L. pneumophila serogroup 10 strains isolated from different sources. In conclusion, macrorestriction analysis is a valuable tool for studying the molecular epidemiology of L. pneumophila serogroup 10.

  8. Epidemiologic investigation by macrorestriction analysis and by using monoclonal antibodies of nosocomial pneumonia caused by Legionella pneumophila serogroup 10.

    PubMed Central

    Lück, P C; Helbig, J H; Günter, U; Assmann, M; Blau, R; Koch, H; Klepp, M

    1994-01-01

    A 67-year-old woman was hospitalized with an acute pneumonia of the left lower lobe. Legionella pneumophila serogroup 10 was cultured from two sputum specimens taken on days 18 and 20 and was also detected by direct immunofluorescence assay by using a commercially available species-specific monoclonal antibody as well as serogroup 10-specific monoclonal antibodies. Antigenuria was detected in enzyme-linked immunosorbent assays by using serogroup 10-specific polyclonal and monoclonal antibodies. In the indirect immunofluorescence test rising antibody titers against serogroups 1, 4, 5, 8, 9, 10, 14, and 15 were found in serum, with the highest titers found against serogroups 8, 9, and 10. L. pneumophila serogroups 10 and 6 and a strain that reacted with serogroup 4 and 14 antisera were cultured from both central and peripheral hot water systems of the hospital. Macrorestriction analyses of the genomic DNAs by pulsed-field gel electrophoresis showed that the isolate from the patient was identical to the serogroup 10 strains from the hospital hot water system. In contrast, the genomic DNAs of 16 unrelated L. pneumophila serogroup 10 strains showed 12 different restriction patterns. Monoclonal antibody subtyping revealed only minor differences in L. pneumophila serogroup 10 strains isolated from different sources. In conclusion, macrorestriction analysis is a valuable tool for studying the molecular epidemiology of L. pneumophila serogroup 10. Images PMID:7852558

  9. Neutrophil and Alveolar Macrophage-Mediated Innate Immune Control of Legionella pneumophila Lung Infection via TNF and ROS

    PubMed Central

    Ziltener, Pascal; Reinheckel, Thomas; Oxenius, Annette

    2016-01-01

    Legionella pneumophila is a facultative intracellular bacterium that lives in aquatic environments where it parasitizes amoeba. However, upon inhalation of contaminated aerosols it can infect and replicate in human alveolar macrophages, which can result in Legionnaires’ disease, a severe form of pneumonia. Upon experimental airway infection of mice, L. pneumophila is rapidly controlled by innate immune mechanisms. Here we identified, on a cell-type specific level, the key innate effector functions responsible for rapid control of infection. In addition to the well-characterized NLRC4-NAIP5 flagellin recognition pathway, tumor necrosis factor (TNF) and reactive oxygen species (ROS) are also essential for effective innate immune control of L. pneumophila. While ROS are essential for the bactericidal activity of neutrophils, alveolar macrophages (AM) rely on neutrophil and monocyte-derived TNF signaling via TNFR1 to restrict bacterial replication. This TNF-mediated antibacterial mechanism depends on the acidification of lysosomes and their fusion with L. pneumophila containing vacuoles (LCVs), as well as caspases with a minor contribution from cysteine-type cathepsins or calpains, and is independent of NLRC4, caspase-1, caspase-11 and NOX2. This study highlights the differential utilization of innate effector pathways to curtail intracellular bacterial replication in specific host cells upon L. pneumophila airway infection. PMID:27105352

  10. Experimental Evolution of Legionella pneumophila in Mouse Macrophages Leads to Strains with Altered Determinants of Environmental Survival

    PubMed Central

    Ensminger, Alexander W.; Yassin, Yosuf; Miron, Alexander; Isberg, Ralph R.

    2012-01-01

    The Gram-negative bacterium, Legionella pneumophila, is a protozoan parasite and accidental intracellular pathogen of humans. We propose a model in which cycling through multiple protozoan hosts in the environment holds L. pneumophila in a state of evolutionary stasis as a broad host-range pathogen. Using an experimental evolution approach, we tested this hypothesis by restricting L. pneumophila to growth within mouse macrophages for hundreds of generations. Whole-genome resequencing and high-throughput genotyping identified several parallel adaptive mutations and population dynamics that led to improved replication within macrophages. Based on these results, we provide a detailed view of the population dynamics of an experimentally evolving bacterial population, punctuated by frequent instances of transient clonal interference and selective sweeps. Non-synonymous point mutations in the flagellar regulator, fleN, resulted in increased uptake and broadly increased replication in both macrophages and amoebae. Mutations in multiple steps of the lysine biosynthesis pathway were also independently isolated, resulting in lysine auxotrophy and reduced replication in amoebae. These results demonstrate that under laboratory conditions, host restriction is sufficient to rapidly modify L. pneumophila fitness and host range. We hypothesize that, in the environment, host cycling prevents L. pneumophila host-specialization by maintaining pathways that are deleterious for growth in macrophages and other hosts. PMID:22693450

  11. Rapid Pathogen-Induced Apoptosis: A Mechanism Used by Dendritic Cells to Limit Intracellular Replication of Legionella pneumophila

    PubMed Central

    Nogueira, Catarina V.; Lindsten, Tullia; Jamieson, Amanda M.; Case, Christopher L.; Shin, Sunny; Thompson, Craig B.; Roy, Craig R.

    2009-01-01

    Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication. PMID:19521510

  12. The YhhN Protein of Legionella Pneumophila is a Lysoplasmalogenase*

    PubMed Central

    Jurkowitz, Marianne S.; Patel, Aalapi; Wu, Lai-Chu; Krautwater, Annalise; Pfeiffer, Douglas R.; Bell, Charles E.

    2014-01-01

    Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or -choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein, and expressed it in E. coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 and a Km of 45 µM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, monoacylglycerophospho-ethanolamine or –choline; the pH optimum is 6.5–7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host. PMID:25445671

  13. The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase.

    PubMed

    Jurkowitz, Marianne S; Patel, Aalapi; Wu, Lai-Chu; Krautwater, Annalise; Pfeiffer, Douglas R; Bell, Charles E

    2015-02-01

    Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 µmol/min/mg protein and a Km of 45 μM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.

  14. Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

    DOE PAGES

    Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.; ...

    2016-12-16

    Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, tomore » query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.« less

  15. Influence of iron-limited continuous culture on physiology and virulence of Legionella pneumophila.

    PubMed Central

    James, B W; Mauchline, W S; Fitzgeorge, R B; Dennis, P J; Keevil, C W

    1995-01-01

    A virulent strain of Legionella pneumophila serogroup 1, subgroup Pontiac, was grown in continuous culture at a constant growth rate under iron-replete and iron-limited conditions. Iron limitation was achieved by the removal of ferrous sulfate and hemin from the chemically defined medium. Residual contaminating iron, 0.45 microM, was sufficient to support iron-limited growth. Typical iron-replete cultures metabolized 3.3 microM iron. Serine provided the principal source of carbon and energy for both cultures, although iron-replete cultures also depleted a number of other amino acids. There was a 40% decrease in culture biomass under iron-restricted conditions. Iron limitation did not significantly affect carbohydrate metabolism, with the molar growth yield for carbon (Ycarbon) comparable for both cultures. However, under iron-limited conditions a sixfold increase in Yiron correlated with a significant decrease in the iron content of the biomass, as the culture utilized the available iron more efficiently. Highly pleomorphic iron-replete cultures became uniform cultures of short fine rods when adapted to iron-deficient conditions. In addition to the morphological and physiological changes, iron limitation had a critical effect on culture virulence. The virulence of this strain was significantly (P < 0.05) reduced when the culture was subjected to iron-limited conditions. This phenomenon was reversible, with a significant increase in culture virulence upon reversion to iron-replete conditions. When compared in an in vitro macrophage assay, the number of culturable avirulent iron-limited cells located intracellularly after infection was significantly lower than for the virulent replete and control cultures. These results further support the role of environmental parameters in regulating the virulence of L. pneumophila. PMID:7591051

  16. Inactivation of Legionella pneumophila by combined systems of copper and silver ions and free chlorine.

    PubMed

    Pianetti, Anna; Sabatini, Luigia; Citterio, Barbara; Sisti, Elivio; Pierfelici, Lucia; Bruscolini, Francesca

    2008-01-01

    The aim of this study was to evaluate the efficacy of copper and silver ions and of free chlorine in different combinations and concentrations (0.4 to 0.8-0.04 to 0.08 mg/l Cu(2+) Ag(+); 0.4 to 0.8-0.04 to 0.08-0.2 mg/l Cu(2+) Ag(+) Cl; 0.4 to 0.8-0.04 to 0.08-2 mg/l Cu(2+) Ag(+) Cl; 0.4 to 0.8-0.04 to 0.08-4 mg/l Cu(2+) Ag(+) Cl; 2-20 mg/l Cl), in inactivating Legionella pneumophila in drinking and distilled water after a contact time of 24-hours. Treatment with chlorine alone at 20 mg/l concentration was found to be the most effective treatment leading to complete killing of bacteria within 4 minutes in all water samples. On the other hand, at 2 mg/l concentration complete inactivation was obtained after 3 hours. The association of copper and silver ions at concentrations of 0.4-0.04 mg/l was found to be less effective and live bacteria could still be identified in all water samples after a 24 hour contact time. When testing copper and silver ions in combination, at concentrations of 0.8-0.08 mg/l and different combinations of the three disinfectants, results varied according to the various concentrations and type of water. The combination of copper and silver with 2 mg/l of chlorine was found to be more effective than 2mg/l of chlorine alone; a synergistic effect can therefore be hypothesized. The physical and chemical properties of drinking water, in particular its chlorine content, may have affected the water disinfection process when disinfecting agents were used in low concentrations. In conclusion, this study confirms the efficacy of shock hyperchlorination in the inactivation of Legionella pneumophila. However, the combination of free chlorine with metal (copper and silver) ions may represent a valid option for reducing the concentration of disinfectants to safer levels for human health and avoiding damage to water distribution systems especially in facilities such as hotels and hospitals.

  17. Epidemiology and Ecology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa

    PubMed Central

    Hilborn, Elizabeth D.; Arduino, Matthew J.; Pruden, Amy; Edwards, Marc A.

    2015-01-01

    Background Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa are opportunistic premise plumbing pathogens (OPPPs) that persist and grow in household plumbing, habitats they share with humans. Infections caused by these OPPPs involve individuals with preexisting risk factors and frequently require hospitalization. Objectives The objectives of this report are to alert professionals of the impact of OPPPs, the fact that 30% of the population may be exposed to OPPPs, and the need to develop means to reduce OPPP exposure. We herein present a review of the epidemiology and ecology of these three bacterial OPPPs, specifically to identify common and unique features. Methods A Water Research Foundation–sponsored workshop gathered experts from across the United States to review the characteristics of OPPPs, identify problems, and develop a list of research priorities to address critical knowledge gaps with respect to increasing OPPP-associated disease. Discussion OPPPs share the common characteristics of disinfectant resistance and growth in biofilms in water distribution systems or premise plumbing. Thus, they share a number of habitats with humans (e.g., showers) that can lead to exposure and infection. The frequency of OPPP-infected individuals is rising and will likely continue to rise as the number of at-risk individuals is increasing. Improved reporting of OPPP disease and increased understanding of the genetic, physiologic, and structural characteristics governing the persistence and growth of OPPPs in drinking water distribution systems and premise plumbing is needed. Conclusions Because broadly effective community-level engineering interventions for the control of OPPPs have yet to be identified, and because the number of at-risk individuals will continue to rise, it is likely that OPPP-related infections will continue to increase. However, it is possible that individuals can take measures (e.g., raise hot water heater temperatures and filter

  18. The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophila.

    PubMed

    Schell, Ursula; Simon, Sylvia; Sahr, Tobias; Hager, Dominik; Albers, Michael F; Kessler, Aline; Fahrnbauer, Felix; Trauner, Dirk; Hedberg, Christian; Buchrieser, Carmen; Hilbi, Hubert

    2016-02-01

    The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI-1 (3-hydroxypentadecane-4-one) for cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs-regulated processes include pathogen-host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI-1 promotes the motility of L. pneumophila by signalling through LqsS/LqsT and LqsR. Upon addition of LAI-1, autophosphorylation of LqsS/LqsT by [γ-(32) P]-ATP was inhibited in a dose-dependent manner. In contrast, the Vibrio cholerae autoinducer CAI-1 (3-hydroxytridecane-4-one) promoted the phosphorylation of LqsS (but not LqsT). LAI-1 did neither affect the stability of phospho-LqsS or phospho-LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the compound positively regulates a number of genes, including the non-coding RNAs rsmY and rsmZ, and negatively regulates the RNA-binding global regulator crsA. Accordingly, LAI-1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI-1 regulates motility and the biphasic life style of L. pneumophila through LqsS- and LqsT-dependent phosphorylation signalling.

  19. Legionella pneumophila DNA in serum samples during Legionnaires' disease in relation to C-reactive protein levels.

    PubMed

    van de Veerdonk, F L; de Jager, C P C; Schellekens, J J A; Huijsmans, C J J; Beaumont, F; Hermans, M H A; Wever, P C

    2009-04-01

    Legionella pneumophila DNA can be detected in serum from patients with Legionnaires' disease (LD). We explored this observation studying the kinetics of L. pneumophila DNA in serum samples in relation to C-reactive protein (CRP). Eleven hospitalized patients with LD were studied. Diagnosis was made by Legionella urinary antigen test in 8 patients and seroconversion in 3 patients. A macrophage infectivity potentiator (MIP) real-time PCR was performed on 31 serum samples, including 20 follow-up serum samples. Serum samples obtained on the day of admission were MIP PCR-positive in 7 (64%) and MIP PCR-negative in 4 (36%) patients. Three (75%) of the 4 patients with a MIP PCR-negative serum sample on the day of admission became positive during follow-up. Overall, L. pneumophila DNA was detected in serum samples from 10 of the 11 patients (91%). CRP levels in the 7 patients with a positive MIP PCR serum sample on day of admission (499 +/- 144 mg/l; median +/- SD) were significantly higher than those in the 4 patients with a negative MIP PCR serum sample on the day of admission (244 +/- 97 mg/l). No difference in the severity of the disease on the day of admission was found between these patients. The presence of L. pneumophila DNA in serum is a common phenomenon in hospitalized patients with LD, although in some cases it is not yet present on the day of admission. L. pneumophila DNA in serum on the day of admission correlates with high CRP lev