Two years' performance of an in-house ELISA for diagnosis of Legionnaires' disease: Detection of specific IgM and IgG antibodies against Legionella pneumophila serogroup 1, 3 and 6 in human serum

2013 ◽  
Vol 94 (2) ◽  
pp. 94-97 ◽  
Author(s):  
P.L. Elverdal ◽  
C.S. Jørgensen ◽  
K.A. Krogfelt ◽  
S.A. Uldum
Keyword(s):  
Human Serum ◽  
Legionella Pneumophila ◽  
Disease Detection ◽  
Igg Antibodies ◽  
Legionnaires Disease

scholarly journals Population analysis of Legionella pneumophila reveals the basis of resistance to complement-mediated killing

2020 ◽  
Author(s):  
Bryan A. Wee ◽  
Joana Alves ◽  
Diane S. J. Lindsay ◽  
Ross L. Cameron ◽  
Amy Pickering ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Human Serum ◽  
Legionella Pneumophila ◽  
Human Disease ◽  
Population Analysis ◽  
Single Gene ◽  
Clinical Isolates ◽  
Classical Pathway ◽  
Evolutionary Analysis ◽  
Legionnaires Disease

AbstractLegionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires’ disease. L. pneumophila is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 900 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We found that although some clones are more commonly associated with clinical infections, the capacity for human disease is representative of the breadth of species diversity. To investigate the bacterial genetic basis for human disease potential, we carried out a genome-wide association study that identified a single gene (lag-1), to be most strongly associated with clinical isolates. Molecular evolutionary analysis showed that lag-1, which encodes an O-acetyltransferase responsible for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. Functional analysis revealed a correlation between the presence of a functional lag-1 gene and resistance to killing in human serum and bovine broncho-alveolar lavage. In addition, L. pneumophila strains that express lag-1 escaped complement-mediated phagocytosis by neutrophils. Importantly, we discovered that the expression of lag-1 confers the capacity to evade complement-mediated killing by inhibiting deposition of classical pathway molecules on the bacterial surface. In summary, our combined population and functional analyses identified L. pneumophila genetic traits linked to human disease and revealed the molecular basis for resistance to complement-mediated killing, a previously elusive trait of direct relevance to human disease pathogenicity.SignificanceLegionella pneumophila is an environmental bacterium associated with a severe pneumonia known as Legionnaires’ disease. A small number of L. pneumophila clones are responsible for a large proportion of human infections suggesting they have enhanced pathogenicity. Here, we employed a large-scale population analysis to investigate the evolution of human pathogenicity and identified a single gene (lag-1) that was more frequently found in clinical isolates. Functional analysis revealed that the lag-1-encoded O-acetyltransferase, involved in modification of lipopolysaccharide, conferred resistance to the classical pathway of complement in human serum. These findings solve a long-standing mystery in the field regarding L. pneumophila resistance to serum killing, revealing a novel mechanism by which L. pneumophila may avoid immune defences during infection.

scholarly journals Efficacy of SCH27899 in an Animal Model of Legionnaires' Disease Using Immunocompromised A/J Mice

2000 ◽  
Vol 44 (5) ◽  
pp. 1333-1336 ◽  
Author(s):  
Joan K. Brieland ◽  
David Loebenberg ◽  
Fred Menzel ◽  
Roberta S. Hare
Keyword(s):  
Animal Model ◽  
Murine Model ◽  
Legionella Pneumophila ◽  
Immunocompromised Host ◽  
Lung Infection ◽  
Cortisone Acetate ◽  
Contrast Administration ◽  
Once Daily ◽  
Legionnaires Disease ◽  
Lung Infections

ABSTRACT The efficacy of SCH27899, a new everninomicin antibiotic, against replicative Legionella pneumophila lung infections in an immunocompromised host was evaluated using a murine model of Legionnaires' disease. A/J mice were immunocompromised with cortisone acetate and inoculated intratracheally with L. pneumophilaserogroup 1 (105 CFU per mouse). At 24 h postinoculation, mice were administered either SCH27899 (6 to 60 mg/kg [MPK] intravenously) or a placebo once daily for 5 days, and mortality and intrapulmonary growth of L. pneumophila were assessed. In the absence of SCH27899, there was 100% mortality inL. pneumophila-infected mice, with exponential intrapulmonary growth of the bacteria. In contrast, administration of SCH27899 at a dose of ≥30 MPK resulted in ≥90% survival of infected mice, which was associated with inhibition of intrapulmonary growth ofL. pneumophila. In subsequent studies, the efficacy of SCH27899 was compared to ofloxacin (OFX) and azithromycin (AZI). Administration of SCH27899, OFX, or AZI at a dose of ≥30 MPK once daily for 5 days resulted in ≥85% survival of infected mice and inhibition of intrapulmonary growth of the bacteria. However, L. pneumophila CFU were recovered in lung homogenates following cessation of therapy with all three antibiotics. These studies demonstrate that SCH27899 effectively prevents fatal replicativeL. pneumophila lung infection in immunocompromised A/J mice by inhibition of intrapulmonary growth of the bacteria. However, in this murine model of pulmonary legionellosis, SCH27899, like OFX and AZI, was bacteriostatic.

Subtyping ofLegionella pneumophilaisolates by arbitrarily primed polymerase chain reaction

1995 ◽  
Vol 41 (9) ◽  
pp. 846-848 ◽  
Author(s):  
E. Ledesma ◽  
J. Llorca ◽  
M. A. Dasí ◽  
M. L. Camaró ◽  
E. Carbonell ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Legionella Pneumophila ◽  
Water Sources ◽  
Chain Reaction ◽  
Single Room ◽  
Legionnaires Disease ◽  
Polymerase Chain ◽  
Resort Hotel ◽  
Different Water Sources

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.Key words: Legionella pneumophila, AP-PCR, subtyping, outbreak.

scholarly journals Immunoglobulin G antibodies directed against protein III block killing of serum-resistant Neisseria gonorrhoeae by immune serum.

1986 ◽  
Vol 164 (5) ◽  
pp. 1735-1748 ◽  
Author(s):  
P A Rice ◽  
H E Vayo ◽  
M R Tam ◽  
M S Blake
Keyword(s):  
Membrane Protein ◽  
Neisseria Gonorrhoeae ◽  
Human Serum ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Immune Serum ◽  
Igg Antibodies ◽  
Porin Protein ◽  
Blocking Activity ◽  
Normal Human

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent serum from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by IgG isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, an antigenically conserved outer membrane protein, contained most of the blocking activity in IgG. Antibodies specific for gonococcal porin (protein I), the major outer membrane protein, displayed no blocking function. In separate experiments, immune convalescent DGI serum which did not exhibit bactericidal activity was restored to killing by selective depletion of protein III antibodies by immunoabsorption. These studies indicate that protein III antibodies in normal and immune human serum play a role in serum resistance of N. gonorrhoeae.

scholarly journals Human Lung Tissue Explants Reveal Novel Interactions during Legionella pneumophila Infections

2013 ◽  
Vol 82 (1) ◽  
pp. 275-285 ◽  
Author(s):  
Jens Jäger ◽  
Sebastian Marwitz ◽  
Jana Tiefenau ◽  
Janine Rasch ◽  
Olga Shevchuk ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Legionella Pneumophila ◽  
Human Lung ◽  
Transcriptional Response ◽  
Human Infection ◽  
Bacterial Invasion ◽  
Human Lung Tissue ◽  
Content Type ◽  
Tissue Explants ◽  
Legionnaires Disease

ABSTRACTHistological 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 forLegionella pneumophilainfection comprising living human lung tissue. We stimulated lung explants withL. pneumophilastrains 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 ofL. pneumophilato 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 ofL. pneumophilainfections. 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.

How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane

2001 ◽  
Vol 114 (24) ◽  
pp. 4637-4650 ◽  
Author(s):  
Lewis G. Tilney ◽  
Omar S. Harb ◽  
Patricia S. Connelly ◽  
Camenzind G. Robinson ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Mitochondrial Membranes ◽  
Macrophage Infection ◽  
Thin Sections ◽  
Stage Process ◽  
Legionnaires Disease ◽  
Rough Er ◽  
Membrane Changes ◽  
Phagosomal Membrane

Within five minutes of macrophage infection by Legionella pneumophila, the bacterium responsible for Legionnaires’ disease, elements of the rough endoplasmic reticulum (RER) and mitochondria attach to the surface of the bacteria-enclosed phagosome. Connecting these abutting membranes are tiny hairs, which are frequently periodic like the rungs of a ladder. These connections are stable and of high affinity - phagosomes from infected macrophages remain connected to the ER and mitochondria (as they were in situ) even after infected macrophages are homogenized. Thin sections through the plasma and phagosomal membranes show that the phagosomal membrane is thicker (72±2 Å) than the ER and mitochondrial membranes (60±2 Å), presumably owing to the lack of cholesterol, sphingolipids and glycolipids in the ER. Interestingly, within 15 minutes of infection, the phagosomal membrane changes thickness to resemble that of the attached ER vesicles. Only later (e.g. after six hours) does the ER-phagosome association become less frequent. Instead ribosomes stud the former phagosomal membrane and L. pneumophila reside directly in the rough ER. Examination of phagosomes of various L. pneumophila mutants suggests that this membrane conversion is a four-stage process used by L. pneumophila to establish itself in the RER and to survive intracellularly. But what is particularly interesting is that L. pneumophila is exploiting a poorly characterized naturally occuring cellular process.

scholarly journals Highlighting the Potency of Biosurfactants Produced by Pseudomonas Strains as Anti-Legionella Agents

2018 ◽  
Vol 2018 ◽  
pp. 1-15 ◽  
Author(s):  
Clémence Loiseau ◽  
Emilie Portier ◽  
Marie-Hélène Corre ◽  
Margot Schlusselhuber ◽  
Ségolène Depayras ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Close Association ◽  
Water Systems ◽  
Antagonistic Activity ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Free Living ◽  
Legionnaires Disease ◽  
Culture Supernatants ◽  
Multispecies Biofilms

Legionella pneumophila, the causative agent of Legionnaires’ disease, is a waterborne bacterium mainly found in man-made water systems in close association with free-living amoebae and multispecies biofilms. Pseudomonas strains, originating from various environments including freshwater systems or isolated from hospitalized patients, were tested for their antagonistic activity towards L. pneumophila. A high amount of tested strains was thus found to be active. This antibacterial activity was correlated to the presence of tensioactive agents in culture supernatants. As Pseudomonas strains were known to produce biosurfactants, these compounds were specifically extracted and purified from active strains and further characterized using reverse-phase HPLC and mass spectrometry methods. Finally, all biosurfactants tested (lipopeptides and rhamnolipids) were found active and this activity was shown to be higher towards Legionella strains compared to various other bacteria. Therefore, described biosurfactants are potent anti-Legionella agents that could be used in the water treatment industry although tests are needed to evaluate how effective they would be under field conditions.

scholarly journals Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. e00128-18 ◽  
Author(s):  
Danka Pavliakova ◽  
Peter C. Giardina ◽  
Soraya Moghazeh ◽  
Shite Sebastian ◽  
Maya Koster ◽  
...  
Keyword(s):  
Human Serum ◽  
Lower Limit ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Correlate Of Protection ◽  
Immune Correlate ◽  
Relative Standard ◽  
Limit Of Quantitation

ABSTRACT A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials. IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

scholarly journals Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB ofLegionella pneumophilaContribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
J. Rasch ◽  
C. M. Ünal ◽  
A. Klages ◽  
Ü. Karsli ◽  
N. Heinsohn ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Temperature Tolerance ◽  
Lung Damage ◽  
Atypical Pneumonia ◽  
Content Type ◽  
Wide Range ◽  
Legionnaires Disease ◽  
Environmental Fitness ◽  
Sliding Motility

ABSTRACTThe gammaproteobacteriumLegionella pneumophilais the causative agent of Legionnaires’ disease, an atypical pneumonia that manifests itself with severe lung damage.L. pneumophila, a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. InL. pneumophilathe surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness ofL. pneumophila. Furthermore, they contribute to infection of the natural hostAcanthamoeba castellaniias well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators ofL. pneumophilawith compensatory action and pleiotropic effects.

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