The HL-60 model for the interaction of human macrophages with the Legionnaires' disease bacterium

The facultative intracellular pathogen, Legionella pneumophila, multiplies within and kills human monocytes and alveolar macrophages. We show that L. pneumophila strain Philadelphia-1 infects, multiplies within and kills the promyelocyte HL-60 cell line after its differentiation into macrophage-like cells. The characteristics of the interaction between L. pneumophila and differentiated HL-60 cells closely resemble those between L. pneumophila and human peripheral blood monocytes. With both cell types, C receptors and serum C mediate attachment of L. pneumophila, which are taken up by coiling phagocytosis. The replicative phagosome is lined with ribosomes; intracellular multiplication is iron-dependent; and replicating bacteria ultimately destroy the host cell. As in human monocytes, an avirulent mutant derivative of L. pneumophila Philadelphia-1, 25D, does not replicate in and is not cytopathic for differentiated HL-60 cells. Differentiated HL-60 cells therefore provide a convenient and faithful model for the study of L. pneumophila-mononuclear phagocyte interaction.     

Investigation of mechanisms involved in phagocytosis of Legionella pneumophila by human cells

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive and multiply efficiently in a variety of mammalian cells. By using in vitro assays, the uptake of L. pneumophila into monocytes has shown to be mediated, at least in part, through attachment of complement-coated bacteria to complement receptors, but complement-independent phagocytosis could also be demonstrated. Since complement levels in the human lung are normally low, the role of complement-dependent phagocytosis in the pathogenesis of Legionnaires' disease is doubtful. However, the contribution of other potential phagocytosis-related host cell surface molecules to the phagocytosis of L. pneumophila has never been investigated. We therefore analyzed the role of complement receptors 1 (CD35) and 3 (CD11b/18), the lipopolysaccharide (LPS) receptor (CD14), the beta(1)-integrin chain of the fibronectin receptor (CD29), the intercellular adhesion molecule 1 (ICAM-1, CD54) and the transferrin receptor (CD71) in the complement-independent uptake of L. pneumophila. To exclude any influence of culture conditions onto phagocytosis rates, we compared a fresh clinical isolate with an agar-adapted isolate of L. pneumophila. In addition, we used three different host cell types (MM6, HeLa and Jurkat cells) expressing different rates of complement receptors. We could show that both strains of L. pneumophila were phagocytized by the three host cell lines to the same extent, but intracellular multiplication was only found in MM6 and, although to a much lesser degree, in Jurkat cells. Preincubation of MM6 cells with monoclonal antibodies directed against the above cited phagocytosis-related receptors did not result in inhibition of L. pneumophila uptake. We therefore conclude that typical phagocytosis-related cell surface receptors are not involved in the complement-independent phagocytosis of L. pneumophila.     

Icm/Dot-dependent upregulation of phagocytosis by Legionella pneumophila

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia. Dependent on the icm/dot loci, L. pneumophila survives and replicates in macrophages and amoebae within a specialized phagosome that does not fuse with lysosomes. Here, we report that phagocytosis of wild-type L. pneumophila is more efficient than uptake of icm/dot mutants. Compared with the wild-type strain JR32, about 10 times fewer icm/dot mutant bacteria were recovered from HL-60 macrophages in a gentamicin protection assay. The defect in phagocytosis of the mutants could be complemented by supplying the corresponding genes on a plasmid. Using fluorescence microscopy and green fluorescent protein (GFP)-expressing strains, 10-20 times fewer icm/dot mutant bacteria were found to be internalized by HL-60 cells and human monocyte-derived macrophages (HMMPhi). Compared with icm/dot mutants, wild-type L. pneumophila infected two to three times more macrophages and yielded a population of highly infected host cells (15-70 bacteria per macrophage) that was not observed with icm/dot mutant strains. Wild-type and icmT mutant bacteria were found to adhere similarly and compete for binding to HMMPhi. In addition, wild-type L. pneumophila was also phagocytosed more efficiently by Acanthamoeba castellanii, indicating that the process is independent of adherence receptor(s). Wild-type L. pneumophila enhanced phagocytosis of an icmT mutant strain in a synchronous co-infection, suggesting that increased phagocytosis results from (a) secreted effector(s) acting in trans.     

Interferon-gamma-activated human monocytes inhibit the intracellular multiplication of Legionella pneumophila

We have examined the interaction between interferon-gamma (IFN-gamma)-activated human monocytes and Legionella pneumophila, the agent of Legionnaires' disease. Human monocytes activated with human recombinant IFN-gamma inhibit the intracellular multiplication of L. pneumophila. The degree of inhibition is proportional to the concentration of IFN-gamma, and maximal inhibition consistently occurs with greater than or equal to 2 micrograms/ml. Monoclonal anti-IFN-gamma antibody completely neutralizes the capacity of IFN-gamma to activate monocytes. Monocytes infected 24 hr after explantation maximally inhibit L. pneumophila multiplication if treated with IFN-gamma before infection or up to 2 hr after infection; treatment 6 hr or more after infection results in submaximal inhibition. Monocytes infected 48 hr after explantation inhibit L. pneumophila multiplication maximally if treated with IFN-gamma up to 12 hr before infection, but submaximally if treated at the time of infection. Once activated, monocytes inhibit L. pneumophila multiplication in the absence of IFN-gamma in the culture. Strikingly, monocytes maximally inhibit L. pneumophila multiplication after treatment with IFN-gamma for as briefly as 1 hr before infection. In the absence of anti-L. pneumophila antibody, neither IFN-gamma-activated monocytes nor nonactivated monocytes kill L. pneumophila. In the presence of specific antibody and complement, IFN-gamma-activated monocytes kill a proportion (0.5 log) of an inoculum but not more than nonactivated monocytes. L. pneumophila forms a specialized phagosome in IFN-gamma-activated monocytes that does not differ ultrastructurally from the L. pneumophila phagosome in nonactivated monocytes. These results demonstrate that IFN-gamma can activate human monocytes to exert a potent antimicrobial effect against a highly virulent intracellular bacterial pathogen. These findings extend previous observations on interactions between activated mononuclear phagocytes and L. pneumophila, and additionally support the hypothesis that cell-mediated immunity plays a major role in host defense against L. pneumophila.     

Intracellular multiplication of Legionella pneumophila depends on host cell amino acid transporter SLC1A5

The infectious agent of Legionnaires' disease, Legionella (L) pneumophila, multiplies intracellularly in eukaryotic cells. This study has been performed to explore the nutrient requirements of L. pneumophila during intracellular replication. In human monocytes, bacterial replication rate was reduced by 76% in defined medium lacking L-cysteine, L-glutamine or L-serine. SLC1A5 (hATB(0,+)), a neutral amino acid transporter, was upregulated in the host cells after infection with L. pneumophila. Inhibition of SLC1A5 by BCH, a competitive inhibitor of amino acid uptake as well as siRNA silencing of the slc1a5 gene blocked intracellular multiplication of L. pneumophila without compromising viability of host cells. These observations suggest that replication of L. pneumophila depends on the function of host cell SLC1A5.     

Virulence factors of the family Legionellaceae.

Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of eukaryotic substrates, including phosphatidylinositol and tubulin. Since the phosphorylation of either phosphatidylinositol or tubulin might compromise phagocyte activation and bactericidal functions, this enzyme may well be a virulence factor. Administration of the L. pneumophila exoprotease induces lesions resembling those of Legionella pneumonia and kills guinea pigs, suggesting that this protein plays a role in the pathogenesis of legionellosis. However, recent work with a genetically engineered strain has convincingly shown that the protease is not necessary for intracellular survival or virulence. As might be expected with a complex process like intracellular parasitism, it appears that the capability of Legionella strains to invade and multiply in host phagocytes is multifactorial and that no single moiety which is responsible for the virulence phenotype will be found.     

Cytokines and legionellosis

Recent studies have led to an enhanced understanding of the role of cell-mediated immunity and cytokines in Legionnaires' disease. In particular, the effect of interferon gamma on human mononuclear phagocyte iron metabolism and the role of iron availability inLegionella pneumophila intracellular multiplication in human monocytes has been elucidated. With this knowledge it is now possible to develop treatment strategies for Legionnaires' disease using interferon gamma and/or agents affecting human mononuclear phagocyte iron metabolism.Abbreviations M-CSF Macrophage colony stimulating factor     

Infection of cultured human endothelial cells by Legionella pneumophila

Legionella pneumophila is a gram-negative pathogen that causes a severe pneumonia known as Legionnaires' disease. Here, we demonstrate for the first time that L. pneumophila infects and grows within cultured human endothelial cells. Endothelial infection may contribute to lung damage observed during Legionnaires' disease and to systemic spread of this organism.     

RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase

Legionella pneumophila replicates within amoebae and macrophages and causes the severe pneumonia Legionnaires' disease. When broth cultures enter the post-exponential growth (PE) phase or experience amino acid limitation, L. pneumophila accumulates the stringent response signal (p)ppGpp and expresses traits likely to promote transmission to a new phagocyte. The hypothesis that a stringent response mechanism regulates L. pneumophila virulence was bolstered by our finding that the avirulent mutant Lp120 contains an internal deletion in the gene encoding the stationary phase sigma factor RpoS. To test directly whether RpoS co-ordinates virulence with stationary phase, isogenic wild-type, rpoS-120 and rpoS null mutant strains were constructed and analysed. PE phase L. pneumophila became cytotoxic by an RpoS-independent pathway, but their sodium sensitivity and maximal expression of flagellin required RpoS. Likewise, full induction of sodium sensitivity by experimentally induced (p)ppGpp synthesis required RpoS. To replicate efficiently in macrophages, L. pneumophila used both RpoS-dependent and -independent pathways. Like those containing the dotA type IV secretory apparatus mutant, phagosomes harbouring either rpoS or dotA rpoS mutants rapidly acquired the late endosomal protein LAMP-1, but not the lysosomal marker Texas red-ovalbumin. Together, the data support a model in which RpoS co-operates with other regulators to induce L. pneumophila virulence in the PE phase.     

Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions

Legionella pneumophila, the Gram-negative bacterium that causes Legionnaires' disease, can be cultured in the laboratory in a variety of fresh-water amoebae and macrophage-like cell lines. None of these hosts, however, is amenable to genetic analysis, which has limited the ability of researchers to analyse the host factors essential for L. pneumophila growth. In this article, we describe a novel method in which L. pneumophila is grown within the soil amoeba Dictyostelium discoideum and how D. discoideum genetics is being used to analyse the host cell factors involved in L. pneumophila pathogenesis.     

Identification of protective B cell antigens of Legionella pneumophila

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires' disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.     

Interactions of invasive and noninvasive strains of Neisseria meningitidis with monkey epithelial cells, mouse monocytes and human macrophages

Adherence and phagocytosis of invasive and noninvasive Neisseria meningitidis strains was investigated using light, fluorescence and electron microscopy. Invasive strains were isolated from the cerebrospinal fluid and/or blood of the patients with invasive meningococcal disease and noninvasive strains from the nasopharynx and/or larynx of healthy carriers. Adherence/endocytosis was studied on monkey kidney cells (the LLC-MK2 cell line) and phagocytosis on mouse monocytes and human macrophages (the P388D1 and U-937 cell lines, respectively). Although invasive and noninvasive meningococci isolated in the same cluster showed identical genotype and phenotype markers, they were found to interact differently with epithelial cells as well as with monocytes/macrophages. Invasive isolates displayed higher adherence to the surface of LLC-MK2 cells compared to noninvasive ones. Phagocytosis by P388D1 cells of noninvasive strains was effective and the bacteria were damaged by cytolysis. In contrast, invasive bacteria frequently persisted in "coiling" vacuoles and in effect could destroy the host cell. This is the first demonstration of coiling phagocytosis induced by meningococci. Efficiency of phagocytosis by U-937 cells was significantly higher for the noninvasive than invasive strains. Different behaviour of invasive and noninvasive strains of N. meningitidis observed during 4 hours of interactions with epithelial cells and monocytes/macrophages reflects well the higher pathogenic potential of invasive bacteria.     

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

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.     

Periplasmic copper-zinc superoxide dismutase of Legionella pneumophila: role in stationary-phase survival.

Copper-zinc superoxide dismutases (CuZnSODs) are infrequently found in bacteria although widespread in eukaryotes. Legionella pneumophila, the causative organism of Legionnaires' disease, is one of a small number of bacterial species that contain a CuZnSOD, residing in the periplasm, in addition to an iron SOD (FeSOD) in their cytoplasm. To investigate CuZnSOD function, we purified the enzyme from wild-type L. pneumophila, obtained amino acid sequence data from isolated peptides, cloned and sequenced the gene from a L. pneumophila library, and then constructed and characterized a CuZnSOD null mutant. In contrast to the cytoplasmic FeSOD, the CuZnSOD of L. pneumophila is not essential for viability. However, CuZnSOD is critical for survival during the stationary phase of growth. The CuZnSOD null mutant survived 10(4)- to 10(6)-fold less than wild-type L. pneumophila. In wild-type L. pneumophila, the specific activity of CuZnSOD increased during the transition from exponential to stationary-phase growth while the FeSOD activity was constant. These data support a role of periplasmic CuZnSOD in survival of L. pneumophila during stationary phase. Since L. pneumophila survives extensive periods of dormancy between growth within hosts. CuZnSOD may contribute to the ability of this bacterium to be a pathogen. In exponential phase, wild-type and CuZnSOD null strains grew with comparable doubling times. In cultured HL-60 and THP-1 macrophage-like cell lines and in primary cultures of human monocytes, multiplication of the CuZnSOD null mutant was comparable to that of wild type. This indicated that CuZnSOD is not essential for intracellular growth within macrophages or for killing of macrophages in those systems.     

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

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.     

Legionella pneumophila: an aquatic microbe goes astray

Legionella pneumophila is naturally found in fresh water were the bacteria parasitize within protozoa. It also survives planctonically in water or biofilms. Upon aerosol formation via man-made water systems, L. pneumophila can enter the human lung and cause a severe form of pneumonia, called Legionnaires' disease. The pathogenesis of Legionnaires' disease is largely due to the ability of L. pneumophila to invade and grow within macrophages. An important characteristic of the intracellular survival strategy is the replication within the host vacuole that does not fuse with endosomes or lysosomes. In recent times a great number of bacterial virulence factors which affect growth of L. pneumophila in both macrophages and protozoa have been identified. The ongoing Legionella genome project and the use of genetically tractable surrogate hosts are expected to significantly contribute to the understanding of bacterium-host interactions and the regulation of virulence traits during the infection cycle. Since person-to-person transmission of legionellosis has never been observed, the measures for disease prevention have concentrated on eliminating the pathogen from water supplies. In this respect detection and analysis of Legionella in complex environmental consortia become increasingly important. With the availability of new molecular tools this area of applied research has gained new momentum.     

一个细菌的致命之旅——嗜肺军团菌分泌系统及效应蛋白的研究进展

嗜肺军团菌是引起军团菌肺炎以及庞蒂亚克热的革兰氏阴性胞内病原细菌,嗜肺军团菌侵染宿主的主要特点是可以通过其IVB型毒力分泌系统,向宿主细胞内分泌超过150种的底物效应蛋白。通过这些效应蛋白的作用,嗜肺军团菌能够调整宿主细胞的胞内运输途径,改变内外环境来伪装自己的吞噬泡,干扰宿主的细胞周期,抑制宿主细胞的凋亡,从而有效逃避宿主细胞的防御功能,创造出理想的胞内增殖环境。最后,效应蛋白还可以帮助军团菌从宿主细胞中逃逸。目前,嗜肺军团菌已经成为"病原菌-宿主相互作用"的重要研究模型,其毒力分泌系统及其底物效应蛋白的功能也成为细胞微生物学的研究热点。对嗜肺军团菌分泌系统及效应蛋白的研究不仅能够帮助阐明病原细菌的致病机理,还有助于推动对宿主免疫机制的更深层次的研究。文章主要针对嗜肺军团菌的毒力分泌系统,尤其是IVB型分泌系统的结构和功能,以及底物效应蛋白的研究进展进行了综述,向读者展示出一个小小的细菌所拥有的那令人惊叹的、如此狡猾的生存策略和它精致的杀伤武器。     

Correlation between phagocytic and membrane surface properties reflected by partitioning of human peripheral blood monocytes in two-polymer aqueous phases

Human peripheral blood monocyte-enriched fractions (identified by staining for peroxidase and by sizing) were obtained by velocity sedimentation at unit gravity of peripheral blood mononuclear cells. They were then fractionated by countercurrent distribution (a multiple-extraction procedure) in a charged Dextran/poly(ethylene glycol) aqueous phase system. The monocytes remained viable after the separation (order of 90%). Cells obtained from different cavities along the extraction train were tested for their ability to phagocytize latex particles. With increasing partition coefficient (presumably higher charge-associated membrane properties) the ratio of monocytes that phagocytized to monocytes that did not phagocytize increased appreciably. When, however, monocytes were permitted to phagocytize particles prior to countercurrent distribution, an increase in partition coefficient was associated with an appreciable decrease in the above-specified ratio. Control experiments indicate that the observed change in partitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitiartitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitioning) and their ability to ingest particles. Furthermore, an alteration in the surface charge-associated properties of monocytes as a consequence of phagocytosis is indicated by the cells' reduced partition coefficient.     

Cell biology of Legionella pneumophila

Legionella pneumophila is the causative agent of a potentially fatal form of pneumonia named Legionnaires' disease. L. pneumophila survives and replicates inside macrophages by preventing phagosome-lysosome fusion. A large number of L. pneumophila genes, called dot or icm, have been identified that are required for intracellular growth. It has recently been shown that the dot/icm genes code for a putative large membrane complex that forms a type IV secretion system used to alter the endocytic pathway.     

Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples

Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.