Serological diversity within the species Legionella spiritensis

A strain of Legionella isolated from the environment which could not initially be identified was shown by restriction fragment length polymorphisms to be a Legionella spiritensis. This was confirmed by DNA homology studies, cell wall fatty acid composition and isoprenoid quinone analysis. This strain, which is only the second reported representative of the species, was shown to be serologically distinct from the type strain of L. spiritensis and all other serogroups of Legionella.     

Problems associated with identification of Legionella species from the environment and isolation of six possible new species

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.     

Problems associated with identification of Legionella species from the environment and isolation of six possible new species.

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.     

In vitro study of the proteolytic activity of rumen anaerobic fungi

Abstract To better define the antigenic structure of the outer cell membranes of Legionellae, a panel of 6 monoclonal antibodies was raised against partially purified outer membranes of Legionella pneumophila serogroup 1, Corby strain. This study describes the purification and characterization of one of these monoclonal antibodies reacting with a 135-kDa protein, which was shown to be common to all 14 serogroups of Legionella pneumophila . It shows no cross-reactivity with 20 other Legionella species, or 9 other Gram-negative species tested by SDS-PAGE and Western blotting procedures. The epitope would appear to be predominantly surface exposed and, from preliminary detergent extraction studies, not peptidoglycan-associated.     

Specific DNA probe for detecting Legionella pneumophila

A fragment of the gene for cytolysin has been cloned. The product of the gene has been earlier identified as an immunoserological marker of Legionella pneumophila. Clones were selected by immunodetection of cytolysin gene product expression. An EcoRI 3.8 kb fragment of the genomic DNA from Legionella pneumophila strain Philadelphia-1 was used as a DNA probe that hybridized with the bacterial colonies of 20 Legionella pneumophila strains but not with the colonies of 10 other Legionella species or 8 other bacterial genera. The cloned fragment has been shown to be unique for Legionella pneumophila. The region of homology is suggested to be longer than a possible dimension of the cytolysin gene.     

The conjugational transfer of plasmids to Legionella strains

In experiments on conjugation the transfer of a number of R-plasmids having a wide range of hosts, such as plasmids RP1, R68.45, RP4, N3, RK2, S-a, those having a narrow range of hosts, such as plasmid R64, to strains of different Legionella species was shown. The frequency of transfer varied from 3.1 X 10(-3) to 9.4 X 10(-7). The fact that the conjugation transfer was confirmed by the reverse transfer of plasmids from Legionella transconjugates to Escherichia coli strain K12, as well as by the detection of the DNA of the transferred plasmid by means of electrophoresis in agar gel. Plasmid RP1 showed different behavior in transconjugates of various Legionella species after several passages in a medium free of antibiotics. In the Legionella strain under study the unstable preservation of plasmid R64 was observed.     

Legionella shakespearei sp. nov., isolated from cooling tower water.

A Legionella-like organism (strain 214T [T = type strain]) was isolated from a cooling tower in Stratford-upon-Avon, England. This strain required L-cysteine and contained cellular branched-chain fatty acids that are typical of the genus Legionella. Strain 214T produced pink colonies on buffered charcoal-yeast extract agar. Ubiquinone Q-12 was the major quinone. Strain 214T was serologically distinct from other legionellae as determined by a slide agglutination test. The results of DNA hybridization studies showed that strain 214T (= ATCC 49655T) is a member of a new Legionella species, Legionella shakespearei.     

Legionella impletisoli sp. nov. and Legionella yabuuchiae sp. nov., isolated from soils contaminated with industrial wastes in Japan

In this study, we tried to isolate legionellae from nine Legionella DNA-positive soil samples collected from four different sites contaminated with industrial wastes in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 22 isolates of legionellae were obtained from five of the nine samples. Identification of species and/or serogroups (SGs), performed by DNA-DNA hybridization and agglutination tests, revealed that the 22 isolates consisted of ten isolates of Legionella pneumophila including five SGs, five Legionella feeleii, and one each of Legionella dumoffii, Legionella longbeachae, and Legionella jamestownensis. The species of the remaining four isolates (strains OA1-1, -2, -3, and -4) could not be determined, suggesting that these isolates may belong to new species. The 16S rDNA sequences (1476-1488bp) of the isolates had similarities of less than 95.0% compared to other Legionella species. A phylogenetic tree created by analysis of the 16S rRNA (1270bp) genes demonstrated that the isolates formed distinct clusters within the genus Legionella. Quantitative DNA-DNA hybridization tests on the OA1 strains indicated that OA1-1 should be categorized as a new taxon, whereas OA1-2, -3, and -4 were also genetically independent in another taxon. Based on the evaluated phenotypic and phylogenetic characteristics, it is proposed that one of these isolates from the soils, OA1-1, be classified as a novel species, Legionella impletisoli sp. nov.; the type strain is strain OA1-1(T) (=JCM 13919(T)=DSMZ 18493(T)). The remaining three isolates belong to another novel Legionella species, Legionella yabuuchiae sp. nov.; the type strain is strain OA1-2(T) (=JCM 14148(T)=DSMZ 18492(T)). This is the first report on the isolation of legionellae from soils contaminated with industrial wastes.     

Legioliulin, a new isocoumarin compound responsible for blue-white autofluorescence in Legionella (Fluoribacter) dumoffii under long-wavelength UV light

Legionella dumoffii is one of the causative agents of Legionnaires' disease. There are 50 species in the genus Legionella, of which 10 species including L. dumoffii are known to exhibit uncharacterized blue-white autofluorescence. We constructed an L. dumoffii strain that exhibited a high intensity blue-white fluorescence, isolated a fluorescent pigment from the strain, and determined its molecular formula to be C(19)H(14)O(3) by high-resolution mass spectrometry. An NMR analysis revealed that this was a new isocoumarin compound, which was named legioliulin.     

橡树岭军团菌的分离及用16S rDNA测序法的鉴定

军团菌属（Ｌｅｇｉｏｎｅｌａ）自１９７１年确定以来,现已有３９个成员。军团菌属主要分布于供水系统中,如空调冷却塔水和浴池淋浴水中。我们曾于１９９４年从北京某宾馆冷却塔水中分离出一株可疑军团菌,但因缺乏标准抗血清而未能定种。由于核酸分析技术和细菌鉴定观念上进步,使我们能根据１６ＳｒＤＮＡ序列比较推测细菌间进化关系,并进而对未知细菌进行鉴定和检测。我们用以１６ＳｒＤＮＡ为靶序列的ＰＣＲ和测定鉴定,确定了该分离株为橡树岭军团菌（Ｌ．ｏａｋｒｉｄｇｅｎｓｉｓ）。     

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.     

Enhanced growth restriction of Legionella pneumophila in endotoxin-treated macrophages.

Macrophages from A/J mice are permissive for growth of Legionella pneumophila, an intracellular opportunistic pathogen that grows preferentially in macrophages. Macrophages from other mouse strains are highly resistant to growth of Legionella. In the present study, it was found that macrophages from A/J mice are readily activated by pretreatment with lipopolysaccharide (LPS), so that the cells do not permit Legionella to replicate in vitro, as occurs when untreated macrophages from A/J mice are cultured with these organisms for 48 hr. The augmentation of Legionella growth inhibition by LPS-activated macrophages from nonpermissive BDF1 mice also occurred. After in vitro infection, there was a 1000-fold increase in the number of Legionella in A/J macrophages and approximately a 10-fold increase in BDF1 macrophages, but LPS treatment of macrophages from either strain resulted in marked growth restrictions. This suppression was both dose dependent as well as dependent upon the time of addition of the LPS to the macrophages. Furthermore, the lipid A component of LPS was found to be as effective as the intact LPS in activating macrophages to inhibit the intracellular growth of Legionella. Further studies concerning the mechanisms involved are clearly warranted and in progress.     

Analysis of the genetic diversity of Legionella by sequencing the 23S-5S ribosomal intergenic spacer region: from phylogeny to direct identification of isolates at the species level from clinical specimens

This study focuses on the interest of the hypervariable 23S-5S ribosomal intergenic spacer region (ISR) of the genus Legionella to analyze the phylogenic diversity of Legionella at the species and subspecies levels and to identify isolates directly from clinical specimens. The method, using a real-time PCR assay with a single primer pair followed by sequencing, was able to identify correctly 49 reference strains of Legionella belonging to 37 different species, including those implicated in human infections, and to clearly differentiate the three subspecies of L. pneumophila. Based on sequence similarities, the 23S-5S ISR sequences were much more variable than the rpoB and mip sequences (P<0.0001 by the Wilcoxon signed rank test). The 23S-5S ISR method was able to cluster Legionella species in accordance with phenotypic traits, such as autofluorescence or fatty acid membrane composition. Using maximum parsimony methods, the rpoB and 23S-5S ISR data sets were shown to be incongruent (P<0.001). In contrast, the 23S-5S ISR and the mip data sets were found to be congruent (P=0.313), suggesting the interest of combining these two regions to demonstrate phylogenetic links between Legionella species. This molecular assay was shown able to both detect Legionella DNA directly in respiratory specimens from patients exhibiting a Legionella infection and provide accurate identification of the bacterium at the species level in the tested specimens. These properties open a wide range of applications to the 23S-5S ISR sequencing method, from taxonomic analyses to clinical and epidemiological investigations.     

Virulence conversion of Legionella pneumophila by conjugal transfer of chromosomal DNA

In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 x 10(9) CFU) and Chicago-2S (approximately 8.9 x 10(9) CFU) were mated typically yielded 10(3) Kmr Smr LacZ+ transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, we showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.     

Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPIase) activity

Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages. The Mip (macrophage infectivity potentiator) protein has been shown to be an essential virulence factor. A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506-binding proteins (FKBPs) of several different eukaryotic organisms. FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. The gene coding for the Mip protein was cloned from the chromosome of L. pneumophila strain Philadelphia I and sequenced. It was synthesized in Escherichia coli K-12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides. Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP-type PPIases of eukaryotes. However, the N-terminal extension of Mip and/or the substitutions of the variable amino acids in the C-terminal FKBP core leads to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc-Ala-Xaa-Pro-Phe-4-nitroanilide. Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins. In view of the activity of PPIases in protein-folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.     

Survival of Legionella pneumophila in a model hot water distribution system

A virulent strain of Legionella pneumophila was inoculated into an enclosed system supplied with unsterilized water from a domestic hot water supply. Growth of bacteria was monitored over 10 weeks. An increase in the number of organisms other than legionellas occurred but few amoebae were observed and none could be cultured. Viable counts of L. pneumophila in the circulation fluid decreased slightly. However, particles of debris which accumulated in the apparatus and which were stained by the indirect fluorescent antibody technique were found to be almost totally composed of L. pneumophila. On dismantling the apparatus Legionella was isolated in moderately high numbers from several different types of surfaces, particularly natural rubber and silicone.     

Phylogenetic characterization of Legionella-like endosymbiotic X-bacteria in Amoeba proteus: a proposal for 'Candidatus Legionella jeonii' sp. nov

The X-bacteria which initiated organismic association with the D strain of Amoeba proteus in 1966 as parasites have changed to obligate endosymbionts on which the host depends for survival. Owing to the difficulty in cultivating the bacteria in vitro, the identity of X-bacteria has not been determined. As the life cycle of X-bacteria is similar to that of Legionella spp. in soil amoebae, we applied the polymerase chain reaction method with specific primers aimed at Legionella spp. for the detection and cloning of 16S rRNA gene. The identity and intracellular localization of the endosymbiont were confirmed by the application of a specific fluorescently labelled 16S rRNA-targeted probe. In addition we cloned RNA polymerase beta-subunit gene (rpoB) of X-bacteria by genomic library tagging. A phylogenetic analysis of the 16S rRNA gene placed the bacterium within a unique monophyletic group containing all other members of the genus Legionella. Phylogeny from rpoB and mip genes further confirmed the taxonomic context of X-bacteria to be a Legionella sp. In all three phylogenic analyses, X-bacterium was placed apart from Legionella-like amoebal pathogens present in soil amoebae. Thus, we propose the name 'Candidatus Legionella jeonii' sp. nov. for the endosymbiotic X-bacteria in Amoeba proteus.     

Temperate Legionella bacteriophage: discovery and characteristics

For the first time, temperate Legionella bacteriophage was isolated from organs of guinea pig infected with Philadelphia 1 strain of Legionella pneumophila. Negative colonies of bactriophage from 1.5 to 2.5 mm in diameter were detected. Central part of them was transparent and surrounded by peripheral zone of partial lysis. Electron microscopy showed that corpuscles of the phage consist from multifaceted elongated head of stretched hexagonal form and short tail. The bacteriophage lyzed bacteria, which cause Legionnaires' disease, and also had certain lytic activity against causative agent of tularemia.