Comparison of Legionella pneumophila, L. micdadei, L. bozemanii, and L. dumoffii by transmission electron microscopy

Clinical isolates of Legionella pneumophila, L. micdadei, L. bozemanii, and L. dumoffii were grown on charcoal-yeast extract agar from a living-medium inoculum and prepared for transmission electron microscopy by three different methods. Cells of all four Legionella species possessed cytoplasmic vacuoles, a gram-negative type of cell envelope with a dense peptidoglycan-like layer, a ruthenium red-positive polysaccharide capsule, and a single subpolar flagellum. The dense polysaccharide capsule seen on cells of L. micdadei was separated from the outer membrane by an extra layer of electron-lucent material that was not present on cells of the other species examined.     

Room temperature storage of Legionella cultures.

Charcoal-yeast extract agar slant cultures of Legionella representing all four named species were stored at room temperature in the dark. Some L. pneumophila strains survived 2 months, L. bozemanii and L. dumoffii strains were viable at 4 months, and L. micdadei strains remained viable after 1 year in storage.     

Prevalence of pneumonias caused by Legionella species among patients on whom autopsies were performed

We wanted to determine the prevalence of pneumonias caused by Legionella species among patients on whom autopsies were performed in two medical centers in St Louis from January 1976 to June 1981. We screened formaldehyde-fixed deparaffinized lung tissue sections with microscopic evidence of pneumonia from 97 patients with use of the direct immunofluorescence antibody technique with a multivalent antilegionella conjugate containing antibodies to Legionella pneumophila serogroups 1 through 4 plus other Legionella species. One patient (1%) had disseminated L pneumophila serogroup 1 infection. We conclude that the prevalence of pneumonias caused by L pneumophila (serogroups 1 through 4), Legionella micdadei, Legionella bozemanii, Legionella dumoffii, or Legionella gormanii is low in the patients studied.     

Legionella jordanis: a new species of Legionella isolated from water and sewage.

Legionella jordanis sp. nov., as found in two cultures, is described. One isolate was from river water in Indiana and the other isolate was from sewage in DeKalb County, Ga. The former is the type strain of the species, and is designated BL-540 (ATCC 33623). L. jordanis had a partial relationship to L. bozemanii by direct fluorescent-antibody tests but was unrelated to L. pneumophila, L. dumoffii, L. micdadei, L. gormanii, or L. longbeachae. Legionella phenotypic characteristics, including large amounts of branched-chain cellular fatty acids, were shown by the isolates. Studies of DNA relatedness showed that the two cultures of L. jordanis were only slightly related to the six previously described species of Legionella but were more than 90% related to each other. Indirect fluorescent-antibody tests with human sera suggested that unrecognized human infections with L. jordanis may be occurring.     

Legionella micdadei and Legionella dumoffii monoclonal antibodies for laboratory diagnosis of Legionella infections.

Two different monoclonal antibodies directed against Legionella micdadei and L. dumoffii (Genetic Systems Corp., Seattle, Wash.) were evaluated for their specificity and ability to detect L. micdadei and L. dumoffii in human and animal clinical samples and bacterial isolates in an indirect immunofluorescence assay. All three frozen sputum samples and all three Formalin-fixed sputum and liver samples from patients with culture-documented L. micdadei pneumonia were positive when tested with the L. micdadei monoclonal antibody. A Formalin-preserved lung sample from a patient with culture-documented L. dumoffii pneumonia was positive with its homologous monoclonal antibody. No cross-staining reactions were found with either monoclonal antibody on any of 25 human sputum samples tested from patients without Legionella infections. A total of 66 Legionella strains and 56 non-Legionella strains including 22 Pseudomonas strains and 34 other bacterial strains were studied. No cross-staining reactions were found except in Staphylococcus aureus Cowan 1 ATCC 12598. The lower limit of detection in seeded sputum samples was about 7 X 10(4) cells per ml for both monoclonal antibodies. Lung and tracheal lavage specimens from L. micdadei- or L. dumoffii-infected guinea pigs showed specific staining only with their respective monoclonal antibodies. The monoclonal antibodies stained homologous bacteria slightly less intensely than did the polyclonal antisera, but the signal-to-noise ratio was considerably higher for the monoclonal antibodies. No differences in sensitivity of staining of clinical specimens or bacterial isolates were noted between the monoclonal antibodies and the polyclonal reagents for L. micdadei and L. dumoffii (Centers for Disease Control, Atlanta, Ga., and BioDx, Denville, N.J. These monoclonal antibodies ae sensitive and specific, making them good candidates for laboratory diagnostic purposes.     

Genetic, immunological, and cytotoxic comparisons of Legionella proteolytic activities.

Several strains of Legionella pneumophila and other species of Legionella with proteolytic activities were compared by assays, including Southern hybridizations and Western immunoblots, to determine their proteolytic, hemolytic, and cytotoxic activities. Only proteases from strains of L. pneumophila were both hemolytic and cytotoxic, and proteolytic activities extracted from other species of Legionella possessed only hemolytic activity. A 4.0-kilobase DNA sequence encoding the 38-kilodalton metalloprotease from L. pneumophila Philadelphia 1 that we showed previously was responsible for the observed hemolytic and cytotoxic phenotypes (F. D. Quinn and L. S. Tompkins, Mol. Microbiol., 3:797-805, 1989) was used in Southern hybridizations to probe chromosomal DNA from several strains of L. pneumophila and other Legionella species. The probe hybridized to the chromosomal DNA of all serogroups of L. pneumophila but not to any strains of L. dumoffii, L. micdadei, L. feeleii, or L. jordanis that we examined. Additionally, Western immunoblots done with rabbit antisera made to the cloned L. pneumophila protease demonstrated cross-reactions among 38-kilodalton proteins from strains of L. pneumophila, but no reactions were observed with proteins from other species of Legionella. Similarly, the cloned protease from L. pneumophila reacted with convalescent-phase sera from patients infected with L. pneumophila, but not with antisera isolated from patients infected with other Legionella species. Thus, despite some similarities among the proteolytic activities of members of the genus Legionella, including proteolytic and hemolytic phenotypes, metal requirements for zinc or iron, sensitivity to EDTA, and temperature and pH optima, we documented distinct genetic, immunological, and cytotoxicity differences among the proteolytic activities produced by Legionella species.     

Legionella micdadei (Pittsburgh pneumonia agent): direct fluoresent-antibody examination of infected human lung tissue and characterization of clinical isolates.

Legionella micdadei (Pittsburgh pneumonia agent) was identified by direct fluorescent-antibody (DFA) examination of lung tissue in six of seven persons diagnosed previously as having L. micdadei pneumonia only by histopathology and in four persons who also had positive cultures of the organism. No cross-reactions occurred with monospecific DFA conjugates prepared against Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, and Legionella gormanii. One person had L. pneumophila serogroup 6 identified by DFA examination of lung tissue and subsequent culture of stored pulmonary secretions. Characterization of the four strains of L. micdadei revealed specific DFA reactions, bacteriological behavior, and cellular fatty acid composition that allow identification of the organism. DFA testing appears to be a sensitive method for identifying L. micdadei prescent in human lung tissue or cultured on artificial media.     

Determination of catalase, peroxidase, and superoxide dismutase within the genus Legionella.

We examined 40 strains of Legionella for reduced-oxygen scavenging enzymes. Using a simple reaction chamber with a Swinney filter for the Beers and Sizer assay, we determined the catalase activity of live cells grown on buffered charcoal-yeast extract agar. For 29 strains of Legionella pneumophila, the apparent first-order rate constants for catalase ranged from 0.000 to 0.005. Similarly, low values ranging from 0.001 to 0.005 were observed for Legionella wadsworthii, Legionella oakridgensis, and Legionella gormanii. High catalase activities were found for Legionella jordanis, Legionella longbeachae, Legionella micdadei, and Legionella bozemanii, with first-order rate constant values of 0.010 to 0.035. Cell-free extracts were analyzed for catalase, peroxidase, and superoxide dismutase. Cell-free extracts of all strains had superoxide dismutase levels ranging from 8.2 to 30.5 U per mg of protein. The species could be characterized by their catalase and peroxidase since L. pneumophila and L. gormanii had only peroxidase (relative molecular weight [Mr], 150,000); L. dumoffii had a peroxidase (Mr, 150,000) plus a catalase (Mr, 174,000); and all remaining species had catalase only (Mr, 300,000, 220,000, or 150,000).     

Identification of Legionella pneumophila-specific genes by genomic subtractive hybridization with Legionella micdadei and identification of lpnE, a gene required for efficient host cell entry

Legionella pneumophila is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of L. pneumophila, we performed genomic subtractive hybridization between L. pneumophila serogroup 1 strain 02/41 and L. micdadei strain 02/42. A total of 144 L. pneumophila-specific clones were sequenced, revealing 151 genes that were absent in L. micdadei strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among L. pneumophila isolates of various serogroups as well as strains of Legionella longbeachae, L. micdadei, Legionella gormanii, and Legionella jordanis. Twelve predicted ORFs were L. pneumophila specific, including the gene encoding the dot/icm effector, lepB, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all L. pneumophila serotypes tested and an isolate of L. gormanii. These included icmD, the 5' end of a pilMNOPQ locus, and two genes known to be upregulated during growth within macrophages, cadA2 and ceaA. Disruption of an L. pneumophila-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed lpnE, for "L. pneumophila entry." In summary, this investigation has revealed important genetic differences between L. pneumophila and other Legionella species that may contribute to the phenotypic and clinical differences observed within this genus.     

Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed which detected soluble antigens from culture extracts of Legionella pneumophila serogroups 1 to 8, L. micdadei, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. longbeachae serogroups 1 and 2, L. wadsworthii, L. oakridgensis, L. anisa, L. feeleii serogroup 1, and L. jordanis. The assay was approximately 10-fold more sensitive for the eight L. pneumophila serogroups than for the other Legionella species tested. The ELISA detected Legionella antigens in the urine specimens of 25 of 35 patients with L. pneumophila serogroup 1, 3, 4, 6, and 8; L. micdadei; and L. longbeachae serogroup 1 infections. None of the 334 urine specimens from patients with either non-Legionella pneumonia or urinary tract infections was positive. For 10 patients from whom sequential urine specimens were available, Legionella antigens were not detectable from 7 to 19 days after laboratory diagnosis. Test sensitivity was not affected by heavy bacterial contamination. This ELISA offers the detection of a broad spectrum of Legionella antigens by a single test.     

Comparative Analysis of Legionella pneumophila and Legionella micdadei Virulence Traits

While the majority of Legionnaire's disease has been attributed to Legionella pneumophila, Legionella micdadei can cause a similar infection in immunocompromised people. Consistent with its epidemiological profile, the growth of L. micdadei in cultured macrophages is less robust than that of L. pneumophila. To identify those features of the Legionella spp. which are correlated to efficient growth in macrophages, two approaches were taken. First, a phenotypic analysis compared four clinical isolates of L. micdadei to one well-characterized strain of L. pneumophila. Seven traits previously correlated with the virulence of L. pneumophila were evaluated: infection and replication in cultured macrophages, evasion of phagosome-lysosome fusion, contact-dependent cytotoxicity, sodium sensitivity, osmotic resistance, and conjugal DNA transfer. By nearly every measure, L. micdadei appeared less virulent than L. pneumophila. The surprising exception was L. micdadei 31B, which evaded lysosomes and replicated in macrophages as efficiently as L. pneumophila, despite lacking both contact-dependent cytopathicity and regulated sodium sensitivity. Second, in an attempt to identify virulence factors genetically, an L. pneumophila genomic library was screened for clones which conferred robust intracellular growth on L. micdadei. No such loci were isolated, consistent with the multiple phenotypic differences observed for the two species. Apparently, L. pneumophila and L. micdadei use distinct strategies to colonize alveolar macrophages, causing Legionnaire's disease.     

Usefulness of Fatty Acid Composition for Differentiation of Legionella Species

Numerical analysis of fatty acid methyl ester (FAME) profiles of 199 isolates and 76 reference strains, belonging to all validly described species of the genus Legionella that can be cultured in laboratory media, was used to differentiate between the species of this genus. With the exception of the strains that autofluoresced red, it was possible to differentiate all the other Legionella species. The strains of the species L. bozemanii, L. dumoffii, L. feeleii, L. gormanii, L. maceachernii, L. micdadei, and L. quinlivanii did not form single clusters, showing some degree of variability in the fatty acid compositions. The strains of the blue-white autofluorescent species had very similar fatty acid compositions and were difficult to distinguish from each other. Nine isolates had fatty acid profiles unlike those of any of the validly described species and may represent different FAME groups of known species or undescribed Legionella species. The method used in this study was useful for screening and discriminating large number of isolates of Legionella species. Moreover, the results obtained can be included in a database of fatty acid profiles, leading to a more accurate automatic identification of Legionella isolates.     

Identification of mip-like genes in the genus Legionella.

The mip gene of Legionella pneumophila serogroup 1 strain AA100 encodes a 24-kilodalton surface protein (Mip) and enhances the abilities of L. pneumophila to parasitize human macrophages and to cause pneumonia in experimental animals. To determine whether this virulence factor is conserved in the genus Legionella, a large panel of Legionella strains was examined by Southern hybridization and immunoblot analyses for the presence and expression of mip-related sequences. Strains representing all 14 serogroups of L. pneumophila contained a mip gene and expressed a 24-kilodalton Mip protein. Although the isolates of the 29 other Legionella species did not hybridize with mip DNA probes under high-stringency conditions, they did so at reduced stringency. In support of the notion that these strains possess mip-like genes, these species each expressed a protein (24 to 31 kilodaltons in size) that reacted with specific Mip antisera. Moreover, the cloned mip analog from Legionella micdadei encoded the cross-reactive protein. Thus, mip is conserved and specific to L. pneumophila, but mip-like genes are present throughout the genus, perhaps potentiating the intracellular infectivity of all Legionella species.     

Use of multiple molecular subtyping techniques to investigate a Legionnaires' disease outbreak due to identical strains at two tourist lodges.

A multistate outbreak of Legionnaires' disease occurred among nine tour groups of senior citizens returning from stays at one of two lodges in a Vermont resort in October 1987. Interviews and serologic studies of 383 (85%) of the tour members revealed 17 individuals (attack rate, 4.4%) with radiologically documented pneumonia and laboratory evidence of legionellosis. A survey of tour groups staying at four nearby lodges and of Vermont-area medical facilities revealed no additional cases. Environmental investigation of common tour stops revealed no likely aerosol source of Legionella infection outside the lodges. Legionella pneumophila serogroup 1 was isolated from water sources at both implicated lodges, and the monoclonal antibody subtype matched those of the isolates from six patients from whom clinical isolates were obtained. The cultures reacted with monoclonal antibodies MAB1, MAB2, 33G2, and 144C2 to yield a 1,2,5,7 or a Benidorm 030E pattern. The strains were also identical by alloenzyme electrophoresis and DNA ribotyping techniques. The epidemiologic and laboratory data suggest that concurrent outbreaks occurred following exposures to the same L. pneumophila serogroup 1 strain at two separate lodges. Multiple molecular subtyping techniques can provide essential information for epidemiologic investigations of Legionnaires' disease.     

Antiserum-agar plate method for simultaneous detection and direct isolation of Legionella species in clinical and environmental specimens.

Colonies of Legionella pneumophila serotypes 1 through 6, L. micdadei, L. bozemanii, L. dumoffii, and L. gormanii, which were developed on filtered yeast extract agar containing polyvalent antiserum, were surrounded by distinct, specific precipitin rings.     

Rapid identification of clinically relevant Legionella spp. by analysis of transfer DNA intergenic spacer length polymorphism

Analysis of PCR-amplified transfer DNA (tDNA) intergenic spacers was evaluated as a rapid method for identification to the species level of 18 species of Legionella known as human pathogens. Type strains (n = 19), reference strains (n = 16), environmental strains (n = 31), and clinical strains (n = 32) were tested. PCR products using outwardly directed tDNA consensus primers were separated on polyacrylamide gels and analyzed with automated laser fluorescence. Test results were obtained in 8 h starting with 72-h-old bacterial growth on solid medium. Species-specific patterns were obtained for all 18 Legionella species tested: Legionella anisa, L. bozemanii serogroups 1 and 2, L. cincinnatiensis, L. dumoffii, L. feeleii serogroups 1 and 2, L. gormanii, L. hackeliae serogroups 1 and 2, L. jordanis, L. lansingensis, L. longbeachae serogroups 1 and 2, L. lytica, L. maceachernii, L. micdadei, L. oakridgensis, L. parisiensis, L. pneumophila serogroups 1 to 14, L. sainthelensi serogroup 2, L. tucsonensis, and L. wadsworthii. Computer-assisted matching of tDNA-intergenic length polymorphism (ILP) patterns identified all 63 environmental and clinical strains to the species level and to serogroup for some strains. tDNA-ILP analysis is proposed as a routinely applicable method which allows rapid identification of environmental and clinical isolates of Legionella spp. associated with legionellosis.     

Legionella prosthetic-valve endocarditis

Since 1982 seven patients at Stanford University Medical Center have been shown to have prosthetic-valve endocarditis caused by Legionella pneumophila or L. dumoffii. We studied the clinical features of legionella endocarditis at the time of diagnosis and performed a case-control study to analyze risk factors for the infection. All patients with endocarditis had a chronic course (3 to 19 months after surgery) of fever, night sweats, weight loss, and anemia, but no embolic events or immune-complex deposition disease. Five patients required surgical replacement of their infected prosthetic valves. The case-control study revealed that during the early postoperative period, patients who later contracted legionella endocarditis were more likely to have had symptoms and signs attributable to postcardiomyotomy syndrome than were patients who did not contract endocarditis (P less than 0.013). Examination of the legionella isolates by means of molecular techniques demonstrated that the Stanford L. pneumophila isolates were genotypically identical to isolates from the hospital drinking water. L. dumoffii isolates from patients with endocarditis were derived from a single strain apparently unique to this medical center. We conclude that legionella infection was nosocomially acquired in the perioperative period. These cases demonstrate an expanding spectrum of illness caused by legionella species and emphasize the need to consider legionella as a cause of "culture-negative" endocarditis.     

Cross-reactions between Legionella pneumophila (serogroup 1) and twenty-eight other bacterial species, including other members of the family Legionellaceae.

Cross-reactions between Legionella pneumophila serogroup 1 and 28 other bacterial species were studied by various quantitative immunoelectrophoretic techniques. A sonicated L. pneumophila antigen and purified homologous rabbit antibody were used as a reference system. Few antigens (0 to 6) cross-reacted with non-Legionellaceae, but two were found in nearly all gram-negative bacteria tested (antigens no. 1 and 66). Antigen no. 66 of the L. pneumophila reference system was shown to be antigenically similar to the "common antigen" of Pseudomonas aeruginosa reported in many gram-negative bacteria. Greater than 85% of the antigens from L. pneumophila serogroup 1 cross-reacted with the other six serogroups of L. pneumophila. By contrast, Fluoribacter (Legionella) bozemanae, F. (L.) dumoffii, F. (L.) gormanii, and Tatlockia (Legionella) micdadei cross-reacted with only 45, 53, 39, and 43% of the reference system antigens, respectively. The antigenic relatedness of members of the Legionellaceae, expressed as a matching coefficient, is discussed in terms of its taxonomic significance. Serogroup-, genus-, and family-specific antigens are identified in the L. pneumophila reference system.     

Aniline blue-containing buffered charcoal-yeast extract medium for presumptive identification of Legionella species.

By utilizing buffered charcoal-yeast extract medium containing 0.01% aniline blue in conjunction with a long-wave UV light, the differentiation of five species of Legionella was facilitated. L. pneumophila, when grown on this medium, did not absorb the aniline blue dye; however, L. micdadei, L. dumoffii, L. bozemanii, and L. gormanii absorbed the dye in varying amounts and produced colonies of various shades of blue.     

Multilocus enzyme analysis of Legionella dumoffii.

Variability among 29 clinical and environmental strains of Legionella dumoffii was investigated by multilocus enzyme analysis by use of starch gel electrophoresis. Based on results of analysis at 20 enzyme loci, the strains were separated into five closely related electrophoretic types (ETs), which were clearly distinguished from 53 strains representing 53 ETs of L. pneumophila. DNA hybridization results (hydroxyapatite method, 60 and 75 degrees C) for representative strains confirmed that all L. dumoffii ETs were a single genetic species. Although multilocus enzyme analysis indicated that L. dumoffii was genetically a quite uniform species, sufficient variability existed to warrant electromorph fingerprinting for epidemiologic studies.