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.     

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.     

Detection of cell-associated or soluble antigens of Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, Legionella gormanii, and Legionella micdadei by staphylococcal coagglutination tests.

Current methods used for the detection of whole-cell isolates of Legionella or for the detection of Legionella soluble antigens are technically impractical for many clinical laboratories. The purpose of this study was to explore practical alternatives. The results showed that whole cell isolates of Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, Legionella gormanii, and Legionella micdadei were identified specifically by a simple slide agglutination test or slide coagglutination test in which the reagent antisera are first bound to staphylococcal protein A. Soluble antigens were also identified specifically by the slide coagglutination test and by a sandwich immunofluorescence assay. The latter test may be useful in detecting antigen in body fluids of patients with legionellosis or in environmental samples.     

Pleomorphism of Legionella Pneumophila

Legionella pneumophila ILp), serogroups 1-6, was grown in vitro on a variety of media, in embryonated hens' eggs, and in guinea pigs. The morphology of the microbe was examined by light, immunofluorescent, and electron microscopy (transmission, scanning, negative staining). The configuration of all serogroups examined differed somewhat on agar media, in liquid media, and in vivo. Each serogroup of Lp showed pleomorphic features indistinguishable from the others. Except for filamentous forms, pleomorphism was least conspicuous on agar. By contrast, pleomorphism was most apparent in yeast extract broth, and it was detected by all of the morphologic techniques employed. Bacilli were seen most commonly, but the spectrum of forms was as follows: cocci, coccobacilli (short bacilli), medium bacilli, bacilli with terminal cocci, filamentous forms, and branches. Diplococci, branches, and stalks were only rarely seen, and the latter form was never visualized by immunofluorescence. In tissue samples from infected guinea pigs and embryonated hens' eggs, Lp was typically a short bacillus, but coc-coid and coccobacillary forms were seen. Lp is clearly a pleomorphic bacterium, particularly when grown in yeast extract broth. The variety of forms described herein might provide clues to taxonomy, ecologic niche, and physiology of Lp.     

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.     

Pleomorphism of Legionella pneumophila

Legionella pneumophila (Lp), serogroups 1-6, was grown in vitro on a variety of media, in embryonated hens' eggs, and in guinea pigs. The morphology of the microbe was examined by light, immunofluorescent, and electron microscopy (transmission, scanning, negative staining). The configuration of all serogroups examined differed somewhat on agar media, in liquid media, and in vivo. Each serogroup of Lp showed pleomorphic features indistinguishable from the others. Except for filamentous forms, pleomorphism was least conspicuous on agar. By contrast, pleomorphism was most apparent in yeast extract broth, and it was detected by all of the morphologic techniques employed. Bacilli were seen most commonly, but the spectrum of forms was as follows: cocci, coccobacilli (short bacilli), medium bacilli, bacilli with terminal cocci, filamentous forms, and branches. Diplococci, branches, and stalks were only rarely seen, and the latter form was never visualized by immunofluorescence. In tissue samples from infected guinea pigs and embryonated hens' eggs, Lp was typically a short bacillus, but coccoid and coccobacillary forms were seen. Lp is clearly a pleomorphic bacterium, particularly when grown in yeast extract broth. The variety of forms described herein might provide clues to taxonomy, ecologic niche, and physiology of Lp.     

Legionella anisa, a possible indicator of water contamination by Legionella pneumophila

Legionella anisa is one of the most frequent species of Legionella other than Legionella pneumophila in the environment and may be hospital acquired in rare cases. We found that L. anisa may mask water contamination by L. pneumophila, suggesting that there is a risk of L. pneumophila infection in immunocompromised patients if water is found to be contaminated with Legionella species other than L. pneumophila.     

Compost facilities as a reservoir of Legionella pneumophila and other Legionella species

Data on the presence of Legionellae outside the aquatic environment are scarce. Alternative ecosystems that could act as a reservoir for Legionella spp. have been investigated to identify unconventional contaminated substrates that are able to produce bioaerosols. We considered eight green waste collection sites including three composting facilities. Legionella pneumophila sg 1–15, Legionella bozemanii, Legionella cincinnatiensis, Legionella jamestowniensis, Legionella micdadei and L. oakridgensis were isolated from samples taken at six of the eight sites. The degree of contamination ranged from 10³ to 10⁸ CFU/g. Compost facilities appear to comprise an important reservoir for Legionellae.     

Outer membrane proteins from Legionella pneumophila serogroups and other Legionella species.

Outer membranes were isolated from eight serogroups of L. pneumophila and five other Legionella species. The protein composition of the membranes was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single, disulfide stabilized protein with a molecular size of 29,000 to 30,000 daltons was found to be the major outer membrane protein (MOMP) of all the serogroups. The equivalent of the L. pneumophila MOMP was not observed in any of the other Legionella species examined. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels revealed distinctive patterns for each serogroup and other Legionella species that were not observed by staining with Coomassie blue and may result from the presence of lipopolysaccharide in the membrane preparations. The MOMP from serogroup 1 was isolated by exposing crude peptidoglycan to detergent in the presence of heat and reducing agent and was found to be tightly associated with lipopolysaccharide. Antibodies to this complex were used to probe the outer membranes of the remaining, L. pneumophila serogroups and other Legionella species by Western blotting. Serogroup 1 anti-MOMP antibodies were found to react with the MOMP from the remaining seven serogroups examined, whereas antibodies directed against the lipopolysaccharide of serogroup 1 only reacted with lipopolysaccharide from two of the remaining seven serogroups.     

Evaluation of Legionella V-TesT for the detection of Legionella pneumophila antigen in urine samples

We evaluated a new immunochromatographic assay (Legionella V-TesT, Coris BioConcept, Gembloux, Belgium) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Test devices were read at various time points to determine the optimum incubation time regarding performance. The results were compared with those obtained with the BinaxNOW urinary antigen test. The sensitivity and specificity were 82.2% and 98.6%, respectively, for the Legionella V-TesT and 83.9% and 100%, respectively, for the BinaxNOW urinary antigen test after 15 min of incubation. When tests were examined after 60 min, the sensitivity for both tests increased to 91.5%.     

Ultrastructure of Legionella pneumophila.

Eleven lung samples positive for Legionnaires' disease, 12 strains of Legionella pneumophila cultured on various bacteriological media, and one strain growth in the yolk sac of fertile hens' eggs were examined by negative staining, thin sectioning, and scanning electron microscopy. All organisms studied were ultrastructurally similar irrespective of strain, source, or method of cultivation, presenting mainly as short rods, 0.6 x 1.5 micrometer, with tapered ends, though long forms and filaments were also evident. In this they resembled typical Gram-negative organisms. Division was by non-septate binary fission, and the cell wall was composed of two triple-unit membranes with morphological evidence of a peptidoglycan layer. The bacterial cytoplasm was rich in ribosomes and nuclear elements and often contained vacuoles. No acid polysaccharides or bacterial appendages were detected surrounding the organisms. In lung tissue and yolk sac membranes, the organisms replicated within the cytoplasm of infected cells and in the intercellular spaces and were specifically identified in thin sections by immunoferritin techniques.     

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.     

Immunoperoxidase staining of Legionella pneumophila

Immmunoperoxidase staining has been applied to sections of pneumonic lung from a previously published case of Legionnaires' disease. Specific staining of Legionella pneumophila was accomplished with sub-group 1 antiserum, which also revealed staining of phagosomes, and in some areas diffuse background staining of 'soluble' antigen. Some organisms remained unstained with the specific antiserum, and these were revealed by progressive haemalum staining. In other sections, some organisms stained specifically with rabbit anti-μ chain serum but not with anti-γ chain serum, this result suggesting that the organisms were coated with patient's IgM specific antibody.     

GyrA sequence-based typing of Legionella

Comparative sequence analysis of a 423-bp segment of the gyrA gene including a region homologous to the quinolone resistance-determining region (QRDR) of other species was evaluated as a novel typing method for Legionella strains. The study was performed with 29 reference strains representing 11 different Legionella species, with various serogroups, and with 13 clinical isolates of L. pneumophila. Pulsed-field gel electrophoresis and serotyping were employed for comparison of the clinical isolates. QRDR sequencing proved to be a highly discriminative tool for typing Legionellae, and permitted identification of species, serogroups and even different strains within serogroup 1. None of the isolates were resistant to quinolones in vitro and this correlated with dissence of mutations in the QRDR region. The data show that comparative sequence analysis of a short fragment of the gyrA gene is a potentially useful tool for typing of Legionella beyond the serogroup level. It is anticipated that mutations of the QRDR may arise in Legionella as a consequence of the introduction of quinolones as the agents of choice for the treatment of infections with this agent in immunocompromised patients. The employment of QRDR-typing maybe helpful in uncovering such mutations. Received: 7 March 2000     

Pathogenicity of Legionella pneumophila.

The bacterium Legionella pneumophila is the principal etiologic agent of Legionnaires' disease, a form of lobar pneumonia. Ubiquitous in aquatic environments, the gram-negative Legionella organism is a facultative, intracellular parasite of protozoa. The pathogenesis of legionellosis is largely due to the ability of L. pneumophila to invade and grow within alveolar macrophages, and it is widely believed that this ability results from a prior adaptation to intracellular niches in nature. Indeed, intracellular legionellae display a remarkable capacity to avoid endosomal and lysosomal bactericidal activities and to establish a unique replicative phagosome. In recent years, much progress has been made toward identifying the bacterial factors that promote intracellular infection and virulence. Surface structures that enhance infection include LPS, flagella, type IV pili, an outer membrane porin, and the Mip propyl-proline isomerase. Both type II and type IV protein secretion systems are critical for L. pneumophila pathogenesis. Whereas the type II (Lsp) system secretes a collection of degradative enzymes, the type IV (Dot/Icm) system likely exports effector proteins that are especially critical for trafficking of the Legionella phagosome. In addition to facilitating pilus formation and type II secretion, the inner membrane prepilin peptidase (PilD) of L. pneumophila appears to mediate a third, potentially novel pathway that is operative in the mammalian host. Periplasmic and cytosolic infectivity determinants include a catalase-peroxidase and the HtrA and Hsp60 stress-response proteins. The stationary phase response and the iron acquisition functions of L. pneumophila also play key roles in pathogenesis, as do a number of other loci, including the pts, mil and enh genes.