Cross-reacting lipopolysaccharide antigens in Legionella pneumophila serogroups 1 to 14.

Immunological cross-reactions among Legionella species were investigated with sonicated, proteinase K-digested cell lysates. The antigens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were either analyzed for lipopolysaccharides (LPSs) by silver staining or transferred to nitrocellulose membranes for serological characterization with rabbit antibodies directed against Legionella pneumophila serogroups 1 and 5. When antiserum prepared against serogroup 5 was used to probe the LPSs from L. pneumophila serogroups 1 to 14, the antibodies recognized a common epitope harbored by all L. pneumophila serogroups but not by other Legionella species or by the gram-negative bacteria tested as controls. Hence, the serogroup 5 antiserum correctly identified all serogroups of L. pneumophila tested in the LPS immunoblot assay. Moreover, the silver-stained profiles of the isolated LPSs revealed characteristic patterns allowing the identification of the individual serogroups of L. pneumophila.     

Heterogeneity of antibody response in infection with Legionella pneumophila serogroup 1.

The antibody response of patients infected with Legionella pneumophila serogroup 1 in a common source outbreak was investigated. Heat-killed antigens from L pneumophila serogroups 1-3 and 6-10, plus several other strains of L pneumophila, together with 13 other species of legionellas were used in an indirect fluorescence antibody test. Formolised yolk sac antigens made from L pneumophila serogroups 1, 6, and 7 were also used. Although antibodies were produced to several L pneumophila serogroups or Legionella species by individuals, there was no constant pattern, suggesting that the response is a characteristic of the infected individual and not of the infecting strain of Legionella. There is evidence that heat-killed antigen made from L pneumophila serogroup 7 may give unreliable results.     

Electrophoretic and serological characterization of the lipopolysaccharides of Legionella pneumophila.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteinase-K-digested cell lysates to provide preliminary data on the LPS chemotypes for 20 strains of Legionella pneumophila (serogroups 1 to 8). The profiles of all strains except Chicago 2 (serogroup 6) were similar in the number, spacing, and size distribution of the bands visualized on silver-stained gels and were indicative of smooth LPS. However, compared with the bands from Salmonella minnesota smooth LPS, their banding pattern was much tighter, with three to four legionella bands for every salmonella band. The proteinase K digest of Chicago 2 was unique in that only two widely separated silver-stained bands were seen. LPS profiles of 10 serogroup 1 strains were identical, and the profile of Knoxville 1 was not altered by extensive in vitro passage. We used immunoblotting to investigate the serological specificities of the LPSs. When a rabbit antiserum prepared against a serogroup 1 strain was used to probe nitrocellulose sheets that bound LPS from strains belonging to eight different serogroups, it recognized only the LPS from the homologous serogroup. Similar results were observed with serogroup 2, 4, and 6 antisera. Our data indicate that L. pneumophila has a smooth-type LPS with an unusual banding pattern and that it is a serogroup-specific antigen.     

Serogroup specificity of Legionella pneumophila is related to lipopolysaccharide characteristics.

We studied the lipopolysaccharide (LPS) of Legionella pneumophila and six other Legionella species to determine whether strain differences were apparent. The LPS was purified by a cold ethanol extraction procedure, and total carbohydrates represented 10 to 20% of LPS weight. 2-keto-3-deoxyoctonate represented 1 to 13% of the total carbohydrate present in the LPS. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all strains except L. dumoffi showed smooth-type LPS with multiple high-molecular-weight complexes. Proteinase K-treated, whole-cell lysates showed profiles similar to those of purified LPS. Each serogroup of L. pneumophila and each Legionella species had a distinct sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile. L. pneumophila lipid A is antigenically related to the lipid A of Enterobacteriaceae. In immunoblot assays with the LPS of L. pneumophila serogroups 1 to 6 as antigens, serogroup-specific immune monkey sera recognized homologous purified LPS, but not the LPS of the five heterologous serogroups. These studies indicate that LPS composition may be a determinant of serogroup specificity as defined by the immunofluorescence-based serogrouping schema for L. pneumophila and other Legionella species.     

Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species

Genomics can provide the basis for understanding the evolution of emerging, lethal human pathogens such as Legionella pneumophila, the causative agent of Legionnaires' disease. This bacterium replicates within amoebae and persists in the environment as a free-living microbe. 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 over 84% of Legionnaires' disease worldwide. Why L. pneumophila Sg1 is so predominant is unknown. Here, we report the first comprehensive screen of the gene content of 217 L. pneumophila and 32 non-L. pneumophila strains isolated from humans and the environment using a Legionella DNA-array. Strikingly, we uncovered a high conservation of virulence- and eukaryotic-like genes, indicating strong environmental selection pressures for their preservation. No specific hybridization profile differentiated clinical and environmental strains or strains of different serogroups. Surprisingly, the gene cluster coding the determinants of the core and the O side-chain synthesis of the lipopolysaccaride (LPS cluster) determining Sg1 was present in diverse genomic backgrounds, strongly implicating the LPS of Sg1 itself as a principal cause of the high prevalence of Sg1 strains in human disease and suggesting that the LPS cluster can be transferred horizontally. Genomic analysis also revealed that L. pneumophila is a genetically diverse species, in part due to horizontal gene transfer of mobile genetic elements among L. pneumophila strains, but also between different Legionella species. However, the genomic background also plays a role in disease causation as demonstrated by the identification of a globally distributed epidemic strain exhibiting the genotype of the sequenced L. pneumophila strain Paris.     

Immunologic response of patients with legionellosis against major protein-containing antigens of Legionella pneumophila serogroup 1 as shown by immunoblot analysis.

Major protein-containing antigens of Legionella pneumophila serogroup 1 were were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with rabbit antisera to 14 different Legionella species or serogroups. Fourteen bands were observed in immunoelectropherograms of whole-cell, sonicated cell, and heated cell preparations, seven of which appeared in the supernatant fluid from the heated cells and three of which were shown in an outer membrane fraction. Immunoblots of whole-cell antigen preparations of 14 Legionella species or serogroups revealed seven major Legionella proteins: antigens with molecular weights of 58,000, 79,000, and 154,000 were present in all Legionella sp. strains, antigens with molecular weights of 44,000 and 97,000 occurred in multiple species, and antigens with molecular weights of 14,000 and 25,000 were present only in L. pneumophila strains. All sera from 15 patients with culture-confirmed L. pneumophila serogroup 1 disease and 14 of 18 (78%) sera from serologically diagnosed patients reacted with the 58-kilodalton (kDa) common antigen. In contrast, less than one-half of the sera reacted with the L. pneumophila-specific proteins (14 and 25 kDa). Absorption of sera with Escherichia coli cells had no effect on their reactivity with the 58-kDa antigen, whereas absorption with L. pneumophila serogroup 1 cells removed reactivity. These data suggest that the 58-kDa antigen may prove useful in serodiagnostic tests for legionellosis.     

Retrospective evaluation of the Du Pont radioimmunoassay kit for detection of Legionella pneumophila serogroup 1 antigenuria in humans.

We used the Du Pont radioimmunoassay kit for soluble Legionella pneumophila serogroup 1 antigenuria (Du Pont Co., Wilmington, Del.) to test 422 urine samples from patients with and without Legionnaires disease (LD). The urine specimens were collected from 23 patients with culture-proven LD and from 346 patients without LD. L. pneumophila serogroup 1 was isolated from 14 patients with culture-proven LD, and other L. pneumophila serogroups or other Legionella species were isolated from 9 patients; 58 urine specimens were tested from these 23 patients. The non-LD group was composed of 75 bacteremic patients (35 gram-negative and 40 gram-positive bacteremias), 7 patients with candidemia, 48 patients with non-LD pneumonia, 90 patients with gram-negative bacteriuria (greater than 10(5) CFU/ml), 23 patients with gram-positive bacteriuria (greater than 10(5) CFU/ml), 14 patients with candiduria (greater than 10(5) CFU/ml), and 89 outpatients with negative urine cultures. All tests were performed in duplicate, including positive and negative controls. Sample results with values greater than or equal to 3.0 times those of the negative controls were considered positive for L. pneumophila serogroup 1 antigenuria. The average sample-to-negative ratios were 19.1 for the L. pneumophila serogroup 1 specimens, and 1.0 for both the non-serogroup 1 legionella group and the non-LD specimens. All but one of the patients who were culture positive for L. pneumophila serogroup 1 had at least one specimen positive for serogroup 1 antigenuria; none of the non-L. pneumophila serogroup 1 patients had a positive urine test. The test was highly specific (100%) and sensitive (93%) for the detection of L. pneumophila serogroup 1 antigenuria. Concentrations of urine by vacuum evaporation increased test sensitivity without apparently affecting specificity.     

Legionella pneumophila serogroup six: isolation from cases of legionellosis, identification by immunofluorescence staining, and immunological response to infection.

Isolates of Legionella pneumophila that are serologically different from strains of serogroups 1 through 5 were obtained from lung biopsy tissue or pleural fluid from three renal transplant recipients in Chicago, Ill. These strains were placed in a newly designated L. pneumophila serogroup, serogroup 6, on the basis of fluorescent-antibody staining characteristics. An L. pneumophila strain obtained from Bethesda, Md., one from Houston, Tex., and one from Oxford, England, also belong to this new serogroup. L. pneumophila serogroup 6 appears to be widely distributed geographically.     

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.     

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.     

Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody.

Serogroup-specificity of Legionella pneumophila is related to lipopolysaccharide (LPS), and few cross-reactions between serogroups have been observed with rabbit or monkey antisera. C57BL/6 mice were sequentially immunized with crude outer membrane fractions of L. pneumophila serogroups 1, 5, and 7, Legionella bozemanii, and Legionella micdadei. Spleen cells from these mice were then fused with the Sp2-0/Ag14 mouse myeloma cell line. Outer membrane-rich fractions and LPS were prepared from L. pneumophila serogroups 1 to 8 and other Legionella and non-Legionella species. Immunoblots of these extracts were performed with monoclonal antibody obtained from these fusions. One of these monoclonal antibodies recognized an epitope common to all tested serogroups of L. pneumophila and attached to the major constituent of the outer membrane, LPS. This antibody did not react with other Legionella species and numerous gram-negative rods other than Pseudomonas fluorescens CDC93. This monoclonal antibody may be useful in preliminary identification of L. pneumophila as an alternative to direct fluorescent-antibody testing.     

Serogrouping and subtyping of Legionella pneumophila with monoclonal antibodies

Monoclonal antibodies directed against Legionella pneumophila serogroups 1 to 6 were produced by fusing splenocytes of BALB/c mice with the Sp 2/0-Ag14 or the NSO mouse myeloma cell lines. Specificity of these antibodies was determined by indirect fluorescent-antibody staining: 8 reacted with L. pneumophila serogroup 1 and, respectively, 13, 6, 6, 5, and 10 reacted with serogroups 2, 3, 4, 5, and 6; all except 5 were serogroup specific, and none presented cross-reactions with six other species of Legionellaceae. Serogroup determination of 35 isolates of L. pneumophila with seven selected monoclonal antibodies resulted in correct serogrouping in all instances; a pool of the same seven monoclonal antibodies stained intensely all strains of L. pneumophila without any staining of the other species of Legionellaceae. When 24 serogroup 1 isolates of L. pneumophila were stained with eight serogroup 1-specific monoclonal antibodies, the staining patterns could be clustered in five distinct groups. These hybridomas thus represent an unlimited source of standard reagent that could be used in the detection and serogrouping of L. pneumophila; differences in staining patterns could be used as epidemiological markers for these bacteria.     

Epidemiologic investigation by macrorestriction analysis and by using monoclonal antibodies of nosocomial pneumonia caused by Legionella pneumophila serogroup 10.

A 67-year-old woman was hospitalized with an acute pneumonia of the left lower lobe. Legionella pneumophila serogroup 10 was cultured from two sputum specimens taken on days 18 and 20 and was also detected by direct immunofluorescence assay by using a commercially available species-specific monoclonal antibody as well as serogroup 10-specific monoclonal antibodies. Antigenuria was detected in enzyme-linked immunosorbent assays by using serogroup 10-specific polyclonal and monoclonal antibodies. In the indirect immunofluorescence test rising antibody titers against serogroups 1, 4, 5, 8, 9, 10, 14, and 15 were found in serum, with the highest titers found against serogroups 8, 9, and 10. L. pneumophila serogroups 10 and 6 and a strain that reacted with serogroup 4 and 14 antisera were cultured from both central and peripheral hot water systems of the hospital. Macrorestriction analyses of the genomic DNAs by pulsed-field gel electrophoresis showed that the isolate from the patient was identical to the serogroup 10 strains from the hospital hot water system. In contrast, the genomic DNAs of 16 unrelated L. pneumophila serogroup 10 strains showed 12 different restriction patterns. Monoclonal antibody subtyping revealed only minor differences in L. pneumophila serogroup 10 strains isolated from different sources. In conclusion, macrorestriction analysis is a valuable tool for studying the molecular epidemiology of L. pneumophila serogroup 10.     

11th serogroup of Legionella pneumophila isolated from a patient with fatal pneumonia.

A Legionella-like organism (strain 797-PA-H; ATCC 43130) was isolated from a specimen taken from an endotracheal tube of a patient 4 days before death and from the left lung at autopsy. Growth characteristics were consistent with those for Legionella species. Strain 797-PA-H gave negative test results with available direct immunofluorescence assay conjugates and with slide agglutination test antisera prepared against the 22 Legionella species and 35 serogroups now recognized. Minimal reactivity (1 to 2 +) was observed with both tests by using reagents prepared against the Legionella-like organism Lansing 3. Reciprocal absorption studies, however, showed that the cross-reactive antibodies could be removed easily. Physiologic, gas-liquid chromatographic, and DNA hybridization tests revealed that the strain belonged to the species Legionella pneumophila. Therefore, strain 797-PA-H was designated as the type strain of a new L. pneumophila serogroup, serogroup 11.     

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.     

Australian isolates of Legionella longbeachae are not a clonal population.

Legionella longbeachae is almost as frequent a cause of legionellosis in Australia as Legionella pneumophila, but epidemiological investigation of possible environmental sources and clinical cases has been limited by the lack of a discriminatory subtyping method. The purpose of this study was to examine the genetic variability among Australian isolates of L. longbeachae serogroup 1. Pulsed-field gel electrophoresis (PFGE) of SfiI fragments revealed three distinct pulsotypes among 57 clinical and 11 environmental isolates and the ATCC control strains of L. longbeachae serogroups 1 and 2. Each pulsotype differed by four bands, corresponding to <65% similarity. A clonal subgroup within each pulsotype was characterized by >88% similarity. The largest major cluster was pulsotype A, which included 43 clinical isolates and 9 environmental isolates and was divided into five subgroups. Pulsotypes B and C comprised smaller numbers of clinical and environmental isolates, which could each be further divided into three subgroups. The ATCC type strain of L. longbeachae serogroup 1 was classified as pulsotype B, subtype B3, while the ATCC type strain of L. longbeachae serogroup 2 was identified as a different pulsotype, LL2. SfiI macrorestriction analysis followed by PFGE showed that the Australian L. longbeachae strains are not a single clonal population as previously reported.     

Characteristics of anti-Legionella antibodies in patients infected with Legionella pneumophila serogroups 1, 6, and 10.

Nosocomial infections with Legionella pneumophila serogroups 1 and 10 in the Leiden University Hospital and infections with L. pneumophila serogroup 6 in neighboring hospitals gave us an opportunity to study the development of opsonizing antibodies against L. pneumophila serogroups 1, 6, and 10 in the serum of 13 patients. Seven of these patients were infected with L. pneumophila serogroup 1, two were infected with serogroup 6, and four were infected with serogroup 10. The opsonic cross-reactivity of antibodies against these serogroups of L. pneumophila and complement involvement in opsonization were also investigated. Convalescent-phase sera from patients infected with L. pneumophila serogroup 1 or 6 were able to promote ingestion of these serogroups by polymorphonuclear leukocytes, whereas ingestion of L. pneumophila serogroup 10 was enhanced only in the presence of convalescent-phase sera from patients infected with this serogroup. Opsonization of L. pneumophila serogroups 1, 6, and 10 was complement dependent.     

Molecular fingerprinting of Legionella species by repetitive element PCR.

Repetitive element PCR (rep-PCR) uses outward-facing primers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphisms of rep-PCR amplification products can serve as strain-specific molecular fingerprints. Primers directed at the repetitive extragenic palindromic element were used to characterize isolates of Legionella pneumophila and other Legionella species. Substantial variation was seen among the rep-PCR fingerprints of different Legionella species and serogroups. More limited, but distinct, polymorphisms of the rep-PCR fingerprint were evident among epidemiologically unrelated isolates of L. pneumophila serogroup 1. Previously characterized Legionella isolates from nosocomial outbreaks were correctly clustered by this method. These results suggest the presence of repetitive extragenic palindromic-like elements within the genomes of members of the family Legionellaceae that can be used to discriminate between strains within a serogroup of L. pneumophila and between different Legionella species. rep-PCR appears to be a useful technique for the molecular fingerprinting of Legionella species.     

Evaluation of the Merifluor-Legionella immunofluorescent reagent for identifying and detecting 21 Legionella species.

We evaluated a 33-valent polyclonal indirect immunofluorescent-reagent kit (Merifluor-Legionella; Meridian Diagnostics Inc., Cincinnati, Ohio) made for the detection of Legionella species by testing bacterial isolates, seeded sputum, and negative sputum samples. Use of the reagent according to the directions of the manufacturer gave false-negative staining of homologous culture isolates due to a prozone phenomenon; this was solved by diluting test strain suspensions. After this change in testing protocol was made, the reagent gave bright fluorescent staining with 31 of the 33 Legionella strains with which it supposedly reacts. Strongly reacting Legionella strains included the type strains of L. pneumophila serogroups 1 to 10, L. longbeachae serogroups 1 and 2, and serogroup 1 of L. anisa, L. bozemanii, L. cherrii, L. dumoffii, L. gormanii, L. hackeliae, L. jamestowniensis, L. jordanis, L. maceachernii, L. micadedi, L. oakridgensis, L. rubrilucens, L. sainthelensi, L. spiritensis, L. steigerwaltii, and L. wadsworthii. Type strains of L. erythra and L. feeleii fluoresced only dimly with the reagent. Of 10 non-Legionella bacteria known to cross-stain with other polyvalent antisera, 5 also cross-reacted with the Merifluor reagent; these included 3 Bacteroides fragilis and 2 Pseudomonas fluorescens strains. The lower limit of detection of L. pneumophila serogroup 1 in seeded sputum was about 5 x 10(4) to 5 x 10(5) cells per ml. None of 21 randomly collected sputum specimens tested contained fluorescing legionellalike organisms, but 6 specimens did contain brightly fluorescing bacteria atypical in morphology for Legionella species. The Merifluor-Legionella kit appears to perform as well as other polyclonal immunofluorescent reagents used for detection of Legionella species. Because of the cross-reactions observed, which are common to all polyclonal reagents, utilization of this reagent for either bacterial identification or detection must be performed in combination with culture.