Characterization of the beta-lactamases of six species of Legionella.

The beta-lactamases of six Legionella species were characterized by isoelectric focusing, gel filtration, and substrate profiles. Fifteen strains of L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, and L. pneumophila produced beta-lactamases active against nitrocefin. L. micdadei enzymes previously reported to be beta-lactamase negative caused a very slow pH-dependent breakdown of nitrocefin and degraded penicillin G at high substrate concentrations. The bioassay revealed predominantly penicillinase activity for all species except L. micdadei, which had no activity in this assay. The apparent molecular weights of enzymes of L. bozemanii, L. gormanii, and L. pneumophila were in the range of 15,000 to 32,000, and those of L. micdadei and L. longbeachae were greater than 250,000. The isoelectric focusing of extracts of Legionella strains in polyacrylamide gels showed beta-lactamase types specific for species (L. bozemanii, L. gormanii, and L. pneumophila) and serotype (L. pneumophila). It demonstrated four different beta-lactamase types in L. pneumophila and revealed close relationships among L. pneumophila serotypes 1, 3, and 6. L. pneumophila enzymes formed band patterns only in polyacrylamide gels containing 6 M urea, whereas L. dumoffii and L. longbeachae enzymes did not form bands in any of the gels. None of the band patterns resembled those of known plasmid-mediated beta-lactamases. These experiments suggest that isoelectric focusing of chromosomal beta-lactamases may be a valuable tool for taxonomic studies.     

Biofilms as major sources of Legionella spp. in hydrothermal areas and their dispersion into stream water

Abstract Water and biofilms from two hydrothermal areas in central Portugal, and one hydrothermal area in New Mexico, USA, were examined for Legionella spp. In general, Legionella spp were isolated in higher numbers from biofilms than from water, although one biofilm with a temperature of 50°C, did not yield isolates of these organisms. In one area L. pneumophila serogroup (sg) 3 constituted the major population in the thermal discharge by the stream and the biofilm below it; however, L. pneumophila sg 1 was predominant in the sediments of the stream bed with minor thermal springs below the main discharge and in the water downstream. No Legionellae were isolated from water upstream of the hydrothermal area indicating that the thermal area was the source of the organisms in the stream water. In the other two hydrothermal areas, L. pneumophila sg 1 constituted the major population isolated, whereas L. pneumophila sg 3 was absent or isolated in low numbers. Isolates of L. micdadei were also recovered from one hydrothermal area, while ‘ L. londoniensis ’ was isolated from another.     

Antibodies isolated by using cloned surface antigens recognize antigenically related components of Legionella pneumophila and other Legionella species

We studied the antigenic cross-reactivity of surface proteins among various strains of Legionella pneumophila and other Legionella species by using a novel method of antibody purification. Anti-bacterial antibodies in hyperimmune sera were adsorbed to and eluted from the surface of recombinant E. coli cells that express individual L. pneumophila antigens on their surface. These affinity-purified antibodies were then used to probe protein immunoblots prepared from the test strains to detect cross-reactive domains. We found that antigenic proteins are generally conserved in all L. pneumophila serogroups. Although some of these antigenic domains are shared with members of other Legionella species, they are associated with proteins of different molecular mass. Our approach to the study of antigenic cross-reactivity has potential advantages over similar studies that use either monoclonal antibodies or monospecific antibodies prepared by immunization with purified antigens.     

Abundance and distribution of Legionellaceae in Puerto Rican waters

Waters in marine and freshwater areas of Puerto Rico were analyzed for the presence of Legionella spp. by direct fluorescent antibody assay with guinea pig confirmation. Several species, including L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, L. micdadei, and L. pneumophila, were widely distributed among all sites. Legionellaceae, including L. pneumophila, were found in high densities in water collected in the rain forest from epiphytes in trees 30 ft. (about 9.25 m) above the ground. Both interspecific and intersite variations were significant. L. pneumophila was the most abundant species at all sites, with average densities of 10(4) cells ml-1, very close to the range which is potentially pathogenic for humans. Densities of L. pneumophila were highest in sewage-contaminated coastal waters. These are the highest densities of Legionella spp. ever reported for marine habitats. Densities of L. pneumophila were positively correlated with concentrations of sulfates, phosphates, and pH. A survey of 88 fatal atypical pneumonia cases at a Puerto Rico hospital showed that 15% of the patients had L. pneumophila infections. This study establishes L. pneumophila as a relatively common cause of atypical pneumonia in Puerto Rico and suggests natural aquatic habitats as possible sources or reservoirs of pathogenic Legionella spp. in the tropics.     

Vaccination with the major secretory protein of Legionella induces humoral and cell-mediated immune responses and protective immunity across different serogroups of Legionella pneumophila and different species of Legionella.

In a previous study, we demonstrated that immunization of guinea pigs with the major secretory protein (MSP) of Legionella pneumophila, serogroup 1 induced humoral and cell-mediated immune responses to MSP and protective immunity against lethal aerosol challenge with this serogroup of L. pneumophila. Although serogroup 1 L. pneumophila cause most cases of Legionnaires' disease, other serogroups of L. pneumophila and species of Legionella cause many cases. In this study, we have examined if immunization with MSP induces humoral and cell-mediated immune responses and protective immunity across different serogroups of L. pneumophila and species of Legionella. By immunoblot analysis, MSP from L. pneumophila serogroup 1 (Lp1 MSP), L. pneumophila serogroup 6 (Lp6 MSP), and Legionella bozemanii (Lb MSP) shared common epitopes recognized by guinea pig anti-Lp1 MSP antiserum. These MSP molecules, however, were not identical as they had different apparent m.w. Immunization of guinea pigs with MSP induced strong cell-mediated immune responses across the different serogroups and species, as indicated by splenic lymphocyte proliferation and cutaneous delayed-type hypersensitivity in response to both homologous and heterologous MSP. Immunization with MSP induced strong protective immunity across two serogroups of L. pneumophila; overall, 9 survived aerosol challenge with L. pneumophila serogroup 1 compared to 0 of 12 (0%) sham-immunized control animals (p = 3 x 10(-4), Cochran-Mantel-Haenzel chi 2 statistic for pooled data). Immunization with MSP also induced protective immunity across species of Legionella but protection was species-specific. Whereas immunization with Lb MSP induced protective immunity against L. pneumophila, neither immunization with Lp1 MSP nor immunization with Lb MSP induced protective immunity against L. bozemanii, which produces MSP. Not surprisingly, immunization with MSP did not induce protective immunity against MSP-negative Legionella micdadei. In the case of both L. bozemanii and L. micdadei, immunization with a sublethal dose did confer protective immunity to aerosol challenge indicating that these species do contain immunoprotective components. This study demonstrates that immunization with MSP induces humoral and cell-mediated immune responses across different serogroups of L. pneumophila and species of Legionella, but that the capacity of MSP immunization to induce protective immunity is species-specific. Nevertheless, an MSP vaccine has the potential to induce protective immunity against the great majority of cases of Legionnaires' disease.     

Distribution of Legionella spp. in hydrothermal areas in continental Portugal and the island of São Miguel, Azores.

Nineteen aquatic environment sites from three hydrothermal areas on continental Portugal and one area on the island of São Miguel, Azores, were examined for the recovery of Legionella spp. Physicochemical and bacteriological parameters were also determined for each site. Water temperatures varied between 22 and 67.5 degrees C, although the majority had temperatures above 40 degrees C; the pH varied between 5.5 and 9.2. The number of Legionella spp. recovered varied between 5.0 x 10(2) and 2.3 x 10(6) CFU/liter. A total of 288 isolates from 14 sites were identified by indirect immunofluorescence assay. The majority of the isolates belonged to Legionella pneumophila (74.3%), of which most belong to serogroup 1, but the relative proportion of L. pneumophila serogroups varied considerably. L. pneumophila serogroup 1 constituted 96.2% of the isolates in area 2 from central Portugal, but no isolates of this serogroup were recovered from São Miguel, where serogroup 6 strains were the predominant isolates. Ninety-six percent of the L. pneumophila serogroup 1 isolates belonged to monoclonal antibody subgroups OLDA and Bellingham. Other species identified were L. bozemanii serogroup 2, L. dumoffii, L. micdadei, L. moravica, L. oakridgensis, L. sainticrucis, and L. sainthelensi. Two undescribed species, which react by indirect immunofluorescence assay to antisera to "L. londoniensis" and "L. nautarum" and a group of isolates with strong cross-reaction to L. cincinnatiensis/L. sainticrucis/L. longbeachae by indirect immunofluorescence assay were also recovered. The latter were the only isolates recovered from area 3, in east central Portugal, over a period of 1 year.     

Abundance and distribution of Legionellaceae in Puerto Rican waters.

Waters in marine and freshwater areas of Puerto Rico were analyzed for the presence of Legionella spp. by direct fluorescent antibody assay with guinea pig confirmation. Several species, including L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, L. micdadei, and L. pneumophila, were widely distributed among all sites. Legionellaceae, including L. pneumophila, were found in high densities in water collected in the rain forest from epiphytes in trees 30 ft. (about 9.25 m) above the ground. Both interspecific and intersite variations were significant. L. pneumophila was the most abundant species at all sites, with average densities of 10(4) cells ml-1, very close to the range which is potentially pathogenic for humans. Densities of L. pneumophila were highest in sewage-contaminated coastal waters. These are the highest densities of Legionella spp. ever reported for marine habitats. Densities of L. pneumophila were positively correlated with concentrations of sulfates, phosphates, and pH. A survey of 88 fatal atypical pneumonia cases at a Puerto Rico hospital showed that 15% of the patients had L. pneumophila infections. This study establishes L. pneumophila as a relatively common cause of atypical pneumonia in Puerto Rico and suggests natural aquatic habitats as possible sources or reservoirs of pathogenic Legionella spp. in the tropics.     

Cytolytic and phospholipase C activity in Legionella species

To examine one possible mechanism of damage to leucocytes and tissue cells in legionellosis, seven species of Legionella were examined for cytolytic activity and for elaboration of phospholipase C, an enzyme that can damage mammalian cell membranes. Cytolysis was assessed using erythrocytes in agar. Phospholipase C was assayed by release of p-nitrophenol from p-nitrophenylphosphorylcholine and of tritiated phosphorylcholine from L-alpha-dipalmitoyl-[choline-methyl-3H]phosphatidylcholine. L. pneumophila, L. bozemanii, L. micdadei, L. dumoffii, L. gormanii, L. longbeachae and L. jordanis all lysed dog red blood cells, which have a high ratio of membrane phosphatidylcholine to sphingomyelin. The same strains hydrolysed varying amounts of p-nitrophenylphosphorylcholine; L. bozemanii exhibited the greatest activity. L. pneumophila, L. bozemanii, L. dumoffii, L. longbeachae and L. jordanis, but not L. micdadei, released tritiated phosphorylcholine from labelled substrate. These results indicate that several species of Legionella possess cytolytic capability; exotoxins with activity may play a role.     

Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever

Abstract Sera from patients suffering from Mediterranean spotted fever (i.e. an infection due to Rickettsia conorii ) were studied by immunoblot to investigate cross-reactivity. A prevalence of IgM antibodies to Proteus OX 19, Proteus 0X 2, to the Rickettsia typhus group, to Legionella pneumophila serovars 4 and 5, to L. bozemanii Wiga and to L. micdadei Tatlock was found. Western blot confirmed that the antibodies were directed against the lipopolysaccharide as demonstrated by proteinase K digestion of the antigens. Cross-adsorptions showed that there is a common cross-reacting epitope among L. bozemanii Wiga, R. typhi and Proteus OX 19 but cross-reacting antibodies to L. micdadei and OX 2 were distinct and independent. This IgM cross-reaction could lead to a misdiagnosis.     

Distribution of Legionella spp. in hydrothermal areas in continental Portugal and the island of S?o Miguel, Azores.

Nineteen aquatic environment sites from three hydrothermal areas on continental Portugal and one area on the island of S?o Miguel, Azores, were examined for the recovery of Legionella spp. Physicochemical and bacteriological parameters were also determined for each site. Water temperatures varied between 22 and 67.5 degrees C, although the majority had temperatures above 40 degrees C; the pH varied between 5.5 and 9.2. The number of Legionella spp. recovered varied between 5.0 x 10(2) and 2.3 x 10(6) CFU/liter. A total of 288 isolates from 14 sites were identified by indirect immunofluorescence assay. The majority of the isolates belonged to Legionella pneumophila (74.3%), of which most belong to serogroup 1, but the relative proportion of L. pneumophila serogroups varied considerably. L. pneumophila serogroup 1 constituted 96.2% of the isolates in area 2 from central Portugal, but no isolates of this serogroup were recovered from S?o Miguel, where serogroup 6 strains were the predominant isolates. Ninety-six percent of the L. pneumophila serogroup 1 isolates belonged to monoclonal antibody subgroups OLDA and Bellingham. Other species identified were L. bozemanii serogroup 2, L. dumoffii, L. micdadei, L. moravica, L. oakridgensis, L. sainticrucis, and L. sainthelensi. Two undescribed species, which react by indirect immunofluorescence assay to antisera to "L. londoniensis" and "L. nautarum" and a group of isolates with strong cross-reaction to L. cincinnatiensis/L. sainticrucis/L. longbeachae by indirect immunofluorescence assay were also recovered. The latter were the only isolates recovered from area 3, in east central Portugal, over a period of 1 year.     

Cytolysin as an immunoserological marker of Legionella pneumophila

Coagglutination test (CoaT), radioimmunoassay (RIA) and ELISA were used for detection of cytolysin produced by L. pneumophila. The sensitivity of RIA was 100 ng/ml, CoaT--10-20 ng/ml, ELISA--1 ng/ml. To determine cytolysin production among various strains and species of Legionella, the authors studied bacterial ultrasonic lysates. All 11 strains of L. pneumophila tested were cytolysin-positive. L. longbeachae, L. bozemanii and L. dumoffii strains failed to produce cytolysin. The authors believe that an antigen of cytolysin can be used for identification of L. pneumophila.     

Rapid detection of the gene of Legionella pneumophila using the fluorescence polarization with the asymmetric PCR.

We attempted the rapid detection method of Legionella pneumophila by the asymmetric PCR and the fluorescence polarization. Eleven extracted DNAs from L. pneumophila serogroup 1 to approximately 6, L. bozemanii, L. dumoffii, L. gormanii, L. micdadei, and Pseudomonas aeruginosa were amplified by asymmetric PCR, and the polarization of those products were measured. Only the polarization of L. pneumophila serogroup 1 to approximately 6 rose within a few minutes after the beginning of measurement. The sensitivity to L. pneumophila using this method was 10(3) cells.     

Humoral immunity indices of chronic alcoholism patients

Sera obtained from 642 patients with chronic alcoholism and 400 sera from healthy donors, collected in different regions of the USSR (Moscow, the Kirghiz SSR, Transcarpathian Province, the northern Caucasus), were studied. Out of all patients with chronic alcoholism whose blood sera were examined, persons aged 20-30 years constituted 22%, 31-40 years, 41.8%, 41-50 years, 27.5%, above 50 years, 8.7%. The results of the study of the immune structure of these patients with respect to the causative agents of respiratory diseases revealed a large proportion of persons seropositive to influenza virus B Leningrad/369/75 (92-93%) and coronavirus OC-43 (78-93%). Patients with chronic alcoholism were shown to belong to a high risk group with respect to Legionella pneumophila infection. The proportion of persons with antibodies to L. pneumophila among such patients (up to 21.1%) was considerably higher than among healthy donors (not more than 6%). In different regions of the USSR large risk groups with respect to diphtheria (42-56%) and tetanus (12-45%) were detected among patients with chronic alcoholism. These patients also showed considerably higher levels of anti-tissue antibodies in comparison with the healthy population.     

A phylogenetic analysis of Legionella

Four species of Legionella, L. pneumophila NCTC 11192, L. bozemanii NCTC 11368, L. micdadei NCTC 11371 and L. jordanis ATCC 33623 have been characterized by oligonucleotide cataloguing of their 16S ribosomal RNA. All four species are phylogenetically closely related, while no specific relationship could be detected with any other group of organisms investigated so far with respect to this method. At a low level of relationship legionellae are members of the broad group of purple photosynthetic bacteria and their non-phototrophic relatives, in which Legionella form an independent line of descent.     

Development and validation of ELISA for detection of antibodies to Legionella pneumophila serogroup 1, 3 and 6 in human sera

The aim of this study was to determine whether separate measurement of immunoglobulin (Ig) M and G antibodies to Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6 as single antigens can facilitate an early diagnosis of Legionnaires' disease. The developed ELISA was evaluated and compared with an in-house indirect Legionella immunofluorescence antibody test (IFAT) measuring Total Ig. A total of 193 sera from 128 patients with confirmed L. pneumophila infections were used to assess the sensitivity of the developed ELISA. The sensitivity was assessed in different time-periods after onset of symptoms. It was found that the sensitivity of the test increased during the first month of infection, IgM being the most sensitive; ranging from 13% in the first week after onset of symptoms, 45% in the second week to 84% in the third week; in the fourth (and beyond) week a drop to 67% was observed. The IFAT detecting L. pneumophila sg 1-6 had a sensitivity of 11%, 27%, 80% and 59%, respectively, during these time-periods. The test with the lowest sensitivity was the IgG ELISA (0%, 21%, 36% and 52%), but by combining the IgG results with the IgM results, the overall sensitivity of the assay was improved (13%, 48%, 88% and 70%).This study confirms that detection of IgG and IgM antibodies by ELISA is an important diagnostic tool especially during the initial phase of the disease, when supported by other tests like the urinary antigen test, PCR or culture.Furthermore, we showed that the ELISA is suitable for the detection of significant changes in antibody levels in paired serum samples. It was found that the sensitivity was higher for the ELISA assays than for the IFAT. Both the in-house IgM ELISA and the IFAT had a low false positive rate, while a 14% false positive rate was found for the IgG ELISA among serum samples from patients with other infections.     

Differentiation of Legionella species by soluble protein patterns in polyacrylamide slab gels

Water-soluble protein extracts were obtained from representative strains of eleven species of Legionella and examined by polyacrylamide slab gel electrophoresis (PAGE). The eleven species included: L. pneumophila (serogroups 1-9), L. micdadei, L. longbeachae (serogroups 1 and 2), L. gormanii, L. dumoffii, L. bozemanii, L. jordanis, L. oakridgensis, L. wadsworthii, L. feeleii and L. maceachernii. Species identification was verified by gas-liquid chromatography of the non-hydroxy, monohydroxy and dihydroxy fatty acids. Strains within each species shared common patterns of protein banding. Each species had a distinctive protein banding pattern. Results indicate that PAGE of water soluble protein extracts is a useful technique for identification of Legionella isolates.     

Cross-reactivity and sensitivity of two Legionella urinary antigen kits, Biotest EIA and Binax NOW, to extracted antigens from various serogroups of L. pneumophila and other Legionella species

Legionella antigen detection kits for diagnosing legionellosis from urine have become widely used, but basic information about reactivity of the kits to non-serogroup (SG) 1 L. pneumophila and other Legionella species remains incomplete. We evaluated Biotest EIA and the most recently developed Binax NOW by using in-vitro extracted antigens of 22 L. pneumophila SG 1 to 15 strains and of 27 other Legionella species. Both kits showed excellent sensitivity to L pneumophila SG 1 antigens, but reacted to different sets of non-SG I L. pneumophila with different sensitivity. No cross-reactivity was observed to Legionella species other than L. pneumophila.     

Legionella spp. in Puerto Rico cooling towers.

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Infection of Tetrahymena pyriformis by Legionella longbeachae and other Legionella species found in potting mixes.

All Legionella longbeachae strains, both serogroups of L. bozemanii, and three strains of L. anisa reproducibly infected washed Tetrahymena pyriformis at 30 degrees C. L. pneumophila serogroup 1 strains infected T. pyriformis less reproducibly than did L. longbeachae. Low-level concentrations of nutrients in cocultures inhibited infection. Four L. micdadei strains and L. anisa ATCC 35292 failed to infect T. pyriformis.     

Legionella spp. in Puerto Rico cooling towers

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.