Distribution of Legionella longbeachae serogroup 1 and other legionellae in potting soils in Australia.

Legionella longbeachae serogroup 1 and other Legionella spp. were isolated from 73% of 45 potting soils made in Australia by 13 manufacturers but were not detected in 19 potting soils made in Greece, Switzerland, and the United Kingdom examined between March 1989 and May 1990. Several Legionella species were isolated from a small number of samples of uncomposted pine sawdusts, but it is not known whether sawdust was the source of some of the legionellae found in potting soils. Legionella spp. persisted for periods ranging from 3 to 10 months in a potting soil held at temperatures between -20 and 35 degrees C. Isolates of L. longbeachae serogroup 1 from soil did not grow at 43 degrees C, a temperature which was also lethal for this species in soil. Most Legionella spp. isolated from potting and natural soils belonged to one distinct group according to analysis of ubiquinones and were serologically related to several known species in this group. A small number of potting soils contained L. pneumophila and L. micdadei.     

Isolation of Legionella longbeachae serogroup 1 from potting mixes

Following a statewide outbreak of legionellosis due to Legionella longbeachae serogroup 1 in South Australia in 1988 and 1989, studies were performed to find a source of the organism. A number of water and soil samples with and without acid decontamination were examined for L. longbeachae by using a selective medium containing vancomycin, aztreonam, and pimafucin. There were no isolations of L. longbeachae from water samples. Organisms resembling L. longbeachae were isolated from a number of samples of potting mixes and from soil surrounding plants in pots collected from the homes of four patients. The organisms were found to persist for 7 months in two potting mixes stored at room temperature. Legionellae were isolated with difficulty from potting mixes which were allowed to dry out. Identification of isolates as L. longbeachae serogroup 1 was confirmed by quantitative DNA hybridization and serological tests. Restriction-fragment-length-polymorphism studies showed minor differences between patient and environmental isolates but differentiated these readily from L. longbeachae serogroup 2 and other antigenically related legionellae. The isolation of L. longbeachae from some potting mixes and the prolonged survival of the organisms in this medium suggest that soil rather than water is the natural habitat of this species and may be the source of human infections.     

Isolation of Legionella longbeachae serogroup 1 from potting mixes.

Following a statewide outbreak of legionellosis due to Legionella longbeachae serogroup 1 in South Australia in 1988 and 1989, studies were performed to find a source of the organism. A number of water and soil samples with and without acid decontamination were examined for L. longbeachae by using a selective medium containing vancomycin, aztreonam, and pimafucin. There were no isolations of L. longbeachae from water samples. Organisms resembling L. longbeachae were isolated from a number of samples of potting mixes and from soil surrounding plants in pots collected from the homes of four patients. The organisms were found to persist for 7 months in two potting mixes stored at room temperature. Legionellae were isolated with difficulty from potting mixes which were allowed to dry out. Identification of isolates as L. longbeachae serogroup 1 was confirmed by quantitative DNA hybridization and serological tests. Restriction-fragment-length-polymorphism studies showed minor differences between patient and environmental isolates but differentiated these readily from L. longbeachae serogroup 2 and other antigenically related legionellae. The isolation of L. longbeachae from some potting mixes and the prolonged survival of the organisms in this medium suggest that soil rather than water is the natural habitat of this species and may be the source of human infections.     

Distribution of legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates

A hospital warm water system was monitored for the presence and distribution of legionellae. Subtyping of ten selected Legionella pneumophila isolates, originating from four different sites in the system by using serogroup specific antisera in an indirect immunofluorescence test, revealed that nine of the ten isolates belong to serogroup 6, while the remaining one was serogroup 10. Two monoclonal antibodies (mAbs) specific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reacted with these mAbs. Genome analysis by elaborating NotI profiles using the pulsed field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates derived from different sites, including a new building connected by a ring pipe, were identical according to restriction fragment patterns. The patterns were distinguishable from those of the two L. pneumophila serogroup 6 reference strains, and from that of the L. pneumophila serogroup 10 isolate. These data argue for a relatively homogeneous L. pneumophila serogroup 6 population in the entire water system.     

Genetic relatedness of Legionella longbeachae isolates from human and environmental sources in Australia.

The genetic relatedness of Legionella longbeachae isolated in Australia since 1987 was investigated by restriction fragment length polymorphism (RFLP) analysis and allozyme electrophoresis. Three radiolabeled probes were used in Southern hybridizations for the RFLP studies. They were Escherichia coli 16S and 23S rRNA and cloned fragments of L. longbeachae selected empirically from genomal banks in lambda and a cosmid. The legionellae included in the study comprised 11 Legionella longbeachae serogroup 1 organisms isolated from humans, 28 L. longbeachae serogroup 1 isolates from environmental sources, and 3 L. longbeachae serogroup 2 environmental isolates. These were compared with the American Type Culture Collection reference strains of both serogroups and some other related Legionella species. Results of allozyme and RFLP analysis showed that all the isolates from humans and all but three of the environmental L. longbeachae serogroup 1 isolates were closely related. They were also closely related to L. longbeachae serogroup 1 ATCC 33462. There was wider variation among the three L. longbeachae serogroup 2 environmental isolates. One of these was closely related to L. longbeachae serogroup 2 ATCC 33484. RFLP studies with the rRNA probe provided the most discrimination among isolates but did not distinguish between the two serogroups.     

Genetic characterization of Legionella pneumophila serogroup 1 associated with respiratory disease in Australia.

Techniques were developed for genetic characterization of Legionella pneumophila serogroup 1 by using restriction fragment length polymorphism analysis. Allozyme analysis provided an index of the discrimination achieved by restriction fragment length polymorphism. Isolates from human cases of legionellosis were examined by both methods, and their profiles were compared with reference strains of L. pneumophila serogroup 1 obtained from the American Type Culture Collection. Eighteen distinct clones were evident among the isolates examined. Both methods could be used to trace the source of an outbreak of legionellosis caused by L. pneumophila serogroup 1.     

Chemical composition of lipopolysaccharides from Legionella bozemanii and Legionella longbeachae

Lipopolysaccharides (LPS) from Legionella bozemanii serogroup 1 and Legionella longbeachae serogroup 1 were subjected to chemical analyses. The lipid A part of both LPSs contained 2,3-dideoxy-2,3-diamino-d-glucose as major constituents and d-glucosamine and glycerol as minor constituents of the sugar backbone structure. Both LPSs exhibited a very complex fatty acid composition. Twenty amide-linked 3-hydroxy fatty acids were detected in LPS of L. longbeachae, whereas seventeen were encountered in LPS of L. bozemanii. Both LPSs contained nine ester-linked nonhydroxy fatty acids and the unique long-chain fatty acids 27-oxo-octacosanoic acid, 29-oxotriacontanoic acid, heptacosane-1,27-dioic acid and nonacosane-1,29-dioic acid. SDS-PAGE showed that L. bozemanii produced smooth-form LPS, whereas L. longbeachae LPS was mainly of the R-type. Composition analyses were in accordance with these electrophoretic patterns. d-Quinovosamine and l-fucosamine constituted 80 mol% of the polysaccharide part of L. bozemanii LPS. Other sugars identified were d-glucosamine, d-mannose, d-glucose, l-rhamnose, d-glycero-d-manno-heptose, l-glycero-d-mannoheptose, 2-keto-3-deoxy-octonic acid and glycerol. The polysaccharide chain from LPS of L. longbeachae appeared to be shorter, but composed of the same sugars except l-fucosamine. Both LPSs contained glycerol phosphate and glucosamine phosphate and L. longbeachae LPS contained in addition glucose phosphate.Abbreviations EI Electron impact - GlcN3N 2,3-Diamino-2,3-dideoxy-d-glucose - HPAEC High pH anion-exchange chromatography - Kdo 2-Keto-3-deoxy-octonic acid - LPS Lipopolysaccharide - PCP Phenol/chloroform/petroleum ether solvent - PED Pulsed electrochemical detection - PS Polysaccharide - TFA Trifluoroacetyl - TMS Trimethylsilyl     

Genetic characterization of Legionella pneumophila serogroup 1 associated with respiratory disease in Australia.

Techniques were developed for genetic characterization of Legionella pneumophila serogroup 1 by using restriction fragment length polymorphism analysis. Allozyme analysis provided an index of the discrimination achieved by restriction fragment length polymorphism. Isolates from human cases of legionellosis were examined by both methods, and their profiles were compared with reference strains of L. pneumophila serogroup 1 obtained from the American Type Culture Collection. Eighteen distinct clones were evident among the isolates examined. Both methods could be used to trace the source of an outbreak of legionellosis caused by L. pneumophila serogroup 1.     

Rapid enumeration of viable Legionella pneumophila serogroup 1

A rapid assay for the detection of viable Legionella pneumophila serogroup 1 was evaluated. A total of 431 environmental water samples were examined using an immunofluorescent assay (IFA) combined with the cell respiration stain iodonitrotetrazolium violet (INT) and the results compared with conventional culture. The IFA/INT assay was at least as sensitive and much quicker than culture for the detection of viable Legionella pneumophila serogroup 1 in most types of sample.     

Production of monoclonal antibodies against Legionella pneumophila serogroup 1

Four monoclonal antibodies to Legionella pneumophila Philadelphia 1 were produced by the fusion of immunized BALB/c lymphocytes to a murine myeloma cell line. Two (Lp1-1 and Lp1-3) of the four monoclonal antibodies reacted with 14 L. pneumophila serogroup 1 strains, and the other (Lp1-2 and Lp1-4) reacted with only three out of 20 strains tested. These four monoclonal antibodies did not bind to any strains of L. pneumophila serogroup 2-7 and other Legionella species. In addition, it has been shown that these monoclonal antibodies may be useful not only for subserotyping of L. pneumophila but also for retrospective diagnosis using immunopathological methods.     

Effects of three oxidizing biocides on Legionella pneumophila serogroup 1

A study was conducted to determine the bactericidal effects of ozone and hydrogen peroxide relative to that of free chlorine on Legionella pneumophila serogroup 1. In laboratory batch-type experiments, organisms seeded at various densities were exposed to different concentrations of these biocides in demand-free buffers. Bactericidal effects were measured by determining the ability of L. pneumophila to grow on buffered charcoal-yeast extract agar supplemented with alpha-ketoglutarate. Ozone was the most potent of the three biocides, with a greater than 99% kill of L. pneumophila occurring during a 5-min exposure to 0.10 to 0.30 micrograms of O3 per ml. The bactericidal action of O3 was not markedly affected by changes in pH or temperature. Concentrations of 0.30 and 0.40 micrograms of free chlorine per ml killed 99% of the L. pneumophila after 30- and 5-min exposures, respectively. A 30-min exposure to 1,000 micrograms of H2O2 per ml was required to effect a 99% reduction of the viable L. pneumophila population. However, no viable L. pneumophila could be detected after a 24-h exposure to 100 or 300 micrograms of H2O2 per ml. Attempts were made to correlate the biocidal effects of O3 and H2O2 with the oxidation of L. pneumophila fatty acids. These tests indicated that certain biocidal concentrations of O3 and H2O2 resulted in a loss or severe reduction of L. pneumophila unsaturated fatty acids.     

Effects of three oxidizing biocides on Legionella pneumophila serogroup 1.

A study was conducted to determine the bactericidal effects of ozone and hydrogen peroxide relative to that of free chlorine on Legionella pneumophila serogroup 1. In laboratory batch-type experiments, organisms seeded at various densities were exposed to different concentrations of these biocides in demand-free buffers. Bactericidal effects were measured by determining the ability of L. pneumophila to grow on buffered charcoal-yeast extract agar supplemented with alpha-ketoglutarate. Ozone was the most potent of the three biocides, with a greater than 99% kill of L. pneumophila occurring during a 5-min exposure to 0.10 to 0.30 micrograms of O3 per ml. The bactericidal action of O3 was not markedly affected by changes in pH or temperature. Concentrations of 0.30 and 0.40 micrograms of free chlorine per ml killed 99% of the L. pneumophila after 30- and 5-min exposures, respectively. A 30-min exposure to 1,000 micrograms of H2O2 per ml was required to effect a 99% reduction of the viable L. pneumophila population. However, no viable L. pneumophila could be detected after a 24-h exposure to 100 or 300 micrograms of H2O2 per ml. Attempts were made to correlate the biocidal effects of O3 and H2O2 with the oxidation of L. pneumophila fatty acids. These tests indicated that certain biocidal concentrations of O3 and H2O2 resulted in a loss or severe reduction of L. pneumophila unsaturated fatty acids.     

Differences in aerosol survival between pathogenic and non-pathogenic strains of Legionella pneumophila serogroup 1

A strain of Legionella pneumophila serogroup 1 known to be virulent for guinea-pigs was found to be least stable at a relative humidity (r.h.) of 60% when stored as a small particle aerosol. Three L. pneumophila serogroup 1 strains of different virulence for guinea-pigs were then tested at a r.h. of 60% at 20°C. The most virulent strain was found to have the best survival and the avirulent strain was least stable. The strain of intermediate virulence did not survive as well as the virulent strain but was more stable than the avirulent strain. Strains of L. pneumophila serogroup epidemiologically associated with legionnaires' disease had better survival in small particle aerosols than strains which were not associated with disease. Subtyping with monoclonal antibodies also showed that the type more commonly associated with disease survived longer in aerosols than the other subtypes.     

Differences in aerosol survival between pathogenic and non-pathogenic strains of Legionella pneumophila serogroup 1

A strain of Legionella pneumophila serogroup 1 known to be virulent for guinea-pigs was found to be least stable at a relative humidity (r.h.) of 60% when stored as a small particle aerosol. Three L. pneumophila serogroup 1 strains of different virulence for guinea-pigs were then tested at a r.h. of 60% at 20 degrees C. The most virulent strain was found to have the best survival and the avirulent strain was least stable. The strain of intermediate virulence did not survive as well as the virulent strain but was more stable than the avirulent strain. Strains of L. pneumophila serogroup epidemiologically associated with legionnaires' disease had better survival in small particle aerosols than strains which were not associated with disease. Subtyping with monoclonal antibodies also showed that the type more commonly associated with disease survived longer in aerosols than the other subtypes.     

A second serogroup of Legionella erythra serologically indistinguishable from Legionella rubrilucens

N.A. SAUNDERS, N. DOSHI AND T.G. HARRISON. 1992. Twenty-two red-autofluorescent Legionella strains were identified serologically as either Legionella rubrilucens or L. erythra. A rRNA probe was used for restriction fragment length polymorphism (RFLP) analysis of the strains and the patterns generated were used as an additional method of identifying the strains to species level. In two instances strains which were identified as L. rubrilucens by serology appeared to belong to the species L. erythra by RFLP analysis. This apparent contradiction was resolved by measurements of DNA/DNA homology which confirmed the existence of a second serogroup of L. erythra serologically indistinguishable from L. rubrilucens.     

Genotyping of Legionella pneumophila serogroup 1 strains isolated in Northern Sicily, Italy

During a three-year period, from April 2002 to May 2005, one hundred-forty-seven samples, taken from technical systems of water distribution at point of use, were repeatedly collected at six different sites in Northern Sicily and assayed for the presence of Legionella pneumophila serogroup 1 and serogroups 2 to 14. At the first samplings, the water distribution systems of all the sites were heavily contaminated, and disinfection treatments by the superheat and flush method were therefore performed. Treatments were always successful against L. pneumophila sg.1, but only in a few cases against all other serogroups. Eighty-six strains of L. pneumophila sg. 1, isolated from 26 of these samples, were characterized by amplified fragment length polymorphism (AFLP) analysis and sequence-based typing (SBT) procedure. Perfectly overlapping results were obtained by both the procedures and four genotypes were identified, accounting for all the isolates. The easy transferability of the SBT data through a web-based database made it possible to identify the presence in Northern Sicily of the two SBT types most commonly circulating in Europe.     

A second serogroup of Legionella erythra serologically indistinguishable from Legionella rubrilucens.

Twenty-two red-autofluorescent Legionella strains were identified serologically as either Legionella rubrilucens or L. erythra. A rRNA probe was used for restriction fragment length polymorphism (RFLP) analysis of the strains and the patterns generated were used as an additional method of identifying the strains to species level. In two instances strains which were identified as L. rubrilucens by serology appeared to belong to the species L. erythra by RFLP analysis. This apparent contradiction was resolved by measurements of DNA/DNA homology which confirmed the existence of a second serogroup of L. erythra serologically indistinguishable from L. rubrilucens.     

Detection of Legionella pneumophila serogroup 1 urinary antigen using an enhanced chemiluminescence ELISA

An enhanced chemiluminescence enzyme-linked immunosorbent assay has been developed for the detection of soluble antigen in the urine of patients with Legionnaires' disease (LD). In the assay antigen(s) in the urine samples are captured by a rabbit anti-L. pneumophila antibody coated onto microtitre strips. A fluorescein-isothiocyanate (FITC) conjugate of the same antibody is then added which binds to the captured antigen. Any immobilized FITC-labelled antibody is then detected with a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody. HRP activity is monitored after oxidation of luminol in the presence of H₂O₂ and iodophenol. The resulting luminescence is recorded using a camera luminometer. Urine specimens were available for testing from 31 patients with evidence of ongoing L. pneumophila serogroup 1 infection. A positive result was obtained in the cases of 12/12 specimens from culture-proven LD patients, and 16/19 specimens from patients with serological evidence of LD. Thus the sensitivity is estimated to be 28/31 (90%) The specificity was estimated using urine specimens from eight patients with non-L. pneumophila pneumonias of known aetiology. All eight specimens gave a negative result.     

Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species

Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light.