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PubMed1985

Effect of non-Legionellaceae bacteria on the multiplication of Legionella pneumophila in potable water.

A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.

Bacterial Physiological Phenomena↗

PubMed1981

Glycine-containing selective medium for isolation of Legionellaceae from environmental specimens.

Glycine, at a final concentration of 0.3%, has been shown to be an excellent selective agent for the isolation of Legionellaceae. Stock cultures of Legionella pneumophila were not inhibited on buffered charcoal-yeast extract agar containing the amino acid. Among the other Legionellaceae tested, only one of two strains of L. dumoffii and two of six strains of L. micdadei were appreciably inhibited. This medium permitted the isolation of L. pneumophila from environmental specimens with marked inhibition of many non-Legionellaceae bacteria. The selectivity of the medium was subsequently improved by the incorporation of vancomycin (5 microgram/ml) and polymyxin B (100 U/ml). This selective medium, glycine-vancomycin-polymyxin B agar, should facilitate the recovery of Legionellaceae from environmental sources.

Culture Media↗

PubMed1984

Agglutinating antibody titers to members of the family Legionellaceae in cystic fibrosis patients as a result of cross-reacting antibodies to Pseudomonas aeruginosa.

The objective of this study was to evaluate the prevalence and significance of antibody titers to organisms in the family Legionellaceae in 128 serum samples collected from cystic fibrosis patients at routine examinations. Antibody titers were determined for 10 antigenic types of Legionellaceae; Legionella pneumophila serogroups 1 to 6, Fluoribacter (Legionella) bozemanae, Fluoribacter (Legionella) dumoffii, Fluoribacter (Legionella) gormanii, and Tatlockia (Legionella) micdadei. The method of antibody titer determination was the microagglutination test. Elevated titers (greater than or equal to 1:64) to one or more antigens were found in 41.3% of cystic fibrosis patients but in only 9.7% of 103 normal control subjects (P less than 0.01). Titers to 8 of the 10 antigens were directly correlated with the number of Pseudomonas aeruginosa precipitating antibodies in patient sera, as determined by crossed immunoelectrophoresis (correlation coefficients, greater than or equal to 0.74). Cross-reactions between P. aeruginosa and L. pneumophila were substantiated by crossed immunoelectrophoresis of hyperimmune rabbit serum as well as patient sera against P. aeruginosa and Legionellaceae antigens. Monospecific antibody to the "common antigen" of P. aeruginosa was used to demonstrate the presence of this antigen in L. pneumophila. The presence of cross-reacting antibodies in cystic fibrosis patients chronically infected with P. aeruginosa emphasizes the need for cautious interpretation of antibody titers to members of the family Legionellaceae.

Adolescent↗

PubMed1984

Ecological distribution of Legionellaceae in the Quebec city area.

One hundred environmental water samples, which were collected in the Quebec city area and cultured on buffered charcoal yeast extract medium and three selective media, were inoculated to guinea pigs and were screened by direct immunofluorescent staining (DFA) for the presence of Legionellaceae. Six isolates were made (four Legionella pneumophila and two Tatlockia (Legionella) micdadei: three by animal inoculation and three by culture). No samples were simultaneously positive by both methods. After screening by DFA, 43 of the 100 samples were positive for Legionellaceae and 27 of those contained more than one serogroup and (or) species of Legionellaceae. Legionella pneumophila (serogroups 1 to 6) was the most frequent species seen by DFA. These results clearly show that Legionellaceae are frequent members of the freshwater microbial flora of the Quebec city area.

Culture Media↗

PubMed1996

Distribution of mip-related sequences in 39 species (48 serogroups) of Legionellaceae.

The macrophage infectivity potentiator gene (mip) from Legionella pneumophila is a major virulence factor of the species. Thus, mip-detection by amplification has been proposed to assess the presence of L. pneumophila in clinical and environmental samples. The distribution of mip-related sequences within the Legionellaceae was studied by DNA amplification using mip-specific primers followed by Southern blot hybridization with an internal probe. Thirty-nine species (48 serogroups) of Legionellaceae were screened in this attempt. Using this approach, sequences related to mip were observed in 89% of the tested species including the most recently described L. fairfieldensis, L. lansingensis and L. shakespearei. In several cases, cloning and sequencing of the amplified products confirmed the high levels of similarity between the sequence found in non-pneumophila species with that of the L. pneumophila mip gene. This confirms previous reports that mip related genes are widespread among Legionellaceae and therefore specific detection of the species L. pneumophila cannot be based on mip-targeted amplification.

Blotting, Southern↗

PubMed1995

In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy.

Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.

Animals↗

PubMed1984

Clinical laboratory differentiation of Legionellaceae family members with pigment production and fluorescence on media supplemented with aromatic substrates.

A systematic study of pigment production (browning) and fluorescence (extracellular yellow-green and intracellular blue-white) by nine Legionellaceae species was performed. A total of 56 strains representing Tatlockia micdadei (Pittsburgh pneumonia agent), Legionella pneumophila, Legionella jordanis, Legionella longbeachae, Legionella oakridgensis, Legionella wadsworthii, Fluoribacter bozemanae, Fluoribacter gormanii, and Fluoribacter dumoffii could be separated on media supplemented with tyrosine plus cystine, 3,4-diaminobenzoic acid, 3,5-diaminobenzoic acid, and 3-aminotyrosine. Parallel testing by hippurate hydrolysis and the bromocresol purple spot test enabled the identification of Legionellaceae species 24 to 72 h after primary isolation. This schema may be a practical alternative to species-specific antisera methods (slide agglutination or direct immunofluorescence) in the identification of members of the family Legionellaceae.

Amino Acids↗

PubMed2000

Sequencing of the rpoB gene in Legionella pneumophila and characterization of mutations associated with rifampin resistance in the Legionellaceae.

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis, was sequenced from 18 rifampin-resistant Legionella isolates representing four species (L. bozemanii, L. longbeachae, L. micdadei, and L. pneumophila), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae.

Amino Acid Sequence↗

PubMed1991

The use of 16S ribosomal RNA analyses to investigate the phylogeny of the family Legionellaceae.

The 16S ribosomal RNA sequences of Legionella pneumophila, L. erythra, L. hackeliae, L. spiritensis, L. longbeachae, L. bozemanii (Fluoribacter bozemanae) and L. micdadei (Tatlockia micdadei) were determined using reverse transcriptase. The sequences were compared with published sequences for Gram-negative bacteria and phylogenetic trees were constructed. The data confirm previous work which showed that the family Legionellaceae forms a monophyletic subgroup within the gamma subdivision of the Proteobacteria. The data show that all of the legionellae studied are highly related (greater than 95%) on the basis of 16S rRNA sequences and do not support the division of the family Legionellaceae into three genera.

Base Sequence↗

PubMed1986

Susceptibility of members of the family Legionellaceae to thermal stress: implications for heat eradication methods in water distribution systems.

To ascertain the feasibility of heat inactivation as an eradication method applicable to all members of the family Legionellaceae, we tested the heat resistance of 75 isolates which represented 19 members of this family of organisms. The ranges of thermal death times at 60, 70, and 80 degrees C were 1.3 to 10.6, 0.7 to 2.6, and 0.3 to 0.7 min, respectively. These data suggest that the method of heat eradication will be effective against all members of the family Legionellaceae.

Hot Temperature↗

PubMed1983

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.

Antigens, Bacterial↗

PubMed1985

[Oligodynamic action of 17 different metals on Bacillus subtilis, Enterobacteriaceae, Legionellaceae, Micrococcaceae and Pseudomonas aeruginosa].

The oligodynamic action of the pure metals aluminium, antimony, bismuth, cadmium, cobalt, copper, gold, iron, lead, manganese, mercury, nickel, platinum, silver, tin, titanium, and zinc on Bacillus subtilis (1 strain), Enterobacteriaceae (26 strains), Legionellaceae (13 strains), Micrococcaceae (6 strains), and Pseudomonas aeruginosa (4 strains) was investigated using an agar diffusion test. B. subtilis and Legionellaceae exhibited the highest susceptibility. The apathogenic micrococci and staphylococci were more susceptible to oligodynamic action of some metals than S. aureus. The group of gramnegative rods was the most resistant. The susceptibility of the different bacteria seems to be very different. Apart from the known oligodynamic action of some heavy metals the activity of antimony, cobalt, gold, and platinum should be mentioned.

Bacillus subtilis↗

PubMed1994

Legionellaceae in the potable water of Nova Scotia hospitals and Halifax residences.

Water was cultured from 39 of 48 hospitals (7 Halifax hospitals and 32 non-Halifax hospitals) in the province of Nova Scotia and from 90 residences (74 private dwellings, 16 apartments) in Halifax to determine the frequency of legionella contamination. Six of seven Halifax hospitals had Legionellaceae isolated from their potable water compared with 3 of 32 non-Halifax hospitals (P < 0.0001). Overall, 19 of 59 (32%) of the water samples from Halifax hospitals were positive for legionellae compared with 5 of 480 (1%) samples from non-Halifax hospitals (P < 0.0000). Five of the six positive Halifax hospitals had Legionella pneumophila serogroup 1 and 1 had L. longbeachae serogroup 2 recovered from their potable water. Legionella contamination was associated with older, larger (> or = 50 beds) hospitals with total system recirculation. These hospitals also had water with a higher pH and calcium content but lower sodium, potassium, nitrate, iron and copper content. Fourteen of the 225 (6.2%) water samples from Halifax residences were positive for legionellae -8% (6/74) of the single family dwellings were positive, compared with 25% (4/16) apartments. The positivity rate of 15.7% for the 19 electric hot-water heaters in Halifax homes was not significantly different from the 32% positivity for Halifax hospitals. L. longbeachae accounted for 2 of the 14 isolates of legionellae from Halifax homes.

Calcium↗

PubMed1995

Intergenic 16S rRNA gene (rDNA)-23S rDNA sequence length polymorphisms in members of the family Legionellaceae.

A method based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions was developed for the identification of species within the family Legionellaceae. The sizes of the PCR products varied from 1,353 to 350 bp. Strains of Legionella pneumophila were characterized as having products of approximately 900 and 530 bp, and L. birminghamensis had products of 1,390, 960, and 380 bp. Of the 38 species of legionellae examined, only 7 were indistinguishable (L. erythra from L. rubrilucens, L. anisa or L. cherrii from L. tucsonensis, and L. quateirensis from L. shakespearei). Two environmental isolates were identified as L. pneumophila. Strain LLAP-3, which was a symbiont of amoebae, could not be associated with any Legionella sp. studied.

Bacterial Typing Techniques↗

PubMed2001

Presence of Legionellaceae in warm water supplies and typing of strains by polymerase chain reaction.

Outbreaks of Legionnaire's disease present a public health challenge especially because fatal outcomes still remain frequent. The aim of this study was to describe the abundance and epidemiology of Legionellaceae in the human-made environment. Water was sampled from hot-water taps in private and public buildings across the area of Göttingen, Germany, including distant suburbs. Following isolation, we used polymerase chain reaction in order to generate strain specific banding profiles of legionella isolates. In total, 70 buildings were examined. Of these 18 (26%) had the bacterium in at least one water sample. Legionella pneumophila serogroups 1, 4, 5 and 6 could be identified in the water samples. Most of the buildings were colonized solely by one distinct strain, as proven by PCR. In three cases equal patterns were found in separate buildings. There were two buildings in this study where isolates with different serogroups were found at the same time.

Colony Count, Microbial↗

PubMed1989

Exposure to Legionellaceae at a hot spring spa: a prospective clinical and serological study.

Following the occurrence of five cases of Legionnaires' disease among patients and therapists at a French hot spring spa, a series of cleansing procedures and an epidemiological study were undertaken. During a 3-month period, the spring water was repeatedly sampled. Serum samples were taken from 689 randomly selected patients, 230 therapists, 134 administrative staff and a control group of 904 blood donors. Legionellaceae were present in the spring water at concentrations of 10(3)-10(5) colony forming units/l. Fifteen different species or serogroups were isolated with Legionella pneumophila serogroups 3 and 1 predominating. No clinical cases of Legionnaires disease were observed during the study. However, 11% of the therapists and 5% of the patients either had a high titre of antibody (greater than or equal to 256) to at least one species or serogroup or seroconverted during the study. Mean antibody titres in the three study groups were significantly higher than those in the blood donors against 11 of the 32 legionella antigens tested. Nine of these 11 antigens corresponded to species or serogroups isolated from the spring water. The highest mean antibody titres in all three study groups were against L. pneumophila serogroup 3, the most common legionella in the spring water. These findings have important implications for the maintenance of adequate standards of hygiene, bacteriological sampling and clinical surveillance in this and similar establishments.

Adult↗

PubMed1989

Disease due to the Legionellaceae (other than Legionella pneumophila). Historical, microbiological, clinical, and epidemiological review.

More than 20 species of the Legionellaceae family of bacteria have been discovered since the discovery of Legionella pneumophila. Thirteen have been implicated as causative agents of pneumonia including the Pittsburgh pneumonia agent (Tatlockia micdadei, Legionella micdadei). Although outbreaks of nosocomial pneumonia in immunosuppressed hosts have been well-described, most cases have occurred sporadically in the community. The spectrum of disease ranges from severe life-threatening pneumonia to a self-limiting febrile illness (Pontiac fever). Isolation from the natural aquatic environment has preceded its discovery as agents of human disease in 6 species, while environmental isolation has not yet been obtained for 3 species implicated in disease. The mode of transmission is uncertain, but cases of dual infection by L. pneumophila and the newer species suggests that the epidemiology of these new organisms will be similar to that of L. pneumophila. The antibiotic of choice appears to be erythromycin. The historical background, epidemiology, microbiology, and clinical manifestations of these newly-discovered organisms are reviewed in comparative fashion.

Humans↗

PubMed1987

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.

Analysis of Variance↗