Legionella-like Organisms Research Articles

Genetic speciation of environmental Legionella isolates in Thailand

Legionella-like organisms were isolated during 2003–2007 from various water resources by culturing on selective media of Wadowsky–Yee–Okuda agar. The 256 isolates were identified as belonging to the Legionella genus based on detection of 108bp PCR product of the 5S rRNA gene, while the inclusion as Legionella pneumophila were confirmed by PCR detection of a specific mip gene region of 168bp. The 50 isolates, identified as non-pneumophila, were then subjected to DNA tree analysis, based on mip gene of ∼650bp and rnpB genes product ranged from 304 to 354bp. Phylogenetic tree was constructed to predict their species in relative to the available database. The isolates of which their speciation, based on those two genes were inconclusive, were then investigated for the almost full-length of 16S rRNA sequences. The isolates were assigned as 16 known Legionella species, and proposed seven novel species based on their unique 16S rRNA sequence.

Studies of environmental water samples for different parameters for isolation of Legionella spp.

Legionella pneumophila has frequently been isolated from patients with Legionnaires disease and in several instances also from epidemic-related environmental samples. A selective medium was developed and used successfully to isolate Legionella pneumophila and Legionella-like organisms from environmental specimens animal inoculation methods. Legionella pneumophila is a Gram-negative, facultative, intracellular bacterium that is the causative agent of Legionnaires' disease. This medium consists of charcoalyeast extract agar to which have been added cephalothin (4µg/mL), colistin (16µg/ mL), vancomycin (0.5µg/mL), and cycloheximide (80µg/mL). Pretreating of the environmental water samples with an acid buffer (pH 2.2), followed by plating on the selective medium, improved the rate of recovery of both Legionella and Legionella-like organisms relative to that with direct plating on selective media.

Rare occurrence of heterotrophic bacteria with pathogenic potential in potable water

Since the discovery of Legionella pneumophila, an opportunistic pathogen that is indigenous to water, microbiologists have speculated that there may be other opportunistic pathogens among the numerous heterotrophic bacteria found in potable water. The US Environmental Protection Agency (USEPA) developed a series of rapid in vitro assays to assess the virulence potential of large numbers of bacteria from potable water to possibly identify currently unknown pathogens. Results of surveys of potable water from several distribution systems using these tests showed that only 50 of the approximately 10,000 bacterial colonies expressed one or more virulence characteristics. In another study, 45 potable water isolates that expressed multiple virulence factors were tested for pathogenicity in immunocompromised mice. None of the isolates infected mice that were compromised either by treatment with carrageenan (CG), to induce susceptibility to facultative intracellular pathogens, or by cyclophosphamide (CY), to induce susceptibility to extracellular pathogens. These results indicate that there are very few potential pathogens in potable water and that the currently developed in vitro virulence screening tests give an overestimation of the numbers of heterotrophic bacteria that may be pathogens. Current efforts are focused on using the animal models to screen concentrated samples of waters known to contain large numbers of heterotrophic bacteria and newly discovered Legionella-like organisms that parasitize amoebae.

Isolation of Legionella anisa from multiple sites of a hospital water system: the eradication of Legionellacontamination.

For the prevention of nosocomial cases of legionellosis in the Ryukyu University Hospital neonatal wards, we examined nine shower units and a sink tap water unit for the presence of Legionella, over a 6-year period. We isolated Legionella-like organisms (LLO) from showerheads by culturing sediments from the water samples on buffered charcoal yeast extracts (BCYE). We used DNA-DNA hybridization to determine that the organisms were L. anisa. A fingerprinting technique called random amplified polymorphism DNA (RAPD) also showed that all the organisms were identical at the genome level. Replacement of the shower heads harboring colonies of L. anisa prevented further contamination. Nosocomial cases of legionellosis have not been reported from the wards during the period of this survey. This is the first description of the isolation of L. anisa from multiple sites within a hospital, and RAPD analysis suggests that these may be the spread of a single clone.

Legionella taurinensis sp. nov., a new species antigenically similar to Legionella spiritensis.

A group of 42 Legionella-like organisms reacting specifically with Legionella spiritensis serogroup 1 antisera were collected throughout Europe by the Centre National de Référence (French National Reference Centre) for Legionella. This group of isolates differed somewhat from L. spiritensis in terms of biochemical reactions, ubiquinone content and protein profile. The latter two analyses revealed that one of these L. spiritensis-like isolates, Turin I no. 1T, was highly related, but not identical to any of the red autofluorescent species of Legionella. In fact, this strain was the first of these particular isolates recognized to emit a red autofluorescence when exposed to UV light. Profile analysis of randomly amplified polymorphic DNA established that the red autofluorescent L. spiritensis-like isolates constituted a homogeneous group distinct from Legionella rubrilucens and Legionella erythra. DNA-DNA hybridization studies involving the use of S1 nuclease confirmed that the indicated group of isolates are a new species of Legionella, for which the name Legionella taurinensis is proposed with strain Turin I no. 1T (deposited as ATCC 700508T) as the type strain.

Legionella waltersii sp. nov. and an unnamed Legionella genomospecies isolated from water in Australia.

Two Legionella-like organisms were isolated from water samples obtained in Adelaide, Australia. One organisms was isolated from a drinking water distribution system, and the other was isolated from a cooling tower at a sewage treatment plant. Both strains required L-cysteine for growth and contained cellular branched-chain fatty acids and ubiquinones typical of the genus Legionella. These strains were serologically distinct from each other as determined by a slide agglutination test. STrain 2074-AUS-ET (T = type strain) was serologically distinct from all previously described Legionella species and serotypes. Strain 2055-AUS-E could not be differentiated biochemically or serologically from Legionella quinlivanii. Both strains were shown by DNA hybridization studies (Hydroxyapatite method) to be members of new Legionella species. Legionella waltersii sp. nov. is the name proposed for strain 2074-AUS-ET (= ATCC 51914T). L. waltersii was less than 10% related to other Legionella species. Strain 2055-AUS-E (= ATCC 51913) was informally named Legionella genomospecies 1, since it could not be phenotypically distinguished from L. quinlivanii. Legionella genomospecies 1 was closely related to L. quinlivanii strains (53 to 69% related with 4.5 to 6.5% divergence at 60 degrees C and 31 to 52% related at 75 degrees C).

Polymerase Chain Reaction法によるクーリングタワー水から分離した<I>Legionella pneumophila</I>の同定

We studied the polymerase chain reaction (PCR) method with 5 strains of Legionella pneumophila serogroup 4, 7 strains of L. pneumophila serogroup 5, 7 strains of L. pneumophila UT and 15 strains of Legionella like organisms (LLO). We used mip-primers reported by Mahbubani et al and LEG-primers reported by Starnbach et al. Positive PCR results were obtained by all strains of L. pneumophila serogroup 4, L. pneamophila serogroup 5, L. pneumophila UT and 11 strains of LLO using mip-primers. Using LEG-primers, positive results were obtained by 2, 6, 5 and 8 strains respectively. All strains that showed positive results using LEG-primer PCR, were contained by the strains that showed positive results using mip-primer PCR. Conversely, there were some strains that were positive in mip-primer PCR and negative in LEG-primer simultaneously. Therefore, mip-primers is recommended to detect L. pneumophila in clinical specimens by PCR method.

Legionella fairfieldensis sp. nov. isolated from cooling tower waters in Australia

Three Legionella-like organisms were isolated from water from the cooling towers of two Australian institutions. The strains grew on buffered charcoal-yeast extract (BCYE) agar but not on BCYE agar in the absence of L-cysteine. Gas-liquid chromatography profiles of the isolates were consistent with those for Legionella spp. They were serologically distinct from other legionellae in a slide agglutination test. DNA hybridization studies showed that the three isolates belong to a new species of Legionella, Legionella fairfieldensis (ATCC 49588).

Problems associated with identification of Legionella species from the environment and isolation of six possible new species

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.

Legionella sainthelensi serogroup 2 isolated from patients with pneumonia

Three Legionella-like organisms isolated from patients with pneumonia are shown to belong to the species Legionella sainthelensi by DNA hybridization studies and to a new serogroup, serogroup 2, by serological studies (ATCC 49322). L. sainthelensi serogroup 2 and L. santicrusis are indistinguishable bu slide agglutination, but are separable on the basis of their cell wall fatty acid profiles.

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.

Isolation of Legionella pneumophila from hotels of Greece.

Twenty water samples collected from 6 hotels situated in various areas of Greece were examined for the presence of Legionella pneumophila and Legionella-like organisms. Five of the six hotels included in this investigation were associated with cases of legionellosis. Legionella pneumophila serogroups 1 and 8 were isolated from four of six hotels, mainly from the hot water supply system. This is the first isolation and identification of L. pneumophila in Greece.

Legionella moravica sp. Nov. and Legionella brunensis sp. Nov. Isolated from cooling-tower water

Two Legionella-like organisms were isolated from cooling-tower water samples in Czechoslovakia. They were presumptively identified as legionellae by their growth on buffered charcoal-yeast extract agar (BCYE) containing L-cysteine and their absence of growth on BCYE without L-cysteine. Both strains contained predominantly branch-chained cellular fatty acids and were therefore definitively placed in the genus Legionella. They were serologically distinct from other described Legionella species and were shown by DNA studies to constitute two new Legionella species, Legionella moravica (type strain 316-36; ATCC 43877) and Legionella brunensis (type strain 441-1; ATCC 43878). Two Legionella-like organisms were isolated from cooling-tower water samples in Czechoslovakia. They were presumptively identified as legionellae by their growth on buffered charcoal-yeast extract agar (BCYE) containing L-cysteine and their absence of growth on BCYE without L-cysteine. Both strains contained predominantly branch-chained cellular fatty acids and were therefore definitively placed in the genus Legionella. They were serologically distinct from other described Legionella species and were shown by DNA studies to constitute two new Legionella species, Legionella moravica (type strain 316-36; ATCC 43877) and Legionella brunensis (type strain 441-1; ATCC 43878).

Legionella moravica sp. Nov. and Legionella brunensis sp. Nov. Isolated from cooling-tower water

Two Legionella-like organisms were isolated from cooling-tower water samples in Czechoslovakia. They were presumptively identified as legionellae by their growth on buffered charcoal-yeast extract agar (BCYE) containing L-cysteine and their absence of growth on BCYE without L-cysteine. Both strains contained predominantly branch-chained cellular fatty acids and were therefore definitively placed in the genus Legionella. They were serologically distinct from other described Legionella species and were shown by DNA studies to constitute two new Legionella species, Legionella moravica (type strain 316-36; ATCC 43877) and Legionella brunensis (type strain 441-1; ATCC 43878).

Legionella pneumophila serogroup 14 isolated from patients with fatal pneumonia.

Two Legionella-like organisms, one isolated from postmortem lung tissue and the other from a bronchial aspirate, were shown by growth, physiologic, and genetic characteristics to belong to the species Legionella pneumophila. Subsequent serologic testing indicated that both strains belonged to a new serogroup, serogroup 14.

Legionella anisa: a new species of Legionella isolated from potable waters and a cooling tower.

Between March 1980 and June 1981, five strains of Legionella-like organisms were isolated from water. Four were recovered from potable water collected from hospitals in Chicago, Ill., and Los Angeles, Calif., during outbreaks of nosocomial legionellosis. The fifth strain was isolated from water collected from an industrial cooling tower in Jamestown, N.Y. The strains exhibited biochemical reactions typical of Legionella species and were gram-negative motile rods which grew on buffered charcoal-yeast extract agar but not on blood agar, required cysteine, and were catalase positive, urease negative, nitrate negative, hippurate negative, and nonfermentative. All strains were positive for oxidase and beta-lactamase and produced a brown, diffusible pigment. Of the five strains, four exhibited blue-white autofluorescence under long-wavelength UV light. The fatty-acid composition and ubiquinone content of these strains were consistent with those of other Legionella species. Direct fluorescent-antibody examination of the five strains with conjugates to previously described Legionella species demonstrated no cross-reactions except with the conjugates to L. longbeachae serogroup 2 and L. bozemanii serogroup 2. Four strains gave a 4+ reaction to the L. longbeachae serogroup 2 conjugate and the fifth strain gave a 1+ reaction. Each of the five strains gave a 4+ reaction with the conjugate to L. bozemanii serogroup 2. DNAs from the five strains were highly related (84 to 99%) and showed 5 to 57% relatedness to other Legionella species. These strains constitute a new species in the genus Legionella, and the name Legionella anisa sp. nov. is proposed. The type strain of L. anisa is WA-316-C3 (ATCC 35292).

Capillary gas chromatographic analysis of carbohydrates of Legionella pneumophila and other members of the family Legionellaceae.

Legionella pneumophila, the causative agent of Legionnaires disease, and related organisms have previously been characterized primarily by conventional bacteriological methods, DNA-DNA hybridization, antigenic analysis, and fatty acid analysis. By capillary gas chromatographic analysis for carbohydrates, we have shown that muramic acid and glucosamine, characteristic markers of bacterial cell walls, were present in samples of L. pneumophila and a group of legionella-like organisms. Some bacterial samples contained two unusual isomeric aminodideoxyhexoses (X1 and X2). L. pneumophila was characterized by the absence of fucose and the presence of the peak X1. Tatlockia micdadei (Legionella micdadei) was distinguishable by the presence of large amounts of rhamnose and fucose and by the absence of X1 and X2. Fluoribacter strains were much more variable in their carbohydrate composition. These data suggest that, in addition to other reported techniques, carbohydrate profiling by capillary gas chromatography can be a valuable diagnostic method in reference microbiology laboratories for differentiating members of the family Legionellaceae.

The influence of surfactant (Tween) to the growth and producing activity of esterase of Legionella species and Legionella-like organisms

The influence of surfactant (Tween) to the growth and producing activity of esterase of Legionella species and Legionella-like organisms

Enhanced primary isolation of Legionella pneumophila from clinical specimens by low-pH treatment.

During the 12-month study period, 919 clinical specimens were submitted to the laboratory for culture of Legionella pneumophila. All specimens were plated onto nonselective and semiselective media both directly and after treatment with a KCl-HCl solution, pH 2.2. Of the 51 specimens (5.5%) positive for L. pneumophila and Legionella-like organisms, 20 were recovered by acid pretreatment only, 5 were recovered by direct plating only, and 26 were recovered by both acid pretreatment and direct inoculation. These data demonstrated that acid pretreatment substantially improved the recovery of L. pneumophila and Legionella-like organisms from the clinical specimens examined.

New members of the Coenzyme Q series from the Legionellaceae

Since the discovery of Legionella pneumophila, the causative agent of Legionnaires disease, two other groups of fastidious bacteria have been isolated which share many phenotypic characteristics with L. pneumophila [1-7] which have been referred to as Atypical Legionella-Like Organisms (ALLO) [2]. One group contains strains known as the Pittsburgh pneumonia agent (PPA) [6] and the Tatlock strain [8], for which the name Tatlockia micdadei has been proposed [4]. Members of the second group are serologically and genetically more diverse [2,3,7]. The name Fluoribacter [4] has been proposed for the latter group, and presently three species F. bozemanae, F. dumoffii and F. gormanii are recognised [4,7]. The taxonomic interrelationships of Legionella, Tatlockia and Fluoribacter are, however, equivocal, and additional criteria are required to tighten the generic and species descriptions. Ubiquinones (Coenzyme Q) are constituents of bacterial plasma membranes, and play an important role in electron transport and oxidative phosphorylation. Furthermore, recent studies have indicated that the structural variations exhibited by these compounds are of value in bacterial classification (see [9] for review). Ubiquinones, 2,3-dimethoxy-5-methyl-6-polyprenyl1,4-benzoquinones form a relatively large homologous series in which the length of the C-6 isoprenyl side-chain may vary from 1 to up to 12 isoprene units (Fig. 1). The major constituents of bacterial ubiquinones have been shown however to possess between 7 and 10 isoprene units (i.e. n = 7 to 10, Fig. 1) [9]. Recently, Finnerty et al. [10] reported Legionella pneumophila possessed ubiquinones as its sole respiratory quinones. Although the detailed structure of the ubiquinones of L. pneumophila was not determined, the authors noted that it possessed a side-chain containing more than 10 isoprene units [10]. This report is of considerable interest, as ubiquinone isoprenologues with more than 10 isoprene units have been found previously only in small amounts in a Pseudomonas sp. [11] and trace amounts in certain photosynthetic bacteria [ 12,13]. In this paper we report the structure of the ubiquinones of L. pneumophila. The opportunity was taken to also determine the ubiquinone content of other Legionella species, and representatives of the genera Fluoribacter and Tatlockia.