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).     

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).     

Characterization of the Legionella anisa population structure by pulsed-field gel electrophoresis

We analysed 38 French isolates of Legionella anisa by means of pulsed-field gel electrophoresis (PFGE) with single or double digestion. Double digestion was more discriminatory than single digestion, and can thus be useful for epidemiological studies of L. anisa. Several isolates from different parts of France clustered together on the basis of their PFGE patterns (similarity cutoff of 80%), suggesting that the L. anisa population structure is homogenous or that a few clones of L. anisa strains have spread widely in France.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis.

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.     

Multiplication of Legionella spp. in tap water containing Hartmannella vermiformis

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.     

Interspecies sequence differences in the Mip protein from the genus Legionella: implications for function and evolutionary relatedness

The nucleotide sequence of the mip genes and their inferred amino acid sequences were determined from 35 Legionella species and compared with the published sequences for L . pneumophila , L . micdadei and L . longbeachae . The sequences were 69–97% conserved at the nucleotide level and 82–99% at the amino acid level, with total conservation of amino acids determined to be associated with sites known to be involved in peptidyl prolyl cis–trans isomerase activity. No apparent difference could be determined in the arrangement of amino acids that would predict a functional difference in Mip from species associated with disease and Mip from species isolated only from the environment. Additionally, a phylogenetic comparison of the sequences with published 16S RNA sequences, using both genetic distance and maximum parsimony methods, was performed. Few relationships were apparent that were well supported by both data sets, the most robust being a clade comprising {[( cincinnatiensis , longbeachae , sainthelensi , santicrucis ) gratiana ] ( moravica , quateirensis , shakespearei , worsleiensis ) anisa , bozemanii , cherrii , dumoffii , gormanii , jordanis , parisiensis , pneumophila , steigerwaltii , tucsonensis , and wadsworthii  }.     

Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis

In a series of experiments, we have determined that Legionella pneumophila will proliferate as an intracellular parasite of the ciliated holotrich Tetrahymena pyriformis in sterile tap water at 35 degrees C. After 7 days of incubation, serpentine chains of approximately 10(3) L. pneumophila cells were observed throughout the cytoplasm of the protozoan infected initially with 1 to 30 L. pneumophila cells. The overall L. pneumophila population increased from ca. 1.0 X 10(2) to ca. 5.0 X 10(4) cells per ml in the coculture within this time frame. The interactions between the protozoan and the bacterium appear to depend upon their concentrations as well as temperature of incubation. L. pneumophila did not multiply in sterile tap water alone, in suspensions of lysed T. pyriformis, or in cell-free filtrates of a T. pyriformis culture. In addition to establishing an ecological model, we found that addition of T. pyriformis to environmental specimens served as an enrichment method that improved isolation of legionella from the specimens.     

Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis.

In a series of experiments, we have determined that Legionella pneumophila will proliferate as an intracellular parasite of the ciliated holotrich Tetrahymena pyriformis in sterile tap water at 35 degrees C. After 7 days of incubation, serpentine chains of approximately 10(3) L. pneumophila cells were observed throughout the cytoplasm of the protozoan infected initially with 1 to 30 L. pneumophila cells. The overall L. pneumophila population increased from ca. 1.0 X 10(2) to ca. 5.0 X 10(4) cells per ml in the coculture within this time frame. The interactions between the protozoan and the bacterium appear to depend upon their concentrations as well as temperature of incubation. L. pneumophila did not multiply in sterile tap water alone, in suspensions of lysed T. pyriformis, or in cell-free filtrates of a T. pyriformis culture. In addition to establishing an ecological model, we found that addition of T. pyriformis to environmental specimens served as an enrichment method that improved isolation of legionella from the specimens.     

Legionella sainthelensi: a new species of Legionella isolated from water near Mt. St. Helens

Six strains of a new species, Legionella sainthelensi, were isolated from freshwater in areas affected by the volcanic eruptions of Mt. St. Helens in the state of Washington. Strains of L. sainthelensi are culturally and biochemically similar to other legionellae. They grow on buffered charcoal yeast agar but not on media that lack cysteine. They are gram-negative, nonsporeforming, motile rods that are positive in reactions for catalase, oxidase, gelatin liquefaction, and beta-lactamase. They are negative in reactions for urease, hydrolysis of hippurate, reduction of nitrates, fermentation of glucose, and blue-white autofluorescence. Their cell wall fatty acid composition is qualitatively similar to those of other legionellae, with 50 to 62% branched-chain fatty acids. They contain the isobranched-chain 14- and 16-carbon acids and anteisobranched-chain 15- and 17-carbon acids and relatively large amounts of straight-chain 16-carbon acid. All strains of L. sainthelensi contain approximately equal amounts of ubiquinones Q9, Q10, Q11, and Q12, a pattern similar to those of Legionella bozemanii, Legionella dumoffi, and Legionella longbeachae. Serological cross-reactions were observed between L. sainthelensi, both serogroups of L. longbeachae, and Legionella oakridgensis. Three strains of L. sainthelensi were greater than 90% related by DNA hybridization. The type strain of L. sainthelensi, Mt. St. Helens 4, was 36% related to the type strain of L. longbeachae and 3 to 14% related to the other nine described Legionella species.     

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.     

Legionella sainthelensi: a new species of Legionella isolated from water near Mt. St. Helens.

Six strains of a new species, Legionella sainthelensi, were isolated from freshwater in areas affected by the volcanic eruptions of Mt. St. Helens in the state of Washington. Strains of L. sainthelensi are culturally and biochemically similar to other legionellae. They grow on buffered charcoal yeast agar but not on media that lack cysteine. They are gram-negative, nonsporeforming, motile rods that are positive in reactions for catalase, oxidase, gelatin liquefaction, and beta-lactamase. They are negative in reactions for urease, hydrolysis of hippurate, reduction of nitrates, fermentation of glucose, and blue-white autofluorescence. Their cell wall fatty acid composition is qualitatively similar to those of other legionellae, with 50 to 62% branched-chain fatty acids. They contain the isobranched-chain 14- and 16-carbon acids and anteisobranched-chain 15- and 17-carbon acids and relatively large amounts of straight-chain 16-carbon acid. All strains of L. sainthelensi contain approximately equal amounts of ubiquinones Q9, Q10, Q11, and Q12, a pattern similar to those of Legionella bozemanii, Legionella dumoffi, and Legionella longbeachae. Serological cross-reactions were observed between L. sainthelensi, both serogroups of L. longbeachae, and Legionella oakridgensis. Three strains of L. sainthelensi were greater than 90% related by DNA hybridization. The type strain of L. sainthelensi, Mt. St. Helens 4, was 36% related to the type strain of L. longbeachae and 3 to 14% related to the other nine described Legionella species.     

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.     

聚合酶链反应-酶切分型鉴定广州地区环境水源军团菌

【目的】探讨聚合酶链反应-酶切分型在快速鉴定环境水源军团菌方面的应用价值,并了解广州地区环境水源军团菌的分布状况。【方法】对广州地区采集的44份环境水样,作军团菌分离培养,再对分离菌株进行16Sr DNA PCR-酶切分型鉴定、16S rDNA基因测序和mip基因测序鉴定。【结果】在广州地区环境水源分离的112株军团菌,经聚合酶链反应-酶切分型鉴定、16S rDNA基因测序和mip基因测序鉴定,检出嗜肺军团菌66株,非嗜肺军团菌46株,其中菲氏军团菌20株,戈氏军团菌17株,橡树岭军团菌7株,长滩军团菌2株。【结论】聚合酶链反应-酶切分型检测环境水源军团菌是一种简便、快速、特异的鉴定方法;在广州地区环境水源中普遍存在军团菌,主要是嗜肺军团菌,其次是菲氏军团菌,戈氏军团菌,橡树岭军团菌和长滩军团菌。     

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.     

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.     

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.     

Biofilm formation by Legionella spp. in experiment

Ability of biofilm formation was studied in 28 strains belonging to 12 species of Legionella. Optimal conditions for formation of biofilms were ascertained using reference strain Legionella pneumophila Philadelphia 1. Comparative assessment of the ability of Legionella spp. to form biofilms was performed by cultivation in proteosopepton broth (for 96 hours) and in water (for up to 2 weeks). Highest rates of biofilm formation were observed for strains of L. pneumophila and L. longbeachae. Between L. pneumophila strains the most prominent ability to form biofilms was observed in newly isolated strains BLR-05 and TOTAL 1. Opportunity to use different ability of Legionella species to biofilm formation as a epidemiologically significant marker and for modeling of biofilms of Legionella in association with other microorganisms was discussed.     

Genomic heterogenicity amongst phenotypically similar Legionella micdadei strains

Abstract Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, β-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both Sfi I and Not I restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei .     

Temperature-dependent expression of flagella in Legionella

Legionella pneumophila, the causative agent of Legionnaires' disease, was analysed by electron microscopy for production of surface structures. Crystalline surface (S-) layers and fimbriae were not detected, but monotrichous flagellation was seen. Polyclonal antibodies specific for the 47 kDa flagellin subunit of L. pneumophila Philadelphia I were used in Western blots to confirm the presence of flagella subunits in various L. pneumophila strains tested, but the antiserum also reacted with flagellin subunits of L. micdadei, L. hackelia [serogroup (SG) 1 and SG2] and L. longbeachae (SG2). Flagellation of Legionellae was shown to be temperature regulated. When the growth temperature of virulent and avirulent of strain L. pneumophila Philadelphia I was shifted from 30 degrees C to either 37 or 41 degrees C, a decrease in the percentage of flagellated bacteria within the population was observed.