A colony based confirmation assay for Legionella and Legionella pneumophila employing the EnviroAmp™Legionella system and seroagglutination

Modifications to the EnviroAmp^™ Legionella detection system are described which permit the rapid analysis of bacterial colonies taken from Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit. When PCR was positive for Leg. pneumophila , this result was confirmed by seroagglutination. The reverse dot blot hybridization assay was only used where PCR indicated a Legionella sp. other than Leg. pneumophila , permitting further savings on detection system components. This technique and standard confirmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except for two isolates which gave a positive result for Legionella spp. by PCR and hybridization but were negative using standard procedures. Raising the annealing/extension temperature of the PCR by 2 °C eliminated the false positive result with these two isolates but did not adversely effect the sensitivity of the assay, as determined by re-testing of 68 environmental isolates and testing of 69 new environmental isolates and 12 Legionella reference species. The modified technique provides a convenient and cost effective alternative to standard confirmation procedures.     

High diversity and abundance of Legionella spp. in a pristine river and impact of seasonal and anthropogenic effects

The diversity and dynamics of Legionella species along a French river watershed subject to different thermal and wastewater discharges during an annual cycle were assessed by 16S rRNA gene sequencing and by a fingerprint technique, single-strand conformation polymorphism. A high diversity of Legionella spp. was observed at all the sampling sites, and the dominant Legionella clusters identified were most closely related to uncultured bacteria. The monthly monitoring revealed that Legionella sp. diversity changes were linked only to season at the wastewater site whereas there was some evidence for anthropogenic effects on Legionella sp. diversity downstream of the thermal bath. Quantification of Legionella pneumophila and Legionella spp. by culture and quantitative PCR (qPCR) was performed. Whereas only L. pneumophila was quantified on culture media, the qPCR assay revealed that Legionella spp. were ubiquitous and abundant from the pristine source of the river to the downstream sampling sites. These results suggest that Legionella spp. may be present at significant concentrations in many more freshwater environments than previously thought, highlighting the need for further ecological studies and culturing efforts.     

The effect of Aeromonas strains on the growth of Legionella

Thirty-one environmental and two type strains of Aeromonas were tested for their ability to inhibit the growth of strains of several Legionella species. Of 10 Legionella spp. tested, only Legionella pneumophila and Leg. longbeachae were able to resist the inhibitory effects of some of the Aeromonas strains. Selected Aeromonas strains were also tested for their ability to inhibit the growth of several non-legionella strains. None of the non-legionella strains were inhibited by any of the selected Aeromonas strains. Attempts were made to isolate Aeromonas strains from cooling towers and Aeromonas hydrophila was isolated from one of the cooling towers tested. The results suggest that many strains of Aeromonas can inhibit the growth of Legionella strains on solid media and could affect the isolation of legionellas from water sources.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer.

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

The action of interferon preparations on strains of Legionella of various serogroups and species

The action of different preparations of interferon on Legionella strains has been studied in vivo and in vitro. The preparations of leukinferon at a concentration of 500 international units (I.U.) and reaferon at a concentration of 10,000 I.U. have been found to produce an inhibiting effect on Legionella strains in vitro, in a medium with carbon-yeast agar. Leukinferon at a concentration of 125 I.U. suppresses the growth of L. pneumophila also in a liquid medium. The preparation of leukinferon at a minimal concentration of 100 I.U. has been found to suppress the development of lethal infection in chick embryos infected with L. pneumophila strain Philadelphia 1.     

High-performance liquid chromatographic analysis of ubiquinones from new Legionella species

Abstract The ubiquinone composition of Legionella feeleii, L. oakridgensis, L. rubrilucens, L. sainthelensi, L. wadsworthii and some environmental legionella isolates was investigated by high performance liquid chromatography. The results of the present study indicate ubiquinones are valuable criteria for the rapid identification of Legionella species and the recognition of new taxa.     

嗜肺军团菌简易鞭毛染色法及在不同培养基上鞭毛的生长观察

本文报道了一种较为简易、快速的嗜肺军团菌 Legionella pneumophila 鞭毛染色法,并探讨了嗜肺军团菌在两种培养基(BCYE、NYE)上鞭毛的生长情况。实验结果提示这种鞭毛染色法有助于军团病的病原学诊断。     

Legionella pneumophila cell envelope: Permeability to hydrophobic molecules

Legionella pneumophila is sensitive to a number of toxic hydrophobic compounds. Suspensions of cells bound large amounts of the dye crystal violet, and disk agar diffusion assays confirmed the marked sensitivity to this compound. Fatty acids were also inhibitory to the growth ofL. pneumophila in liquid media, and growth inhibition increased with increasing chain length to a maximum with myristic acid. Oxygen uptake by respiring cells was inhibited by similar concentrations of fatty acids.L. pneumophila was also sensitive to low concentrations of progesterone. These results indicated thatL. pneumophila has an outer membrane with unusual permeability to hydrophobic compounds. This characteristic was accompanied by a measurable cell surface hydrophobicity as determined by adherence of the bacterium to the hydrocarbon hexadecane.     

Novel phospholipase A activity secreted by Legionella species

Bacterial phospholipases are regarded as a major virulence factor in infection. In bacteria associated with pneumonia, destruction of lung surfactant and host cell membranes by bacterial phospholipases secreted during infection is thought to contribute to the disease. Phospholipase C (PLC) activity has been described in several Legionella species (W. B. Baine, J. Gen. Microbiol. 134:489-498, 1988; W. B. Baine, J. Gen. Microbiol. 131:1383-1391, 1985). By using detection methods such as thin-layer chromatography and mass spectrometry, PLC activity could not be detected in several strains of Legionella pneumophila. Instead, phospholipid degradation was identified to be caused by a novel PLA activity. We could demonstrate that PLA secretion starts at the mid-exponential-growth phase when bacteria were grown in liquid culture. Several Legionella species secreted different amounts of PLA. Legionella PLA may act as a powerful agent in the mediation of pathogenicity due to destruction of lung surfactant and epithelial cells.     

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.     

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.     

Specific DNA probe for detecting Legionella pneumophila

A fragment of the gene for cytolysin has been cloned. The product of the gene has been earlier identified as an immunoserological marker of Legionella pneumophila. Clones were selected by immunodetection of cytolysin gene product expression. An EcoRI 3.8 kb fragment of the genomic DNA from Legionella pneumophila strain Philadelphia-1 was used as a DNA probe that hybridized with the bacterial colonies of 20 Legionella pneumophila strains but not with the colonies of 10 other Legionella species or 8 other bacterial genera. The cloned fragment has been shown to be unique for Legionella pneumophila. The region of homology is suggested to be longer than a possible dimension of the cytolysin gene.     

Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses

Legionella pneumophila is the causative pathogen of Legionnaires' disease, which is characterized by severe pneumonia. In regard to the pathophysiology of Legionella infection, the role of inflammatory phagocytes such as macrophages has been well documented, but the involvement of dendritic cells (DCs) has not been clarified. In this study, we have investigated the immune responses that DCs generate in vitro and in vivo after contact with L. pneumophila. Heat- and formalin-killed L. pneumophila, but not live L. pneumophila, induced immature DCs to undergo similar phenotypic maturation, but the secreted proinflammatory cytokines showed different patterns. The mechanisms of the DC maturation by heat- or formalin-killed L. pneumophila depended, at least in part, on Toll-like receptor 4 signaling or on Legionella LPS, respectively. After transfer to naive mice, DCs pulsed with dead Legionella produced serum Ig isotype responses specific for Legionella, leading to protective immunity against an otherwise lethal respiratory challenge with L. pneumophila. The in vivo immune responses required the Ag presentation of DCs, especially that on MHC class II molecules, and the immunity yielded cross-protection between clinical and environmental strains of L. pneumophila. Although the DC maturation was impaired by live Legionella, macrophages were activated by live as well as dead L. pneumophila, as evidenced by the up-regulation of MHC class II. Finally, DCs, but not macrophages, exhibited a proliferative response to live L. pneumophila that was consistent with their cell cycle progression. These findings provide a better understanding of the role of DCs in adaptive immunity to Legionella infection.     

Assessment of bactericidal activity of disinfectants against Legionella on the biofilm model

Comparative assessment of bactericidal activity of different disinfectants against Legionella biofilms was conducted. Monospecies biofilms of 3 strains of Legionella pneumophila obtained on plastic plates in stable conditions were used as models. It has been shown that for degradation of biofilms as well as for prophylactic action of disinfectants in preventing formation of biofilms on plastic surfaces, higher concentrations of preparations were needed as compared to their bactericidal concentrations for culture of Legionella determined by method of serial dilutions.     

Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay

The aim of this study was to develop a sensitive, cultivation-independent analytical method for Legionella in man-made water systems which can be performed within one day in crude sample extracts. The new assay for the genus Legionella is a paramagnetic bead based fluorescence sandwich hybridization assay (SHA) for the 16S rRNA based on two oligonucleotide probes which makes the method highly specific. An advantage over RT-PCR is the exclusive detection of viable cells and, due to the high number of 16S RNA molecules, the possibility to apply the method directly in crude cell extracts without prior purification of the nucleic acids. A high sensitivity was obtained by modifying the probe chemistry and hybridization conditions. The most sensitive assay uses a 3'-end biotin-labelled capture probe and a 3'-end DIG tailed detection probe and has a detection limit of 20 amol target molecules corresponding to 1.2x10(7) molecules of 16S rRNA and approximately 1800 Legionella cells. Using this assay type the number of Legionella cells was determined in Legionella contaminated water samples. The results show that the developed SHA can be applied for estimation of the approximate number of Legionella cells based on the number of 16S rRNA molecules in a water sample.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal.

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.     

Virulence factors of the family Legionellaceae.

Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of eukaryotic substrates, including phosphatidylinositol and tubulin. Since the phosphorylation of either phosphatidylinositol or tubulin might compromise phagocyte activation and bactericidal functions, this enzyme may well be a virulence factor. Administration of the L. pneumophila exoprotease induces lesions resembling those of Legionella pneumonia and kills guinea pigs, suggesting that this protein plays a role in the pathogenesis of legionellosis. However, recent work with a genetically engineered strain has convincingly shown that the protease is not necessary for intracellular survival or virulence. As might be expected with a complex process like intracellular parasitism, it appears that the capability of Legionella strains to invade and multiply in host phagocytes is multifactorial and that no single moiety which is responsible for the virulence phenotype will be found.     

Growth, respiration and survival of Legionella pneumophila at high temperatures

The optimum temperature for multiplication of legionella strains in culture media is around 37°C. The effect of high temperatures on the growth of strains isolated from various environments is poorly known. We studied the growth (cell multiplication, respiration) of clinical and environmental Legionella pneumophila strains in liquid media at intervals of 0.5°C in the temperature range from 41.6 to 51.6°C using a temperature gradient incubator. Cell multiplication and CO₂ production decreased markedly with all the strains at temperatures above 44–45°C. CO₂ continued to be produced up to 51.6C even if cell multiplication generally stopped at around 48.4–50.0C. Thus, legionella retained its metabolic activity beyond the maximum temperature for cell multiplication. The CO₂ production per bacterial cell (metabolic quotient, qCO₂) increased with increasing temperature up to 45°C, whereafter it decreased, the turning point being almost at the same at which the rate of cell multiplication decreased. The difference in qCO₂ between the strains may reflect their different physiological capacities for tolerating high temperatures.