Gentamicin-Containing Peptone-Yeast Extract Medium for Cocultivation of Hartmannella vermiformis ATCC 50256 and Virulent Strains of Legionella pneumophila

We evaluated the use of peptone-yeast extract (PY) medium, different strains of Hartmannella vermiformis, and gentamicin in a coculture system to improve the discrimination of virulent and avirulent strains of Legionella pneumophila. H. vermiformis ATCC 50256 was unique among four strains of H. vermiformis, in that it multiplied equally well in Medium 1034 and PY medium (Medium 1034 without fetal calf serum, folic acid, hemin, and yeast nucleic acid and with a 50% reduction of peptone). However, both a virulent strain of L. pneumophila and its avirulent derivative strain multiplied in cocultures when PY medium was used. The multiplication of this avirulent strain was greatly reduced by incorporating gentamicin (1 (mu)g/ml) into the cocultivation system. Five virulent-avirulent sets of L. pneumophila strains were then tested for multiplication in cocultures with H. vermiformis ATCC 50256 and the gentamicin-containing PY medium. Only the virulent strains multiplied. The modified cocultivation system can discriminate between virulent and avirulent strains of L. pneumophila.     

Interferon-gamma-activated human monocytes inhibit the intracellular multiplication of Legionella pneumophila

We have examined the interaction between interferon-gamma (IFN-gamma)-activated human monocytes and Legionella pneumophila, the agent of Legionnaires' disease. Human monocytes activated with human recombinant IFN-gamma inhibit the intracellular multiplication of L. pneumophila. The degree of inhibition is proportional to the concentration of IFN-gamma, and maximal inhibition consistently occurs with greater than or equal to 2 micrograms/ml. Monoclonal anti-IFN-gamma antibody completely neutralizes the capacity of IFN-gamma to activate monocytes. Monocytes infected 24 hr after explantation maximally inhibit L. pneumophila multiplication if treated with IFN-gamma before infection or up to 2 hr after infection; treatment 6 hr or more after infection results in submaximal inhibition. Monocytes infected 48 hr after explantation inhibit L. pneumophila multiplication maximally if treated with IFN-gamma up to 12 hr before infection, but submaximally if treated at the time of infection. Once activated, monocytes inhibit L. pneumophila multiplication in the absence of IFN-gamma in the culture. Strikingly, monocytes maximally inhibit L. pneumophila multiplication after treatment with IFN-gamma for as briefly as 1 hr before infection. In the absence of anti-L. pneumophila antibody, neither IFN-gamma-activated monocytes nor nonactivated monocytes kill L. pneumophila. In the presence of specific antibody and complement, IFN-gamma-activated monocytes kill a proportion (0.5 log) of an inoculum but not more than nonactivated monocytes. L. pneumophila forms a specialized phagosome in IFN-gamma-activated monocytes that does not differ ultrastructurally from the L. pneumophila phagosome in nonactivated monocytes. These results demonstrate that IFN-gamma can activate human monocytes to exert a potent antimicrobial effect against a highly virulent intracellular bacterial pathogen. These findings extend previous observations on interactions between activated mononuclear phagocytes and L. pneumophila, and additionally support the hypothesis that cell-mediated immunity plays a major role in host defense against L. pneumophila.     

Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila.

At the site of a legionellosis outbreak, amoebae and two ciliates, Tetrahymena sp. and Cyclidium sp., were isolated from cooling-tower water containing Legionella pneumophila. The Tetrahymena sp. and the amoebae repeatedly showed the ability to support intracellular multiplication of L. pneumophila. Both were isolated from cooling towers specifically implicated as the source for the spread of legionellosis. These protozoa may be reservoirs supporting the survival and multiplication of virulent legionellae in cooling-tower water.     

Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila

At the site of a legionellosis outbreak, amoebae and two ciliates, Tetrahymena sp. and Cyclidium sp., were isolated from cooling-tower water containing Legionella pneumophila. The Tetrahymena sp. and the amoebae repeatedly showed the ability to support intracellular multiplication of L. pneumophila. Both were isolated from cooling towers specifically implicated as the source for the spread of legionellosis. These protozoa may be reservoirs supporting the survival and multiplication of virulent legionellae in cooling-tower water.     

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

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

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

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

Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellanii.

Survival and distribution of legionellae in the environment are assumed to be associated with their multiplication in amoebae, whereas the ability to multiply in macrophages is usually regarded to correspond to pathogenicity. Since most investigations focused on Legionella pneumophila serogroup 1, we examined the intracellular multiplication of different Legionella species in Mono Mac 6 cells, which express phenotypic and functional features of mature monocytes, and in Acanthamoeba castellanii, an environmental host of Legionella spp. According to the bacterial doubling time in Mono Mac 6 cells and in A. castellanii, seven clusters of legionellae could be defined which could be split further with regard to finer differences. L. longbeachae serogroup 1, L. jordanis, and L. anisa were not able to multiply in either A. castellanii or Mono Mac 6 cells and are members of the first cluster. L. dumoffi did not multiply in Mono Mac 6 cells but showed a delayed multiplication in A. castellanii 72 h after infection and is the only member of the second cluster. L. steigerwaltii, L. gormanii, L. pneumophila serogroup 6 ATCC 33215, L. bozemanii, and L. micdadei showed a stable bacterial count in Mono Mac 6 cells after infection but a decreasing count in amoebae. They can be regarded as members of the third cluster. As the only member of the fourth cluster, L. oakridgensis was able to multiply slight in Mono Mac 6 cells but was killed within amoebae. A strain of L. pneumophila serogroup 1 Philadelphia obtained after 30 passages on SMH agar and a strain of L. pneumophila serogroup 1 Philadelphia obtained after intraperitoneal growth in guinea pigs are members of the fifth cluster, which showed multiplication in Mono Mac 6 cells but a decrease of bacterial counts in A. castellanii. The sixth cluster is characterized by intracellular multiplication in both host cell systems and consists of several strains of L. pneumophila serogroup 1 Philadelphia, a strain of L. pneumophila serogroup 2, and a fresh clinical isolate of L. pneumophila serogroup 6. Members of the seventh cluster are a strain of agar-adapted L. pneumophila serogroup 1 Bellingham and a strain of L. pneumophila serogroup 1 Bellingham which was passaged fewer than three times on BCYE alpha agar after inoculation and intraperitoneal growth in guinea pigs. In comparison to members of the sixth cluster, both strains showed a slightly enhanced multiplication in Mono Mac 6 cells but a reduced multiplication in amoebae. From our investigations, we could demonstrate a correlation between prevalence of a given Legionella species and their intracellular multiplication in Mono Mac 6 cells. Multiplication of members of the genus Legionella in A. castellanii seems to be dependent on mechanisms different from those in monocytes.     

Legionella pneumophila inhibits acidification of its phagosome in human monocytes

We used quantitative fluorescence microscopy to measure the pH of phagosomes in human monocytes that contain virulent Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in these phagocytes. The mean pH of phagosomes that contain live L. pneumophila was 6.1 in 14 experiments. In the same experiments, the mean pH of phagosomes containing dead L. pneumophila averaged 0.8 pH units lower than the mean pH of phagosomes containing live L. pneumophila, a difference that was highly significant (P less than 0.01 in all 14 experiments). In contrast, the mean pH of phagosomes initially containing live E. coli, which were then killed by monocytes, was the same as for phagosomes initially containing dead E. coli. The mean pH of L. pneumophila phagosomes in activated monocytes, which inhibit L. pneumophila intracellular multiplication, was the same as in nonactivated monocytes. To simultaneously measure the pH of different phagosomes within the same monocyte, we digitized and analyzed fluorescence images of monocytes that contained both live L. pneumophila and sheep erythrocytes. Within the same monocyte, live L. pneumophila phagosomes had a pH of approximately 6.1 and sheep erythrocyte phagosomes had a pH of approximately 5.0 or below. This study demonstrates that L. pneumophila is capable of modifying the pH of its phagocytic vacuole. This capability may be critical to the intracellular survival and multiplication of this and other intracellular pathogens.     

A mathematical model of the growth of a population of Legionella pneumophila in the presence of Tetrahymena pyriformis protozoa]

The mathematical model describing the dynamics of the growth of L. pneumophila in aqueous environment in the presence of protozoa has been worked out. The model has demonstrated considerable heterogeneity of the initial population of virulent L. pneumophila strains. The number of bacteria capable of multiplication in Infusoria is no more than 0.1% of the initial population. The time of the generation of the infective agent inside Tetrahymena pyriformis is 2.8 hours.     

A note on the survival of Legionella pneumophila in stagnant tap water

Tap water, from an experimental hot water system, containing a known virulent strain of Legionella pneumophila was stored in screw-capped bottles for 14 months. Viable counts showed survival of L. pneumophila and at least three other bacterial species. This reinforces the view that L. pneumophila can survive in stagnant water foatively long periods of time.     

A note on the survival of Legionella pneumophila in stagnant tap water

Tap water, from an experimental hot water system, containing a known virulent strain of Legionella pneumophila was stored in screw-capped bottles for 14 months. Viable counts showed survival of L. pneumophila and at least three other bacterial species. This reinforces the view that L. pneumophila can survive in stagnant water for relatively long periods of time.     

An oxaloacetate decarboxylase homologue protein influences the intracellular survival of Legionella pneumophila

Abstract Legionella pneumophila is a facultative intracellular parasite which is able to survive in various eukaryotic cells. We characterised a Tn5-mutant of the L. pneumophila Corby strain and were able to identify the insertion site of the transposon. It is localised within an open reading frame which shows high homology to the α-subunit of the oxaloacetate decarboxylase (OadA) of Klebsiella pneumoniae . The OadA homologous protein of L. pneumophila was detected in the wild-type strain by Western blotting. Since the intracellular multiplication of the oad A⁻ mutant strain is reduced in guinea pig alveolar macrophages and human monocytes, it is concluded that the oad A gene product has an effect on the intracellular survival of L. pneumophila .     

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.     

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.     

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.     

The HL-60 model for the interaction of human macrophages with the Legionnaires' disease bacterium

The facultative intracellular pathogen, Legionella pneumophila, multiplies within and kills human monocytes and alveolar macrophages. We show that L. pneumophila strain Philadelphia-1 infects, multiplies within and kills the promyelocyte HL-60 cell line after its differentiation into macrophage-like cells. The characteristics of the interaction between L. pneumophila and differentiated HL-60 cells closely resemble those between L. pneumophila and human peripheral blood monocytes. With both cell types, C receptors and serum C mediate attachment of L. pneumophila, which are taken up by coiling phagocytosis. The replicative phagosome is lined with ribosomes; intracellular multiplication is iron-dependent; and replicating bacteria ultimately destroy the host cell. As in human monocytes, an avirulent mutant derivative of L. pneumophila Philadelphia-1, 25D, does not replicate in and is not cytopathic for differentiated HL-60 cells. Differentiated HL-60 cells therefore provide a convenient and faithful model for the study of L. pneumophila-mononuclear phagocyte interaction.     

The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum.

Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum. This compartment may constitute a rich source of nutrients for the bacteria and is probably recognized as cellular compartment. The remarkable similarity of the intracellular infections of macrophages and protozoa by L. pneumophila strongly supports the hypothesis that adaptation of the bacterium to the intracellular environment of protozoa may be the mechanism for its ability to adapt to the intracellular environment of human alveolar macrophages and causes pneumonia.     

Colonization of components of a model hot water system by Legionella pneumophila

A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.     

Legionella pneumophila infection induces programmed cell death,caspase activation,and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

Background

Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells.

Methods

Nuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila.

Results

The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells.

Conclusion

Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.