1.1. Intracellular Multiplication of Legionellae Within Human Monocytes
Legionellae are important etiological agents of pneumonia. Legionella pneumophila (predominantly serogroup 1) is detected in most cases of legionellosis; other species only occasionally cause infections, predominantly in immunocompromized patients (1-4). Aquiferous technical systems are the primary source of infection (air-conditioning systems, refrigerators, showers, whirlpools, springs, taps, moisturizing equipment, medical nebulizers, and swimming pools). Legionellae are present in the water in these systems, within the amoebae, flagellates, and ciliates in which they replicate (2,5,6). After inhalation of contaminated aerosols, the bacteria multiply intracellularly within alveolar macrophages (7). The ability to multiply within monocytic host cells is usually considered to correspond to pathogenicity (8-13). The mechanisms of intracellular replication have been only partially characterized (recently summarized in ref. 14).
Analysis of the molecular pathogenesis of Legionella infection, both in the pathogen itself and in the host cell, is the subject of current research and may lead to new options in prophylaxis and treatment. We have established the human Mono Mac 6 cell line (MM6) (15) instead of the previously used histiocytic lymphoma cell line U 937 or the promyelocytic leukemia cell line HL-60 (16-18) to investigate the intracellular replication of legionellae and the molecular pathogenesis of Legionella infection within human monocytic host cells (19). MM6 cells represent a more mature mac-
rophage-like cell line that expresses phenotypic and functional properties of mature monocytes and that does not need to be stimulated by phorbol esters or 1,25-dihydroxyvitamin D3 (15,19). A good correlation between the prevalence of a given Legionella species and its intracellular multiplication in MM6 cells could be demonstrated.
In addition to Legionella, MM6 cells were found to support the intracellular growth of Mycobacterium tuberculosis (20) and Chlamydia pneumoniae (21), two other important bacterial agents involved in induction of pneumonia. Therefore, the MM6 model might be adaptable to investigations of the molecular pathogenesis of other intracellu-lar bacteria that can replicate within human monocytes and induce disease.
1.2. Intracellular Multiplication of Legionellae
Within Acanthamoeba castellanii as an Environmental Host
In the environment, legionellae survive by intracellular replication in protozoa. This intracellular replication seems to play a major role in bacterial ecology and pathogen-esis. Some intracellular events following infection are common to both amoebae and monocytes. To analyze the intracellular multiplication of different Legionella species within a typical environmental host, axenic cultures of A. castellanii are preferentially used. We were able to show that only certain Legionella species are able to multiply in A. castellanii, possibly because of a specialized adaption to the amoebal host (19).
Apart from legionellae, amoebae in the environment have also been found to harbor bacteria of several other pathogenic genera, including Burkholderia pickettii (22), Burkholderia cepacia (23), and Burkholderia pseudomallei (24), Chlamydia pneumoniae (25), Listeria (26), Mycobacterium avium (27,28), Pseudomonas aeruginosa (29), Vibrio cholerae (30), and E. coli (31). Rickettsiae are also presumed to be endosymbionts of amoebae (32). As shown for L. pneumophila, M. avium is more virulent in an mouse model after intracellular growth within A. castellanii (27). Therefore, the A. castellanii model to study the behavior of L. pneumophila in an environmental host might also be useful to investigate the interaction between other bacterial pathogens and protozoa.
The virulence of legionellae for human alveolar macrophages is considered to be a consequence of their evolution as a parasite of amoebae. In 1980, Rowbotham (33) published the first report on intracellular multiplication of L. pneumophila within Acanthamoeba spp. and Naegleria spp. (33). Thereafter, several reports described replication of Legionella culture isolates from clinical samples within protozoa isolated from the presumed source of infection (34-41). Intracellular growth within protozoa enhances the ability of L. pneumophila to infect human monocytes (42), induces phenotypic modulation (43,44), and causes resistance to chemical disinfectants, biocides, and antibiotics (45,46). Inhalation of legionellae packaged in amoebae results in induction of more severe clinical cases of legionellosis (33,47).
This speculation was supported by a recently published mouse model of coinhalation of L. pneumophila and Hartmanella vermiformis. Coinhalation with H. vermiformis significantly enhanced intrapulmonary growth of L. pneumophila, resulting in greater mortality in comparison with inhalation of legionellae alone (48). Intrapulmonary growth of mutant strains of L. pneumophila with reduced virulence for H. vermiformis but preserved virulence for monocytes was not significantly enhanced by coinhalation (49). Intrapulmonary growth of L. pneumophila was significantly greater in mice inoculated with L. pneumophila--infected H. vermiformis than in mice inoculated with an equivalent number of bacteria or coinoculated with L. pneumophila and uninfected H. vermiformis (50).
The mechanism of intrapulmonary growth enhancement of legionellae by amoebae remains to be determined. Amoeba-induced inhibition of proinflammatory cytokine production could be excluded, since coinhalation as well as inhalation of L. pneumophila-infected H. vermiformis induced significantly enhanced levels of interferon-y and tumor necrosis factor-a in A/J mice similar to the levels induced during replicative L. pneumophila infection alone (48). Three possible explanations for the potentiating effect of coinhalation for Legionella infection were discussed: modification of the host response to L. pneumophila infection by amoebae, the function of amoebae as implanted host cells, or the enhanced virulence of amoeba-associated bacteria (48).
We established an in vitro coculture model of MM6 cells, A. castellanii and Legionella species of different intracellular growth rates within human monocytes, to analyze the underlying molecular and biochemical mechanisms (51). In this coculture model, reactants were separated using a cell culture chamber system that separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximum 4-log multiplication within MM6 cells, which could not be further increased by coculture with A. castellanii, significantly enhanced replication of L. gormanii, L. micdadei, L. steigerwaltii, L. longbeachae, and L. dumoffii was noted after coculture with amoebae. This effect was seen only with uninfected but not with Legionella-infected amoebae. The supporting effect for intracellular multiplication in MM6 cells could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment of uninfected A. castellanii and Legionella-infected MM6 cells, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms that are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.
2.1. Culture of Legionella Bacteria
1. BCYE-a agar (Oxoid). Store at 4°C; pay attention to expiration date.
2. Legionella species can be obtained from the American Type Culture Collection (ATCC, Rockville, MD). Process bacteria according to the instructions of ATCC.
2.2. Culture of Host Cells
1. MM6 cells can be obtained from the Department of Human and Animal Cell Cultures (Mascheroder Weg 1b, D-38124 Braunschweig, Germany, Phone: +49-531-2616-161; fax: +49-531-2616-150). For details of culture conditions, see the homepage of Prof. Ziegler-Heitbrock (www.monocytes.de).
2. 75-cm3 vented culture flasks (Costar).
4. Fetal calf serum (FCS; Myoclone super plus; Gibco). The serum has to be inactivated (30 min, 56°C). After inactivation, every batch of serum must be checked for cytotoxicity (by Trypan Blue exclusion) since some batches may be cytotoxic for MM6 cells. After inactivation and cytotoxicity testing, store serum at -20°C until use. Compatible batches of sera should be ordered in sufficient amounts (e.g., 20 bottles).
5. 2 mM l-glutamine (Gibco). Store at -20°C until use. After defrosting, store at 4°C.
6. 1% Nonessential amino acids (Gibco). Store at 4°C.
7. Oxalacetate, pyruvate, and bovine insulin (OPI; Sigma). Store at -20°C until use.
8. Columbia blood agar (Oxoid). Store at 4°C; pay attention to expiration date.
9. Mycoplasma plus™ PCR (Stratagene).
2.2.2. Acanthamoeba castellanii
1. A. castellanii can be obtained from the ATCC (cat. no. 30234).
2. 75-cm3 Vented culture flasks (Costar).
4. Yeast extract (Difco).
8. Sodium citrate dihydrate (Sigma).
10. NaH2PO3 (Sigma).
12. Columbia blood agar (Oxoid). Store at 4°C; pay attention to expiration date.
2.3. Infection of Host Cells
1. 6-Well tissue culture plate (Greiner).
2. 24-Well tissue culture plate (Costar).
3. 2-mL Syringes and 27-gage needles.
4. Sterile Eppendorf tubes (capacity 2 mL).
5. Gentamicin (pay attention to the different storage conditions according to the manufacturer's instructions).
2.4. Coculture of MM6 and A. castellanii
1. Transwell® insert (Costar).
2. Columbia blood agar (Oxoid). Store at 4°C; pay attention to expiration date.
3. Millex®-GS 0.22-^m syringe-driven filter unit (Millipore).
Legionellae are fastidious, slow-growing bacteria. They are grown on BCYE-a agar at 35°C in 3% CO2. After 3-5 d, small, round glistening colonies with a defined edge appear on the agar. The edge usually displays a pink or blue-green iridescence; the center of the colonies is usually grayish, with a characteristic speckled opalescence, the appearance of which resembles cut or ground glass. Later on, the centers of the colonies become creamy white and often lose the ground-glass appearance. At this time, large and small colonies are common and are therefore not evidence for contamination with other bacteria. Since legionellae do not grow on blood agar, this type of agar can be used for contamination control. For contamination of BCYE-a agar with molds (see Note 1).
3.2. Culture of Host Cells
MM6 cells are cultured as replicative nonadherent monocytes under lipopolysac-charide-free conditions in 75-cm3 vented culture flasks in 25 mL of RPMI-1640 medium supplemented with 10% fetal calf serum (Myoclone super plus), 2 mM l-glutamine, 1% nonessential amino acids, and OPI (containing 150 ^g oxalacetate, 50 ^g pyruvate, and 8.2 ^g of bovine insulin per mL) at 37°C in 5% CO2 (MM6 medium) and are diluted 1:3 twice a week in fresh medium (15). We recommend checking the cell culture regularly for sterility using Columbia blood agar and for Mycoplasma contamination using Mycoplasma plus™ polymerase chain reaction (PCR) according to the manufacturer's instructions (see Notes 2 and 3).
A. castellanii are grown in 75-cm3 vented culture flasks in 25 mL PYE broth (2% protease pepton no. 3, 0.1% yeast extract, 0.1 M glucose, 4 mM MgSO4, 0.4 M CaCl2, 0.1% sodium citrate dihydrate, 0.05 mM Fe[NH4]2[SO4]2, 2.5 mM NaH2PO3, and 2.5 mM K2HPO3, pH 6.5) at 35°C in 5% CO2 (52) and are diluted 1:2 twice a week. Sterility testing should be performed as described for MM6 cells.
3.3. Infection of Host Cells
1. Infection and incubation of MM6 cells after infection is performed in MM6 medium without FCS to exclude phagocytosis of bacteria via Fcy receptors.
2. Nonadherent MM6 cells are harvested by centrifugation at 400g for 10 min.
3. The pellet is washed twice in MM6 medium without FCS.
4. Legionellae are harvested from BCYE-a agar, suspended in MM6 medium without FCS, and adjusted to an OD578nm of 0.2 (by a spectrophotometer) corresponding to a concentration of approx 3 x 108 legionellae/mL. For this concentration, bacteria can be centrifuged at 3000g for 10 min.
5. 2 x 107 MM6 cells are pelleted and resuspended with legionellae in a volume of 1.5 mL in a well of a 6-well tissue culture plate.
6. The bacteria-to-cell ratio can be selected from 100:1 to 0.1:1. It should be taken into account that legionellae are cytotoxic for monocytes at higher multiplicities of infection (53). Therefore, lower bacteria-to-cell ratios are recommended.
7. Cocultures of bacteria and monocytes are then incubated at 35°C in 5% CO2 for 2 h.
8. After this period, nonphagocytized bacteria are killed by addition of 4.5 mL MM6 medium without FCS containing 100 ^g/mL gentamicin for 1 h at 35°C in 5% CO2.
9. After three washes by centrifugation at 400g for 10 min, the cells are resuspended in 10 mL MM6 medium without FCS and distributed in 1-mL aliquots into the wells of a 24-well tissue culture plate, giving a concentration of 2 x 106 infected MM6 cells per well. This time point is defined as 0.
10. The cells are then incubated for an additional 72 h at 35°C in 5% CO2.
11. Every 24 h, the contents of two wells are aspirated and pelleted by centrifugation at 400g for 10 min.
12. The supernatant is transferred into sterile tubes of approx 2 mL capacity.
13. 1 mL of sterile distilled water is added to the pellet, and final disruption of the cells is performed by aspirating the suspension through a 27-gage needle.
14. The supernatant and lysis fluid are pooled, and serial 10-fold dilutions are made.
15. 100 ^L of each dilution is inoculated onto BCYE-a agar to determine the number of viable legionellae after multiplication in MM6 cells.
16. Colonies on the agar are counted on d 5 after incubation at 35°C in 5% CO2.
17. For control, 1 mL of the original Legionella suspension in MM6 medium without FCS and without cells is incubated in a well of the tissue culture plate, and serial 10-fold dilutions are made at the same time points as indicated above.
18. Viability of MM6 cells is determined by Trypan Blue exclusion at the same time points as indicated above.
19. We recommend testing for contamination of the infected cell culture by subculture onto Columbia blood agar plates.
Coculture of A. castellanii and bacteria is performed in an identical manner except that legionellae are suspended in PYE broth without glucose and the culture is incubated for 96 h.
1. Infected MM6 cells (see Subheading 3.3.) are resuspended in a mixture of 50% MM6 medium without FCS and 50% PYE broth without glucose. NaCl must be added to yield an NaCl concentration of 6.5 g/L (coculture medium) identical to the MM6 medium (see Note 4). This mixture was found to support growth of MM6 cells as well as A. castellanii in a manner comparable to the original cell culture media for these cells described in Subheading 3.2.
2. Infected MM6 cells are distributed in 1 mL aliquots into the wells of a 24-well tissue culture plate, giving a concentration of 1 x 106 infected MM6 cells per well.
3. 1 x 106 A. castellanii organisms are resuspended in coculture medium and are added using a Transwell® insert, which separates both cell types by a microporous polycarbonate membrane with a pore size of 0.1 ^m, impervious to bacteria, amoebae, and MM6 cells.
4. Absence of legionellae from the upper chamber (A. castellanii) is determined by means of culture on BCYE-a agar, and sterility of the coculture is checked by culture on Columbia blood agar.
5. The coculture is incubated for 72 h.
6. Intracellular replication of bacteria within MM6 cells is detected as described above in Subheading 3.3.
7. Multiplication within MM6 cells in coculture with A. castellanii is compared with multiplication within MM6 cells in coculture medium without amoebae.
8. To demonstrate the presence of amoeba-derived effector molecule(s) in the coculture, a cell-free Transwell® supernatant is obtained by harvesting the content of a Transwell® insert (i.e., noninfected amoebae in coculture with infected MM6 cells, with both host cells separated by a microporous membrane), subsequent centrifugation (400g for 10 min), and filtration through a Millex®-GS 0.22-^m syringe-driven filter unit to obtain sterility.
9. 1 x 106 infected MM6 cells per well are then coincubated for 72 h with this cell-free supernatant. Intracellular multiplication of legionellae in MM6 cells is determined as described above (see Subheading 3.3.).
10. Multiplication within MM6 cells in coculture with the supernatant is compared with multiplication within MM6 cells in coculture medium.
1. BCYE-a agar is an ideal medium for growth of molds, especially Aspergillus fumigatus. Therefore, laboratories and equipment (especially incubators) should be free of molds.
2. Contamination of the cell culture is the main source of failure in using the in vitro models presented above. Common contaminants are fast-growing bacteria such as staphylococci and streptococci, especially when people having short-term or inadequate experience with cell cultures are working in the laboratory. Good staff training can prevent most contamination events. We recommend checking the cell cultures routinely (at least twice a week before dilution) as well as the in vitro infection experiments (parallel inoculation of lysis fluid onto BCYE-a agar and Columbia blood agar).
A serious and often undetected problem is the contamination of cell cultures with Mycoplasma organisms. The origins are bovine serum, laboratory personnel, and mycoplasma-infected cell cultures; the latter spread the infection to other cell cultures within the same laboratory. It is important to keep in mind that Mycoplasma organisms, particularly in continuous cell lines such as MM6 cells or A. castellanii, grow slowly and do not destroy the host cells. However, the influence of Mycoplasma organisms on various parameters such as changes in metabolism, growth, viability, and morphology of the cells should not be underestimated. Mycoplasma organisms can seriously interfere with experiments. We therefore recommend checking the cell cultures at least quarterly for Mycoplasma contamination. A variety of techniques are available to detect Mycoplasma in cell cultures. The most reliable are enzyme immunoassays and PCR. We can recommend the Mycoplasma plus™ PCR (Stratagene). In case of Mycoplasma contamination, cell cultures should be discarded and the incubator should be disinfected. A new batch of cells should be cultured and used for in vitro infection. If no new batch is available, cell cultures contaminated with Mycoplasma can be treated with BM-Cyclin (Roche), an antibiotic combination of a pleuromutilin and a tetracycline derivative, without marked cytotoxic side effects. This treatment requires about 2-3 wk. In vitro infections should not be performed during this time since legionellae are sensitive to these antibiotics.
3. Intracellular multiplication of legionellae is a function of intact host cells. Monocytes or amoebae maintained under inadequate conditions may not support the intracellular growth of bacteria, although crude tests for viability (such as Trypan Blue exclusion) do not indicate cell death. Therefore, cells should be diluted twice a week in fresh medium as described above. During washing procedures, cells should not be centrifuged faster than 400g to avoid disruption.
4. As described in Subheading 3., the cell culture medium for coculture of MM6 cells and A. castellanii resembles a mixture of 50% MM6 medium without FCS and 50% PYE broth without glucose. This mixture is hypotonic, and it is necessary to add NaCl to achieve a salt concentration of 6.5 g/L identical to MM6 medium since hypotonic medium induces intracellular growth of non-replicative Legionella species owing to swelling of the host cells (51).
1 Brenner, D. J. (1987) Classification of the legionellae. Semin. Respir. Infect. 2, 190-205.
2 Davis, G. S. and Winn, W. C. (1987) Legionnaire's disease: respiratory infection caused by Legionella bacteria. Clin. Chest Med. 8, 419-439.
3 Fang, G. D., Yu, V. L., and Vickers, R. M. (1989) Disease due to the Legionellaceae (other than Legionella pneumophila). Medicine 68, 116-132.
4 Nguyen, H. M., Stout, J. E., and Yu, V. L. (1991) Legionellosis. Infect. Dis. Clin. North Am. 5, 561-584.
5 Hart, C. A. and Makin, T. (1991): Legionella in hospitals: a review. J. Hosp. Infect. 18(Suppl A), 481-489.
6 Winn, W. C. (1988) Legionnaires disease: historical perspective. Clin. Microbiol. Rev. 1, 60-81.
7 Horwitz, M. A. (1980) The Legionnaires' disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. J. Clin. Invest. 66, 441-450.
8. Ciancotto, N. P., Eisenstein, B. I., Mody, C. H., Toews, G. B., and Engleberg, N. C. (1989) A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection. Infect. Immun. 57, 1255-1262.
9 Dowling, J. N., Saha, A. K., and Glew, R. H. (1992) Virulence factors of the family Legionellaceae. Microbiol. Rev. 56, 32-60.
10 Horwitz, M. A. (1987) Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes. J. Exp. Med. 166, 1310-1328.
11 Pearlman, E., Jiwa, A. H., Engleberg, N. C., and Eisenstein, B. I. (1988) Growth of Legionella pneumophila in a human macrophage-like (U 937) cell line. Microb. Patho-genesis 5, 87-95.
12. Yamamoto, Y., Klein, T. W., Newton, C. A., Widen, R., and Friedman, H. (1988) Growth of Legionella pneumophila in thioglycolate-elicited peritoneal macrophages from A/J mice. Infect. Immun. 56, 70-375.
13 Yoshida, S. and Mizuguchi, Y (1986) Multiplication of Legionella pneumophila Philadelphia 1 in cultured peritoneal macrophages and its correlation to susceptibility of animals. Can. J. Microbiol. 32, 438-442.
14 Swanson, M. S. and Hammer B. K. (2000) Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu. Rev. Microbiol. 54, 567-613.
15 Ziegler-Heitbrock, H. W. L., Thiel, E., Futterer, A., Herzof, V., Wirtz, A., and Riethmuller, G. (1988) Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int. J. Cancer 41, 456-461.
16 Marra, A., Horwitz, M. A., and Shuman, H. A. (1990) The HL-60 model for the interaction of human macrophages with the Legionnaires' disease bacterium. J. Immunol. 144, 2738-2744.
17 Pearlman, E., Jiwa, A. H., Engleberg, N. C., and Eisenstein, B. I. (1988) Growth of Legionella pneumophila in a human macrophage-like (U 937) cell line. Microb. Pathogen. 5, 87-95.
18 Watanabe, M., Shimamoto, Y., Yoshida, S., et al. (1993) Intracellular multiplication of Legionella pneumophila in HL-60 cells differentiated by 1,25-dihydroxyvitamin D3 and the effect of interferon y. J. Leukocyte Biol. 54, 40-46.
19 Neumeister, B., Schöniger, S., Faigle, M., Eichner, M., and Dietz, K. (1997) Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellani. Appl. Environ. Microbiol. 63, 1219-1224.
20. Barrow, E. L. W., Winchester, G. A., Staas, J. K., Quenelle, D. C., and Barrow, W. W. (1998) Use of microsphere technology for targeted delivery of rifampin to Mycobacterium tuberculosis-infected macrophages. Antimicrob. Agents Chemother. 42, 2682-22689,
21. Heinemann, M., Susa, M., Simnacher, U., Marre, R., and Essig, A. (1996) Growth of Chlamydia pneumoniae induces cytokine production and expression of CD14 in a human monocytic cell line. Infect. Immun. 64, 4872-4875.
22. Michel, R. and Hauröder, B. (1997) Isolation of an Acanthamoeba strain with intracellular Burkholderia pickettii infection. Zentralbl. Bakt. 285, 541-557.
23. Landers, P., Kerr, K. G., Rowbotham, T. J., et al. (2000) Survival and growth of Burkholderia cepacia within the free-living amoeba Acanthamoeba polyphaga. Eur. J. Clin. Microbiol. Infect. Dis. 19, 121-123.
24. Inglis, T. J. J., Rigby, P., Robertson, T. A., Dutton, N. S., Henderson, M., and Chang, B. J. (2000) Interaction between Burkholderia pseudomallei and Acanthamoeba species results in coiling phagocytosis, endamebic bacterial survival, and escape. Infect. Immun. 68, 1681-1686.
25. Essig, A., Heinemann, M., Simnacher, U., and Marre, R. (1997) Infection of Acanthamoeba castellanii by Chlamydia pneumoniae. Appl. Environ. Microbiol. 63, 1396-1399.
26. Ly, T. M. C. and Müller, H. E. (1990) Ingested Listeria monocytogenes survive and multiply in protozoa. J. Med. Microbiol. 33, 51-54.
27. Cirillo, J. D., Falkow, S., Tompkins, L. S., and Bermudez, L. E. (1997) Interaction of Mycobacterium avium with environmental amoebae enhances virulence. Infect. Immun. 65, 3759-3767.
28. Steinert, M., Birkness, K., White, E., Fields, B., and Quinn, F. (1998) Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls. Appl. Environ. Microbiol. 64, 2256-2261.
29. Michel, R., Burghardt, H., and Bergmann, H. (1995) Acanthamoeba, naturally intracellu-larly infected with Pseudomonas aeruginosa, after their isolation from a microbiologi-cally contaminated drinking water system in a hospital. Zentralbl. Hyg. Umweltmed. 196, 532-544.
30. Thom, S., Warhurst, D., and Drasar, B. S. (1992) Association of Vibrio cholerae with fresh water amoebae. J. Med. Microbiol. 36, 303-306.
31. Barker, J., Humphrey, T. J., and Brown, M. W. (1999) Survival of Escherichia coli 0157 in a soil protozoan: implications for disease. FEMS Microbiol. Lett. 173, 291-295.
32. Fritsche, T. R., Horn, M., Seyedirashti, S., Gautom, R. K., Schleifer, K. H., and Wagner, M. (1999) In situ detection of novel bacterial endosymbionts of Acanthamoeba spp. phy-logenetically related to members of the order Rickettsiales. Appl. Environ. Microbiol. 65, 206-212.
33. Rowbotham, T.J. (1980) Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J. Clin. Pathol. 33, 1179-1183.
34. Barbaree, J. M., Fields, B. S., Feeley, J. C., Gorman, G. W., and Martin, W. T. (1986) Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila. Appl. Environ. Microbiol. 51, 422-424.
35. Breiman, R.F., Fields, B.S., Sanden, G.N., Volmer, L.J., Meier, A., and Spika, J.S. (1990) Association of shower use with Legionnaires' disease. JAMA 263, 2924-2926.
36. Fields, B. S., Sanden, G. N., Barbaree, J. M., et al. (1989) Intracellular multiplication of Legionella pneumophila in amoebae isolated from hospital hot water tanks. Curr. Microbiol. 16, 131-137
37. Fields, B. S., Nerad, T. A., Sawyer, T. K., et al. (1990) Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis. J. Protozool. 37, 581-583.
38. Nahapetian, K., Challemel, O., Beurtin, D., Dubrou, S., Gounon, P., and Squinazi, F. (1991) The intracellular multiplication of Legionella pneumophila in protozoa from hospital plumbing systems. Res. Microbiol. 142, 677-685.
39. Rowbotham, T. J. (1983) Isolation of Legionella pneumophila from clinical specimens via amoebae, and the interaction of those and other isolates with amoebae. J. Clin. Pathol. 36, 978-986.
40. Sanden, G. N., Morrill, W. E., Fields, B. S., Breiman, R. F., and Barbaree, J. M. (1992) Incubation of water samples containing amoebae improves detection of legionellae by the culture method. Appl. Environ. Microbiol. 58, 2001-2004.
41. Wadowsky, R. M., Butler, L. J., Cook, M. K., et al. (1988) Growth supporting activity for Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors. Appl. Environ. Microbiol. 54, 2677-2682.
42. Cirillo, J. D., Falkow, S., and Tompkins, L. S. (1994) Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infect. Immun. 62, 3254-3261.
43. Abu-Kwaik, Y., Fields, B. S., and Engleberg, N. C. (1994) Protein expression by the protozoan Hartmanella vermiformis upon contact with its bacterial parasite Legionella pneumophila. Infect. Immun. 62, 1860-1866.
44. Barker, J., Lambert, P. A., and Brown, M. R. (1993) Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila. Infect. Immun. 61, 3503-3510.
45. Barker, J., Brown, M. R., Collier, P. J., Farrell, I., and Gilbert, P. (1992) Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation. Appl. Environ. Microbiol. 58, 2420-2425.
46. Barker, J., Scaife, H., and Brown, R. W. (1995) Intraphagocytic growth induces an antibiotic resistant phenotype of Legionella pneumophila. Antimicrob. Agents Chemother. 39, 2684-2688.
47. O'Brien, S. J. and Bhopal, R. S. (1993) Legionnaires' disease: the infective dose paradox. Lancet 342, 5-6.
48. Brieland, J., McClain, M., Heath, L., et al. (1996) Coinoculation with Hartmanella vermiformis enhances replicative Legionella pneumophila infection in a murine model of Legionnaires' disease. Infect. Immun. 64, 2449-2456.
49. Brieland, J., McClain, M., Legendre, M., and Engleberg, C. (1997) Intrapulmonary Hartmanella vermiformis: a potential niche for Legionella pneumophila replication in a murine model of legionellosis. Infect. Immun. 65, 4892-4896.
50. Brieland, J. K., Fantone, J. C., Remick, D. G., Legendre, M., McClain, M., and Engleberg, C. (1997) The role of Legionella pneumophila-infected Hartmanella vermiformis as an infectious particle in a murine model of Legionnaires' disease. Infect. Immun. 65,5330-5333.
51. Neumeister, B., Reiff, G., Faigle, M., Dietz, K., and Lang, F. (2000) Influence of Acanthamoeba castellanii on intracellular growth of different Legionella species in human monocytes: establishment of an in-vitro coculture system. Appl. Environ. Microbiol. 66, 914-919.
52. Moffat, J. F. and Tompkins, L. S. (1992) A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infect. Immun. 60, 296-301.
53. Gao, L. Y. and Abu-Kwaik, Y. (1999) Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopatho-genicity. Infect. Immun. 67, 862-870.
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All you need is a proper diet of fresh fruits and vegetables and get plenty of exercise and you'll be fine. Ever heard those words from your doctor? If that's all heshe recommends then you're missing out an important ingredient for health that he's not telling you. Fact is that you can adhere to the strictest diet, watch everything you eat and get the exercise of amarathon runner and still come down with diabetic complications. Diet, exercise and standard drug treatments simply aren't enough to help keep your diabetes under control.