Science Research: Legionella

Rebecca Traub

Legionella Literature Review

Legionellae are gram negative bacilli which do not grow on routine media used in Bacteriology to grow organisms. It requires cysteine and other nutrients to grow, so it is often difficult to isolate and identify. There are 41 species of Legionella (personal communication, B. Fields), but Legionella pneumophila is the usual cause of disease. Other strains such as L. micdadei, L. bozemanii, L. longbeachae, and L dumofii are usually less virulent and are mostly found within the immunocomprimised community.(Hoge and Breiman, 1991)

Identification: There are two known manifestations of Legionella: Pontiac Fever and Legionnaires' Disease which usually involves pneumonia. Both results cause anorexia, weakness and discomfort, pain in the muscles, headache, and fever with chills, though Legionnaires' Disease is the manifestation which is most often diagnosed and can be fatal if not treated. The hardest part of curing Legionella infections is not the treatment, but recognizing that the source of pneumonia is the Legionella bacteria.

Occurrence: The identification of Legionella occurred in 1976 when there was an outbreak of pneumonia in a hotel in Philadelphia (Fraser, Tsai, Orenstein et al., 1977). Most of the people who died were members of the American Legion. The first known outbreak of the disease, however, was in 1965 in a hospital in Washington DC (Thacker, Bennet, Tsai et al., 1978), while the first isolation of the bacteria actually occurred in 1947 from a guinea pig (McDade and Brenner and Bozeman, 1979). Usually, in diagnosed cases, the bacteria causes Legionnaires' Disease, but there have been recorded cases of Pontiac Fever which does not cause pneumonia and requires no treatment. Legionella has been recovered from kidney and heart infections, though non-pneumonia infection is rare.

Reservoir: Legionella's reservoir is thought to be mostly aquatic. Hot water sources, water heaters, cooling towers and air conditioners are common breeding sites for the bacteria. Legionella has been detected in mud from natural sources like rivers and ponds but never from dry dirt.(Hoge and Breiman, 1991)

Protozoa in water sources have been shown to increase Legionella proliferation. The bacteria multiplies intracellularly, and therefore requires protozoa such as amoebae to aid in reproduction. One amoebae can hold enough Legionella bacteria to cause infection in a human being and can be contained in one droplet of aerosolized water.(Breiman, 1993; Fields, 1993; Panikov, Merkurov and Tartakovskii, 1993; Naphaetian et al., 1991; Fields et al. 1993)

Mode of Transmission: Evidence (Hoge and Breiman, 1991) seems to point towards the use of aerosol devices such as hot tubs and showers as the mode of transmission. No reports of human to human transmission have been proven.

Method of Infection: Infection usually begins with inhalation of the bacteria. Once the Legionella reaches the alveoli, assuming the virulence is great enough to overcome the host's immune reactions, the bacteria comes into contact with an alveolar macrophage. Through phagocytosis (coiling phagocytosis), the macrophage takes the bacteria into a food vacuole inside the cell. Legionella inhibits monocyte lysosome fusion, allowing the bacteria to multiply within the macrophage and ultimately lyse the cell and infect new cells.(Nash, Libby and Horwitz, 1984) Studies (Moffat and Tompkins, 1992; King et al., 1991) indicate that while amoebae use pinocytosis or receptor-mediated uptake to take in the bacteria (personal communication, B. Fields), human cells use both phagocytosis and pinocytosis in bringing the bacteria inside the monocyte.
 

Incubation Period: Legionnaires' Disease has an incubation period of 2-10 days, with a usual incubation of 5-6 days. Pontiac Fever has an incubation period of 5-66 hours, with a normal range of 24-48 hours.

Susceptibility and Resistance: According to Marston et al, (1994), anyone can acquire Legionella related disease, but there are some risk factors. There is increased susceptibility with age, smokers are especially in risk, as are immunocomprimised patients resulting from either cancer, kidney failure, diabetes, organ transplant or HIV.

Treatment: Erythromycin is the drug of choice, though there have been recent studies (Dunn and Barradell, 1996) confirming the effectiveness of the antibiotic Azithromycin.

Genetic Study: Recently, most research involving Legionella has focused on the genetic roots of Legionella, it's functions, and the genetic loci of host organisms that can suppress Legionella growth.

Some studies (Dumais and O'Connell and Cianciotto, 1996; O'Connell, Hickey and Cianciotto, 1996; Pope and O'Connell and Cianciotto, 1996) have begun to focus on the genetic loci of Legionella. Genetic technology has aided in beginning to understand, at the nucleotide level, the roots of Legionella function and phenotype.

Mutagensesis is a approach to understanding genetic function. Southern hybridization or RFLP analysis have allowed scientists to compare mutants created with plasmids and phages to wild type DNA. (Pope, O'Connell, Cianciotto, 1996, O'Connell, Hickey, Cianciotto, 1996) Through these processes, gene loci, necessary for iron acquisition and assimilation (Pope, O'Connell, Cianciotto, 1996) and hemin binding have been defined. Through PCR and nucleotide sequencing analysis, it has been possible to find the reading frames responsible for specific traits within the Legionella genome, including amino acid sequences, promoter sequences, and iron interaction sequences (O'Connell, Hickey, Cianciotto, 1996). Southern Hybridization analysis and database searches have additionally determined that hbp, the locus responsible for hemin-binding promotion is close to restricted for Legionella (O'Connell, Hickey, Cianciotto, 1996). Also, Electrophoresis has helped in comparing different species of Legionella in their genetic diversity (O'Connell, Hickey, Cianciotto, 1996).

Several other loci besides the hbp have been identified. The mip locus helps survival after entry into macrophages and protozoa, icm and dot loci are necessary for intracellular replication, and the hel locus helps with killing the host cell (Pope and O'Connell and Cianciotto, 1996).

According to Miyamoto et al, (1996), Intracellular multiplication that was controlled by mice was identified as a single mouse gene, Lgn 1. The gene allowed macrophages to suppress the intracellular multiplication of Legionella.

The genetic aspect of Legionella is the future of the bacteria's research. Everything that one might want to understand about Legionella and similar pathogens is coded in the genetic makeup of the bacteria. Only through better understanding a pathogen can we attempt to stop it. Determining further the genetic loci which control Legionella reproduction and survival will help us to comprehend not only Legionella related disease, but future bacteria that share similar traits.

References

Breiman, Robert F., In Legionella - Current Status and Emerging Perspectives, Eds. Barbaree, Breiman, Dufour, (1993), pp30-35. American Society for Microbiology, Washington, D.C.

Dunn, C. J., Barradell, L. B. (1996). Azithromycin - A Review of its Pharmacological Properties and Use as 3-day Therapy in Respiratory Tract Infections. Drugs. 51(3) : 483-505.

Fields, B.S., Fields, S.R., Utley, Loy, Janine N. Chin, White, Elizabeth H., Steffens, W.L., Shotts, Emmett B. (1993). Attatchment and Entry of Legionella pneumophila in Hartmeannella vermiformis. The Journal of Infectious Diseases. 167 : pp 1146- 1150.

Fields, B.S. In Legionella - Current Status and Emerging Perspectives, Eds. Barbaree, Breiman, Dufour, (1993), pp129-136. American Society for Microbiology, Washington, D.C.

Fraser, D.W., Tsai, T,. Orenstein, W., et al. (1977). Legionnaires' disease: description of an epidemic of pneumonia. New England Journal of Medicine. 297: 1186-1196.

Hoge, C.W., and Breiman, R.F. (1991). Advances in the Epidemiology and Control of Legionella infections. Epidemiologic Reviews. 13: 329-340.

King, H.C., Fields, B.S., Shotts Jr., E.B., White, E.H. (1991). Effects of Cytochalasin D and Methylamine on Intracellular Growth of Legionella pneumophila in Amoebae and Human Monocyte-Like Cells. Infection and Immunity. 59(3) : pp 758 - 763.

Marston, B.J., Lipman, H.B., Breiman, R.F. (1994). Surveillance for Legionnaires' Disease. Arch Intern Med. 154: 2417-2422.

McDade, J.E., Brenner, D.J., Boeman, F.M.. (1979). Legionnaires' disease bacterium isolated in 1947. Ann Intern Med. 90: 659-661.

Miyamoto, H., Maruta, K., Ogawa, M., Beckers, M.C., Gros, P., and Yoshida, S. (1996). Spectrum of Legionella Species Whose Intracellular Multiplication in Murine Macrophages Is Genetically Controlled by Lgn 1. Infection and Immunity. 64(5) : 1842 - 1845.

Moffat, J.F., Tompkins, L.S. (1992). A Quantitative Model of Intracellular Growth of Legionella pneumophila in Acanthamoeba castellanii. Infection and Immunity. 60(1) : p 296-301.

Naphaetian, K., Challemel, O., Beurtin, D., Dubrou, S., Gounon, P., Squinazi, R. (1991). The Intracellular multiplication of Legionella pneumophila in Protozoa from Hospital Plumbing Systems. Research in Microbiology. 142 p 677-686.

Nash, T.W., Libby, D., Horwitz, M.A.. (1984). Interaction between the Legionnaires' disease bacterium (L. pneumophila) and human alveolar macrophages: influence of antibody, lymphokines, and hydrocortisone. J Clin Invest. 74: 771-782.

O'Connell, W.A., Hickey, E.K., and Cianciotto, N.P. (1996). A Legionella pneumophila Gene That Promotes Hemin Binding. Infection and Immunity. 64(3) : 842-848.

Panikov, N.S., Merkurov, A.E., and Tartakovskii, I.S. In Legionella - Current Status and Emerging Perspectives, Eds. Barbaree, Breiman, Dufour, (1993), pp153-159. American Society for Microbiology, Washington, D.C.

Pope, C.D., O'Connell, W.A., and Cianciotto, N.P. (1996). Legionella pneumophila Mutants That Are Defective for Iron Acquisition and Assimilation and Intracellular Infection. Infection and Immunity. 64(2) : 629-636.

Thacker, S.B., Bennet, J.V., Tsai, T., et al. (1978). An outbreak in 1975 of severe respiratory illness caused by Legionnaires' disease bacterium. Journal of Infectious Disease. 238: 512-519.

Legionella Photos
 

Legionella, Current Status and Emerging Perspectives, eds. James M. Barbaree, Robert F. Breiman, and Alfred P. Dufour. ASM, American Society for Microbiology Press, Washington DC.
 

"FIG. 1. Scanning electron micrographs of virulent L. pneumophila cell attached to H. vermiformis (x20,000). Bacteria were frequently seen attached to the ends of amoeba processes (filopodia). The micrographs were taken from H. vermiformis cultures that had been coincubated with L. pneumophila for 8 h."

(Fields, Barry S., Legionella and Protozoa: Interaction of a Pathogen and Its Natural Host, Legionella, Current Status and Emerging Perspectives, Washington DC: ASM Press)
 

"FIG 2. Scanning electron micrograph of a virulent L. pneumophila cell entering an H. vermiformis cell (x6.600; insert, x20,000). Pseduopodia were not observed in the uptake of the bacteria. The micrograph was taken from an H. vermiformis culture that had been coincubated with L. pneumophila for 12 h.)"

(Fields, Barry S., Legionella and Protozoa: Interaction of a Pathogen and Its Natural Host, Legionella, Current Status and Emerging Perspectives, Washington DC: ASM Press)
 

"FIG 1. Immunogold labelling for TDP is guinea pig alveolar macrophages infected in vitro with L. pneumophila."

(Williams, Rechnitzer, Lever, and Fitzgeorge, Intracellular Production of Legionella pneumophila Tissue-Destructive Protease in Alveolar Macrophages, Legionella, Current Status and Emerging Perspectives, Washington DC: ASM Press)

"Scanning EM of a U937 cell 1 h postinfection. Note numerous microvilli and adherent legionellae (arrows) (x4,200)."
 

(Gibson and Rodgers, Ultrastructure of the Adherence and Intracellular Replication of Legionella pneumophila in Macrophage-Like U937 Cells, Legionella, Current Status and Emerging Perspectives, Washington DC: ASM Press)

Legionella, the bacteria that causes Legionnaires' disease in human being, is found naturally in water sources such as lakes and ponds as well as man made sources such as cooling towers and humidifiers. The bacteria is an intracellular pathogen, meaning that it requires other cells, whether they be amebic or human, for survival.

Recently, genetic study has determined a number of virulence genes encoded in Legionella DNA. Using transposon vectored DNA mutations, strains lacking these genes determined which are required for Legionella virulence. The Icm genes have been identified as necessary for Legionella intracellular mutliplication, the mip genes for increasing Legionella infection (macrophage infectivity potentiator), and ompS, the gene for the major outer membrane protein (MOMP), essential in Legionella binding to macrophages, stimulating a unique coiling phagocytosis.

The question now arises as to how these bacterial genes are regulated. How does the Legionella organism know when it has entered a phagosome inside the cell as a cue for intracellular multiplication (expression of the Icm genes)? The answer lies within 2-component signal transduction pathways that regulate gene control. Essentially, outer membrane proteins send messengers to intracellular proteins in response to extracellular stimuli, and these proteins inside the cell consequently regulate the gene transcription/expression of the bacteria.
 

This summer I will be further investigating genes for Legionella virulence plus the extracellular stimuli (like salt, osmolarity, pH and temperature) that result in the regulation of gene expression.

Comparison of Guinea Pig and Protozoan Models for Determining Virulence of Legionella Species

A Quantitative Model of Intracellular Growth of Legionella pneumophila in Acanthamoeba castellanii

Attachment and Entry of Legionella pneumophila in Hartmannella veriformis

Effects of Cytochalasin D and Methylamine on Intracellular Growth of Legionella pneumophila in Amoebae and Human Monocyte-Like Cells

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PageUpdated 15th March 1999
 
 

11th September 2000