Higher Order Taxa:
Bacteria; Proteobacteria; Gammaproteobacteria; Legionellales; Legionellacaea; Legionella; Legionella pneumophila
Description and significance
The Legionellaceae are fastidious gram-negative bacteria that reside in aquatic environments all over the globe. In their natural environment, the Legionellaceae are intracellular parasites of free-living protozoa. These organisms may also inhabit man-made water distribution systems. The family Legionellaceae consists of a single genus, Legionella. More specifically, this genus includes the species Legionella pneumophila, which are non-encapsulated, aerobic bacilli. In addition to L. pneumophila, there are 41 other species in the genus and together these species divide into 64 serogroups. Within the species L. pneumophila, human infection is caused primarily (but not exclusively) by a limited number of serogroups—serogroups 1, 4, and 6. L. pneumophila is the most frequent cause of human legionellosis, better known as Legionnaire’s disease in the Legionellaceae family. It is also a relatively common cause of community-acquired and nosocomial pneumonia in adults. (1) And L. pneumophila serogroup 1 alone is responsible for 70-90% of cases. (2)
The Legionellaceae were not documented until 1976, when a detrimental outbreak of pneumonia occurred in Philadelphia at an American Legion Convention. (Fraser et al., 1977; Tsai et al., 1979). Thirty four of the 221 people who became ill after exposure died within the first few weeks after the convention. The culprit, L. pneumophila, was isolated first by inoculation of postmortem lung tissue into guinea pigs and then by subculture into a rich artificial medium (McDade et al., 1977). Then by indirect immunofluorescent antibody assay, it was found that over 90% of those that fell ill had at least four times the concentration of antibody in the blood (fourfold rise in titer) against this organism. The same method was used to screen previously saved sera from earlier outbreaks of unexplained respiratory disease and they discovered that a number of them were associated with seroconversion to L. pneumophila, including a “rickettsia-like” organism, isolated by guinea pig inoculation from the blood of a feverish patient in 1947, which today is recorded as the earliest known isolate of L. pneumophila. (1)
The Legionella genome structure continues to undergo research. Three different genomes of L. pneumophila were completed over a span of three years. First, Legionella pneumophila ssp. Pneumophila str. Philadelphia 1 (3,397,754 bp, 3002 genes) was completed in October 2001. Plasmid pLPP of Legionella pneumophila str. Paris was completed in October 2004; in addition to the full genome (3,503,610 bp, 3136 genes). Plasmid pLPL of Legionella pneumophila str. Lens was also completed in October 2004, in addition to the full genome (3,345,687 bp, 3001 genes). (3)
The genome of Legionella pneumophila str. Paris was found to contain 3,503,610 base pairs and contains about 3027 predicted protein-encoding genes. The genome is a circular chromosome with an average GC content of 38%. In 2004, researchers analyzed the complete genome sequences of L. pneumophila Paris, an endemic strain that is predominantly found in France and L. pneumophila Lens, another endemic strain. The Lens strain is 3,345,687 base pairs long and contains about 2878 protein-encoding genes and like the Paris strain its genome is a circular chromosome with an average GC content of 38%. The two strains differed in about 13% of their genome sequences having 3 different plasmids. The Paris strain is unique because it contains a type V secretion system and a 36 Kb sequence carried on a multicopy plasmid or integrated into a chromosome encodes its type IV secretion system. This ability for genes to move around increases the versatility in L. pneumophila. To alter host cell functions to its advantage L. pneumophila contains many genes that encode eukaryotic-like proteins or motifs. As a result, the genome reveals co-evolution of L. pneumophila as a fresh-water amoebae and a human pathogen. (4)
Cell Structure and Metabolism
L. pneumophila is a gram-negative, non-encapsulated, aerobic bacillus with a single, polar flagellum. The organism is approximately 2µm in length and 0.3-0.9µm in width, but in nutrient-deficient media, it may become long and filamentous. It is surrounded by a gram-negative cell wall and pili are sometimes identified. The cell envelope is composed of branched-chain fatty acids and distinctive ubiquinones, whose structural differences have been used to classify different Legionella species. The outer-membrane is comprised of a lipopolysaccharide (LPS), which is “fully sequenced and found to have several novel features which have pathophysiologic consequence” and is noticeably less endotoxic than enterobacterial LPS since it has weak interactions with the CD14 receptor on monocytes, The interactions are probably inhibited by the long-chain fatty acids of L. pneumophila lipid A, which are two times the length of enterobacterial lipid A. A single, major protein also makes up the outer membrane and functions as a porin and as a target for human complement fixation. L. pneumophila serogroup 1LPS also has a repeating O antigen. It is a homopolymer of an uncommon sugar, called legionaminic acid. ”LPS is the immunodominant antigen of the Legionellaceae, and the O antigen is the determinant of serogroup specificity within the genus.” (5)
All organisms interact with the environment they are in and with the organisms they share that environment with. In one research study, the potable water distribution system was determined to be the culprit behind hospital-acquired Legionnaires’ disease. Researches hypothesized that the natural microbial population and sediment (aerosols or particulate mater) were, together, growth-promoting factors of L. pneumophila. A series of in vitro experiments proved that many factors supported and even improved the survival of L. pneumophila. Results showed that water from the hot-water storage tank was a constant sustainer for the survival of L. pneumophila, as was the concentration of sediment in the water system. Also, both the presence of bacteria and the combination of bacteria and sediment working together improved the survival of L. pneumophila. And lastly, the sediment was found to have nutritional value—it stimulated the growth of environmental microflora, which then stimulated the growth of L. pneumophila. The results coincide with experimental observations that L. pneumophila prefer to grow and localize in hot, moist environments where there is a supply of nutrition from the sediment. Concluding, it was not surprising that L. pneumophila found their preferable environment to be the potable water system, where they formed interrelationships between temperature, sediment, and microflora. From this data, it is clear that L. pneumophila interact with their environment and the organisms they share that environment with. (6)
L. pneumophila can only be acquired from an environmental source; therefore, infection never occurs between humans or humans and animals. Another interesting thing is, unlike other pathogens that cause bacterial pneumonia, L. pneumophila do not inhabit the upper respiratory tract. Once inhaled, they are small enough to avoid the defenses of the upper airway.
In the lung, pulmonary alveolar macrophages (macrophages of the air sacs in the lung) and sometimes type II alveolar epithelial cells take up L. pneumophila where it begins to grow intracellularly. It seems intracellular infection is necessary for producing infection because mutants of L. pneumophila are unable to cause disease. This is true also because L. pneumophila is not sensitive to antimicrobials (e.g., penicillin, cephalosporin, aminoglycosides) that are excluded by the plasma membrane, and therefore, it is treated only with antibiotics that can enter the host cells (e.g., macrolides, quinolones, tetracyclines). (7)
Then the bacteria undergo a series of events that begins with phagocytosis. Most, but not all strains of L. pneumophila are taken up by “coiling phagocytosis”—when the macrophage coils around the bacteria to take it up, which is mediated by the CR1 and CR3 receptors (complement receptors of the macrophage) with or without fixation of complement serum factors like C3. Also during uptake the plasma membrane engulfing the bacteria is altered; some membrane proteins, like MHCI and II (both molecules that aid in immune response), are specifically excluded whereas others, like CR3 and 5’-nucleotidase are preserved. (7) Two hours later, the phagosome (the vacuole formed around the pathogen) is found near mitochondria, smooth vesicles, or the nuclear membrane. Normally, phagosomes will fuse with lysosomes and pathogenic microorganisms are killed, but in this case acidification of the phagosome does not take place, and its membrane does not get late endosomal markers, like rab7 and LAMP-1. This results in the failure of phagosolysosomal fusion which allows bacteria to avoid intracellular killing. Mutants of L. pneumophila on the other hand cannot avoid it and are transported to the lysosome within 30 minutes of uptake. (7) Fully virulent organisms on the other hand will take up an endosome that has become enclosed by the rough ER (a response to cellular amino acid starvation i.e. autophagy) 4 hours after uptake. It is by this endosome enclosure that bacterial multiplication proceeds. While the bacteria are multiplying inside their host cells, they also “begin to express flagella, motility, cellular toxicity, and sensitivity to physiologic concentrations of sodium chloride.” (7) The genes that mediate intracellular trafficking and necrosis (accidental cell death) are the same genes that introduce the cytotoxic phenotype of L. pneumophila. Introduction of cytotoxicity in response to cellular amino acid starvation in the host may be a key mechanism that signals the bacteria to exit host cells and infect new cells. (7)
A particular feature of Legionella is its dual host system allowing the intracellular growth in protozoa (amoebae), and during infection in human pulmonary alveolar macrophages. (8) Like macrophages, amoebae ingest L. pneumophila by phagocytosis, which can be mediated by amoebic-specific receptors. After ingestion, the bacteria evade phagosolysosomal fusion and localize to a membrane-vesicle surrounded by endoplasmic reticulum where it grows and acquires motility before release from the cell. This growth process of L. pneumophila to infect mammalian phagocytes strongly supports the theory that it evolved from protozoa. “Since L. pneumophila are not transmitted between mammalian hosts or return to the natural environment by infected individuals, this hypothesis explains why this pathogen is adapted to intracellular life in the human lung without any apparent selection for pathogenic traits in the microenvironment.” (7)
The Mip gene, a 24 kDa bacterial envelope protein, was the first virulence determinant of L. pneumophila. The consequence of a Mip mutation Is a 1.5 to 3 log reduction in infectivity of explanted alveolar macrophages, alveolar epithelial cells, and amoebae. (7)
Iron is essential for all pathogenic bacteria. Without it, the growth of intracellular L. pneumophila becomes restricted. It was found that gamma-interferon restricts the growth of L. pneumophila by downregulating the expression transferring receptors on macrophages and the cellular concentration of ferritin. Naturally, L. pneumophila taken from iron-deficient cultures grown are defective in cellular infection. L. pneumophila has a fur homolog, which regulates the expression of an aerobactin synthetase homolog. Even though siderophores have yet to be identified in L. pneumophila, a mutation of this iron acquisition gene will cut intracellular infection by 80-fold. (7)
Studies on intracellular survival of L. pneumophila have shown two chromosomal regions to encode functions that are essential for establishing intracellular infection. The genes have been designated icm (intracellular multiplication) by one group and dot (defect in organelle trafficking) by the other. Mutation in almost any of these genes results in either a complete loss of cellular infectivity. In all cases, the loss of infectivity is associated with a failure to evade phagolysosmal fusion, as well as a loss of the immediate contact cytotoxicity of L. pneumophila at high rates of infections. Mutations in several of the dot-icm genes—not just those with sequence homology to known conjugation genes have been shown to terminate plasmid transfer. These same mutations also result in loss of this toxicity coupled with the insertion of a pore into host cell membranes. The proteins encoded by these loci probably either combine to form the pore or participate in its transfer or both. (7)
Lastly, legionellosis is the infection caused by L. pneumophila and can cause either: Legionnaires' disease, which is characterized by fever, myalgia, cough, pneumonia, or Pontiac fever, a milder illness without pneumonia. The symptoms of Legionnaire’s disease range from a mild cough and low fever to rapidly progressive pneumonia, coma, and death. Early symptoms include slight fever, headache, aching joints and muscles, lack of energy or tiredness, and loss of appetite. Later symptoms include high fever, cough, difficulty breathing/shortness of breath, chills, chest pain, common gastrointestinal symptoms including vomiting, diarrhea, nausea, and abdominal pain. The symptoms of Pontiac fever include flu-like symptoms such as fever, headache, tiredness, loss of appetite, muscle and joint pain, chills, nausea, and a dry cough. Patients usually reach full recovery within two to five days without medical attention and no deaths have been reported. (9)
Much of the research done on Legionella pneumophila is related to ecology and how it relates to the environment. Some studies compare L. pneumophila to other microorganisms. Other studies look at the cases of Legionellosis and investigate the causes of the outbreaks. In this first study, scientists investigate the largest outbreak of Legionellosis associated with spa baths. In July 2002, a large outbreak of legionellosis occurred in a bathhouse with spa facilities in Miyazaki Prefecture. 295 of the costumers had pneumonia and/or symptoms of fever, coughing, etc., 7 of the 295 died, and 37% were hospitalized. Clinical samples were taken from 95 patients, mostly inpatients, and were tested microbiologically. 46 confirmed cases of legionellosis were diagnosed in laboratories. Urine antigen was detected more frequently by Binax NOW immunochromatographic assay than by Biotest EIA. But both assays only detected urine antigen in samples collected within 4 weeks of the outbreak. Also 3 more cases were brought to attention after an administrative report was published. L. pneumophila DNA was detected in 5 of 17 patients by nested PCR. In general, urinary antigen detection and PCR were more effective in laboratory diagnostic tests than culture and serology. But still, culture along with molecular epidemiology is vital for verifying the infection’s source. (10)
The second study compares Streptococcus pneumoniae and Legionella pneumophila pneumonia in HIV-infected patients. The epidemiological data, clinical features and mortality of community-acquired pneumonia (CAP) by both microorganisms were compared and it was concluded that Legionella pneumophila had a higher morbidmortality in HIV+ patients. A comparative study of 15 HIV patients with CAP by L. pneumophila to 46 patients with CAP by S. pneumoniae that took almost 11 years (January 1994 to December 2004) resulted in many observations supporting the fact that L. pneumophila has a higher morbidmortality. First, nothing was observed until antibiotic treatment began. Second, L. pneumophila patients were more frequent smokers, cancer patients, and receivers of chemotherapy. Third, L. pneumophila patients had a significantly higher CD4 count, an untraceable viral load, and were treated more often with highly active antiretroviral therapy. Fourth, interestingly, more S. pneumoniae patients had AIDS. Fifth, L. pneumophila pneumonia was more severe. Sixth, L. pneumophila patients had an increased amount of extrarespiratory symptoms, hypoatraemia, and creatine phosphokinase. Seventh, they also underwent respiratory failure, and required bilateral chest X-rays more. All these results support the fact that there is a higher mortality rate for L. pneumophila pneumonia patients compared to S. pneumoniae patients. (11)
A third study made an interesting discovery that Acanthamoeba polyphaga resuscitates viable non-culturable (VNC) Legionella pneumophila after disinfection. “Amoebae are the natural hosts for Legionella pneumophila and play essential roles in bacterial ecology and infectivity to humans.” (12) Researchers hypothesized that freshwater amoebae play a vital role in bacterial resistance to disinfectants and also in successive revival of VNC L. pneumophila resulting in reemergence of the virulent strain into sterile water sources. With treatment of sodium hypochlorite (NaOCl) as a variable in the experiment, it was found that, indeed, “amoebic trophozoites protect intracellular L. pneumophila from eradication by NaOCl, and play an essential role in resuscitation of VNC L. pneumophila in NaOCl-disinfected water sources”. (12) First, they demonstrated that in the absence of Acanthamoeba polyphaga, treatment of 256 p.p.m. of NaOCl caused 7 L. pneumophila strains to become non-culturable (unable to grow). Then, in the presence of A. polyphaga, an increase of resistance to NaOCl was shown. Furthermore, when disinfected waters were co-cultured with A. polyphaga, the non-culturable L. pneumophila were resurrected, which resulted a dramatic increase in its numbers. In the end, to make sure VNC L. pneumophila is completely eliminated, a co-culture of Legionella-protozoa is the essential tool. (12)
1. Lederberg, Joshua et al. Legionella. Encyclopedia of Microbiology. Second Edition. Volume 3. San Diego, 2000. p. 19
2. Rathore, Mobeen, MD.Legionella Infection. eMedicine. 2006.
3. National Center for Biotechnology Information site: http://www.ncbi.nlm.nih.gov/sites/entrez?db=Genome&itool=toolbar
4. Cazalet C, Rusniok C, Bruggemann H, Zidane N, Magnier A, Ma L, Tichit M, Jarruaud S, Bouchier C, Bandenesch F, Kunst F, Etienne J, Glaser P, Buchrieser C. Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet. 2004. Volume 11. p. 1165-1173.
5. Lederberg, Joshua et al. Legionella. Encyclopedia of Microbiology. Second Edition. Volume 3. San Diego, 2000. p. 19-20
6. Stout JE, Yu VL, Best MG. Ecology of Legionella pneumophila within water distribution systems. Appl Environ Microbiol. 1985. Volume 1. p. 221-228.
7. Lederberg, Joshua et al. Legionella. Encyclopedia of Microbiology. Second Edition. Volume 3. San Diego, 2000. p. 21-24
8. Jules M., Buchriser C. Legionella pneumophila adaptation to intracellular life and the host response: Clues from genomics and transcriptomics. FEBS Lett. 2007.
9. Centers for Disease Control and Prevention site: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/legionellosis_g.htm
10. Kawano K, Okada M, Kura F, Amemura-Maekawa J, Watanabe H. Largest outbreak of legionellosis associated with spa baths: comparison of diagnostic tests. Kansenshogaku Zasshi. 2007. Volume 2. p. 173-182.
11. Pedro-Botet ML, Spoena N, Garcia-Cruz A, Mateu L, Garcia-Nunez M, Rev-Joly C, Sabria M. Streptococcus pneumoniae and Legionella pneumophila pneumonia in HIV-infected patients. Scand J Infect Dis. 2007. Volume 2. p. 122-128.
12. Garcia MT, Jones S, Pelaz C, Millar RD, Abu Kwaik Y. Acanthamoeba polyphaga resuscitates viable non-culturable Legionella pneumophila after disinfection. Environ Microbiol. 2007. Volume 5. p. 1267-1277.