Legionella pneumophila: Overview

▶The History of Discovery

  • The first mass outbreak of legionellosis designated later as “Legionnaires’ disease” was registered in Philadelphia in 1976 among the participants of convention of American Legion (US veterans’ organization).
  • This disease manifested as severe pneumonia. The outbreak demonstrated high case-fatality ratio above 15% – from 4400 delegates attending the meeting 182 have become ill and 29 died.
  • The causative agent of this disease was discovered in 1977 by J.E. McDade and C.C. Shepard after its isolation from the lung tissue of patients died from pneumonia.


  • The bacteria pertain to the order Legionellales and family Legionellaceae. This family includes the single genus Legionella.
  • Near 60 legionella species are known to date. Despite more than 20 species are encountered as human pathogens in certain clinical conditions, Legionella pneumophila is responsible for more than 90% of cases of legionella-associated infections including the most severe clinical forms like Legionnaires’ disease.

▶Structure and Properties of Legionella pneumophila


  • The bacteria are small polymorphic gram-negative rods with tapered ends; coccobacteria and filamentous forms can be observed.
  • Microbial cells have no capsule or spore, albeit possess 1-3 polar flagella.
  • These bacteria are facultative intracellular parasites. In natural conditions they replicate inside water protozoa, e.g. amoebae cells.
  • Life cycle of Legionella pneumophila includes two basic phases – replicative and transmissive; each represents distinct morphological forms of bacteria.
  • In replicative phase under nutrient-rich conditions the bacteria actively propagate intracellularly within Legionella-containing vacuole (or LCV). Microbial cells look like non-flagellated long rods, which are low-cytotoxic and low-virulent.
  • When the conditions become worsened, Legionella pneumophila transforms into short thick motile rods, which are stress-resistant, cytotoxic and demonstrate enhanced virulence. These bacteria leave the host cell being capable of infecting new cells (transmissive phase).
  • If replicated inside free-living amoeba cells, transmissive phase results in almost dormant spore-like but motile and virulent mature infectious forms (MIFs). In case of prolonged stay in water they turn into viable but non-culturable (VBNC) morphological forms.


  • Legionella pneumophila grows in aerobic conditions in atmosphere, supplemented with 5% CO2.
  • The bacterium needs special media for culture like buffered charcoal yeast extract (BCYE) agar with cystein, iron salts (ferric pyrophosphate) and antibiotics, or blood agar with various supplements.
  • The optimum temperature for growth is 35-37oС. In 3-5 days “opal-like” gray-white colonies appear. Sometimes they may produce brownish pigment.
  • Blood agar culture may show hemolysis. Some strains produce autofluorescence.
  • As facultative intracellular parasites, legionellae grow well in cell culture lines and yolk sac of chicken embryos.

Biochemical properties

  • Legionella pneumophila is aerobic bacterium that produces oxidase and catalase.
  • As primary source of carbon and energy the bacteria largely use amino acids (e.g., serine). To lesser extent the bacteria metabolize glucose.
  • L. pneumophila has no urease, but possesses the number of proteases. Microbial cells liquefy gelatin and slowly hydrolyze starch.

Antigenic structure

  • L. pneumophila are divided into at least 16 serogroups by their thermostable somatic polysaccharide O-antigen.
  • Nevertheless, about 85% of all cases of Legionnaires’ disease are related with bacteria of serogroup 1.
  • Bacterial H-Ag is lack of diagnostic value.

Virulence factors

  • L. pneumophila has powerful systems of protein secretion that ensure the translocation of virulent effector proteins into the host cells.
  • The structures of type IV secretion system (T4SS) generally termed as translocon deliver almost 300 microbial effector proteins into eukaryotic cell.
  • They govern all the process of bacterial habitation inside the host cells – from microbial entry and its replication in legionella-containing vacuole up to bacterial egress and infection of new host cells.
  • These proteins account for microbial long-term survival within phagocytes inhibiting phagosome-lysosome fusion.
  • Many bacterial effector proteins share evident similarity with proteins of eukaryotic host cells thus emphasizing the unique capacity of L. pneumophila to interkingdom gene exchange.
  • Additional type II secretion system of legionellae (LSP – Legionella secretion pathway) stimulates the secretion of virulent microbial enzymes.
  • Among them are numerous phospholipases A and C that destroy the membranes of cells. Also the bacteria produce metalloproteases, phosphatases and other enzymes.
  • Bacterial exotoxins legiolysin and cytolysin contribute to the membrane pore formation, lysis of host cells and hemolytic activity of legionellae.
  • Outer membrane proteins participate in adhesion. The flagella foster microbial entry into the cells.
  • When living outside the natural hosts, L. pneumophila indispensably creates tough biofilm, firmly attached to the underlying surface. Within biofilm the bacteria remain highly protected against natural and artificial biocides.


  • As the bacteria normally live in freshwater reservoirs, they are markedly resistant in watery environment.
  • They stay viable for years in tap water, artificial systems of water supply, cooling towers, fountains, spa baths, etc.
  • Protozoans, harboring the bacteria, protect them from the action of biocides. Nevertheless, microbial cells are generally sensitive to conventional disinfectants (e.g., chlorine-containing substances, phenol, aldehydes, ethanol, etc.).
  • For water disinfection chloramine and calcium hypochlorite are commonly used.

▶Pathogenesis and Clinical Findings in Legionelloses

  • Legionellae are broadly distributed in nature. They are normal habitants of freshwater sources, where they predominantly live inside the ciliated protozoa (like amoebae Acanthamoeba or Naegleria) or in slime moulds.
  • Dwelling in protozoan cells is beneficial for bacterial survival protecting them from harsh environmental influences and providing with nutrients.
  • Generally present in low amounts in natural freshwater habitats, L. pneumophila intensively colonizes human-made acquatic systems that operate in temperature range 25-55ºC and produce large amounts of water aerosol.
  • They are found in hot-water supplies, air-conditioning cooling towers, baths, shower-rooms, whirlpool and thermal spas, etc. The bacteria form poorly permeable biofilm on plastics and other artificial surfaces.
  • As the environmental conditions play a decisive role in microbial propagation and spread, the infections caused by L. pneumophila are regarded as typical sapronoses.
  • Humans are occasionally infected with L. pneumophila being exposed to infected water aerosol. Overall, humans are the “dead ends” for legionellae replication.
  • There are two main forms of human L. pneumophila infections – Legionnaires’ disease and Pontiac fever. The individuals with healthy immune status usually demonstrate self-limiting illness or remain asymptomatic.
  • Pontiac fever is relatively benign infection of upper respiratory tract with favorable prognosis.
  • By contrast, Legionnaires’ disease is severe lung disorder manifesting like atypical pneumonia with serious prognosis and high fatality rate especially in cases of epidemic outbreaks.
  • It is opportunistic infection predominantly affecting males with chronic lung diseases, smokers, immunocompromised or elderly persons, cancer patients, etc. The disease may arise as hospital outbreaks.
  • Transmission route for the infection – airborne via infected aerosol. Human-to-human transmission is not observed.
  • Incubation period varies from 2 to 14 days.
  • When appeared in the airways, L. pneumophila is captured by alveolar macrophages and epithelial cells. The bacteria enter the macrophages by macropinocytosis or coiling phagocytosis, thereby making Legionella-containing vacuole (LCV) isolated from cytoplasm by membrane.
  • All this process is controlled by effector proteins of type IV secretion system. These proteins also inhibit phagosome-lysosome fusion, thus preventing microbial digestion and vacuole acidification.
  • Inside LCV the bacteria come into replicative phase and propagate. When the nutrients are exhausted, they undergo transformation into motile transmissive virulent forms.
  • By the action of cytotoxins and enzymes legionellae penetrate vacuole, move into cytoplasm and finally leave the cell through the pores, created in cytoplasmic membrane.
  • This leads to the destruction of respiratory epithelium and macrophages and stimulates inflammatory response.
  • Newly generated bacteria commence to infect neighbouring host cells leading to microbial dissemination.
  • Legionnaires’ disease affects lower respiratory tract – terminal bronchioli and alveoli – resulting in severe lobar pneumonia.
  • The disease has sharp onset with fever, chills and headache. This is followed by cough, tachypnea, and chest pain.
  • Necrosis of lung tissue may stimulate further microbial spread. It results in systemic infection and septic shock with lung hemorrhages, damage of gastrointestinal tract, kidneys and CNS.
  • Lethality in Legionnaires’ disease strongly depends on initial patient’s state, comorbidity and quality of treatment. Usually it falls into the range 8-25% but in case of hospital outbreaks it may exceed 50% in persons with immunosuppression.
  • Humoral and cellular post-infectious immunity is type-specific, protective and relatively stable.

▶Laboratory Diagnosis of Legionellosis

  • The specimens are taken from sputum, pleural exudate, blood, urine, samples of lung tissue on autopsy.
  • Rapid detection of bacteria is elaborated by immunofluorescence test; microbial antigens are determined by ELISA.
  • DNA of L. pneumophila in clinical samples is detected by genetic tests (PCR).
  • Microbial culture isolation is performed in blood agar and buffered charcoal yeast extract (BCYE) agar with cystein, iron salts and antibiotics.
  • After incubation for 3-5 days characteristic “opal-like” gray-white colonies are determined. The growth on blood agar is followed by hemolysis.
  • Microscopy of culture reveals small polymorphic gram-negative rods.
  • Identification of microbial serogroup is made by agglutination test – most of virulent L. pneumophila pertain to serogroup 1.
  • Additional biochemical tests for utilization of amino acids, proteins and carbohydrates are elaborated.
  • Besides agar plating, L. pneumophila can be cultured in various cell lines (macrophage or epithelial cultures) and in laboratory animals (e.g., guinea pigs).
  • Serological testing is performed by indirect immunofluorescence and ELISA. The diagnostic titer of patients’ antibodies in single immunofluorescence test is 1:128 and higher.
  • Serological testing can be also carried out with paired sera tests, where fourfold rise in antibody titer should be observed.

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▶Treatment and Prophylaxis of Legionellosis

  • Favorable prognosis of Legionnaires’ disease strongly depends on timely administrated adequate antibiotic treatment.
  • Macrolides and azalides (e.g. azithromycin and clarithromycin) as well as respiratory fluoroquinolones are the drugs of choice for legionellosis treatment.
  • Additional cure includes fluid resuscitation, administration of glucocorticoids, mechanical lung ventilation if required.
  • Prophylaxis of infection is non-specific. Sanitary control measures should prevent microbial contamination of public and private water systems as well as exclude the possibility of hospital outbreaks of Legionnaires’ disease.
  • A proper strategy of prevention of disease spread in health care settings comprises efficient disinfection of systems of water supply, air conditioning, and patient management; laboratory testing of patients with hospital-acquired pneumonia for legionellosis; epidemiological investigations of disease outbreaks with clarification of transmission routes.
  • High-efficacy measure resulting in eradication of L. pneumophila from artificial water systems is the increase of temperature of circulating water above 60ºC.
  • From commonly available disinfectants chloramine demonstrates elevated biocidal activity against legionellae.