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.

▶Classification

  • 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

Morphology

  • 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.

Cultivation

  • 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.

Resistance

  • 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.

▶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.