Legionella pneumophila, the primary causative agent for Legionnaires’ disease, is an aerobic, pleomorphic, flagellated, non-spore-forming, Gram-negative bacterium. L. pneumophila is an intracellular bacterium that preferentially infects soil amoebae and freshwater protozoa for replication. Due to L. pneumophila's ability to thrive in water, it can grow in water filtration systems, leading to faucets, showers, and other fixtures. Aerosolized water droplets containing L. pneumophila originating from these fixtures may be inhaled by humans. Upon entry to the human respiratory tract, L. pneumophila is able to infect and reproduce within human alveolar macrophages. L. pneumophila infections can be diagnosed by a urine antigen test. The infections caused by the bacteria can be treated with fluoroquinolones and azithromycin antibiotics. It is a Gram-negative, aerobic bacterium that is non-fermentative. It is oxidase- and catalase-positive. L. pneumophila colony morphology is gray-white with a textured, cut-glass appearance; it also requires cysteine and iron to thrive. It grows on buffered charcoal yeast extract agar as well as in moist environments, such as tap water, in "opal-like" colonies.
L. pneumophila is an intracellular bacterium. As an intracellular bacterium, L. pneumophila has a preferential parasitic relationship with protozoa, which serve as a reservoir for the bacterium. This relationship allows the bacterium to shield itself from environmental stressors contributing to its ability to grow quickly. Disinfectants are also often ineffective against the bacterium once an environment has been infected due to their intracellular nature. Resistance to these environmental stressors contributes to the pathogenicity and virulence of the microbe. This form also corresponds with infection, and this virulence is partially characterized by the flagellation of the microbe and a decrease in sodium ion resistance.
Cell membrane structure
Since L. pneumophila is a Gram-negative bacterium, its unique outer membrane composed of lipoproteins, phospholipids, and other proteins is the distinguishing feature of Legionella Legionella spp. possess unique lipopolysaccharides (LPS) extending from the outer membrane leaflet of the outer cell membrane that play a role in pathogenicity and adhesion to a host cell. Lipopolysaccharides are the leading surface antigen of all Legionella species including L. pneumophila. The unique attributes are key factors in its serological identity and biological function.
L. pneumophila is notably resistant to chlorine derivatives that are commonly used to control water borne pathogens. This resistance allows infiltration and persistence in water systems even when standard disinfectant processes are employed. Of note, this bacterium can form and reside in biofilms within water system pipes, allowing it be aerosolized through fixtures such as faucets, showers, and sprinklers. Exposure to these aerosols can lead to infection in susceptible individuals.
Biofilms
Biofilms are specialized, surface attachment communities that can consist of one or multiple microbes, ranging from bacteria, algae, and protozoa. L. pneumophila attaches well to plumbing plastics, whereas copper inhibits L. pneumophila attachment. Carbon-rich environments are favorable for L. pneumophila biofilms, as these environments provide nutrients for replication. Studies suggest that L. pneumophila prefers to adhere to pre-established biofilms instead of being the primary microbe to attach to uncolonized surfaces.
The biofilm-forming capabilities of L. pneumophila are heavily correlated with outbreaks in Legionnaires’ disease and stronger virulence. When taking part in multispecies biofilm formation, L. pneumophila persistence is heavily influenced by other microorganisms. Protozoa is one of the most notable microorganisms that influences L. pneumophila persistence, since L. pneumophila utilizes protozoa to replicate itself.
Metabolism
L. pnemophila metabolize both carbohydrates and complex polysaccharides minimally, but amino acids are the main carbon and energy source for the bacterium. Amino acids are imported from the host organism and used by L. pneumophila to generate energy through the citric acid cycle (Krebs cycle) and as sources of carbon and nitrogen.
L. pneumophila metabolizes glucose using the Entner-Doudoroff (ED) pathway and pentose phosphate pathway (PP) as well as glycolysis and the citric acid cycle (TCA). Though glucose is not the main carbon source for the bacterium, glucose generates poly-3-hydroxybutyrate (PHB) through the ED pathway, which is a storage molecule converted to acetyl-CoA for use by the TCA cycle (Krebs cycle) when the microbe is nutrient deprived. Even though L. pneumophila primarily uses amino acids as a carbon source, the bacteria does contain multiple amylases, such as LamB which hydrolyzes polysaccharides into glucose monomers for metabolism.
Genomics
There are 15 known serogroups of L. pneumophila, but serogroup 1 is most commonly causes Legionnaires' disease. Serogroups 5 and 10 may also cause rare co-infections. The genomes of six strains, L. pneumophila Philadelphia, L. pneumophila Paris, L. pneumophila Lens, L. pneumophila Corby, L. pneumophila Alcoy, and L. pneumophila 130b were isolated from 2004-2010 which paved the way for understanding the molecular biology of the bacteria.
Subspecies, which are commonly defined by geographical location, share about 80% of their genome with variation between strains that account for the difference in virulence between subspecies. The genome is relatively large of about 3.5 mega base pairs (mbp) which reflects a higher number of genes, corresponding with the ability of Legionella to adapt to different hosts and environments. The bffA gene is associated with biofilm formation, and it is seen that strains without this gene form biofilms both quicker and thicker which aids in resistance to environmental stressors. In-depth comparative genome analysis using DNA arrays to study the gene content of 180 Legionella strains revealed high genomic plasticity and frequent horizontal gene transfer events.
Pathogenesis
thumb|225x225px|The life cycle of L. pneumophila within a [[amoeba.]]thumb|Image of alveoli filled with leukocytes and fibrin due to pneumonia caused by Legionella bacteria.
L. pneumophila is able to invade and replicate within human alveolar macrophages. Internalization of the bacteria appears to occur through phagocytosis or coiling phagocytosis and is reliant on Dot/Icm type 4B secretion system (T4BSS). Once internalized, the Dot/Icm system begins secreting bacterial effector proteins that recruit host factors to the Legionella containing vacuole (LCV). This process prevents the LCV from fusing with the lysosomes that would otherwise degrade the bacteria. Vesicles of the host cell's rough endoplasmic reticulum are attracted to the LCV, and these vacuoles supply the LCV with necessary lipids and proteins. L. pneumophila replication occurs within the LCV. Once nutrients are depleted, the bacteria gain flagella and cytoxicity. To exit the host cell, L. pneumophila lyses the LCV and resides in the cytoplasm. In the cytoplasm, L. pneumophila inhibit organelle and plasma membrane function and structure which ultimately leads to osmotic lysis of the host cell.
Virulence factors
Membrane based
L. pneumophila exhibits a unique lipopolysaccharide (LPS) structure that is highly hydrophobic due to its being densely packed with branched fatty acids, and elevated levels of O-acetyl and N-acetyl groups. This structure helps prevent interaction with a common LPS immune system co-receptor, CD14. There is also a correlation between an LPS with a high molecular-weight and the inhibition of phagosome-lysosome fusion. L. pneumophila produces pili of varying lengths. The two pili proteins: PilE and Prepilin peptidase (PilD) are responsible for the production of type IV pili and subsequently, intracellular proliferation. L. pneumophila possesses a singular, polar flagellum that is used for cell motility, adhesion, host invasion, and biofilm formation. The same regulators that control flagellation also control lysosome avoidance and cytotoxicity.
Another key virulence factor of L. pneumophila is iron acquisition, the microbe utilizes two methods of iron uptake. Ferrous iron is collected through the use of a transport system involving an inner-membrane protein known as protein FeoB. Optimal intracellular infection is achieved in amoebae and macrophages via this transport system. The second form of uptake, involving ferric iron, is achieved through an iron chelator known as legiobactin. This is secreted by L. pneumophila when the microbes are being grown in a low iron chemically designed media.
Significant progress has also been made in identifying surface structures of Legionella pneumophila that contribute to its virulence and intracellular infection. One of the most notable of these surface features is the flagellum. Although the flagellum is not necessarily required for intracellular replication, it promotes host cell invasion through mechanisms that are independent of adherence. A flagellum's expression is regulated by growth conditions and is typically observed only in the motile, virulent phase of the bacterium. It is considered a potential virulence factor, as non-flagellated mutants exhibit reduced infectivity. This is further supported by observations that 1` in the exponential growth phase is non-motile and displays decreased virulence.
Protozoan interaction
L. pneumophila is capable of infecting and multiplying within various species of free-living protists and amoebas, which further enhances their survivability. Protozoa that are associated with this bacteria are Acanthamoeba , Saccamoeba, and Platyamoeba. These protozoa provides nutrients to the bacteria allowing its longevity to be increased. Furthermore under stress, protozoa plays an essential role in keeping L. pneumophila alive and can bring them back to life. This bacteria first enters into protozoa in a unique way in order to survive. They build vesicles in order to enter into the host and use them to replicate. This same process is seen when they infect humans as well. In fact scientists believe that L. pneumophila ability to survive in human macrophages is due to how they adapted to survive in protozoa.[https://www.mdpi.com/2076-2607/11/1/74]
Legionella-containing vacuole
240px|thumbnail|right|TEM image of L. pneumophila within a [[phagocytic cell]]
For Legionella to survive within macrophages and protozoa, it must create a specialized compartment known as the Legionella-containing vacuole (LCV). Through the action of the Dot/Icm secretion system, the bacteria are able to prevent degradation by the normal endosomal trafficking pathway and instead replicate. Shortly after internalization, the bacteria specifically recruit endoplasmic reticulum-derived vesicles and mitochondria to the LCV while preventing the recruitment of endosomal markers such as Rab5a and Rab7a. Formation and maintenance of the vacuoles are crucial for pathogenesis; bacteria lacking the Dot/Icm secretion system are not pathogenic and cannot replicate within cells, while deletion of the Dot/Icm effector SdhA results in destabilization of the vacuolar membrane and no bacterial replication.
Detection and treatment
Antisera have been used both for slide agglutination studies and for direct detection of bacteria in tissues using immunofluorescence via fluorescent-labelled antibodies. Specific antibodies in patients can be determined by the indirect fluorescent antibody test. ELISA and microagglutination tests have also been successfully applied. Levofloxacin and azithromycin have great intracellular activity and are able to penetrate into Legionella-infected cells. The Infectious Diseases Society of America recommends 5–10 days of treatment with levofloxacin or 3–5 days of treatment with azithromycin; however, patients that are immunocompromised or have a severe disease may require an extended course of treatment. Roughly 2 out of 100,000 people are infected each year in the European Union (EU), with an infection rate of approximately 5 per 100,000 in Italy. The highest reported amount of cases in the US, EU, and Italy have been among men over the age of 50. Several large outbreaks of Legionnaires’ Disease have come from public hot tubs due to the temperature range of the water being ideal for the bacteria's growth.
thumb|Image shows x-rays of the lungs that depicts a case of pneumonia caused by L. pneumophila.
Legionnaires’ disease gained globally recognition after an outbreak in 1976 at a hotel in Philadelphia, Pennsylvania. The causative agent of the outbreak was L. pneumophila, which had contaminated the hotel's air conditioning water supply, allowing the microbe to be dispersed within the hotel's environment. A prominent mode of transmission for the disease is the inhalation of contaminated water aerosols.
Besides Legionnaires’ disease, L. pneumophila is also responsible for Pontiac fever. Pontiac fever occurs when L. pneumophila is in a non-pneumatic form. The first outbreak of Pontiac fever was reported in 1968 in Pontiac, Michigan with approximately 144 cases. During the first outbreak, there was a mandatory 48 hour incubation period. Common symptoms of Pontiac fever include fever and headaches and the illness itself last between 2-5 days. In recent decades, Pontiac fever and Legionnaires’ disease cases have increased.[https://www.mdpi.com/2077-0383/11/20/6126]
Using a OneHealth framework is essential to understanding the prevalence of this L. pneumophila. This is because its transmission is entirely driven by interactions between humans, water infrastructure, and environmental conditions. Since the bacterium naturally persists in freshwater and man-made water systems, the risk of human infection greatly increases when environmental management or public health monitoring breaks down. It is because of this interconnectedness that it remains vital that surveillance of water systems and environmental controls are intact and used as clinical detection and treatment.