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. This causes the onset of Legionnaires' disease, also known as legionellosis. Infected humans may display symptoms such as fever, delirium, diarrhea, and decreased liver and kidney function. 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.
Characterization L. pneumophila is a coccobacillus. 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. pneumophilia is a generally mesophilic bacterium preferring environments between 20 °C and 59 °C and tolerating environments up to 66 °C; death is observed at temperatures at or above 70 °C. The bacterium is also generally tolerate of high NaCl solute concentrations enduring 1.5% sodium chloride in laboratory conditions and up to 3% sodium chloride in vivo.
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. Predators of bacteria, such as amoeba and ciliates, are natural hosts for L. pneumophila while humans are accidental hosts, as evidenced by there being only one reported case of L. pneumophila human-to-human transmission. Rather than through contagious spread, it infects the alveolar macrophages in human lungs when inhaled as aerosol.
A special characteristic allows for the microbe to thrive in extracellular environments, such as various freshwater environments. This is achieved through its two forms: transmissive and replicative. The transition between the two is activated by changes in the availability of metabolic/nutritional resources in its current environment. The transmissive phase is favored in nutrient-low environments where the bacterium exhibits spore-like character. 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. The replicative form follows to carry out proliferation.
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 bases for the somatic antigen specificity of this organism are located on the side chains of its cell wall. The chemical composition of these LPS side chains both with respect to components and arrangement of the different sugars, determines the nature of the somatic or O-antigenic determinants, which are important means of serologically classifying many Gram-negative bacteria. L. pneumophila exhibits distinct chemical characteristics in its LPS structure that distinguish it from other Gram-negative bacteria. The unique attributes are key factors in its serological identity and biological function.
Ecology
L. pneumophila is able to live in a diverse range of environmental conditions, tolerating temperatures from 0 °C-63 °C, a pH range of 5.0–8.5, and in dissolved oxygen concentrations of 0.2-15.0 mg/liter. However, it multiplies only within a narrower temperature range of 25 °C to 42 °C.
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. Water supply networks are the main source of L. pneumophila contamination, with the microbe commonly found in places such as cooling towers, water systems of hospitals, hotels, and cruise ships. 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. These protective matrixes enable the microbe to live for extended periods of time in low-nutrient environments and in the presence of biocides.
L. pneumophila is highly present in water systems, both natural and manmade. 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.
Multispecies biofilm on plumbing systems and in water distribution systems facilitate L. pneumophila growth due to the presence of freshwater protozoa. L. pneumophila has the ability to live independently in biofilms, or can grow intracellularly in protozoa.
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. In addition to glucose, L. pneumophila can use both glycogen and starch as a carbon source using the glucoamylase GamA. The microbes main carbon source is amino acids as it preferentially metabolizes glutamic acid, serine, threonine, and tyrosine. The glutamate-aspartate transaminase reaction was also found to be connected with gluconeogenesis and the citric acid cycle within L. pneumophila. Glycerol is also used as a substrate, as indicated by transcriptome analysis.
Nutrient acquisition Legionella is auxotrophic for seven essential amino acids: cysteine, leucine, methionine, valine, threonine, isoleucine, and arginine. Ala, Asp, and Glu are also key amino acids required by the microbe, but they are co-metabolized from host catabolites. Inside the vacuole, nutrient availability is low; the high demand of amino acids is not covered by the transport of free amino acids found in the host cytoplasm. To improve the availability of amino acids, the parasite promotes the host mechanisms of proteasomal degradation using the bacterium's AnkB protein. This generates an excess of free amino acids in the cytoplasm of L. pneumophila-infected cells that can be used for intravacuolar proliferation of the parasite. Amino acids are imported into the LCV through various amino acid transporters such as the neutral amino acid transporter B(0). 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. The ED pathway is mainly used for glucose metabolism, but the L. pnemophila genome contains genes encoding for a multitude of alternative pathways.
Protein degradation to recycle amino acids and hydrolyzing polysaccharides are not the only methods by which L. pneumophila obtains carbon and energy sources from the host. Type II–secreted degradative enzymes may provide an additional strategy to generate carbon and energy sources. L. pneumophila is the only known intracellular pathogen to have a Type II Secretion System (secretome).
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. There is a relatively high abundance of genes encoding eukaryotic-like proteins (ELPs). ELPs are beneficial for mimicking the bacteria's eukaryotic hosts for pathogenicity. Other genes of L. pneumophila encode for Legionella-specific vacuoles, efflux transports, ankyrin-repeat proteins, and many other virulence related characteristics. 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. Horizontal gene transfer allows L. pneumophila to evolve at a rapid pace and commonly is associated with drug resistance.
Pathogenesis