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Legionnaires’ Disease and Pontiac Fever
Reviewed for currency January 14, 2021
History
Legionnaires’ disease (LD) is an acute pneumonic illness caused by gram-negative bacilli of the genus Legionella , the most common of which is Legionella pneumophila (Lp). Pontiac fever (PF) is a febrile, nonpneumonic, systemic illness closely associated with, if not caused by, Legionella spp. Legionellosis is the term that encompasses all diseases caused by, or presumed to be caused by, the Legionella bacteria, including LD, focal nonpulmonary infections, and PF.
LD was first recognized when it caused an epidemic of pneumonia at a Pennsylvania State American Legion convention in Philadelphia in 1976; 221 people were affected, and 34 died. Despite intensive laboratory investigation, the cause of the outbreak went undetected for many months. Epidemiologic investigation concluded that the disease was most likely airborne, and focused primarily at one convention hotel, which closed because of adverse publicity. About 6 months later, Joseph McDade and Charles Shepard, investigators at the Centers for Disease Control and Prevention, discovered the etiologic agent, a fastidious gram-negative bacillus. Because of the historical association with the American Legion convention, this disease is now called “legionnaires’ disease,” and the etiologic agents belong to the family Legionellaceae, with Lp being the agent responsible for the 1976 Philadelphia epidemic. Several past unsolved outbreaks of pneumonia in the 1950s to the early 1970s had been LD. An unsolved epidemic of a nonpneumonic febrile illness in Pontiac, Michigan, was found to be associated with Lp exposure; this illness was termed “Pontiac fever”. Prior epidemics of PF occurred as early as 1949. Bacterial isolates from the 1940s through the 1960s, previously thought to be rickettsial agents, were found to be Legionella bacteria. Both the organism and the disease had been studied decades before, but major advances in technology were required to properly determine its cause.
Even with identification of Lp in 1977 as the cause of LD, how best to diagnose the disease and ways to abort epidemics of LD remained uncertain for several years. Epidemics of the disease, especially nosocomial ones, commonly lasted for years. It was discovered that Lp and other Legionella spp. were naturally occurring aquatic bacteria that grew in warm water, in cooling towers, water heaters, and potable water plumbing. These discoveries led to the end of several multiyear outbreaks of the disease. It is now unusual for LD outbreaks to last more than a week or two.
LD occurs in both sporadic and epidemic forms, sometimes involving many hundreds of victims. The disease, while a relatively rare (1%–5%) cause of community-acquired pneumonia that is often easily treated and relatively mild, can cause severe, and fatal, disease.
The Etiologic Agent
The Legionella spp. are small gram-negative bacilli with fastidious growth requirements. Amino acids are the main energy source for extracellular growth, with glycerol or glucose used during intracellular growth. Obligate aerobes, the bacteria grow at temperatures ranging from 20°C to 42°C. Coxiella burnetii, an obligate intracellular parasite and the etiologic agent of Q fever, is the closest relative of the Legionellaceae. l -Cysteine is required for the growth of all but one of the clinically important Legionella spp., and this amino acid is needed for the initial growth of all described Legionella spp. from environmental or clinical sources. Soluble iron is required for optimal growth, and for initial isolation of the bacterium from both clinical and environmental sources. A yeast extract agar containing iron, l -cysteine, α-ketoglutarate, and charcoal and buffered with an organic buffer (buffered charcoal-yeast extract [BCYEα] agar) is the preferred growth medium for clinical isolation. Clinically important Legionella spp. grow best at 35°C in humidified air on BCYEα medium, usually in 3 to 5 days after inoculation of plates. Up to 14 days’ incubation may very rarely be required for the isolation of unusual Legionella spp.
More than 60 different Legionella spp. have been described, about half of which have been reported to infect humans. Lp serogroup 1 ( Lp 1) caused the 1976 Philadelphia outbreak, and is the cause of 65% to 90% of all cases of LD for which there is a bacterial isolate. Lp 1 dominance is not universal. For example, Lp 1 constituted only 66% of Legionella spp. isolates in LD patients in Ontario Province, Canada, with Lp serogroup 6 being a relatively common isolate in that province. Also, L. longbeachae (Llb) causes 50% to 85% of LD cases in New Zealand, and a similar fraction in Australia. Lp 1 can be divided into multiple subtypes using a variety of serologic, other phenotypic, and genetic methods. One particular subtype of Lp 1 causes 55% to 76% of cases of LD due to Lp, and 85% of cases due to Lp 1. This “Pontiac” subtype reacts with a specific monoclonal antibody and contains the lag-1 gene encoding a lipopolysaccharide acetyl transferase. The most common non- Lp species that are isolated from humans are Llb, L. micdadei, L. bozemanae, and L. dumoffii, which, with the exception of Australasia, constitute fewer than 5% of culture-proven cases.
Most clinical microbiology laboratories should be able to identify Legionella bacteria to the genus level ( Fig. 232.1 ). Identification of Lp and Lp 1 can be accomplished by sophisticated laboratories. Identification of other Lp serogroups and other Legionella spp. is more difficult, requiring molecular testing for accurate identification. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identification of bacterial colonies can be useful for the identification of common Legionella spp., but may not be completely accurate depending on the validity of the database, with custom databases increasing the accuracy of bacterial identification.
Microbial Ecology
The Legionella bacteria are found in our natural aqueous environment, in lakes, streams, and even coastal oceans, at temperatures ranging from 5°C to greater than 50°C. Warm water (25°C–40°C) supports the highest concentration of these bacteria, with warm water being the major bacterial reservoir leading to LD. Free-living amebae in the same waters support the intracellular growth and survival of the Legionella bacteria ( Fig. 232.2 ). When faced with inimical environmental factors the Legionella -infected amebae encyst, allowing the survival of both the host and parasite. In both natural and man-made waters, Legionella -infected amebae are found in consortia of many different microorganisms, all of which exist in a biofilm.
In addition to intraamebal survival, free-living Legionella bacteria can enter a low metabolic state termed “viable but not cultivatable”, making them difficult to recover from the environment and biocide-resistant. The Legionella bacteria, amebae, and other microorganisms constantly escape from the biofilm because of pressure fluxes into a freely moving phase. Environmental changes that disrupt the biofilm can result in the sudden and massive release of Legionella bacteria into the surrounding water. If this water is then aerosolized or aspirated, the bacteria can cause illness in a susceptible host. Almost all cases of LD result from Legionella contamination of warm man-made water sources. Legionella is one of several opportunistic pathogens that are found in the built plumbing environment. Some recently reported novel water sources include rain puddles in tropical regions, tsunami-related water exposure, windshield wiper fluid, water from truck tankers used to clean or maintain roads, and corroded domestic water pipes. One exception to water as a risk factor is that Llb appears to be transmitted mainly through potting soil used by gardeners. Water treatment of source water to make potable water may increase the concentration of Legionella bacteria (and mycobacteria) in the water. Legionella bacteria are present in very low concentrations in disinfectant-treated cold potable water, usually at levels of less than 1 bacterium per liter. The bacterial density can be amplified by growth in biofilm within water distribution pipes, especially older pipes with low or no water flow. The bacteria can be further amplified in the presence of warm conditions, such as those found in many buildings or heat rejection devices. Legionella bacteria concentrations in air-conditioning cooling towers range from 10 2 to 10 8 colony-forming units (CFU)/L. Up to 80% of air-conditioning cooling towers tested contain the bacterium, as do 5% to 30% of home and industrial water heaters and hot water plumbing. Contaminated water that is aerosolized serves as a disseminator of the bacteria into the environment. The concentration of Legionella bacteria in a particular environmental site may spontaneously fluctuate over a wide range.
Pathogenesis
Overview
LD is initiated by inhalation, and probably microaspiration, of Legionella bacteria into the lungs. Although Legionella bacteria are ubiquitous in our environment, they rarely cause disease. A confluence of a number of factors must occur simultaneously before LD is possible. These factors include the presence of virulent strains in an environmental site; a means for dissemination of the bacteria, such as by aerosolization; and proper environmental conditions allowing the survival and inhalation of an infectious dose of the bacteria by a susceptible host. Strains of different virulence exist for the same species, and some species and serogroups are more virulent than others. Possible strain virulence factors include aerosol stability, ability to grow within macrophages, possession of eukaryotic gene homologs, and surface hydrophobicity.
The infectious form of the bacterium is not known, but in all cases the bacteria originate from water or soil. Several possibilities exist for the infectious particle, including bacteria contained within an amebal cyst, a sporelike form, a biofilm particle containing Legionella bacteria and other bacteria, and freely dispersed extracellular Legionella bacteria. Virulence increases when the bacterium is grown in amebae, in the late stationary phase in vitro, or as the sporelike form. The bacterial inoculum required to cause LD is unknown. Guinea pigs develop asymptomatic infection, disease, and death with inocula of 10 to 100, 1000, and 10,000 bacteria, respectively. Bacteria in amebal cysts or in a biofilm fragment contain greater than 1000 bacteria, making it possible that inhalation of an infected amebal cyst or biofilm fragment could cause disease. Survival of aerosolized extracellular Lp is dependent on relative humidity. Relative humidity may be a key factor in disease transmission. LD is a seasonal disease in some regions, with most cases occurring in the warmer months. For example, in the northeastern United States, the majority of cases occur from June through early October. This is probably because of elevated ambient temperature and humidity.
After bacteria enter the lung, they are phagocytosed by alveolar macrophages. The bacteria produce virulence factors that enhance phagocytosis and promote intracellular survival and replication (see “ Legionella pneumophila Virulence Factors ” below). After sufficient intracellular replication, the bacteria kill the macrophage, escape into the extracellular environment, and are then rephagocytosed by macrophages. The bacterial concentration in the lung increases due to amplification of the bacteria within macrophages.
Following this intracellular multiplication, neutrophils, additional macrophages, and erythrocytes infiltrate the alveoli, and capillary leakage results in edema. Chemokines and cytokines released by infected macrophages help trigger the severe inflammatory response. In the mouse model of LD, the relevant proinflammatory chemokines and cytokines include keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor-α (TNF-α), interleukin (IL)-12 and IL-18, and interferon-γ (IFN-γ). Humans with LD had elevated levels of TNF-α and IL-8 in relation to other bacterial pneumonias in one study. Systemic spread of the bacteria may be accomplished by infection of circulating monocytes.
The mechanisms for systemic toxicity of the disease are unclear, but it involves the severe inflammatory response to virulent Lp. Cytokine production is mediated by detection of microbial products by receptors of the innate immune system. Toll-like receptors (TLRs) on macrophages and other cells initiate host responses to both virulent Lp and avirulent mutants by detecting common pathogen-associated molecular patterns (PAMPs). TLR2 detects lipoproteins and lipopeptides, TLR5 flagellin, and TLR9 bacterial DNA. Replication of Lp is greater in the lungs of mice deficient for TLR2 or the TLR adapter protein MyD88, resulting in higher mortality; and mice lacking TLR5 or TLR9 have delayed innate immune responses but disease susceptibility is not as pronounced as in TLR2-deficient mice. Epidemiologic data indicate that humans with a TLR5 stop polymorphism are more susceptible to LD; for unclear reasons human TLR4 polymorphisms are protective even though the lipid A moiety produced by Lp has an atypical structure and is less stimulatory toward TLR4 than the classic lipid A molecule produced by Enterobacteriaceae.
The delivery of microbial products into the macrophage cytosol by virulent Lp contributes significantly to the robust inflammatory response. The mouse protein caspase 11 and the human homologues caspase 4 and caspase 5 bind directly to cytosolic lipopolysaccharide (LPS) during Lp infection of macrophages, and caspase 11 has been shown to be essential for septic shock, implicating this pathway as being critical for LD pathology. Many responses are activated by host nucleotide-binding oligomerization domain (NOD)–like receptor (NLR) proteins. The mouse NLR protein NAIP5 detects bacterial flagellin to activate caspase-1–dependent processing and secretion of the cytokines IL-1b and IL-18, and animals that are defective for NAIP5 are significantly more susceptible to Lp infection. The NLR proteins NOD1 and NOD2 respond to peptidylglycan fragments delivered into the cytosol by virulent Lp, and sustain activation of innate immune signaling pathways. Lastly, the proteins RIG-I and MAVS respond to bacterial nucleic acid delivered into the cytosol during Lp infection to activate type I interferon production.
Although the severe Th1 response induced by Lp can be detrimental to the host under high bacterial loads, these cytokines are crucial for the clearance of Legionella organisms. Cytokines produced by infected alveolar macrophages are essential for the recruitment of neutrophils and for stimulating IFN-γ production by natural killer cells, which are both needed for sterilization in the lung. Neutrophils are efficient at clearing extracellular Lp from the lungs. Macrophages activated by IFN-γ kill Lp. This change in macrophage permissiveness involves, among other things, a reduction in intracellular iron, a factor that is necessary for Lp replication. Indeed, the majority of legionellae seen in lung samples are associated with alveolar macrophages. Furthermore, the susceptibility of an animal species correlates with the ability of Lp to infect its macrophages, and bacterial mutants that are impaired for in vitro infection of macrophages have reduced virulence. Antibodies develop during the course of Lp infection, but the humoral immune response does not appear to be critical for host defense.
It is widely believed that the adaptation of Lp to protozoan niches in nature engendered it with the ability to infect mammalian phagocytes. Lp enters the macrophage by conventional or coiling phagocytosis, processes that utilize the host cell actin cytoskeleton. Opsonization with the C3 component of complement can promote phagocytosis, but entry by this pathway dampens the oxidative burst and thereby may enhance bacterial intracellular survival. However, opsonin-independent phagocytosis also appears to be important. Even in the event that the oxidative burst is triggered, Lp strains may be resistant to hydrogen peroxide, superoxide anion, and hydroxyl radicals.
After entry, legionellae reside within a nascent phagosome ( Fig. 232.3 ) that does not fuse with endosomes or lysosomes, thereby avoiding acidification and degradative enzymes. The phagosome subverts host vesicles in the early secretory pathway using proteins produced by Lp. The vacuole containing Lp rapidly recruits membrane from the endoplasmic reticulum and develops into an organelle that resembles the host rough endoplasmic reticulum. This specialized Legionella -containing vacuole supports intracellular replication. This vacuole expands during replication and ultimately fills the host cell. Upon nutrient depletion (e.g., amino acid depletion), Lp enters stationary phase and converts to a flagellated form that is primed to seek out and infect new host cells. Egress of Lp from the expended host cell is not well understood, but in macrophages this involves pathogen-induced apoptosis that leads to cellular necrosis.
Neither the pathogenesis nor the etiology of PF is known with certainty. PF is caused by inhalation of a disease-causing environmental aerosol derived from water containing microorganisms including Legionella bacteria. Thirty percent to 85% of patients with this disease have serum anti- Legionella antibody in higher concentrations than is found in the normally healthy population. The prevalent assumption is that the illness is caused by inhalation of the Legionella bacteria. Since the aerosols contain a mixture of microorganisms and endotoxins, it is unclear whether the disease is due to inhalation of endotoxin, of a polymicrobial aerosol, or of Legionella bacteria alone or to a combination of all these agents. A study of the virulence for guinea pigs of the Lp strain that caused the 1976 Philadelphia LD epidemic and the PF environmental isolate from the Pontiac, Michigan, epidemic showed no differences between the two bacterial strains. The incubation period of 4 to 6 hours of some PF patients supports a toxin-mediated illness, while the median incubation period of around 35 hours is more suggestive of initial bacterial multiplication causing illness. Infections with non- Legionella bacteria can produce Legionella antibodies, so the presence of such antibodies does not prove that PF is due solely to Legionella infection or intoxication. Bath water fever, a clinical syndrome thought to be due to endotoxin inhalation, is very similar to PF, suggesting that PF may also be caused by endotoxin inhalation. Several reports exist in which exposure to the same environmental source led to PF in most exposed people, but to LD in a few people; whether the milder illness was really PF or mild LD is open to question, and does not help answer the question of etiology and pathogenesis. Perhaps the strongest evidence implicating systemic infection with Legionella bacteria as the cause of PF has been the very rare reports of positive Lp urinary antigen tests or positive cultures in patients with PF. The rarity of such cases and the rapid recovery without antibiotic therapy argue against systemic infection as the cause of PF.
Legionella pneumophila Virulence Factors
Studies of Lp grown in mammalian eukaryotic cells and in its natural environmental host, free-living amebae, show that the bacterium changes dramatically within cells as the intracellular infection progresses, becoming very small and highly motile as the cell loses its ability to sustain bacterial multiplication. These small motile forms, termed the “transmissive” type of bacteria, have increased virulence for eukaryotic cells. Once inside the cells, a nonmotile “replicative” phase predominates. The transmissive bacteria can be produced in vitro under nutrient-limiting conditions. Multiple bacterial factors control this transition between the transmissive and replicative phases, with some of the most important ones being production of ppGpp and activation of LetA/S and RpoS. CsrA plays an important role in the posttranscriptional regulation of this process.
A variety of surface structures have been implicated in Lp pathogenesis. Type IV pili modestly promote bacterial attachment to macrophages and epithelial cells, and flagella promote invasion independent of adherence. The major outer membrane protein is a porin that also serves as a binding site for complement components and thus mediates opsonophagocytosis. The Mip protein is a surface-exposed peptidyl prolyl isomerase that is required for the early stages of intracellular infection and for full virulence in animals. Legionella LPS contains some endotoxic activity, and changes in LPS have correlated with increases in serum resistance, intracellular growth, and virulence. Finally, the rcp gene, which appears to encode a lipid A–modifying enzyme, confers resistance to cationic peptides and promotes macrophage and lung infection.
Lp secretes a variety of proteins, degradative enzymes, and putative toxins. The release of proteins by Lp into the extracellular milieu is mediated primarily by a type II secretion system. Genome sequencing also suggests the existence of type I and type V secretion systems. A number of enzymes and novel proteins are all secreted via the Legionella type II system. Mutations within the genes encoding the type II secretion system diminish infectivity for macrophages, protozoa, and animals. A secreted zinc protease is produced during infection and promotes pathology in the guinea pig model of disease as well as intracellular infection of some amebae hosts.
A Legionella type IVb secretion system called Dot/Icm is essential for intracellular replication and virulence in animal models of disease. It is essential for the ability of the Legionella parasite to modulate host vesicular transport to avoid delivery to lysosomes and promote vacuole biogenesis. Mutations in dot/icm loci lead to loss of virulence. More than 300 proteins are secreted by the Dot/Icm system and in most cases these “effector” proteins translocate from the Legionella -containing vacuole into the host cell cytoplasm. Some of these have been implicated in bacterial evasion of lysosome fusion. Others, such as RalF, DrrA (SidM), LepB, LidA, and SidJ, play roles in the recruitment of endoplasmic reticulum–derived membranes to the Legionella -containing vacuole. The effector SdhA is important for maintaining the integrity of the vacuole in which Lp resides, and effectors SidF, SidP, and LepB directly modulate phosphatidylinositol phosphate signatures on the cytosolic surface of the vacuole that are important for the binding of other effectors. Lp expresses several effectors that function as glucosyltransferases that are capable of inhibiting host cell protein synthesis (elongation factor 1A). Although Dot/Icm is essential for Lp infection, a second type IV secretion system known as Lvh is found in addition to Dot/Icm in many strains; however, this system does not appear to deliver effector proteins into host cells but retains the ability to promote DNA conjugation, which could facilitate horizontal transfer of genes encoding effectors. Interestingly, many of the type II and type IV secreted proteins as well as other mediators of infection (e.g., LpnE and Lpg0971) bear striking sequence similarity to eukaryotic proteins, suggesting that they were acquired by horizontal gene transfer from a eukaryotic host and use similar biochemical activities to facilitate Lp infection.
Several infectivity factors have been localized to the Lp periplasm or cytoplasm. A Cu-Zn superoxide dismutase resides in the periplasm, affording resistance to toxic superoxide anions, and the KatB catalase-peroxidase is needed for optimal intracellular infection. The Legionella phosphoenolpyruvate phosphotransferase and HtrA protein promote intracellular growth and virulence, and the phosphotransferase regulates expression of the transcriptional activator PmrA. Lp iron acquisition, important for intra- and extracellular replication, involves, among other things, a secreted ferric iron chelator (the siderophore legiobactin), a secreted pyomelanin with ferric reductase activity, and an inner membrane ferrous iron transporter (FeoB).
In addition to the identification of eukaryotic-like proteins in the Lp genome, another outcome of sequencing bacterial genomes is the realization that there are large segments of DNA, including plasmids and chromosomal “islands”, that can vary between Lp strains. It is possible that these variable regions of the genome will help explain differences in virulence that may exist between strains. Indeed, large deletions of the chromosome that remove individual islands can decrease Lp replication in specific protozoan hosts.
Virulence Factors and Pathogenesis of Other Legionella Species
Relatively little is known about the virulence mechanisms, molecular pathogenesis, and cell biology of infections caused by Legionella spp. other than Lp. With the major exception of Llb, infections caused by the other Legionella spp. are rare and found almost exclusively in severely immunocompromised patients. Genome sequencing of 38 different Legionella species revealed that there are over 5000 different effector proteins encoded by the genus and only seven Dot/Icm effector proteins that are shared by all species. Thus effector plasticity, which facilitates adaptation of Legionella to different environmental hosts, likely influences human virulence potential. Despite unrestricted growth in otherwise nonpermissive macrophages, some non- Lp species fail to promote inflammatory cell death, which could be correlated to a failure to cause disease in nonimmunocompromised people, although this is unstudied. Llb resides within a ribosome-studded phagosome, albeit with markers of late endosomal maturation, unlike Lp, which is able to block endosomal maturation at an early stage. In contrast, L. micdadei resides in a smooth phagosome, and morphologic data suggest L. dumoffii grows within the cytoplasm rather than in a phagosome. Analysis of the genome of several different Llb strains has shown that this species has and utilizes the Dot/Icm system for causing infection, that it is nonflagellated, and that it possesses a wide range of eukaryotic effectors different from those found in Lp. Many of the Llb effectors appear to have been derived from plants and soil organisms, suggesting that it has evolved as primarily a soil-adapted bacterium. Experimental virulence of Llb for macrophages appears to be relatively independent of growth phase, in contrast to Lp, and studies in mice and explanted human macrophages show that host antibacterial cytokines are suppressed.
Epidemiology
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