Legionella pneumophila is a thin, pleomorphic, gram-negative rod that may show elongated, filamentous forms up to 20 μm long. In clinical specimens, the organism stains poorly or not at all by Gram stain or the usual histologic stains; however, it can be demonstrated by certain silver impregnation methods (Dieterle stain) and by some simple stains without decolorization steps. Polar, subpolar, and lateral flagella may be present. Most species of Legionella are motile. Spores are not found.
✺ Gram-negative rod that stains with difficulty
Structurally, L pneumophila has features similar to those of gram-negative bacteria with a typical outer membrane, thin peptidoglycan layer, and cytoplasmic membrane. The toxicity of L pneumophila lipopolysaccharide (LPS) is significantly less than that of other gram-negative bacteria such as Neisseria and the Enterobacteriaceae. This has been attributed to chemical makeup of the LPS side chains that renders the cell surface highly hydrophobic, a property which may promote distribution in aerosols.
✺ LPS is less toxic than that of most gram-negative species
Side chains are hydrophobic
Legionella is a facultative intracellular pathogen multiplying to high numbers inside free-living amoebas, other protozoa, and macrophages. In human-made water systems the organisms persist in a low metabolic state imbedded in biofilms. In vitro L pneumophila fails to grow on common enriched bacteriologic media such as blood agar due to requirements for certain amino acids (L-cysteine), ferric ions, and slightly acidic conditions (optimal pH 6.9). Even when these requirements are met, growth under aerobic conditions is slow, requiring 2 to 5 days to produce colonies that have a distinctive surface resembling ground glass. Although a few enzymatic actions (catalase, oxidase, β-lactamase) are demonstrable, the classification of Legionella depends largely on antigenic features, chemical analysis, and nucleic acid homology comparisons. The closest relative among pathogenic bacteria is Coxiella burnettii (see later).
✺ Intracellular parasite of protozoa
✺ Biofilms form in water systems
✺ Growth requires L-cysteine, ferric ions, and low pH
Classification based on antigenic structure and nucleic acid homology
Legionella pneumophila has multiple serogroups (16) and there are over 50 other Legionella species (eg, Legionella longbeachae, Legionella bozemanii, Legionella dumoffii, Legionella micdadei). The original Philadelphia strain (serogroup 1) is still the most common, and a limited number of L pneumophila serogroups account for 80% to 90% of cases. This suggests enhanced virulence for humans, since the frequency of L pneumophila among species found in the environment is below 30%. Less than half of the non-L pneumophila species have been isolated from human infections.
Multiple L pneumophila serogroups and other Legionella species exist
The widely publicized outbreak of pneumonia among attendees of the 1976 American Legion convention in Philadelphia led to the isolation of a previously unrecognized infectious agent, L pneumophila. The event was unique in medical history. For months the American public entertained theories of its cause that ranged from chemical sabotage to viroids and fears that something like Michael Crichton’s 1969 novel The Andromeda Strain was ahead. It was almost a letdown to find that a gram-negative rod that could not be stained or grown by the common methods was responsible. The Centers for Disease Control investigation was an outstanding example of the benefits of pursuing sound epidemiologic evidence until it is explained by equally sound microbiologic findings. We now know the disease had occurred for many years. Specific antibodies and organisms have been detected in material preserved from the 1950s, and a mysterious hospital outbreak in 1965 has been solved retrospectively by examination of preserved specimens. Today, most cases of Legionnaires disease in the United States are caused by just a few L pneumophila serogroups, including the original Philadelphia strain, but there is considerable variation worldwide. In Australia, New Zealand, and Japan L longbeachae and L pneumophila are found with similar frequency.
1976 outbreak led to discovery of new bacterium
Earlier outbreaks have been solved
In nature, Legionella species are ubiquitous in freshwater lakes, streams, and subterrestrial groundwater sediments. They are also found in moist potting soil, mud, and riverbanks. In these sites, they also exist as parasites of protozoa including numerous species of amoebas, which appear to be the environmental reservoir. Transmission to humans occurs when aerosols are created in manmade water supplies that harbor Legionella. Most outbreaks have occurred in or around large buildings such as hotels, factories, and hospitals with cooling towers or some other part of an air-conditioning system as the dispersal mechanism. Some hospital outbreaks have implicated respiratory devices and potable water coming from parts of the hot water system such as faucets and showerheads. Even the mists used in supermarkets to make the vegetables look fresh have been the source of outbreaks. Legionella can persist in a water supply despite standard disinfection procedures, particularly when the water is warm and the pipes contain scale or low-flow areas that compromise the effectiveness of chlorine compounds.
✺ Amoebas in freshwater habitat act as reservoir
✺ Infections are associated with aerosols distributed by humidifying and cooling systems
It is difficult to ascertain the overall incidence of Legionella infections because most information has been from outbreaks that constitute only a small part of the total cases. Estimates based on seroconversions suggest approximately 25 000 cases in the United States each year. The attack rate among those exposed is estimated at less than 5% and serious cases are generally limited to immunocompromised persons. Person-to-person transmission has not been documented, and the organisms have not been isolated from healthy individuals. Growth in free-living amoebas produces Legionella cells that are more resistant to environmental stress (acid, heat, osmotic) and have enhanced infectivity.
✺ Person-to-person transmission or carriers are unknown
✺ Disease rate among exposed is low
Legionella pneumophila is striking in its propensity to attack the lung, producing a necrotizing multifocal pneumonia. Microscopically, the process involves the alveoli and terminal bronchioles, with relative sparing of the larger bronchioles and bronchi (Figure 34–1). The inflammatory exudate contains fibrin, neutrophils, macrophages, and erythrocytes. A striking feature is the preponderance of bacteria within phagocytes and the lytic destruction of inflammatory cells.
✺ Strong tropism for the lung
✺ Necrotizing multifocal pneumonia with intracellular bacteria
Legionella pneumonia. Note the filling of alveoli with exudate. Some of the alveolar septa are starting to degenerate. (Reproduced with permission from Connor DH, Chandler FW, Schwartz DQ, et al: Pathology of Infectious Diseases. Stamford CT: Appleton & Lange, 1997.)
Inhaled Legionella bacteria reach the alveoli, where they attach to their pathogenic target the alveolar macrophage. In this process, they are aided by flagella, pili, and a variety of other proteins. Following attachment the bacteria enter the macrophage in an endocytic vacuole. Inside the cell L pneumophila initiates a process which prevents fusion with the lysosome and instead recruits ribosomes, mitochondria, and elements of the host cell endoplasmic reticulum (ER) into its own phagosome called the Legionella-containing vacuole (LCV) (Figure 34–2 A, B). In the LCV niche protected from lysosomal digestion, the organisms multiply to high numbers (Figures 34–2 C, 34–3). They eventually kill the macrophage releasing new cells to repeat the cycle. The multiple enzymes released in this process lead to inflammation, destructive lesions in the lung, and a systemic toxicity that may be related to cytokine release.
✺ Organisms invade alveolar macrophages
✺ Lysosomal fusion is blocked
✺ Host ER incorporated into LCV
Multiplication of Legionella pneumophila in human macrophages. A. L pneumophila enters the cell in a phagosome which sometimes has a coiling pattern. B. The new Legionella-containing vacuole (LCV) is lined by ribosomes and mitochondria from the endoplasmic reticulum (ER). C. The bacteria multiply within the macrophages to reach very high numbers. (Courtesy of Dr. Marcus Horwitz.)
Legionnaires disease. Imprint smear of lung shows L pneumophila (stained red) mostly inside alveolar macrophages. (Reproduced with permission from Connor DH, Chandler FW, Schwartz DQ, et al: Pathology of Infectious Diseases. Stamford CT: Appleton & Lange, 1997.)
Legionella pneumophila accomplishes this control of the phagocyte through the complex deployment of over 200 proteins. Only a few of these proteins have functions which are known or have been inferred by genomic analysis. It is known that the majority of these proteins are produced by an injection secretion system (type IV) which in contrast to those described in other gram-negative pathogens operates from inside the unfortunate macrophage. As the intracellular population grows, the virulence protein deployment shifts to products facilitating egress from the LCV and macrophage with some causing pore-forming membrane lysis. The entire process in environmental protozoa is similar to that in the macrophage. In both amoebas and humans this rapid growth takes place under nutrient-rich conditions. Similar to other intracellular bacterial pathogens (Chlamydia, Chlamydophila, and Coxiella), L pneumophila also has a nutrient-restricted phase in which elements which mediate resistance to environmental stress and facilitate future infectivity are produced. This appears to be the situation in the low metabolic state of biofilm-imbedded cells, which lurk in the pipes of human-constructed water systems.
Many proteins injected through secretion system inside host cell
✺ Macrophage and amoeba replication similar
✺ Nutrient-restricted phase facilitates environmental survival, infectivity
Just as intracellular multiplication is the key to L pneumophila virulence, its arrest by innate and adaptive mechanisms is the most important aspect of immunity. The high level of innate immunity to Legionella infection in most persons is related to brisk pattern recognition responses triggered by toll-like receptors (TLRs) in macrophages and dendritic cells that recognize Legionella LPS. The activation of the TH1 adaptive immune response and its associated cytokines (IFN-γ, IL-12, IL-18) completes the process of macrophage activation and intracellular killing of the invading Legionella. Failure of this aspect of the immune response is the primary reason for most cases of progressive Legionnaires disease in the immunocompromised. Antibodies formed in the course of Legionella infection are useful for diagnosis, but do not appear to be important in immunity. It is unknown whether humans who have had Legionnaires disease are immune to reinfection and disease.
Innate defenses triggered by TLRs
✺ Cytokine-activated macrophages limit intracellular growth
Antibody is less important
LEGIONNAIRES DISEASE: CLINICAL ASPECTS
Legionnaires disease is a severe toxic pneumonia that begins with myalgia and headache, followed by a rapidly rising fever. A dry cough may develop and later become productive, but sputum production is not a prominent feature. Chills, pleuritic chest pain, vomiting, diarrhea, confusion, and delirium all may be seen. Radiologically, patchy or interstitial infiltrates with a tendency to progress toward nodular consolidation are present unilaterally or bilaterally. Liver function tests often indicate some hepatic dysfunction. In the more serious cases, the patient becomes progressively ill and toxic over the first 3 to 6 days, and the disease terminates in shock, respiratory failure, or both. The overall mortality rate is about 15%, but it has been higher than 50% in some hospital outbreaks. Mortality is particularly high in patients with serious underlying disease or suppression of cell-mediated immunity.
✺ Severe toxic pneumonia occurs in 5% of those exposed
✺ Mortality is high among the immunocompromised
A less common form of disease called Pontiac fever (named for a 1968 Michigan outbreak), is a nonpneumonic illness that resembles influenza with fever, myalgia, dry cough, and a short incubation period (6-48 hours). Pontiac fever is a self-limiting illness and may represent a reaction to endotoxin or hypersensitivity to components of the Legionella or their protozoan hosts.
The established approach to diagnosis combines direct fluorescent antibody (DFA) with culture of infected tissues. PCR is an alternative method. For this purpose, a high-quality specimen such as lung aspirates, bronchoalveolar lavage, or biopsies are preferred, because the organism may not be found in sputum. Typically, the Gram smear fails to show bacteria owing to poor staining, but organisms are revealed by DFA using L pneumophila-specific conjugates. These conjugates use monoclonal antibodies, which bind to all serogroups of L pneumophila, but not the non-L pneumophila species. DFA is rapid, but it yields a positive result in only 25% to 50% of culture-proved cases.
✺ High-quality specimens are needed
✺ DFA is rapid but only 50% sensitive
Cultures must be made on buffered charcoal yeast extract (BCYE) agar medium that includes supplements (amino acids, vitamins, L-cysteine, ferric pyrophosphate), which meets the growth requirements of Legionella. It is buffered to meet the acidic conditions—optimal for Legionella growth. The isolation of large gram-negative rods on BCYE after 2 to 5 days that have failed to grow on routine media (blood agar, chocolate agar) is presumptive evidence for Legionella. The diagnosis is confirmed by DFA staining of bacterial smears prepared from the colonies. BCYE also allows isolation of species of Legionella species other than L pneumophila.
Culture on BCYE is required for isolation
Cultures will isolate other species
The difficulty and slow speed of culture together with the low sensitivity of DFA have spurned searches for other methods. This has led to the development of nucleic acid amplification (NAA) procedures for use in respiratory specimens and immunoassay methods for the detection of antigen in urine. NAA methods such as the polymerase chain reaction (PCR) have proved to be rapid and much more sensitive than DFA. A simple card-based antigenuria detection test has also proved to be sensitive for the common L pneumophila serogroup 1, but does not detect other serogroups or other Legionella species. The primary barrier to making these methods more widely used is that Legionnaires disease is uncommon except in immunocompromised populations. This tends to limit their availability to reference laboratories and hospitals serving immunocompromised patients. Demonstrating a significant rise in serum antibody is used primarily for retrospective diagnosis and in epidemiologic studies.
PCR is rapid and sensitive
✺ Antigenuria detects serogroup 1
The best information on antimicrobial therapy is still provided by the original Philadelphia outbreak. Because the cause of Legionnaires disease was completely obscure at the time, the cases were treated with many different regimens. Patients treated with erythromycin clearly did better than those given the penicillins, cephalosporins, or aminoglycosides. Subsequently, it was shown that most Legionella produce β-lactamases. In-vitro susceptibility tests and animal studies have confirmed the activity of erythromycin and have shown that azithromycin, fluoroquinolones, doxycycline, rifampin, and trimethoprim-sulfamethoxazole are also active. Currently a fluoroquinolone or azithromycin is preferred.
A fluoroquinolone or azithromycin is the treatment of choice
The prevention of legionellosis involves minimizing production of aerosols in public places from water that may be contaminated with Legionella. Prevention is complicated by the fact that, compared with other environmental bacteria, Legionella bacteria are relatively resistant to chlorine and heat. The bacteria have been isolated from hot water tanks held at over 50°C. Methods for decontaminating water systems are still under evaluation. Some outbreaks have been terminated by hyperchlorination, by correcting malfunctions in water systems, or by temporarily elevating the system temperature above 70°C. The installation of silver and copper ionization systems similar to those used in large swimming pools has been effective as a last resort in hospitals plagued with recurrent nosocomial legionellosis. An outbreak reported from a neonatal intensive care unit in Cyprus was traced to free-standing humidifiers which had been filled with tap water. This underscores both the ubiquity of Legionella and the need to at least start with sterile water wherever possible.
✺ Preventing Legionella aerosols is primary goal
✺ Heat, hyperchlorination, and metal ions may be needed in institutions