Legionella Spp and Legionnaires Disease Introduction and History: *Legionnaires disease is an acute pneumonic illness caused by Gram-negative

bacilli of the genus Legionella. *Legionnaires disease was first recognized as a distinct clinical entity when it caused an epidemic of pneumonia at an American Legion convention in Philadelphia in 1976; *221 people were affected, and 34 died. Taxonomy: *According to Oligonucleotide sequence of 16S rRNA, they are classified within the -subdivision of the Proteobacteria, within the Legionellaceae group. *The main member of the Legionellaceae group is the Legionellaceae family. *Forty Legionella species have been validly published. *All of the Legionella species that have been isolated from clinical sources also have been isolated from the environment, with the exceptions of: *L. hackeliae and *L. tucsonensis. *There are 40 species in the family of legionella bacteria in the world. *Of these species, 12 have been implicated in human disease. *90% of these disease cases are caused by Legionella Pneumophila. General Description: *The Legionellaceae are: *Mesophilic, *Assaccharolytic, *Obligately aerobic *Gram-negative bacilli. *All are weakly catalase-positive. *The oxidase reaction is usually weak and may be negative. *They are nonsporeforming and unencapsulated. *All but a few species are motile by means of one to three polar or lateral flagellae, although motility may be lost. *Amino acids rather than carbohydrates are used as an energy source. *L-Cysteine is required for growth, and iron is required for initial isolation from the environment or clinical specimens; iron-free cultivation of multiple passage isolates has been reported. -L. pneumophila, L. micdadei, and probably many other, if not all, Legionella spp. are facultative parasites of many eukaryotic cells, including some free-living amoebae. *So far most of these have not been cultured outside of their protozoal host, making their characterization difficult. *The parasitic relationships Legionella form with amebas and other aquatic

eukaryotes play a major role in their virulence. *The amebas provide the bacteria with an ideal environment for reproduction and development. *In addition, the hosts make Legionella more resistant to adverse environmental conditions. *The bacterial length is highly variable, depending upon: *Growth Conditions, *Stage Of Growth, *Whether the bacterium is grown: * In eukaryotic cells or * Extracellular environments. *The bacterium can vary from highly motile tiny (1 m) coccoid forms during the last stages of intra-amoebal growth to very long and nonmotile filaments (23 20 m) during stationary phase on solid media. *When grown on solid media the bacterium is usually a rod 540 m in length, with a cell width of 0.30.9 m; *Bacteria incubated on plates for many days may become shorter and motile. *When grown to early log phase in broth, the bacterium is usually 510 m in length and single-celled, but some may be filamentous. -Bacteria grown to early log phase are nonmotile short bacilli, in broth, on solid media, and in the early stages of intra-amoebal and intra-macrophage growth. -In late stationary phase on solid media, and in broth, there can be a mixture of filamentous forms, medium-length rods, and short motile coccoid forms. *When grown to late stationary phase (OD600 >1.8) in broth many of the bacteria become coccobacillary and highly motile, whereas some forms are filamentous. *Bacteria grown in amoebae, macrophages, or macrophage-like cell lines are short coccobacilli. -Ultrastructural studies of the bacteria show an arrangement characteristic of Gram-negative rods, with typical trilaminar inner and outer membranes. -A definite peptidogylcan structure also is seen in the cell wall. -Several species, except L. micdadei, appear to have an extracellular polysaccharide capsule or slime layer that can be visualized by ruthenium red staining, even though the bacteria appear unencapsulated by other means. -Both flagellae and pili have been observed in some ultrastructure studies. -Staining characteristics depend to some degree on the source of the bacteria. -Typically, Gram stain does not reliably stain the legionellae in clinical specimens, although bacteria cells from artificial media stain somewhat better. -The substitution of basic fuchsin for the saffranin counterstain in the Gram technique makes the legionellae more readily visible. -The legionellae can also be readily stained by the Gimenez stain and the Dieterle

silver stains, but neither technique is specific for these bacteria. *The pH and temperature optima for In vitro growth are 6.8 7.0 and 2542C, respectively, with optimal growth occurring In vitro between 35 and 37C. *The growth optimum in nature is unknown, but may have a very wide range (10 45 C). *Branched chain fatty acids are the predominant cellular fatty acids. *The G +C content of the Legionella genus is 3852 mol%. *The size of the L. pneumophila genome is approximately 3.9 Mbp. *Among the three means of bacterial genetic exchange, Conjugation and Transformation are known to function within the Legionella genus. *Thus far, there have been no reports of transduction or bacteriophage in the legionellae. *Plasmids have been detected in some strains of Legionella, including representatives of L. pneumophila and at least five other species, and range in size from 2195 MDa. Genus and Species Identification: *Identification of Legionella spp. to the genus level is usually quite easy, as is identification of L. pneumophila. *This is because the combination of: *L-cysteine dependence, *Colonial morphology *Growth rate *is very specific for Legionella spp., and because a very sensitive and specific monoclonal antibody for L. pneumophila is available for identification. Ecology: *Legionella are aquatic organisms, inhabiting freshwater environments; *Streams, *Lakes, *Warm springs, *Rivers, *Riverbanks. *Humans are accidental hosts. *Legionella occur in high amounts in thermally polluted water. *In addition, the bacteria can be found in: *Cooling towers, *Air conditioners, *Spa equipment, *Fountains, *Humidifiers, or *Showers. *In their natural aquatic environment, legionella feed on various nutrients from the water, but are most adept in the role of an intracellular parasite on other

bacteria. *Once it is uptaken by a larger bacterium, it resists bacterial defences and then multiplies. *Environmental conditions which promote the growth of legionella are: *Water temperature between 20 50 C. *Stagnant water. *pH range of 2.0 8.5 *Sediment in water which supports the growth of supporting microbiota. *Presence microbiota including algae, protozoa, and others. *L-Cysteine-HCL and Iron salts to promote growth. Culture Media: -L. pneumophila grows best on rich media containing: *Yeast extract, *L-cysteine, *Iron and *Organic buffers. *Buffered charcoal yeast extract medium supplemented with -ketoglutarate (BCYE ) is the primary culture medium used by most clinical laboratories. *The medium pH must be 6.806.90, *The -ketoglutarate, is a growth stimulant, and is used in the BCYE medium of almost all clinical microbiology laboratories. *The charcoal inactivates toxic peroxides produced during autoclaving of the yeast extract, and toxic compounds present in agar. *The BCYE medium can be made selective for L. pneumophila by the addition of: *Cephamandole (4 mg/liter), *Polymyxin B (80,000 U/liter) *Anisomycin (80 mg/liter); *This medium is known as BMPA or PAC medium. *A variety of broth media can be made for the growth of L. pneumophila. *The optimal growth medium is the liquid equivalent of BCYE medium, made without agar; this charcoal containing broth is termed BCYE broth. Growth Rate and Colonial Morphology: *When plated from primary clinical or environmental specimens, colonies of legionellae appear on selective or nonselective BCYE agar within 314 days (average 34 days) of incubation. *Growth from a fresh colony generally requires 1836 h of incubation, depending on the density of the inoculum; *a heavy inoculum usually grows overnight, and *a light inoculum may take several days to grow on subculture. *Some of the more fastidious species, such as L. oakridgensis, L. sainthelensi and L. rubrilucens may require up to several more days of incubation for growth to

appear on subculture; *these and some other Legionella spp. may grow better in the presence of 35% CO2. *Recognition and preliminary identification of the bacteria are facilitated by the use of a dissecting microscope for the examination of the media. *Legionella colonies display a characteristic appearance resembling ground glass, or an opal, when viewed under obliquely transmitted light. *Very young colonies are gray, flat, round, entire and about 0.250.5 mm in diameter; at this stage they are very difficult to see with the naked eye. *Within a day the colonies become raised, round, entire and convex, and are 12 mm in diameter. *The colony edge usually displays a birefringent band, which is either greenish blue or pink depending on the species; *this coloration may be pleomorphic but does not breed true. *Up to this stage, the colonial morphology is distinctive for experienced observers.

Growth of Legionella species on selective differential medium.

L. pneumophila colonies appear light green; L. micdadei colonies appear blue. *As the colonies become several days older, the ground-glass appearance and the birefringent edge tend to disappear and the colonies become umbonate and sometimes tuberculated; *The maximum diameter is usually about 34 mm. *Eventually the colonies flatten completely *Very old colonies are impossible to distinguish with certainty from other bacteria, even for experts. *This makes it especially important to examine plates for early growth when plating from primary specimens, or when contamination with other bacteria is a possibility. *When the colonies are illuminated with a UV lamp the colonies of most species will exhibit a dull yellow fluorescence. *The colonies of L. bozemanii, L. gormanii, L. dumoffli and several other species, display a very striking blue-white autofluorescence under similar conditions of illumination, *whereas colonies of L. rubrilucens, L. erythra and some strains of L. taurinensis produce a red fluorescence. -Most Legionella spp. growing on BCYE medium have a characteristic musty smell. -This odor is specific enough to aid in the identification of the bacterium. -The legionellae are chemoorganotropic do not possess a glucose transport system, nor do they ferment or oxidize other carbohydrates. -Serine, glutamate and perhaps threonine appear to serve as the primary carbon

and energy sources for the legionellae and are catabolized via the Krebs cycle. -Carbohydrate synthesis occurs through gluconeogenesis via the EmbdenMeyerhof pathway. - -Ketoglutaric acid is a growth stimulant and a component of most growth media for the bacterium. -Members of the genus Legionella cannot be identified with any degree of certainty using traditional biochemical testing. -This is because of the general biochemical inertness of the bacterium when using conventional biochemical tests, and because many of the species can only be correctly identified using molecular methods. -The tests that appear to be most useful for bacterial identification include demonstration of: -L-cysteine growth dependence, -Serotyping to detect L. pneumophila and L. pneumophila serogroup 1, -Determination of cellular fatty acids and ubiquinones, -Determination of the macrophage infectivity potentiator (mip) gene sequence. -All species are motile via one to three polar or subpolar flagella and the flagella of all legionella species appear to be antigenically identical. -Only L. oakridgensis appears to be nonmotile. -Determination of flagellation may be difficult as flagellar production is growthcondition dependent. -It is much easier to demonstrate flagellae in plate-grown than broth-grown bacteria, especially from plates that have been incubated at room temperature. -The Ryu stain is a convenient method for staining the flagellae. -Tests for nitrate reductase and urease are negative for all species. -Most strains are reported to be catalase positive when whole cells are tested for the ability to decompose hydrogen peroxide (H2O2); -The catalase reaction can be very weak. -Methods that enhance the sensitivity of the catalase reaction include performance of the test using capillary tubes and the use of 3% H2O2 in 10% Tween 80. -Most species liquefy gelatin, using an assay medium in which gelatin replaces agar in the formulation of BCYE medium. -L. pneumophila and a few other species hydrolyze hippurate. -Most strains produce a -lactamase that is active against cephalosporins and can readily be demonstrated using nitrocefin. -Browning of tyrosine-containing medium is also a helpful phenotypic test, using BCYE medium made without charcoal and containing tyrosine. Identification Techniques: L-Cysteine Growth Dependence

-Establishing the L-cysteine growth dependence is usually the first step for a Gramnegative bacterium growing on BCYE medium with colonial morphology typical of Legionella spp. -In most laboratories, this is done by inoculating the isolate onto a cysteine deficient BCYE agar plate. -Other cysteine-deficient media (such as sheep blood agar) may be used for this purpose, but occasional strains of other heterotrophic environmental bacteria may be encountered that will grow on BCYE and not on blood agar. -Placement of an L-cysteine- containing disk on cysteine-deficient BCYE , or on tryptic soy blood agar, will result in growth of the Legionella bacteria around the disk. -In addition, Brucella blood agar will support the growth of at least some Legionella bacteria, as will several other rich media. -After serial passage, several Legionella species can grow on media without Lcysteine, but generally still exhibit L-cysteine dependence on the first or second passage on artificial media.