Course: Medical Microbiology, PAMB 650/720 Lecture 43: Mycobacteria /Corynebacteria and Legionellae

2006/2007

Faculty: Dr. Fox, Phone: 733 3288, E-mail: afox@med.sc.edu, Office: C-19, Blg. 2 Suggested reading: Murray, Fifth Edition Chapter 29 (Mycobacteria) Chapter 27 (Corynebacterium diphtheriae) and Chapter 38 (Legionella) Key words Acid Fast Tuberculosis M. tuberculosis M. avium-M. intracellulare complex M. leprae M. bovis Atypicals Tubercle PPD Tuberculin Mycobactin Cord factor BCG Leprosy (Hansen's Disease) Runyon groups Mycolic acids Diphtheria C. diphtheriae Loeffler's agar Tellurite agar Metachromatic bodies Diphtheria toxin Schick test Diphtheroids Legionella pneumophila Legionnaires disease Pontiac fever Legionella micdadei Pittsburg pneumonia Charcoal yeast extract agar

MYCOBACTERIA In the 1980's many experts felt that the days of tuberculosis (TB, consumption) as a threat to the US had passed and the incidence of new cases (<20,000 a year) was slowly decreasing. However, TB remains the leading infectious cause of death world-wide. The situation in the 1990's in the US changed dramatically. The incidence of tuberculosis has slightly increased and certainly the disease is not going away (primarily due to the AIDS epidemic). At the same time multiple drug-resistant strains of M. tuberculosis have appeared. The M. avium-M. intracellulare complex (often simply referred to as M. avium), long considered a group of organisms that only rarely infects man, is now recognized as one of the leading opportunists associated with AIDS. M. leprae is the causative agent of leprosy which remains a major disease in the third-world. Due to eradication of infected cattle and pasteurization of milk M. bovis (a zoonotic cause of tuberculosis) is rarely seen in the US. Mycobacteria are obligate aerobic, acid-fast rods.

M. tuberculosis Pathogenesis of tuberculosis The organism primarily infects the lung, but is distributed systemically within macrophages and survives intra-cellularly. Inhibition of phagosome-lysosome fusion and resistance to lysosomal enzymes occur. Cell mediated immunity develops which causes infiltration of macrophages and lymphocytes with development of granulomas (tubercles). The disease can be diagnosed by skin testing for delayed hypersensitivity with tuberculin (protein purified derivative, PPD). A positive test does not indicate active disease; merely exposure to the organism. Other pathogenesis factors (of considerably less importance than delayed hypersensitivity) include mycobactin (a siderophore) and cord factor that damages mitochondria. Diagnosis, Identification and treatment The presence of acid-fast bacteria in sputum is a rapid presumptive test for tuberculosis. Subsequently, when cultured, M. tuberculosis will grow very slowly producing distinct non-pigmented colonies (2 weeks or so currently). M. tuberculosis can be differentiated from most other mycobacteria by the production of niacin. A rapid alternative to culture is polymerase chain amplification. Tuberculosis is usually treated for extensive time periods (9 months or longer) since the organism grows slowly and may become dormant. By using two or more antibiotics (including rifampin, isoniazid, ethionamide and ethambutol) the possibility of resistance developing during this extended time is minimized. M. tuberculosis causes disease in healthy individuals and is transmitted man-man in airborne droplets. Vaccination The BCG vaccine (Bacillus de Calmette et Guerin, an attenuated strain of M. bovis) has not been effective. In the US, where the incidence of tuberculosis is low, widespread vaccination is not practiced. Indeed immunization (resulting in a positive PPD test) is felt to interfere with diagnosis. M. leprae M. leprae is the causative agent of leprosy (Hansen's Disease) a chronic disease often leading to disfigurement. Rarely seen in the US but common in the third world. The organism infects the skin, because of its growth at low temperature. It also has a strong affinity for nerves. In "tuberculoid" leprosy there are few organisms due to control by active cell mediated immunity. In "lepromatous" leprosy, due to immuno-suppression by the organism, the opposite is found. Although uncommon in the U.S. millions of cases occur worldwide. Treatment with 2

antibiotics (initially dapsone and now multi-drug) is effective and the overall disease incidence worldwide is down. The organism does not grow in lifeless media. However, it grows well in the armadillo (which has a low body temperature), allowing production of M. leprae antigens and pathogenesis studies. M. leprae has traditionally been identified on the basis of acid-fast stains of skin biopsies and clinical picture. Lepromin is used in skin testing.

Atypicals The "atypicals" generally infect the immuno-compromised host and are thus not transmitted man-man. With the AIDS epidemic the atypical mycobacteria have taken on new importance with the recognition that the M. avium complex (MAC) are the most commonly associated systemic bacterial infection. Atypical mycobacteria can cause tuberculosis-like or leprosy-like, diseases, and are not susceptible to certain common anti-tuberculous antibiotics. M. tuberculosis, M. avium and AIDS M. avium generally infects AIDS patients when their CD4 (helper T cell) count decreases greatly (below 100/mm3). M. tuberculosis infects AIDS patients much earlier in the disease than M. avium. This clearly demonstrates the much greater virulence of M. tuberculosis. The incidence of systemic disease (versus primarily pulmonary) is much greater in tuberculosis associated with AIDS than in it's absence. Furthermore, histologically lesions often appear lepromatous (non-granulomatous with many organisms). It is rare to find a case of M. avium infection that is not AIDS associated. Approximately 20% of the total tuberculosis cases in the country are associated with AIDS. This helps explain why TB is no longer on the decline. Treatment of M. avium also involves a long-term regimen of multiple drug combinations. However, this organism does not always respond to the regimens used to treat M. tuberculosis. Since M. tuberculosis is the more virulent organism the drug regimen selected is primarily against M. tuberculosis. If M. avium is suspected other agents effective against this organism are included. Other atypicals Presence or absence of pigmentation (and its dependence on growth in the light) and slow or fast growth rates of atypical mycobacteria allow some differentiation - the "Runyon Groups". Modern techniques allow ready speciation of mycobacteria based on their cellular fatty acid and/or mycolic acid profiles. This is only performed in reference laboratories. Genetic tests are taking over here as elsewhere. Mycolic acids are components of a variety of lipids found only in mycobacteria, nocardia and corynebacteria. The chain length of these mycolic acids is longest in mycobacteria, intermediate in nocardia and shortest in corynebacteria. This explains why mycobacteria are generally acid fast; nocardia less acid fast; and corynebacteria are non-acid fast.

CORYNEBACTERIUM DIPHTHERIAE Grows best under strict aerobic conditions, a Gram positive rod that is pleomorphic (e.g. club-shaped). Colonization of the upper respiratory tract (pharynx and nose) and less commonly skin with C. diphtheriae leads to diphtheria. The organism does not produce a systemic infection. However, in addition to a pseudo-membrane being formed locally (which can cause chocking) systemic and fatal injury results primarily from circulation of the potent exotoxin (diphtheria toxin). The latter begins over a period of a week. Thus treatment involves rapid therapy with anti-toxin. Treatment in these non-immune individuals also involves injection of antibiotics. The gene for toxin synthesis is encoded on a bacteriophage (the tox gene). Corynebacteria, not infected with phage, thus do not generally cause diphtheria. Diphtheria is now a disease of almost historic importance in the US due to effective immunization of infants (in conjunction with pertussis and tetanus, DPT) with a toxoid (inactive toxin) that causes production of neutralizing antibodies. However, colonization is not inhibited and thus C. diphtheriae is still found in the normal flora (i.e. a carrier state exists). If we let our guard down and dont vaccinate diphtheria will come back. Immunity can be monitored with the Schick skin test, observing the ability to neutralize toxin. Diphtheria toxin is an A-B exotoxin. Binds to epidermal growth factor (the receptor) on host cells; and then the A component becomes internalized and inhibits protein synthesis. This receptor is common in cells of the heart and lung, explaining why these sites are particularly affected. The exotoxin catalyses the covalent attachment of the ADP-ribose moiety of NADH to a rare amino acid, diphthamide, present in EF2 (elongation factor 2). The toxin is not synthesized in the presence of iron as an iron-repressor complex forms that inhibits expression of the tox gene. C. diphtheriae are identified by growth on Loeffler's medium followed by staining for metachromatic bodies (polyphosphate granules, Babes-Ernst bodies). The term "metachromatic" refers to the color difference of the intracellular polyphosphate granules (pink) compared to the rest of the cell (blue). Characteristic black colonies are seen on tellurite agar from precipitation of tellurium on reduction by the bacteria. Production of exotoxin can be determined by in vivo or in vitro tests. Other organisms that morphologically resemble C. diphtheriae ("diphtheroids" which include other corynebacteria and also propionibacteria) are found in the normal flora. Isolates should not be confused with these organisms. LEGIONELLAE In 1976/77, Legionella pneumophila was recognized as a newly described pathogen after an investigation by the Centers for Disease Control (CDC) of an outbreak of pneumonia, with several fatalities, among a group of Legionnaires at a convention. The disease was subsequently referred to as Legionnaires disease. Another milder self-limiting form of the disease (primarily 4

characterized by myalgia [muscle pain]) but no pneumonia is referred to as Pontiac fever. Quickly L. pneumophila filled the world stage as a major pathogen and as rapidly as interest appeared it waned within the next 10 years; probably, because legionellosis was found to be treatable with erythromycin. Occasional small epidemics of Legionnaire's disease in apparently healthy people still occur. However, the organism is more frequently observed in sick or elderly individuals whose immune responses may be compromised. This particularly occurs in the hospital setting. The causative agent was not recognized previously, since it does not grow on conventional agar such as sheep blood agar. Nowadays L. pneumophila is cultured on media that contains iron and cysteine vital for growth. However primary isolation is still difficult from clinical specimens. The organism stains poorly as a Gram negative rod. Some legionellae (including L. micdadei) also stain slightly acid fast. However, they are NOT genetically related to mycobacteria. Legionella pneumophila is an organism that resides in the environment in pools of stagnant water. It is found as an intracellular agent within protozoa. In man it also survives as a facultative intracellular pathogen. It often infects hot water towers and air conditioning systems. Heat shock proteins are involved in heat survival. Indeed it is the only bacterial agent whose presence is of major concern in indoor air quality. When found in buildings, anti-bacterial treatment of the water supply is recommended. The organism is transmitted in contaminated air but not spread person-person. After recognition of their unique culture characteristics, a large number of other species of Legionella were isolated from environmental and clinical samples. These organisms are only occasional causes of human disease and the vast bulk of legionellosis is caused by Legionella pneumophila (most are serogroup 1). The second most common is Legionella micdadei. The "hot" period of study of these organisms was when it was no longer popular to study bacteria using conventional physiological means. The techniques of fatty acid profiling and DNA-DNA hybridization had just been introduced and were in wide use at the CDC and elsewhere. 16S rRNA sequence analysis has sorted out much of the taxonomy of this group of organisms. However, the techniques are far too complex for routine use in the clinical laboratory. Probes have been used to detect L. pneumophila in clinical specimens and commercial PCR tests are available. Another technique detects the Legionella lipopolysaccharide antigen in urine. Serology is also employed. However, all approaches (including culture) have limited sensitivity in clinical specimens. The clinical importance of this organism is thus difficult to judge.