Todar's Online Textbook of Bacteriology

Tuberculosis

Mycobacterium tuberculosis

Mycobacterium bovis is the etiologic agent of TB in cows and rarely in humans. Both cows and humans can serve as reservoirs. Humans can also be infected by the consumption of unpasteurized milk. This route of transmission can lead to the development of extrapulmonary TB, exemplified in history by bone infections that led to hunched backs.

Other human pathogens belonging to the Mycobacterium genus include Mycobacterium avium which causes a TB-like disease especially prevalent in AIDS patients, and Mycobacterium leprae, the causative agent of leprosy.

History and Present Day Importance

Mycobacterium tuberculosis (MTB) was the cause of the "White Plague" of the 17th and 18th centuries in Europe. During this period nearly 100 percent  of the European population was infected with MTB, and 25 percent of all adult deaths were caused by MTB (Note: The White Plague is not to be confused with the "Black Plague", which was caused by Yersinia pestis and occurred about 3 centuries earlier).

General Characteristics

Mycobacterium tuberculosis is a fairly large nonmotile rod-shaped bacterium distantly related to the Actinomycetes. Many non pathogenic mycobacteria are components of the normal flora of humans, found most often in dry and oily locales. The rods are 2-4 micrometers in length and 0.2-0.5 um in width.

Mycobacterium tuberculosis is an obligate aerobe. For this reason, in the classic case of tuberculosis, MTB complexes are always found in the well-aerated upper lobes of the lungs. The bacterium is a facultative intracellular parasite, usually of macrophages, and has a slow generation time, 15-20 hours, a physiological characteristic that may contribute to its virulence.

Two media are used to grow MTB Middlebrook's medium which is an agar based medium and Lowenstein-Jensen medium which is an egg based medium. MTB colonies are small and buff colored when grown on either medium. Both types of media contain inhibitors to keep contaminants from out-growing MT. It takes 4-6 weeks to get visual colonies on either type of media.

Colonies of Mycobacterium tuberculosis on Lowenstein-Jensen medium. CDC.

Chains of cells in smears made from in vitro-grown colonies often form distinctive serpentine cords. This observation was first made by Robert Koch who associated cord factor with virulent strains of the bacterium.

MTB is not classified as either Gram-positive  or Gram-negative because it does not have the chemical characteristics of either, although the bacteria do contain peptidoglycan (murein) in their cell wall. If a Gram stain is performed on MTB, it stains very weakly Gram-positive or not at all (cells referred to as "ghosts").

Mycobacterium species, along with members of a related genus Nocardia, are classified as acid-fast bacteria due to their impermeability by certain dyes and stains. Despite this, once stained, acid-fast bacteria will retain dyes when heated and treated with acidified organic compounds. One acid-fast staining method for Mycobacterium tuberculosis is the Ziehl-Neelsen stain. When this method is used, the MTB. smear is fixed, stained with carbol-fuchsin (a pink dye), and decolorized with acid-alcohol. The smear is counterstained with methylene-blue or certain other dyes. Acid-fast bacilli appear pink in a contrasting background.

In order to detect Mycobacterium tuberculosis in a sputum sample, an excess of 10,000 organisms per ml of sputum are needed to visualize the bacilli with a 100X microscope objective (1000X mag). One acid-fast bacillus/slide is regarded as "suspicious" of an MTB infection.

Mycobacterium tuberculosis. Acid-fast stain. CDC.

Cell Wall Structure

The cell wall structure of Mycobacterium tuberculosis deserves special attention because it is unique among procaryotes, and it is a major determinant of virulence for the bacterium. The cell wall complex contains peptidoglycan, but otherwise it is composed of complex lipids. Over 60% of the mycobacterial cell wall is lipid. The lipid fraction of MTB's cell wall consists of three major components, mycolic acids, cord factor, and wax-D.

Mycolic acids are unique alpha-branched lipids found in cell walls of Mycobacterium and Corynebacterium. They make up 50% of the dry weight of the mycobacterial cell envelope.  Mycolic acids are strong hydrophobic molecules that form a lipid shell around the organism and affect permeability properties at the cell surface.  Mycolic Acids are thought to be a significant determinant of virulence in MTB. Probably, they prevent attack of the mycobacteria by cationic proteins, lysozyme, and oxygen radicals in the phagocytic granule. They also protect extracellular mycobacteria from complement deposition in serum.

Cord Factor is responsible for the serpentine cording mentioned above. Cord factor is toxic to mammalian cells and is also an inhibitor of PMN migration. Cord factor is most abundantly produced in virulent strains of MTB.

Wax-D in the cell envelope is the major component of Freund's complete adjuvant (CFA).

The high concentration of lipids in the cell wall of Mycobacterium tuberculosis have been associated with these properties of the bacterium:
• Impermeability to stains and dyes
• Resistance to many antibiotics
• Resistance to killing by acidic and alkaline compounds
• Resistance to osmotic lysis via complement deposition
• Resistance to lethal oxidations and survival inside of macrophages

The Disease Tuberculosis

TB infection means that MTB is in the body, but the immune system is keeping the bacteria under control. The immune system does this by producing macrophages that surround the tubercle bacilli. The cells form a hard shell that keeps the bacilli contained and under control. Most people with TB infection have a positive reaction to the tuberculin skin test.  People who have TB infection but not TB disease are NOT infectious, i.e., they cannot spread the infection to other people. These people usually have a normal  chest x-ray. TB infection is not considered a case of TB disease. Major similarities and differences between TB infection and TB disease are given in the table below.

Predisposing factors for TB infection include:
• Close contact with large populations of people, i.e., schools, nursing homes, dormitories, prisons, etc.
• Poor nutrition
• iv drug use
• Alcoholism
• HIV infection is the #1 predisposing factor for MTB infection. 10 percent of all HIV-positive individuals harbor MTB. This is 400-times the rate associated with the general public

Only 3-4% of infected individuals will develop active disease upon initial infection, 5-10% within one year. These percentages are much higher if the individual is HIV+.

Stages of the Disease

The following stages that will be explained are for a MTB - sensitive host. It should be realized that, as stated previously, only a small percent of MTB infections progress to disease and even a smaller percent progress all the way to stage 5. Usually the host will control the infection at some point.

Disease progression depends on:
• Strain of MTB
• Prior exposure
• Vaccination
• Infectious dose
• Immune status of the host

Stage 1

Droplet nuclei are inhaled. One droplet nuclei contains no more than 3 bacilli. Droplet nuclei are so small that they can remain air-borne for extended periods of time. The most effective (infective) droplet nuclei tend to have a diameter of 5 micrometers. Droplet nuclei are generated by during talking coughing and sneezing.  Coughing generates about 3000 droplet nuclei. Talking for 5 minutes generates 3000 droplet nuclei but singing generates 3000 droplet nuclei in one minute. Sneezing generates the most droplet nuclei by far, which can spread to individuals up to 10 feet away.
 

Spread of droplet nuclei from one individual to another. CDC. After droplet nuclei are inhaled, the bacteria are nonspecifically taken up by alveolar macrophages. However, the macrophages are not activated and are unable to destroy the intracellular organisms.
 
 

Tuberculosis begins when droplet nuclei reach the alveoli. When a person inhales air that contains droplets most of the larger droplets become lodged in the upper respiratory tract (the nose and throat), where infection is unlikely to develop. However, the smaller droplet nuclei may reach the small air sacs of the lung (the alveoli), where infection begins.

Stage 2

Begins 7-21 days after initial infection. MTB multiplies virtually unrestricted within unactivated macrophages until the macrophages burst. Other macrophages begin to extravasate from peripheral blood. These macrophages also phagocytose MTB, but they are also unactivated and hence can not destroy the bacteria.

Stage 3

At this stage lymphocytes begin to infiltrate. The lymphocytes, specifically T-cells, recognize processed and presented MTB antigen in context of MHC molecules. This results in T-cell activation and the liberation of cytokines including gamma interferon (IFN). The liberation of IFN causes in the activation of macrophages. These activated macrophages are now capable of destroying MTB.

It is at this stage that the individual becomes tuberculin-positive. This positive tuberculin reaction is the result of the host developing a vigorous cell mediated immune (CMI) response. A CMI response must be mounted to control an MTB infection. An antibody mediated immune (AMI) will not aid in the control of a MTB infection because MTB is intracellular and if extracellular, it is resistant to complement killing due to the high lipid concentration in its cell wall.

Although a CMI response is necessary to control an MTB infection, it is also responsible for much of the pathology associated with tuberculosis. Activated macrophages may release lytic enzymes and reactive intermediates that facilitate the development of immune pathology. Activated macrophages and T-cells also secrete cytokines that may also play a role in the development of immune pathology, including Interleukin 1 ( IL-l), tumor necrosis factor (TNF), and gamma IFN.

It is also at this stage that tubercle formation begins. The center of the tubercle is characterized by "caseation necrosis", meaning it takes on a semi-solid or "cheesy" consistency. MTB cannot multiply within these tubercles because of the low pH and anoxic environment. MTB can, however, persist within these tubercles for extended periods.

Stage 4

Although many activated macrophages can be found surrounding the tubercles, many other macrophages present remain unactivated or poorly activated. MTB uses these macrophages to replicate, and hence, the tubercle grows.

The growing tubercle may invade a bronchus. If this happens, MTB infection can spread to other parts of the lung. Similarly the tubercle may invade an artery or other blood supply line. The hematogenous spread of MTB may result in extrapulmonary tuberculosis otherwise known as milliary tuberculosis. The name "milliary" is derived from the fact that metastasizing tubercles are about the same size as a millet seed, a grain commonly grown in Africa.

The secondary lesions caused by milliary TB can occur at almost any anatomical location, but usually involve the genitourinary system, bones, joints, lymph nodes and peritoneum. These lesions are of two types:

1. Exudative lesions result from the accumulation of PMN's around MTB. Here the bacteria replicate with virtually no resistance. This situation gives rise to the formation of a "soft tubercle".

2. Productive or granulomatous lesions occur when the host becomes hypersensitive to tuberculoproteins. This situation gives rise to the formation of a "hard tubercle".

Stage 5

For unknown reasons, the caseous centers of the tubercles liquefy. This liquid is very conducive to MTB growth, and the organism begins to rapidly multiply extracellularly. After time, the large antigen load causes the walls of nearby bronchi to become necrotic and rupture. This results in cavity formation. This also allows MTB to spill into other airways and rapidly spread to other parts of the lung.

As stated previously, only a very small percent of MTB infections result in disease, and even a smaller percentage of MTB infections progress to an advanced stage. Usually the host will begin to control the infection at some point. When the primary lesion heals, it becomes fibrous and calcifies. When this happens the lesion is referred to as the Ghon complex. Depending on the size and severity, the Ghon complex may never subside. Typically, the Ghon complex is readily visible upon chest X-ray.

Small metastatic foci containing low numbers of MTB may also calcify. However, in many cases these foci will contain viable organisms. These foci are referred to Simon foci. The Simon foci are also visible upon chest X-ray and are often the site of disease reactivation.

Virulence Mechanisms and Virulence Factors

Mycobacterium tuberculosis does not possess the classic bacterial virulence factors such as toxins, capsules and fimbriae. However, a number of structural and physiological properties of the bacterium are beginning to be recognized for their contribution to bacterial virulence and the pathology of tuberculosis.

MTB has special mechanisms for cell entry. The tubercle bacillus can bind directly to mannose receptors on macrophages via the cell wall-associated mannosylated glycolipid, LAM, or indirectly via certain complement receptors or Fc receptors.

MTB can grow intracellularly. This is an effective means of evading the immune system. In particular, antibodies and complement are ineffective. Once MTB is phagocytosed, it can inhibit phagosome-lysosome fusion. The exact mechanism used by MTB to accomplish this is not known but it is thought to be the result of a protein secreted by bacterium that modifies the phagosome membrane. The bacterium may remain in the phagosome or escape from the phagosome, in either case finding a protected environment for growth in the macrophage.

MTB interferes with the toxic effects of reactive oxygen intermediates produced in the process of phagocytosis by two mechanisms:

1. Compounds including glycolipids, sulfatides and LAM down regulate the oxidative cytotoxic mechanism.

2. Macrophage uptake via complement receptors may bypass the activation of a respiratory burst.

Antigen 85 complex. This complex is composed of a group of proteins secreted by MTB that are known to bind fibronectin. These proteins may aid in walling off the bacteria from the immune system and may facilitate tubercle formation although evidence of this is lacking.

Slow generation time. Because of MTB's slow generation time, the immune system may not readily recognize the bacteria or may not be triggered sufficiently to eliminate them. Many other chronic disease are caused by bacteria with slow generation times, for example,  slow-growing M. leprae causes leprosy, Treponema pallidum causes syphilis, and Borrelia burgdorferi causes Lyme disease.

High lipid concentration in cell wall, as mentioned previously, accounts for impermeability and resistance to antimicrobial agents, resistance to killing by acidic and alkaline compounds in both the intracellular and extracellular environment, and resistance to osmotic lysis via complement deposition and attack by lysozyme.

Cord factor. The cord factor is primarily associated with virulent strains of MTB. It is known to be toxic to mammalian cells and to be an inhibitor of PMN migration. However, its exact role in MTB virulence is unclear.
 

Clinical Identification and Diagnosis of Tuberculosis

The diagnosis of tuberculosis requires detection of acid-fast bacilli in sputum via the Ziehl-Neelsen method as previously described.

The organisms must then be cultured from sputum. First, the sputum sample is treated with NaOH. This kills other contaminating bacteria but does not kill the MTB present because cells are resistant to alkaline compounds by virtue of their lipid layer.

The media used for growth of MTB and the the resulting colony morphology have been described previously. However, methods of culturing can take 4-6 weeks to yield visible colonies. As a result, another method is commonly used call the BACTEC System. The media used in the BACTEC system contains radio-labeled palmitate as the sole carbon source. As MTB multiplies, it breaks down the palmitate and liberates radio-labeled CO₂. Using the BACTEC system, MTB growth can be detected in 9-16 days vs 4-6 weeks using conventional media.

Skin Testing is performed as the tuberculin or Mantoux test.  PPD (purified protein derivative) is employed as the test antigen in the Mantoux test. PPD is generated by boiling a culture of MTB, specifically Old Tuberculin (OT). 5 TU (tuberculin units), which equals 0.000lmg of PPD, in a 0.1 ml volume is intracutaneously injected in the forearm. The test is read within 48-72 hours.

Administering the Mantoux test. CDC.
 

The test is considered positive if the diameter of the resulting lesion is 10 mm or greater. The lesion is characterized by erythema (redness) and swelling and induration (raised and hard). 90% of people that have a lesion of 10 mm or greater are currently infected with MTB or have been previously exposed to MTB. 100% of people that have a lesion of 15 mm or greater are currently infected with MTB or have been previously exposed to MTB.

False positive tests usually manifest themselves as lesser reactions. These lesser reactions could indicate prior exposure or infection with other mycobacteria or vaccination with BCG. However, in places were the vaccine is not used, lesser reactions should be regarded as highly suspicious.

False negatives are more rare than false positives but are especially common in AIDS patients as they have an impaired CMI response. Other conditions such as malnutrition, steroids, etc., can rarely result in a false negative reaction.
 

Tuberculosis Treatment

Because administration of a single drug often leads to the development of a bacterial population resistant to that drug, effective regimens for the treatment of TB must contain multiple drugs to which the organisms are susceptible. When two or more drugs are used simultaneously, each helps prevent the emergence of tubercle bacilli resistant to the others. However, when the in vitro susceptibility of a patient's isolate is not known, which is generally the case at the beginning of therapy, selecting two agents to which the patient's isolate is likely to be susceptible can be difficult, and improper selection of drugs may subsequently result in the development of additional drug-resistant organisms.

Hence, tuberculosis is usually treated with  four different antimicrobial agents The course of drug therapy  usually lasts from 6-9 months. The most commonly used drugs are rifampin (RIF) isoniazid (INH), pyrazinamide (PZA ) and ethambutol (EMB) or streptomycin (SM). When adherence with the regimen is assured, this four-drug regimen is highly effective. Based on the prevalence and characteristics of drug-resistant organisms, at least 95% of patients will receive an adequate regimen (at least two drugs to which their organisms are susceptible) if this four-drug regimen is used at the beginning of therapy. Furthermore, a patient who is treated with the four-drug regimen, but who defaults therapy, is more likely to be cured and not relapse when compared with a patient treated for the same length of time with a three-drug regimen.
 

Drugs used to treat TB disease. From left to right isoniazid, rifampin, pyrazinamide, and ethambutol. Streptomycin (not shown) is given by injection. CDC.

Prevention

A vaccine against MTB is available. It is called BCG (Bacillus of Calmette and Guerin, named after the two Frenchmen that developed it). BCG consists of a live attenuated strain derived from Mycobacterium bovis. This strain of Mycobacterium has remained avirulent for over 60 years.

The vaccine is not 100% effective. Studies suggest a 60-80% effective rate in children.

The vaccine is not administered in the U.S. for several reasons:
• The vaccine cannot circumvent disease reactivation in previously exposed individuals.
• The vaccine does not prevent infection, only disease. Therefore, the entire population would have to be vaccinated if the vaccine was to be considered efficacious.
• Vaccination may complicate the way the tuberculin skin test is read in this country. In places that do not vaccinate, the skin test may be used to monitor the effectiveness of antibiotic therapy.

Multidrug-Resistant Tuberculosis (MDR TB) and Extensively Drug-Resistant Tuberculosis  (XDR TB)

Resistance to anti-TB drugs can occur when these drugs are misused or mismanaged. Examples include when patients do not complete their full course of treatment; when health-care providers prescribe the wrong treatment, the wrong dose, or length of time for taking the drugs; when the supply of drugs is not always available; or when the drugs are of poor quality.

Multidrug-resistant tuberculosis (MDR TB) is TB that is resistant to at least two of the best anti-TB drugs, isoniazid and rifampicin. These drugs are considered first-line drugs and are used to treat all persons with TB disease.

Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB. XDR TB is defined as TB which is resistant to isoniazid and rifampin, plus resistant to any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin). Because XDR TB is resistant to first-line and second-line drugs, patients are left with less effective treatment options, and cases often have worse treatment outcomes.

Both MDR TB and XDR TB are more common in TB patients that do not take their medicines regularly or as prescribed, or who experience reactivation of TB disease after having taken TB medicine in the past.

Persons with HIV infection or other conditions that can compromise the immune system are at highest risk for MDR TB and XDR TB. They are more likely to develop TB disease once infected and have a higher risk of death from disease.

The risk of acquiring XDR TB in the United States appears to be relatively low. However, it is important to acknowledge the ease at which TB can spread. But long as XDR TB exists, the U.S. is at some risk.

The risk of acquiring MDR TB in the United States is < 0.7% in U.S.-born persons but higher in foreign-born persons. Since 1998, the percentage of U.S.-born patients with MDR TB has remained fairly constant. However, of the total number of reported primary MDR TB cases, the proportion occurring in foreign-born persons increased from 25% (103 of 407) in 1993 to 80% (73 of 91) in 2006.

The Centers for Disease Control (CDC) Division of Tuberculosis Elimination advises how to prevent or minimize the development of MDR strains of M. tuberculosis.

• The most important thing a patient can do is to take all of their medications exactly as prescribed by a health care provider. No doses should be missed and treatment should not be stopped early. Patients should tell their health care provider if they are having trouble taking the medications. If patients plan to travel, they should talk to their health care providers and make sure they have enough medicine to last while away.

• Health care providers can help prevent MDR TB by quickly diagnosing cases, following recommended treatment guidelines, monitoring patients’ response to treatment, and making sure therapy is completed.

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Written and edited by Kenneth Todar  University of Wisconsin-Madison  Department of Bacteriology  All rights reserved