Mycobacterium and Tuberculosis
Myco = waxy ★★★
Myco means “waxy” in Latin — the genus is named for its thick, complex, lipid-rich, waxy cell wall. That wall allows the germ to spread through the air and survive outside the body. Unit 2 §5 built this wall; here is what it does.
| Classification | Species |
|---|---|
| M. tuberculosis complex | M. tuberculosis · M. bovis |
| M. leprae | Leprosy — §8 |
| Nontuberculosis mycobacteria (NTM) | M. kansasii · M. marinum · M. scrofulaceum · M. avium complex (MAC) · M. fortuitum |
| Morphology | Detail |
|---|---|
| Shape | Slender, rod-shaped, non-motile |
| Staining | Ziehl–Neelsen acid-fast stain — carbolfuchsin (15 min) → acid-alcohol (decolour) → methylene blue (counterstain) |
| Key property | High content of lipids — a special biological property AND a virulence factor |
History for context: Robert Koch proved in March 1882 in Berlin that bacilli caused TB, becoming the Father of Bacteriology and receiving the Nobel Prize in 1905. Waksman discovered streptomycin in 1944 — the first specific agent effective in the treatment of tuberculosis.
Scale: one-third of the world's population is infected. Every year 1.5 million people die of tuberculosis and 10 million are newly infected, 87% in 30 high-burden countries. Why is TB increasing? The HIV/AIDS epidemic · the emergence of multidrug-resistant M. tuberculosis · immigration from countries where TB is common · inadequate institutional control.
- What does 'myco' mean, and why? → Waxy — for the thick, lipid-rich cell wall, which lets the organism spread through air and survive outside the body
- Name the Ziehl–Neelsen reagents in order. → Carbolfuchsin (15 min), acid-alcohol, methylene blue
- Name the four reasons TB is increasing. → HIV/AIDS · multidrug resistance · immigration from high-burden countries · inadequate institutional control
Culture and resistance ★★★
| Property | Detail |
|---|---|
| Requirements | Fastidious · obligate aerobes |
| Medium | Löwenstein–Jensen (L-J) medium — egg yolks, potato flour, glycerol, malachite green |
| Growth | Slow. Generation time 18–24 hours; colonies take 3–4 weeks |
| Resistant to | Sensitive to |
|---|---|
| Drying — survives in sputum | Moist heat |
| Acid and alkali | UV radiation |
| Dyes, e.g. malachite green | Alcohol |
Look at what the organism resists — acid, alkali and malachite green — and then at how a specimen is processed: treated with NaOH to kill other contaminating bacteria, and cultured on a medium containing malachite green.
That is not coincidence. Sputum swarms with normal flora that would overgrow a plate long before a colony appeared at three to four weeks. So the laboratory exploits the two things M. tuberculosis tolerates and its competitors do not: it drowns the specimen in alkali, and it puts a dye in the medium.
The organism's defence against the outside world becomes the means of isolating it — the same reversal seen with SPA in Unit 8.
And the 18–24 hour generation time is Unit 3 §3 again: slow culture, chronic disease, months of therapy.
- What medium, and of what? → Löwenstein–Jensen — egg yolks, potato flour, glycerol, malachite green
- Generation time and colony time? → 18–24 hours; colonies in 3–4 weeks
- What is it resistant and sensitive to? → Resistant to drying (survives in sputum), acid and alkali, dyes such as malachite green; sensitive to moist heat, UV radiation and alcohol
- Why is the specimen treated with NaOH? → To kill contaminating bacteria — the mycobacterium tolerates alkali
Variation — the L form, drug resistance and ⭐ BCG ★★★
Variation in shape and structure — the L form
M. tuberculosis forms L forms — pleomorphic, spherical or filamentous — which revert to the normal form of the bacteria. Isoniazid induces this. Unit 2 §6 defined the L form and warned of exactly this consequence: no wall, so no target for a wall-active drug, then reversion after treatment stops — relapse.
Variation in drug resistance
| Detail | |
|---|---|
| MDR-TB | Multidrug-resistant M. tuberculosis — resistant to the first-line drugs isoniazid and rifampicin |
| XDR-TB | Extensively drug-resistant — a new strain first reported in March 2006 by the WHO |
| Genetics | M. tuberculosis H37Rv: 4.4 million bp, 3924 expected genes, 65.6% GC. Resistance genes: rifampicin — rpoB; isoniazid — katG, inhA, KasA; streptomycin — rpsL, rrS |
| Cause | Improper or incomplete tuberculosis treatment |
⭐ Variation in virulence — BCG
A form of tuberculosis vaccine that is a freeze-dried preparation of a live, attenuated bovine organism. Made by Calmette and Guérin, since 1908. Recommended in immunisation programmes and TB control programmes in China.
For the remaining marks add its use and its consequence: it is given in national immunisation programmes to prevent tuberculosis, particularly the severe childhood forms; and — the point that ties this unit together — BCG vaccination converts the tuberculin test to positive, which is why a positive tuberculin test in a vaccinated person does not prove infection (§6).
Note the three words the marker is looking for: live, attenuated, bovine.
- Define BCG. → Bacille Calmette-Guérin — a freeze-dried preparation of a live, attenuated bovine organism, made by Calmette and Guérin since 1908, used as a TB vaccine
- What are MDR-TB and XDR-TB? → Multidrug-resistant (to first-line isoniazid and rifampicin) and extensively drug-resistant TB, first reported by WHO in March 2006
- What causes drug resistance? → Improper or incomplete treatment
- What induces the L form, and why does it matter? → Isoniazid; the L form has no wall, escapes wall-active drugs, and reverts afterwards — causing relapse
Pathogenesis ★★★
| Definition | |
|---|---|
| TB infection | TB bacilli live inside the person, but do not cause pathological destruction of organs. No signs or symptoms of disease |
| TB disease | TB bacilli progressively invade an organ; signs and symptoms appear |
Transmission is through the respiratory tract, and less commonly the alimentary tract or injured skin. The deck's arithmetic is worth remembering: one droplet = 3 bacilli; talking for five minutes = 3000 droplets = 9000 bacilli.
⭐ Virulence factors — note what is ABSENT
M. tuberculosis has NEITHER endotoxin NOR exotoxin NOR invasive enzymes. It is an intracellular pathogen, and its virulence is entirely in its chemical components.
| Component | Effect |
|---|---|
| Lipids | Chronic granulomatosis, hypersensitivity |
| Protein — tuberculin | Induces hypersensitivity of the host; the basis of the tuberculin test |
| Polysaccharides | Stimulate non-specific inflammation |
The lipids in detail
| Lipid | Action |
|---|---|
| Mycolic acid | Acid-fastness |
| Phospholipids | Proliferation of mononuclear cells |
| Cord factor | In virulent strains; microscopic “serpentine cords”. Inhibits migration of leukocytes; chronic granuloma |
| Sulfatide and LAM (lipoarabinomannan) | Suppress the combination of phagosome with lysosome |
| Waxes (wax D) | Adjuvant; delayed-type hypersensitivity |
Tubercle formation
A tubercle in the lung is a “granuloma”, consisting of a central core of TB bacteria inside an enlarged macrophage, and an outer wall of fibroblasts, lymphocytes and neutrophils.
| Type of pulmonary TB | Detail |
|---|---|
| Primary tuberculosis | Infected for the first time; in young children; usually mild and asymptomatic |
| Post-primary (secondary) tuberculosis | From reactivation of bacilli that survived the primary infection, or inhalation of new bacilli from the environment. Localised and severe |
Hosts: M. tuberculosis — only humans; M. bovis — humans and cattle. Disease is pulmonary or extrapulmonary.
This is the single most important idea in the unit, and the deck's own multiple-choice asks it: pathogenic mechanisms in tuberculosis are primarily attributable to… — cell-mediated hypersensitivity.
The organism has no toxin and no invasive enzyme. It cannot destroy tissue. What it does is survive inside the macrophage that engulfed it — sulfatide and LAM prevent the phagosome fusing with the lysosome, so the cell cannot digest it.
The immune system responds by walling the infected macrophage in: lymphocytes and fibroblasts pile around it and form a granuloma — the tubercle. The centre, starved of blood, undergoes caseous necrosis.
The cavity in a chest radiograph is immunological damage, not bacterial damage. That is why Unit 5 §1 insisted that disease is bacterial damage plus the consequences of the immune response — and why TB is the textbook example.
It also explains HIV. Destroy CD4 T cells and the granuloma cannot form, so the infection is not contained: worldwide, half of HIV-positive individuals die from tuberculosis.
- TB infection vs TB disease? → Infection: bacilli live in the person without organ destruction and without signs or symptoms. Disease: bacilli progressively invade an organ and symptoms appear
- Which virulence factors does M. tuberculosis NOT have? → No exotoxin, no endotoxin, no invasive enzyme — it is an intracellular pathogen
- Name the three classes of virulence component and their effects. → Lipids (chronic granulomatosis, hypersensitivity) · protein/tuberculin (hypersensitivity, the tuberculin test) · polysaccharides (non-specific inflammation)
- What do cord factor, sulfatide/LAM and wax D do? → Cord factor inhibits leukocyte migration and forms chronic granuloma; sulfatide and LAM block phagosome–lysosome fusion; wax D is an adjuvant causing delayed-type hypersensitivity
- Define a tubercle. → A granuloma with a central core of TB bacteria inside an enlarged macrophage, walled by fibroblasts, lymphocytes and neutrophils
- Primary vs post-primary TB? → Primary: first infection, in young children, usually mild and asymptomatic. Post-primary: reactivation or reinfection, localised and severe
Immunity ★★★
- Cell-mediated immunity — appearing 3–6 weeks after infection
- Humoral immunity: circulating antibodies have NO defensive role
- Infection immunity: the immunity to the bacilli depends on the existence of the organisms
- Cell-mediated immunity and hypersensitivity develop at the same time, are both mediated by T cells, but are caused by DIFFERENT components of the bacillus — the protein antigens (tuberculin together with wax D) cause the hypersensitivity
The immunity to the bacilli depends on the existence of the organisms.
Ordinary immunity outlives the infection — you had measles once and you are protected for life, organism long gone. Tuberculosis is not like that. Protection lasts only while living bacilli remain somewhere in the body, holding the T-cell response awake. Clear the organism entirely and the immunity fades.
Two consequences follow. It explains why BCG protection wanes and why boosters were once given. And it explains the paradox of TB: the patient who contains the infection carries live bacilli in a healed granuloma for decades — which is both what protects them and what reactivates as post-primary disease if their immunity ever falters.
Point 4 is the second thing to hold: immunity and hypersensitivity arise together, from the same T cells, but from different bacterial components. That is precisely why the tuberculin test — which measures hypersensitivity — is an indirect and imperfect reading of immunity, and why §6 needs so many caveats.
- What kind of immunity, and when? → Cell-mediated, 3–6 weeks after infection
- What do circulating antibodies do? → Nothing defensive
- Define infection immunity. → Immunity to the bacilli depends on the continued existence of the organisms
- How do immunity and hypersensitivity relate? → They develop at the same time and are both T-cell mediated, but are caused by different bacterial components — tuberculin protein with wax D causes the hypersensitivity
⭐ The tuberculin test ★★★
A skin test to determine the presence of immunity and hypersensitivity of the host to M. tuberculosis, based on the positive reaction of the subject to tuberculin. Tuberculin is a mixture known as purified protein derivative (PPD) from TB bacilli.
| Method | Detail |
|---|---|
| Administration | Intradermal injection of tuberculin |
| Reading | Read at 48–72 hours |
| Measurement | Measure the INDURATION — not the redness |
⭐ Results and interpretation — the exam answer
| Induration | Interpretation |
|---|---|
| ≥ 5 mm in diameter | POSITIVE — previous infection, but not necessarily active disease; also after BCG vaccination |
| ≥ 15 mm in diameter | INTENSELY POSITIVE — active disease |
| < 5 mm | NEGATIVE — no previous contact with M. tuberculosis, OR a false reaction: the early stage of infection, the elderly, patients with severe tuberculosis or another infectious disease such as AIDS, or the use of immunosuppressive drugs |
Principle: it is a type IV delayed-type hypersensitivity reaction, mediated by T cells — the response caused by tuberculin protein together with wax D.
Method: intradermal injection; read at 48–72 hours; measure the induration.
Interpretation: ≥5 mm positive — previous infection but not necessarily active disease, and also seen after BCG. ≥15 mm intensely positive — active disease. <5 mm negative — no previous contact, or a false negative.
The false negatives are where the marks are. Name all four: the early stage of infection (before the 3–6 weeks CMI takes to develop), the elderly, severe tuberculosis or another infectious disease such as AIDS, and immunosuppressive drugs.
Uses: to detect whether a suspected patient has been infected, and to test the host's cell-mediated immune function.
The deck opens and closes with the same patient, and every line is a teaching point.
A 32-year-old man, intravenous drug user, with dry persistent cough, fever, malaise, anorexia, 15 pounds lost in four weeks, chills and sweats. That is textbook tuberculosis.
The first acid-fast smear was negative — then positive after concentration by centrifugation. Smear microscopy needs roughly 10 000 organisms per millilitre; concentration is what makes it work.
HIV positive. The reason he has tuberculosis at all.
The tuberculin test was NEGATIVE — and this is the contradiction the case exists for. He plainly has tuberculosis; the culture proved it. The negative test is a false negative, third row of the table: severe tuberculosis or another infectious disease (AIDS). The test measures a T-cell response, and HIV has destroyed his T cells.
Never exclude tuberculosis on a negative tuberculin test in an immunosuppressed patient. That is the whole case in one sentence.
Cultures positive after three weeks — §2's growth rate, exactly as predicted.
- Define the tuberculin test. → A skin test determining the presence of immunity and hypersensitivity of the host to M. tuberculosis, based on the reaction to tuberculin (PPD)
- Give the method. → Intradermal injection of tuberculin, read at 48–72 hours, measuring the induration
- Interpret the three results. → ≥5 mm positive (previous infection, not necessarily active; also after BCG) · ≥15 mm intensely positive (active disease) · <5 mm negative (no contact, or false)
- Name the four causes of a false negative. → Early stage of infection · the elderly · severe TB or another infection such as AIDS · immunosuppressive drugs
- Why can a positive test not prove infection? → BCG vaccination also converts it to positive
Diagnosis, prevention and treatment ★★
- Decontamination and concentration of the specimen — treated with NaOH to kill other contaminating bacteria, then concentrated by centrifugation
- Smears — acid-fast stain
- Culture — used to confirm the diagnosis of TB
- DNA detection, serology and antigen detection — PCR and enzyme immunoassay (EIA)
| Detail | |
|---|---|
| Prevention | BCG — see §3 |
| Treatment | MULTIPLE drugs: rifampicin, isoniazid, streptomycin, pyrazinamide, para-aminosalicylic acid (PAS). Usually lasts 6–9 months |
The reason for multiple drugs and long duration is now assembled from three earlier sections: an 18–24 hour generation time (§2), an intracellular location behind a waxy wall (§4), and the L form induced by isoniazid itself (§3). Improper or incomplete treatment produces MDR-TB.
- Outline the laboratory diagnosis. → NaOH decontamination and centrifugal concentration → acid-fast smear → culture to confirm → PCR, EIA, serology and antigen detection
- Name five antituberculous drugs. → Rifampicin, isoniazid, streptomycin, pyrazinamide, para-aminosalicylic acid
- How long is treatment, and why multiple drugs? → 6–9 months; slow growth, intracellular location and L-form induction, and to prevent resistance
M. leprae and the nontuberculous mycobacteria ★★
| M. leprae | Detail |
|---|---|
| Biology | Intracellular parasite. Does NOT grow on artificial media or in cell culture. Grows in the footpads of mice. Optimal temperature 30 °C |
| Transmission | Respiratory tract (main) · contact |
| Disease | Leprosy (Hansen's disease) — infects skin and nerves; chronic; causes disfigurement |
⭐ The two types of leprosy
| Lepromatous type | Tuberculoid type | |
|---|---|---|
| CMI | Deficient | Normal |
| Lepromin skin test | Negative | Positive |
| Skin lesion | Abundant acid-fast bacilli — nodular | Few bacilli — macular |
| Course | Progressive and malignant | Benign and non-progressive |
| Infectivity | High | Low |
Read the table from the top row down and it is a single fact repeated.
CMI deficient → the organism is not contained → abundant bacilli in the skin → highly infectious → progressive and malignant → and the lepromin test is negative, because that test, like the tuberculin test, measures a T-cell response that this patient cannot make.
CMI normal → the organism is contained in granulomas → few bacilli → low infectivity → benign and non-progressive → lepromin positive. The nerve damage in this form is done by the granulomatous response itself.
Which is §4's lesson once more: with an intracellular mycobacterium, the host's immune status determines the disease more than the organism does.
Nontuberculous mycobacteria are much less virulent than M. tuberculosis; opportunistic pathogens in humans; often a combined infection with M. tuberculosis, mostly the Mycobacterium avium-intracellulare complex — a very prominent cause of disease in AIDS patients; and difficult to treat.
- How is M. leprae cultured? → It is not — it does not grow on artificial media or in cell culture; it grows in mouse footpads at an optimum of 30 °C
- Compare the two types of leprosy. → Lepromatous: CMI deficient, lepromin negative, abundant bacilli, progressive and malignant, highly infectious. Tuberculoid: CMI normal, lepromin positive, few bacilli, benign and non-progressive, low infectivity
- Describe the nontuberculous mycobacteria. → Much less virulent, opportunistic, often co-infecting with M. tuberculosis (mainly MAC), prominent in AIDS, difficult to treat
Revision
The deck's own multiple-choice questions
| Question | Answer |
|---|---|
| Virulence factors of M. tuberculosis do NOT include… | b. exotoxin — it has none |
| The most common route of acquisition is… | c. respiratory tract |
| After digestion of sputum, isolation is best on… | c. Löwenstein–Jensen medium |
| Pathogenic mechanisms are primarily attributable to… | c. cell-mediated hypersensitivity |
The whole unit on one screen
| Question | Answer |
|---|---|
| Why 'myco'? | Latin for waxy — the lipid-rich wall |
| Stain? | Ziehl–Neelsen: carbolfuchsin → acid-alcohol → methylene blue |
| Medium? | Löwenstein–Jensen — egg yolk, potato flour, glycerol, malachite green |
| Growth? | 18–24 h generation; colonies at 3–4 weeks |
| Toxins? | None — no exotoxin, endotoxin or invasive enzyme |
| Key lipids? | Mycolic acid (acid-fast) · cord factor · sulfatide/LAM (block phagolysosome) · wax D (DTH) |
| Tubercle? | A granuloma — TB inside an enlarged macrophage, walled by fibroblasts, lymphocytes, neutrophils |
| Immunity? | Cell-mediated, 3–6 weeks; antibody useless; infection immunity |
| ⭐ BCG? | Live, attenuated, bovine, freeze-dried; Calmette and Guérin, 1908 |
| ⭐ Tuberculin test? | Intradermal PPD, read 48–72 h, measure induration: ≥5 mm +, ≥15 mm intense, <5 mm − |
| Treatment? | Multiple drugs (rifampicin, isoniazid, streptomycin, pyrazinamide, PAS), 6–9 months |
- Define BCG for 4 marks. → A TB vaccine that is a freeze-dried preparation of a live, attenuated bovine organism, made by Calmette and Guérin since 1908; it converts the tuberculin test to positive
- Describe the tuberculin test and interpret it. → Intradermal PPD read at 48–72 h measuring induration; ≥5 mm positive (previous infection or BCG), ≥15 mm intensely positive (active disease), <5 mm negative — but false negatives occur in early infection, the elderly, severe TB or AIDS, and with immunosuppressive drugs
- Why does M. tuberculosis cause disease without a toxin? → It survives inside macrophages (sulfatide and LAM block phagolysosome fusion); the tissue damage is cell-mediated hypersensitivity — the granuloma and caseation
- Why 6–9 months of multiple drugs? → 18–24 h generation time, intracellular location behind a waxy wall, L-form induction, and prevention of resistance
- Compare lepromatous and tuberculoid leprosy. → Deficient vs normal CMI; lepromin negative vs positive; abundant vs few bacilli; progressive/malignant vs benign; high vs low infectivity