myobacteria

Question 1

descrinbe the classification of myobacteria.

Answer 1

• can grow on artificial media
• cell wall
• single cells
• rods
• ziehl-neelsen/acid fast positive (doesnt fit into gram-positive or gram-negative)

Question 2

what are the myobacteria species of medical importance and the medical condition in which they cause?

Answer 2

M. tuberculosis - Tuberculosis

M. avium complex (MAC) - Disseminated infection in AIDS,
chronic lung infection

M. kansasii - Chronic lung infection

M. marinum - Fish tank granuloma

M. ulcerans - Buruli ulcer

Rapidly growing mycobacteria
(M. fortuitum complex) - Skin and soft tissue infections

M. leprae - Leprosy

Question 3

outline the structure of myobacteria and why this makes it resistant to gram stains.

Answer 3

Slightly curved, beaded bacilli

High lipid content with mycolic acids
in cell wall makes Mycobacteria
resistant to Gram stain

Question 4

Ziehl-Neelsen stain

Answer 4

❖ Carbol fuchsin
❖ Acid alcohol (AFB are resistant to de-
staining)
❖ Methylene blue
* Need 10,000 bacilli per ml sputum to
diagnose

Question 5

outline the basic microbiological structure and features of myobacteria.

Answer 5

• rich in mycolic acids (long-chain fatty acids), making the wall hydrophobic and resistant to desiccation, antibiotics and staining
• contains arabinogalactan and peptidoglycan for structural rigidity
• classified as acid-fast bacteria as its impermeable to gram staining
• slow growth
• slightly aerobic - requires oxygen for growth
• non spore fomring
  non motile
  bacillis

Question 6

what are the characteristics of mycobacterium tuberculosis which makes it difficult to treat?

Answer 6

Challenges:
* Thick lipid rich cell wall making immune cell killing and penetration
of drugs challenging
* SLOW growth
– Gradual onset of disease
– Takes much longer to diagnose
– Takes longer to treat

Question 7

name the tests which can be used to distinguish TB from other myobacterium?

Answer 7

• chest radiograph and sputum
• can use solid and liquid culture to kill off any other rapid growing bacteria

then you can either use a lab test or a NAATs test.
lab tests:
- niacin test - Mtb yellow - other white
- nitrate reduction test - Mtb red - other yellow
- aryl sulphatase test - Mtb pink - other yellow
- pyrazinamide test - Mtb pink - other yellow/colourless

nucleic acid amplification test (NAATs):
- detect TB DNA in a sample, such as sputum, using a process that copies (amplifies) small amounts of genetic material to make it easier to identify. The test looks for genes specific to Mycobacterium tuberculosis, confirming its presence quickly.

tuberculin skin test (mantoux)
- myobacteria lipids stimulate T-cell response in 4-6 weeks after exposure to M. tuberculosis
- this is measured in the tuberculin skin test - an epidermal injection of purified protein derivitives

Question 8

outline the positives and negatives of NAATs compared to lab tests used to differenciate TB from other myobacterium.

Answer 8

• NAATs is rapid whereas solid culture takes 2-8 weeks and liquid culture takes 1-3 weeks
• NAATs can detect 131bacilli/ml
• NAATs sensitivity 88% and specificity 98%

Question 9

outline the treatment for TB

Answer 9

• Standard combination therapy: isoniazid (INH),
  rifampicin (RIF), pyrazinamide (PZA) and ethambutol
  (ETH) x 2 months followed by isoniazid and rifampicin for
  further 4 months
• Second line: injectable agents (streptomycin,
  cycloserine, capreomycin)
• Side effects are wide-ranging and severe, include liver
  damage
  TB treatment- 4-9 months of combination therapy!

Question 10

outline the resistance mechanisms of TB to TB drugs.

Answer 10

Drug inactivation:
– Mtb produces beta-
lactamase
* Drug titration:
– Target overproduction
* Alteration of drug
target:
– Missense mutations
* Altered cell envelope:
– Increased permeability and drug efflux

Question 11

Multi-drug resistant TB

Answer 11

• XDR-TB: resistant to four commonly used TB drugs. 6%
  of all TB cases
• Resultant from inadequate TB therapy and failure to
  clear patients of bacteria
• Treatment is lengthy and expensive

Question 12

Treating XDR-TB (multi-drug resistant)

Answer 12

• BPaL regimen:
  – Bedaquiline
  – Pretomanid
  – Linezolid
• All oral treatments for 6 months
• These can fail too with totally drug
  resistant (TTR) TB. No known solution as
  yet.

Question 13

Bacillus Calmette-Guérin vaccine

Answer 13

• A live attenuated vaccine
• Protects children best
• Does not prevent infection, but allows a
  quick immune response
• Largely replaced by prophylaxis in the
  UK with 3 months of rifampicin and
  isoniazid.

Question 14

how is tb spread.

Answer 14

• droplets in breath
• spit
• mucus

then goes to alveolar macrophages which takes it into the lymphatics to hil;ar lymph nodes

Question 15

latent TB

Answer 15

• Cell mediated
  immune (CMI)
  response from
  T-cells
• Primary
  infection
  contained but
  CMI persists
• Latent TB;
• no clinical disease
• detectable CMI to TB on
  tuberculin skin test

Question 16

pulmonary TB

Answer 16

• Granulomas forms
  around bacilli that have
  settled in apex
• In apex of lung there
  Is more air and less
  blood supply
  (fewer defending
  white cells to fight)
• TB may spread in lung
  causing other lesions
• Could occur immediately following
  primary infection (post-primary)
  or months later after reactivation
• necrosis results in abscess
  forming and caseous material
  coughed up

Question 17

Tb spreads beyond the lungs, name some places it can spread to.

Answer 17

• TB meningitis
• pleural TB
• bone and joint TB
• the lungs
• genito urinary TB
• bacilli in lymph nodes
• bacilli in lung apex

Question 18

how does the immune system react to TB?

Answer 18

• Mycobacteria are phagocytosed by macrophages and traffic to a phagolysosomes.
• The bacterium has adapted to the intracellular environment and aims to withstand
  phagolysosomal killing and escape to the cytosol
• Effective immunity requires CD4 T-cells which generate interferon gamma and this helps activate intracellular killing by macrophages.

Question 19

outline the immune process of granulosoma

Answer 19

Initial Infection and Macrophage Activation:
Mycobacterium tuberculosis infects macrophages in the alveoli
- they survive and replicate within the macrophages
- macrophages release cytokines (e.g., TNF-α, IL-12) that recruit other immune cells
- APC present TB antigens to T-cells, activating CD4+ Th1 cells, which release IFN-γ to activate macrophages.
- macrophages aggregate, forming a core with surrounding lymphocytes and fibroblasts.
- Some macrophages differentiate into epithelioid cells and multinucleated giant cells.

Caseation Necrosis (Optional):
The granuloma’s center may undergo necrosis due to immune-mediated tissue destruction, forming a characteristic caseous (cheese-like) core.
Containment or Reactivation:
Granulomas either contain the infection (latent TB) or break down, releasing bacteria and causing active disease.

Question 20

explain if the formation of a granuloma in tb is advantageous for the bacterium or disadvantageous.

Answer 20

Advantageous for the Bacterium
- Immune Evasion:
The granuloma provides a niche where TB can survive and persist inside macrophages, avoiding complete clearance by the immune system.
- Latent Infection:
By remaining dormant within granulomas, the bacteria evade host immunity and can reactivate later when the immune system is weakened.
- Protection from Environmental Stress:
The granuloma structure shields the bacteria from oxygen, antibiotics, and host enzymes.

Disadvantageous for the Bacterium
- Containment:
A well-formed granuloma can trap the bacteria, preventing their spread and replication, leading to latent rather than active disease.
- Immune Activation:
Granulomas recruit T-cells and produce IFN-γ, which activates macrophages to kill the bacteria.

Net Effect:
The granuloma initially serves as a defense mechanism to contain the infection, but it also creates a niche where M.tuberculosis can persist long-term, contributing to its survival and transmission strategy.

Question 21

what controlls the reactivation of TB once contained in a granuloma?

Answer 21

the stability of the granuloma
- CD4 depletion
- TNFα depletion

Question 22

what are the ways in which scientists study tb?

Answer 22

• Animal models of TB - mice
• Mycobacterium marinum model
• In vivo imaging

Question 23

Describe how mycobacterial growth characteristics influence the use of anti-tuberculous agents

Answer 23

• slow growth rate - drugs must be active for extended period
• dormancy and latent infection - penetrate granulomas
• intracellular growth - penetrate host cells
• complex lipid-rich cell wall