L38: Clinical Microbiology And Infectious Diseases

Question 1

Classification of microbes

Answer 1

(largest to smallest)
Worms (parasites) —> Hand lens
Protozoa —> light microscope
Fungi
Bacteria —> prokaryotes
Virus —> Electron microscope
Prion —> molecular techniques

Question 2

Historical landmarks

Answer 2

Antoine van Leeuwenhoek: microscope
Pasteur and Henle: Germ theory 
Joseph Lister: aseptic technique
Robert Koch: Koch’s postulate
Edward Jenner: immunisation
Gram’s stain
Oncogenicity of virus
Bacteriophage: transmit genetic information (transduction)
Frederick Griffith: S and R strain experiment
Penicillin
Conjugation: plasmids —> antibiotic resistance
Double helical structure of DNA
Genetic engineering
Prions discovery
Complete genome sequence of bacteria
Human microbiome project

Question 3

Pathogenesis of infectious disease

Answer 3

1. Cytolysis by replication / toxins —> apoptosis
2. Immunopathological damage by upregulation of inflammation / molecular mimicry, cross-reactive host antigen
3. Oncogenesis (integration of viral genome into host chromosome) / chronic inflammation e.g. H. pylori and gastric cancer, Hepatitis B and hepatocellular carcinoma

Question 4

Diagnosis of infectious disease

Answer 4

Microbial factors

1. Visualisation: Gram stain + Light microscope, Electron microscopy (need large no. of microbes)
2. Culture (Bacteria: Agar; Virus: need living cells to culture e.g. living mice, embryonated eggs)
3. Detection of components:
   - ELISA: enzyme-linked immunosorbent assay —> antigen
   - Immunofluorescence
   - gas liquid chromatography —> volatile fatty acid profile
   - PCR / RT-PCR / probe hybridisation + Gel electrophoresis —> DNA + RNA sequence

Host factors

1. Serology: Antibody response of host towards component (esp in convalescent phase), need paired sera test —> detect for 4 fold IgG increase
2. Cellular immune response (Mantoux test, lymphocyte proliferation)
3. Histopathology (viral inclusion bodies e.g. Negri bodies in rabies)

Question 5

Bacteria structure / virulence factors

Answer 5

• prokaryotes: no nuclear membrane
• single chromosome (1-6 megabase of nucleotide) attached to Mesosome on cell membrane
• 30s + 50s ribosomal subunits
• energy production: oxidative phosphorylation enzyme system on cell membrane
• Gram +ve: thick peptidoglycan (trap crystal violet + iodine) —> deep blue
• Gram -ve: thin peptidoglycan + LPS outer membrane (endotoxin) —> light pink

LPS outer membrane:

• Lipid A + polysaccharide (Core + O-antigen)
• potent stimulus of host cytokine production —> inflammatory —> sepsis and shock

Peptidoglycan layer:

• 1-4 linked N-acetyl glucosamine + N-acetyl muramic acid (transglycosylation)
• cross link via pentapeptide bond on NAM (transpeptidation)

Polysaccharide / polypeptide Capsules:

• antiphagocytic
• adhesive

Plasmids: double stranded circular coiled DNA replicate independently

Flagella: spread

Pili / Fimbriae:

• adhesion
• colonising factors
• receptors for phages —> conjugation —> transfer of genes

Spores:

• highly resistant
• thick cortex of peptidoglycan and protein coat

Question 6

Growth of bacteria

Answer 6

• Binary fission

- 3 phases: lag, log (generation time: time to double the number), stationary

Question 7

Classification of bacteria (atmospheric requirement & morphology)

Answer 7

Atmospheric requirements

1. Strict aerobes
   - oxygen as final electron acceptor
   - P. aeruginosa
2. Strict anaerobes
   - inorganic substance as final electron acceptor
   - low level of detoxification enzyme —> catalase
   - oxygen radical toxic to them
   - foul smelling volatile fatty acids
3. Facultative anaerobes
   - organic substance / oxygen as final electron acceptor
   - S. aureus, S. pyogenes, E. coli, E. faecalis
4. Microaerophilic
   - grow under low oxygen tension / presence of CO2
   - H. pylori, Campylobacter jejuni

Morphology

1. Staphylo: cluster of grapes
2. Strepto: chain
3. Entero: gut
4. Haemo: blood
5. Helico: spiral

Question 8

Bacterial genetics transformation

Answer 8

Genetic changes

1. Bacteriophages:
   - lysogenic cycle: bacteria latently infected with phage, phage DNA incorporated into bacterial DNA and allowed to replicate as bacteria divide (suppressor gene expressed)
   - phage conversion / induction: phage DNA undergo lytic cycle: toxin gene allowed to expressed
2. Plasmids: conjugation through sex pili
3. Point mutation: drug treatment failure in TB infection

Gene regulation

1. Catabolite induction in nutrient utilisation: lactose binding to repressor detaching from operator —> transcription of enzyme gene
2. Alternative promoter function: antigenic switching —> phase transition —> distinct population of bacteria

Question 9

Gram +ve bacteria examples

Answer 9

Staphylococcus
Streptococcus
Enterococcus
Bacillus
Clostridium
Corynebacterium
Listeria

Question 10

Gram -ve bacteria examples

Answer 10

Neisseria
Haemophilus
Pseudomonas
Campylobacter
Helicobacter
Bordetella
Escherchia
Salmonella
Shigella
Proteus