Bacteriology Flashcards
1
Q
Antibiotic Resistance
A
- Conjugation
- Transposition
- Integrons
- Gene cassettes
2
Q
Conjugative R Plasmid
A
- Circular
- Structured
- Stable
3
Q
Transposable Elements
A
- Jump between positions on DNA
- Insertion sequences (IS)
- Transposons
4
Q
Insertion sequences
A
- Smallest and simplest
- 1-3 kb
- Contain transposase protein (endonuclease and integrase activity)
- Inverted repeat sequence at ends that is recognised by transposase
5
Q
Transposons
A
- > 1 genes unrelated to transposition
- Insert into chromosomes or plasmids
- Flanked by insertion sequences
- Often contain MDR genes
6
Q
Conservative transposition mechanism
A
- cut + paste
- transposable element excised from one location
- Reinsert at second location
- Copy number of conservative transposon = 1
7
Q
Conservative transposition example
A
- IS1
- Transposase cuts target DNA (staggered nick)
- IS integrates
- Gaps filled by DNA polymerase and DNA ligase
- Formation of direct repeats (DR)
- DR: replications of target sequence, replicated on each side
- DR flanks IS
8
Q
Replicative transposition mechanism
A
- Copy + paste
- New copy of transposon produced
- Inserted at another location
- One copy remains at original site
9
Q
Replicative transposition mechanism
A
- Tn3
- 4957 bp
- Inverted terminal repeats: 38 bp
- Transposase binds to IR
- Initiates transposition (staggered nick)
- Ligation of Tn3 ends to target ends
- 3’ ends prime replication
- Cointegrate formed between transposon and target DNA
- One big figure 8
- Revolvase binds to ‘res’ sequences of duplicated transposon and resolves cointegrate by site-specific recombination
10
Q
Mobile Antibiotic Resistance Genes
A
- DNA containing antibiotic resistance genes moves from cell to cell via conjugative plasmids
- Conjugative plasmids acquire gene via transposons
11
Q
The Integron
A
- Genetic unit
- Capture and expression of genes in mobile elements called gene cassettes
- Also provides a promoter, acting as natural cloning and expression vector for the genes cassettes (primarily antibiotic resistance genes)
- Usually found within a transposon and serves as the mechanism transposons use to accumulate multiple antibiotic resistance genes
12
Q
Features of Integron
A
- Attachment site (att)
- att recognised by integrase + acts as acceptor site for cassettes
- Gene encoding site-specific recombinase
- Promoter that drives expression of incorporated sequence
- Cassettes encoding a gene followed by integrase-specific recombination site
- These cassettes can be excised as circles and moved from integron to integron
- Multiple gene cassettes can insert at att site
13
Q
Staphylococcus aureus
A
- Gram +ve cocci
- Part of normal skin flora
- “Golden staph” bc yellow
14
Q
MRSA
A
- Resistant to broad range of penicillin-analogues
- Especially common in hospitals
- Antibiotic sensitivity testing first –> optimal therapy options
- Doctors over-prescribe Vancomycin instead –> VRSA
15
Q
Virulence Factors
A
Bacterial product or strategy that contributes to virulence or pathogenicity
Colonisation of host
- Adhesins (pili/fimbriae)
- Iron binding proteins
- Invasins
Evade host immune system
- Surface polysaccharides (capsule), lipopolysaccharide (LPS)
Damage host
- Exotoxins
16
Q
Measuring Virulence
A
- Estimated from experimental studies of LD50
- Highly virulent pathogens show little difference in number of pathogenic cells required to kill 100% of population vs 50% population
17
Q
Virulence Factors
A
- Adhere to host cells and resist physical removal
- Invade host cells
- Contact host cells (motility)
- Resist phagocytosis by macrophages and complement
- Evade immune defences
- Compete for nutrients
18
Q
Adhere to host cells and resist physical removal
A
- Pili of uropathogenic E. coli adheres to urinary epithelium –> UTI
- Pili, adherens
Adhering to “stick around”
19
Q
Invade host cells
A
- Invasins of Shigella species permit entry into epithelial cells of colon
- Invasins
- Invasins are surface proteins that allow penetration of host cells
- Inside cytoplasm: nutrients, protection from complement, antibodies, other defences, multiplication
20
Q
Contact host cells (motility)
A
- Helicobacter pylori
- Motility and flagella
- Mucosal surfaces of bladder and intestines flush bacteria away to prevent colonisation
- Motile bacteria: contact, attach, colonise
21
Q
Resist phagocytosis by macrophages and complement
A
- Streptococcus pneumoniae evade phagocytosis
- Pneumonia, sinusitis, otitis media, meningitis
- Urinary catheter
22
Q
Evade immune defences
A
- Neisseria gonorrhoeae: phase and antigenic variation of surface proteins
- Phase variation of surface structures
○ N. meningitidis: capsule made of sialic acid
○ S. Pyogenes: capsule made of hyaluronic acid - Both resemble carbs found in human tissue
- Therefore not recognised as foreign by immune system
23
Q
Compete for nutrients
A
- Siderophores
- Siderophores
- Bacteria compete with host tissue and normal flora for limited nutrients
- Iron is essential for both bacterial and human cell growth
- Bacteria synthesise iron chelators (siderophores)
24
Q
Endotoxin
A
- Not secreted
- Structural component of OM of Gram -ve bacteria
- Lipid portion of LPS is endotoxin (lipid A)
- Produces same symptoms, regardless of organism
Some organisms (Neisseria meningitidis) secrete vesicles of LPS in large quantities
25
Q
Exotoxin
A
- Actively secreted and soluble
- Some toxin genes carried on plasmids
- Often cause of disease, not the bacteria
- Therefore, killing of bacteria is insufficient; must clear toxin
- Requires host production of antibodies that neutralise toxin- antitoxin
- Immunisation against disease caused by toxins use inactivated toxins- toxoids
Damage host cells by inhibiting specific metabolic function:
- Cytotoxins: kills host cells or inhibit function
- Neurotoxins: interfere with normal nerve impulse transmission
Enterotoxins: affect epithelial cells of GIT
26
Q
Ulcers
A
- “Excess acid hypothesis”: prevailing theory for gastric ulceration
- Thought to be related to stress, age, diet (alcohol, spicy)
- Treated using antacids
27
Q
Antacids
A
- Antacid treatments –> less symptoms
- Stop antacids –> ulcer
- Antacids = treat symptoms, not cause of ulcers
28
Q
Discovery of H. pylori
A
- Barry J. Marshall and Robin Warren
Peptic ulcer disease: hypothesis that bacteria cause ulcers
29
Q
Helicobacter pylori
A
- Gram -ve
- Spiral shaped
- Multiple (4-6) flagella
- Urease (+) Catalase (+) Oxidase (+)
- Culture (pH 7), microaerophilic (O2 2-8%, CO2 10%)
- Inhabits mucosal layer of human stomach (noninvasive)
- Not cleared by hose immune response
30
Q
H. pylori mode of transmission
A
- Person to person
- Prevalence increased in elderly, some ethnic groups, domestic crowding (large families)
- Route of transmission is mainly oral-oral (family often carry same strain)
31
Q
H. pylori Infection
A
- Adheres to gastric epithelium, lives in mucous gel layer
- H. pylori lives in close association with gastric epithelial cells
- pH more neutral in mucus gel layer, and closer to epithelial cells
- Evades host immune defenses (chronic infection)
- Secretes urease to produce ammonia (protection from gastric acid)
- Produce cytokines that disrupt intercellular junctions (CagA): inflammatory response
- Penetrates intracellular junctions
- Increases permeability of mucus layer
- Gastric acid seeps in –> ulcer
32
Q
Urease
A
Urease: structural subunits
Assembly: accessory genes for active urease synthesis
Ure1: acid activated urea transporter (embedded in membrane of bacteria)
- Urea in gastric juice is transported into periplasm through porin in OM
- Urea is transported into cytoplasm through Ure1 in IM
- Urease (cytoplasm) –> convert urea into basic (higher) pH
- NH3 NH4+ acts as buffer in periplasm
33
Q
Rapid Urease Test
A
- pH indicator: phenol red
- Reading at 1-24 hours
34
Q
CagA (delivery into epithelial cells)
A
- cagA pathogenicity island encodes for type IV secretion system
- CagA is injected into epithelial cells by type IV secretion system
- Inflammation in host cell –> immune response –> ulcer
35
Q
Treatment and Prevention
A
- Acid lowering drugs
- Antibiotics (single-antibiotic treatment is ineffective)
- Amoxicillin, tetracycline, metronidazole (not for pregnancy)
- No effective vaccine
36
Q
Group A Streptococci
A
- Tthroat and skin
- Endemic human diseases
37
Q
Group A Streptococci Diseases
A
- Pharyngitis (strep sore throat)
- Localised common infections: cellulitis, impetigo
- Less common invasive infections: bacteraemia, toxic shock syndrome, necrotising fasciitis
- Post streptococcal sequelae (diseases that develop upon repeated infection): acute post-streptococcal glomerulonephritis (kidney failure), acute rheumatic fever (hear failure)
- Immune sequelae (triggered by inappropriate immune response to infectious agent): rheumatic heart disease
○ M protein: antiphagocytic activity
○ Both M protein and heart myosin are coiled-coil alpha helices - Thus, antibodies against heart valve myosin may direct immunity against heart valve material
38
Q
Epidemiological Categorisation of Pathogens
A
- Pulse field gel electrophoresis (PFGE) –> golden standard
- DNA isolated, cut with restriction enzyme
- Run on gel with alternating current to resolve large pieces of DNA
- Banding patterns can be compared to known strains
39
Q
Prevention of Diseases
A
- Antibiotic treatment
- Endemic regions –> prophylactic treatment may be unavailable or “needle avoidance”
- Aim of vaccine research –> one shot vaccines that prevent infection and do not cause post streptococcal sequelae
40
Q
Koch’s Postulates
A
- Microorganism may not be able to be grown in pure culture
- There may be no animal model of infection for that microorganism
41
Q
Koch’s Postulates for GAS
A
- The bacteria must be present in every case of the disease and absent from healthy animals.
- Present in healthy individuals. - The bacteria must be isolated from the host with the disease and grown in pure culture.
- Grow in pure culture + quick tests to differentiate from other Strep - The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host.
- Different strains of GAS cause pharyngitis and necrotizing fasciitis. Bacteria will also be absent in auto-immune sequelae. - The bacteria must be recoverable from the experimentally infected host and shown to be the same as the original.
- Bacteria can be same species, but belong to different strains
42
Q
Koch’s Molecular Postulates
A
- Identify gene (or gene product) responsible for virulence determinant
- Show gene present in strains of bacteria that cause the disease
- Not present in avirulent strains
- Disrupting the gene reduces virulence and complementation restores virulence
- Introduction of cloned gene into avirulent strain confers virulence
- The gene is expressed in vivo
Specific immune response to gene protects
43
Q
Case Study in Complementation: HtrA
A
- HtrA- protease involved in folding and maturation of secreted proteins
- Lyon and Caparon (2004), hypothesized HtrAis involved in protecting GAS proteins during thermal stress, and is therefore involved in virulence
- Did not demonstrate Koch’s postulates: giving back gene didn’t bring back virulence
44
Q
Case Study in Complementation: HtrA
A
CHECK NOTES
45
Q
Fibronectin Binding Proteins and Colonisation
A
- GAS fibronectin binding proteins bind the ECM component fibronectin
- Fibronectin binds to the human cell surface via the a5b1 integrin receptor
- Fibronectin is efficiently bound to the surface of S. pyogenes
- Numerous fibronectin binding proteins (FBP) have been identified on the surface of S. pyogenes
- Gene knock-out studies have concluded these proteins are “essential” for colonisation in some strains, and absent in other strains.
- Different FBP combinations can contribute to different Group A Strep strains infecting specific tissues (tissue tropism)
- Redundancy in FBPs may allow Group A Strep to infect more than one tissue helps to explain the broad spectrum of Group A Strep infections
46
Q
Tuberculosis
A
- Pulmonary (lungs) and extra-pulmonary (others)
- Symptoms: chronic cough, fever, night sweats, fatigue, weight loss
- Global
- Airborne infection
- Slow growers
47
Q
Tuberculosis Characteristics
A
- Rods, variable in size
- Not motile
- Acid-fast
- Fast and slow growers
- Many saprophytes, several obligate and opportunistic pathogens
- Resistant to common disinfectants
- Heat susceptible
48
Q
Mycobacterium tuberculosis
A
- Single circular chromosome
- Complex regulatory potential
- Varied metabolic and respiratory potential:
○ Aerobic, microaerophilic, anaerobic
○ Most anabolic pathways present
○ Specialised pathways allow adaptation to host environment - Abundance of genes involved in lipid metabolism
49
Q
Tuberculosis Infection description
A
- Resides within macrophages (intracellular pathogen)
- Thick waxy wall
- Active interference with phagosome maturation and lysosome fusion
- Genes allow for low pH
- Genes help detoxify reactive radicals
- DNA repair mechanisms
- Mtbin cytosol
- Adaptable carbon metabolism
- Iron binding proteins (siderophores)
- Modulation of cytokine production and antigen presentation
50
Q
Tuberculosis Mechanism
A
- Inhale into lungs
- Macrophage engulf (around alveolar walls)
- Infected macrophage sends out signals
- Try to contain infection –> develop early cellular infiltrate
- Monocytes, macrophages, neutrophils
- Attract T cells
- Granuloma formation
- Central necrosis –> high density of bacteria in centre
- Rupture of bronchiole wall –> access airways
51
Q
Tuberculosis Pathogenesis
A
- Granuloma: localised imflammatory mononuclear cell infiltrate
- 5-10% active primary disease, 90-95% latent infection
Latency: infection without disease but with potential - Post-primary (secondary) TB
5-10% of latent infected
○ Reactivation
○ Extensive cell death and tissue destruction
○ Immunopathology - Liquefaction of granulomas (cavitation)
- Rupture of granulomas, dissemination and transmission of bacteria
52
Q
Tuberculosis Diagnosis
A
- Skin reactivity to mycobacterial antigens (+ =/= active)
- Chest X-ray
- Lab tests
- Ziehl-Neelsen stain (acid fast?), culture, interferon gamma release assay (IGRA), PCR, molecular epidemiology
53
Q
Drug-resistant Tuberculosis
A
- MDR TB –> resistant to isoniazid and Rifampin
- Extensively drug resistant (XDR) TB –> + multiple second-line drugs
54
Q
Tuberculosis Prevention
A
- Albert Calmette and Camille Guerin
- Bacille Calmette Guérin= BCG
- Attenuated strain of M. bovis by serial passage in artificial media over 13 years
- Protected against TB
- Accepted vaccine for TB
- Some protection for small children
- Effectiveness not confirmed in all studies
- No protection of adults
- Not used in low endemic areas
