MICR2000 MOD 3 BACTERIOLOGY

Conjugation

Transfer of DNA via direct contact between cells

Plasmid in the donor cell is either cut & copied or copied & pasted

Transposable Elements

DNA sequence that can jump from 1 position to another or from 1 DNA molecule to another

Insertion sequences

• have a gene encoding a transposase protein
  - endonuclease: cut and copy function
  - intergrase: copy and past function

• has an inverted repeated sequence at its end that the transposase recognises

Transposons

• contain genes unrelated to transposition that can be mobilised along with the transposable element

• can be inserted into chromosome or plasmid

• often contains multiple antibiotic resistant genes

Transposition mechanisms

Conservative: transposable element is excised from 1 location and reinserted into another → copy number is 1

• transposase cuts target DNA → IS intergrates → DNA pol fills gaps and direct repeats flanking intergrated IS are formed

Replicative: A new copy of a transposon is produced during transposition and inserted at another location → copy number is 2

• transposable element binds to inverted repeats → initiates transposition

• Tn3 is ligated to target ends

• 3’ ends prime replication through Tn3

• cointergrate between transposon and target DNA formed

• resolvase binds to ‘res’ sequence of duplicated transposon and resolves cointergrate by site specific recombination

Mobile Antibiotic Resistant Genes

DNA containing resistant genes move from cell to cell via conjugative plasmids

Conjugative plasmids acquire resistant genes via transposons

Transposons acquire multiple resistant genes via integrons

Integrons

Capture and express genes contained in mobile elements called gene cassettes

• has a promoter → cloning expression vector for gene cassettes

• found within transposons

• features
  - attachment site
  → recognised by intergrase
  → acceptor sites for cassettes
  - intergrase gene encoding a site specific recombinase
  - promoter → drives expression of incorporated sequence
  - cassettes must have an intergrase specific recombination site “59 base element” → can be excised as circles and move from integron to integron

Reduce Antibiotic Resistant Spread

• reduce selective pressures → stop inappropriate use of antibiotics

• remove ineffective antibiotics from use

• monitoring, isolation and treatment programs to prevent resistant pathogens from establishing and spreading

Measure for Microbial Virulence

Can be measured using Lethal Dose 50 which is the dose of an agent that kills 50% of a test group
A lower LD50 is associated with higher virulence or pathogenicity as a smaller amount is needed to kill

Virulence Factors

Virulence factors aid in:

Colonisation

• Adhesins resist physical removal and adhere to host

• Invasins invade host

• Motility allows movement through mucous

Evasion of host immune system

• Biofilms and capsules resist phagocytosis

• Phase and antigenic variation of surface proteins trick host immune system into thinking it is not foreign

Damage to host

• Competes for nutrients (sequests iron using siderophores)

• Endotoxins from lipid A damage host

• Exotoxins inhibit host metabolic function

Staphylococcus aureus
 

• G+ cocci

• MRSA and VRSA

• Adhesins to attach to host and resist removal

• Capsule to resist phagocytosis

• Protein A used as an immunological disguise

• Coagulase secreted to create small blood clots in host to impair immune system

• Secreted enzymes break down DNA, fibrin and hyaluronic acid

• Secretes exotoxins, particularly TSST a toxin

• Produces catalase to neutralise host immune system hydrogen peroxide defence

H. pylori Epidemiology and Modes of Transmission

• Gram negative, spiral shaped and has multiple flagella

• Inhibits mucosal layer of human stomach in close association with gastric epithelial cells

Transmission

• Person to person and usually orally

• Often same strain spreads amongst members of the same family

• Crowding increases prevalence

H. pylori Gastric colonisation and ulcers

Has many virulence factors that aid survival and colonisation with in the gastric environment

• Urease creates a neutral bubble in the acidic gastric environment

• Adhesins bind to the host cells

• Flagella penetrate gastric mucous

• Mucinase degrades the gastric mucus

• CagA induces the host cell to release pro inflammatory cytokines
  - the cagA pathogenicity island encodes the T4 secretion system that acts as a molecular syringe and injects CagA into host cell causing inflammation that disrupts host cell junctions
  - this increases mucousal layer permeability and gastric acid contacting the epithelial cells creates gastric ulcers 

Group A Streptococcus diseases

Localised:

• Cellulitis

• Impetigo

Invasive:

• Bacteraemia

• Toxic shock syndrome

• Necrotising faciitis

Lingering - immune sequelae

• Heart failure

• Kidney failure

Group A Streptococcus virulence factors

M protein

• Believed to be responsible for immune sequelae post strep infection

• It is an anti-phagocytic protein

• Has conserved, variable and hypervariable regions

• M protein and heart myosin are both coil coil alpha helixes, therefore the immune system may mistakenly attack heart myosin, leading to inflammation and thickening

Fibronectin Binding Protein (FBP)

• Host cell fibronectin which are liberally available on cell surface

• Allows colonisation of host cell

• Tissue tropism: different FBP combinations can colonise different tissues

• FBP redundancy may allow for more than 1 tissue to be infected

  

Koch’s molecular postulates

Issues with Koch's postulates

• Some microbes can't be grown in culture

• No appropriate animal models

Koch's molecular postulates - updated for the modern world

• Gene is present in strains that cause disease

• Gene is not present in avirulent strains

• Disrupting a gene decreases virulence and complementation restores virulence

• Introduction of cloned gene into avirulent strain confers virulence

• Gene can be expressed in vivo

• Specific immune response to gene protects

  K