MICR2000 - Bacteriology

Conjugation

Conjugative R plasmid

DNA transfer from one cell to another

• Attach two cells via pilus

• transfer one strand of plasmid to recipient cell

• synthesis of complementary strands in both cells

• cells separate

Can carry AMR genes

Transposable elements

DNA sequences that can move positions within or between DNA strands

Two types:

• Insertion sequences (IS)

  • smallest, encode transposase (endonuclease and integrase activities)

    • this cuts it out and puts it in somewhere else

    • IS does not encode any other genes

  • flanked by inverted repeat (IR) that is recognised by transposase

• Transposons

  • contain transposase and IR as well as non transposition related genes - often AMR

  • often contain integrons

    • contain promotor and attachment site for gene cassettes (free floating DNA)

      • cassettes encoding gene followed by integrase specific recombination site - 59 base element recognised by integrase

    • integrase integrates cassettes to attachment site and promoter tests what they do

    • cassettes excised and transferred btwn integrons

Transposition methods

Conservative - cut and paste

• transposase cuts target DNA (staggered nick)

• transposon integrates, DNA synthesis to fill in gaps = direct repeats

Replicative - copy and paste

• TnpA gene transcribed to make transposase

• transposase binds to IR and initiates transposition

  • cuts donor plasmid at ends and makes staggered nicks on target plasmid

• ligation of transposon to target ends

  • 3’ ends replicate through DNA polymerase, replicating the carried genes of the transposon

  • this forms a cointegrate - a single molecule of DNA between the donor and target plasmid (looks like 8)

    • contains 2x copies of transferred DNA

• Resolvase binds to ‘res’ regions on transposon

  • cuts and recombines forming 2x plasmids with the transposon

How to stop spread of AMR

stop inappropriate use of antibiotics to reduce selective pressure

remove ineffective antibiotics from use

monitoring, isolation and treatment programs to prevent establishment and spread of multiple resistant pathogens

Normal flora

microbes that live on and in our body without causing infection under healthy conditions

• balance btwn enough for microbes to survive and not enough to cause infection

• pathogens can be transient members of normal flora

Virulence factors

Bacterial product or strategy contributing to virulence or pathogenicity

• colonisation of host

• evade immune system

• damage host

6 categories

• motility

  • motile bacteria can target host cells in dynamic environments like mucosal environments

  • ability to contact host cells

• adhere to host cells and resist physical removal

  • pili/fimbriae and adhesins

• invade host cells

  • invasins allow penetration

  • inside the cell access to nuritents, hide from immune system, divide and multiply

• resist phagocytosis and complement

  • capsules hard for macrophages to attach and engulf, biofilms

• evade immune defenses

  • phase variation of surface

    • vary surface structures to evade detection

  • capsules can resemble human tissue

  • don’t evade - KILL

    • endotoxin

      • lipid A in gram -ve outer membrane

      • released when bacteria attacked (membrane breached) and can be secreted

    • exotoxin

      • soluble excreted toxins

      • toxin genes spread on plasmids

      • cytotoxins kill or inhibit cells

      • neurotoxins interfere with nerves

      • enterotoxins affect epithelial cells of GI tract

    • requires production of antitoxins

• ability to compete for nutrients

  • compete with host tissue and normal flora for limited nutrients

Staphylococcus aureus virulence factors

g+ve cocci

adhesins - adhere to host cells

secrete exotoxins that kill host cells

secrete enzymes that deteriorate red blood cells and immune system enzymes

neutralise hydrogen peroxide from macrophages - resist phagocytosis

protein a - evade immune system

capsule - resist phagocytosis

coagulase - slow down immune system through blood clot

Helicobacter pylori and virulence factors

Host adapted pathogen that colonises human stomach and duodenum - inhabits mucosal layer )noninvasive) so not cleared by immune response (persistent infection), can be treated by antibiotics

symptomatic or asymptomatic infection - virulent strains have cag a pathogenicity island

Virulence factors:

Urease

• bacteria imports urea from gastric juice (through porin to periplasm, UreI to cytoplasm) 

• urease in cytoplasm catalyses urea → ammonia reaction

• ammonia makes gastric acid more basic allowing H pylori to survive

Flagella

• motility

• lophotrichous arrangement

• move in mucosal lining

Adhesins

• BabA and SabA allow adherence to gastric epithelium

Mucinase

• degrades gastric mucus locally for easier motility

CagA - cagA pathogenicity island confers high virulence

• CagA protein and type IV secretory system transcribed and translated

• injected into host cells via type IV secretory system (syringe) to release pro-inflammatory cytokines

• increases acid which wears away mucous

Testing for H. pylori

Rapid urease test

• pH test for urease catalysing ammonia production

Treatment and prevention of H. pylori infection

Acid lowering drugs

Antibiotics

Group A Streptococcus (Streptococcus pyogenes) diseases, location

Location

• skin and throat

Range of diseases

• sore throat

• localised common infections

  • cellulitis

  • impetigo (skin infection)

• less common invasive infections

  • bacteraemia

  • toxic shock systems

  • necrotising fascilitis

• post streptococcal sequelae - diseases after repeated infection with GAS

  • kidney failure and acute rheumatic fever (heart failure)

  • immune sequelae

    • GAS makes M protein which has anti phagocytic activity

    • similar to heart myosin - autoimmunity against heart myosin causing rheumatic heart disease

How do GAS infections align/misalign with Koch’s old postulates?

1 - Bacteria present in every case of disease and absent in healthy animals (NO FIT)

• GAS present in normal flora

2 - Bacteria must be isolated from host with disease and grown in pure culture (FITS)

• GAS can be cultured

3 - Specific disease must be reproduced when pure culture of bacteria is inoculated into a healthy susceptible host (NO FIT)

• bacteria absent from post streptococcal sequelae so this doesn’t hold

• different strains produce different things

4 - Bacteria must be recoverable from experimentally infected host and found to be same as original (NO FIT)

• different strains of GAS

Koch’s molecular postulates

Identifying the gene or gene product responsible for virulence rather than the pathogen

Postulates

• shows gene present in strains of bacteria that cause disease and not present in avirulent strains

• disrupting the gene reduces virulence and reintroduction restores virulence

• introduction of cloned gene into avirulent strain congers virulence

• gene is expressed (not methylated)

• specific immune response to gene protects against virulence 

Is HtrA involved in GAS virulence?

thought HtrA involved in protecting GAS proteins during thermal stress

did test and found virulence disappeared when DNA added but didn’t return when it was returned to normal.

• polar effect - DNA downstream affected (frame shift)

double crossover recombination

• keeps reading frame the same

• deleted mutant had no effect on virulence

Therefore HtrA doesn’t affect virulence

GAS virulence factors

M protein

• helps resist phagocytosis

• similar to heart myosin - immune sequelae

Fibronectin binding proteins (FBP)

• allows GAS to bind to fibronectin in ECM of tissues and colonise that tissue

• Phase variation

  • swap out FBPs

  • different combinations can contribute to different tissue binding - tissue tropisms

  • redundancy in FBPs allows infection of more than one tissue