practical 9- gene transfer & antibiotic resistance in E. coli

why is bacteria an increasing problem in hospitals

proportion of them thats resistant to antibiotics in increasing

how to most bacteria gain resistance to antibotics

receiving genes from other bacteria

what is vertical gene transfer

• bacteria evolve by mutations over time

• mutations results in useful traits (resistance)

• new genes are passed onto daughter cells & are fixed in the population

what is horizontal transfer

• bacteria receive genes from other bacteria/plants/animals

• neighbouring organisms in the environment

3 methods of horizontal transfer

• transformation: bacteria takes up endogenous DNA from the environemnt & add it to their own genes

• transduction: foreign DNA is transferred from donor cell to recipient cell via viruses (bacteriaphages)

• conjucation: regulated movement of DNA from one bacteria cell to another via direct contact (involves conjugative plasmids)

function of conjugative plasmids

direct bacteria to form pili & conjugative bridge for DNA

what does hortizontal transfer allow gene transfer between

• bacteria to yeast cells

• bacteria to plant cells

• bacteria to animal cells (including humans)

why are some bacteria resistant to antibiotics

• innate resistance

• lacks structures targeted by antibiotics

  eg: microplasmas don’t have cell walls

what is the organism called that contains chromosomal DNA from donor & DNA from the recipiant

transconjugant

function of conjugative pili

• attaches to specific receptors on the recipient cell

• pili retract so the donor and recipient cells can be in close contact

• allows a conjugative bridge to form

what does the type of conjugative pili on donor bacteria determine

frequency of plasmid transfer

pili encoded by F plasmid

• long & flexible

• forms durable contacts/high frequency transfer with recipient bacteria in both liquid environments and on solid surfaces

pili encodede by R plasmid

• short & brittle

• forms durable contacts/high frequency transfer with recipient bacteria when cells are immobilised on solid surfaces

4 steps to to detect a transconjugant

• antibiotic resistance selection

  1. known resistance of donor bacteria

  2. known resistance of recipient bacteria

  3. use different antibiotic plates using the known antibiotics + a combination of them

  4. if recipient bacteria has taken up donor plasmid it will grow on the combination plate

how to work out conjucation frequencies

• need to do serial dilutions to count bacteria as single colonies

• mark out areas on each antibiotic plate of each serial dilution sample (with transconjugants mixed in)

• divide transconjugant CFU/ml (concentration) by recipient CFU/ml (concentration)

what are selective plates

plates containing known antibiotics so that type of bacteria (donor/recipient/transconjugant) can be detected

steps for performing antibiotic resistance profiling of an isolate using a plate

1. spread a thin layer of bacteria on an agar plate

2. add a Kirby-Bauer disc to the middle of plate

3. measure zones of inhibition to each antibiotic/concentration of antibiotic

relationship between zone of inhibition diameter & antibiotic effectiveness

larger zone of inhibition = antibiotic more effective (bacteria less resistant)

how do Kirby-Bauer discs work to show antibiotic resistance

• antibiotics diffuse through agar

• concentration gradient is established

• inhibits or kills senesitive bacteria (bactericidal/bacteriostatic)

bactericidal effect

antibiotics kills bacteria

bacteriostatic effect

antibiotics inhibit bacteria but doesn’t kill them

gram-positive bacteria

• thicker peptidoglycan layer in cell wall

• no outer membrane

• more sensitive to antibiotics

gram-negative bacteria

• thinner peptidoglycan layer in cell wall

• has an outer membrane

• more resistant to antibiotics

what is competence

• bacteria’s ability to take up extracellular DNA from the environment & incorporate it into its own genome

• 2 types: natural & artificial (competent via chemical treatments)

is E.coli naturally competent

no

how to calculate a 10 fold dilution

1. calculate final total volume after dilution (sample volume + dilutent volume)

2. final total volume should be 10 times more than sample/original volume)

when analysing data what is the reliable range of colonies counted

5-400

how to calculate CFU/ml (measure of the number of viable bacterial cells in a sample)

number of reliable colonies counted on the plate x dilution factor of the plate x (total volume of sample/volume of sample used)