Practical 1. Microbial Offence and Defense Flashcards
What are bioassays used for ?
Used to estimate the concentration of antibiotics in a solution
Why do we need to be able to estimate the concentration of antibiotics in a solution ?
- During the manufacture of antibiotics
2. In a hospital setting, to measure the concentration of the antibiotic in a patients bloodstream
Where does a zone of growth of inhibition occur ?
Around the well
When does the edge of the zone of growth of inhibition form ?
When the antibiotic is too dilute to inhibit growth of the bacteria
What does a more concentrated antibiotic in the well result in ?
A wider zone of growth of inhibition
What can the standard curve be used for ?
To determine the antibiotic concentration in an unknown sample
What can antibiotics be synthesised by ?
- Fungi
2. Bacteria
What did the discovery of antibiotics for chemotherapy result in ?
The emergence of resistant organisms
How would you determine the sensitivity of an organism to antibiotics ?
A disc diffusion method
What must standardised methods for antibiotic disc diffusion consider the effects of ?
- Culture medium
- Inoculum density
- Antibiotic discs
- Incubation conditions
- Interpretation
What does susceptible mean ?
Implies that an infection caused by the strain may be treated with the antibiotic at the dosage recommended for the type of infection and the infecting species of bacterium, unless otherwise contra-indicated
What is meant by resistant ?
Encompasses strains incompletely inhibited within the usual therapeutic range
What is meant by the term intermediate ?
Strains exhibiting an in between diameter of zone of growth inhibition
How might resistance to antibiotics be acquired ?
- Chromosomal mutations
2. Transfer of plasmid determined resistance
What effect might a chromosomal mutation have on an antibiotic and give the example ?
Effect the functionality for example through alteration of the antibiotics target so that it is no longer susceptible
What is still retained in a chromosomal mutation ?
The functionality of the target molecule is retained
What is Rifampicin ?
An antibiotic
How does Rifampicin work ?
Binds to the enzyme RNA polymerase and blocks the ability of the bacterium to transcribe DNA into mRNA
How do mutations in chromosomal genes and Rifampicin work ?
Mutations in the chromosomal genes coding for RNA polymerase result in an altered enzyme to which rifampicin no longer binds, while maintaining functioning of the enzyme
Do chromosomal mutations occur frequently in bacteria populations ?
They occur spontaneously and at low frequencies
How can rifampicin resistant mutants be distinguished from rifampicin sensitive cells ?
By plating on agar containing rifampicin
Does plating on rifampicin containing media induce rifampicin resistant mutants ?
No, they are already present in the population
How can the frequency of rifampicin resistant mutants be calculated ?
Number of rifampicin resistant mutant colonies per ml/ starting culture viability count
What is another name for disc diffusion method ?
Kirby- Bauer Method
What are the zones of growth inhibition for the test organism compared with in disc diffusion ?
Standard sensitive strain
Why cant zones of growth inhibition obtained with different antibiotics be directly compared ?
Different antimicrobial agents diffuse at different rates
What do the organisation such as EUCAST or CLSI do ?
The establish breakpoints to classify a microorganism as resistant or susceptible
What is the purpose of testing an internal control or reference strain ?
This is to make sure that the technique is correct and the antibiotic disc load also since the profile of this strain is known
What is E. coli strain R- ?
A sensitive strain, lacking a resistance plasmid
What is E. coli strain R+ ?
A resistant strain, containing a resistance plasmid
What is conjugation ?
The transfer of genetic material between bacterial cells by direct cell to cell contact
What does the R plasmid carry ?
The gene for chloramphenicol resistance
What does the gene for chloramphenicol resistance confer ?
Resistance to the antibiotic on bacteria harboring this mobile genetic element
What is the host bacterium sensitive to ?
Nalidixic acid
What does the recipient strain carry ?
A chromosomal mutation conferring nalidixic acid resistance
What is plasma encoded chloramphenicol acetyltransferase responsible for ?
Chloramphenicol resistance in R factor bearing E. coli
How does chloramphenicol acetyltransferase inactivate the antibiotic ?
It catalyses the 3-O-acetylation of chloramphenicol in the presence of acetyl coenzyme A
What is nalidixic acid ?
A 4-quinolone antibacterial drug
How does bacterial DNA exist ?
A supercoiled form
What is the enzyme DNA glyrase responsible for
It is a topoisomerase that is responsible for introducing negative supercoils into the structure
How does the nalidixic acid act ?
By inhibiting the activity of the bacterial gyrase, preventing the normal functioning of DNA
What are quinolones ?
Broad spectrum agents that rapidly kill and are well absorbed after oral administration
What is the minimum inhibitory concentration (MIC) ?
The lowest concentration of a substance that inhibits growth of an organism
What is the agar dilution method convenient for ?
Simultaneously testing several strains or for testing different doses of bacteria
In the clinical microbiology what dilutions are normally used ?
2 fold
What is the end point of the titration affected by ?
The inoculum size
