Lab7 Flashcards
How does antibiotic susceptibility vary?
- According to species
2. According to genetic components like resistance (R) plasmids, transposons
Why is it necessary to test bacteria resistance to antibiotics?
So the patient can be treated with an antibiotic which the infecting organism is sensitive to for the therapy to be effective.
What are the four basic methods used to test bacteria for antibiotic resistance
- Disc diffusion (Kirby Bauer) test
- E test
- Dilution method
- Enzyme tests
How is a disc diffusion test conducted?
- Either Mueller Hintin or Isosensitest agar plate is seeded with bacteria via LAWN streak
- Paper disc impregnated with antibiotic is applied to the surface
- during incubation the antibiotic will diffuse through the agar forming a concentration gradient
- A zone of inhibition will form, which reflects the:
1) susceptibility of bacteria,
2) The ability of the antibiotic to diffuse
3) the concentration of antibiotic in the disc - Tables are used to assess whether diameter of zone of inhibition indicates susceptibility or resistance to that particular antibiotic.
How is an E test conducted?
- Same principal as disc diffusion EXCEPT
- The strip that is used is impregnated with a concentration gradient of antibiotic.
- E test measures the minimum inhibitory concentration of the antibiotic
- MIC is shown by the point where the zone of inhibition bisects the graduated strip
What kind of test is the E test?
A quantitative test
How is the Dilution Method conducted?
- Doubling dilutions (e.g. 0.5, 1, 2, 4, 8, 16) of the antibiotic are made in tubes of Mueller Hintin broth
- Each dilution is inoculated with a STANDARD QUANTITY of bacteria
- After incubation, tubes are examined by growth which is indicated by TURBIDITY
- MIC = HIGHEST dilution showing NO GROWTH
- MBC is determined by inoculating agar plates with tubes showing no growth. MBC is the HIGHEST dilution factor with NO GROWTH on the plate
- Test shouled always include a known, sensitive control.
What are the clinical importance of MIC and MBC?
- MIC = minimum inhibitory concentration, the minimal concentration of antibiotic required to inhibit the growth of bacteria,
- does not kill bacteria, only inhibits their growth
-MBC = minimum bactericidal concentration, the minimal concentration of antibiotic required to kill the bacteria.
- In practice, MIC and MBC are often the same
- With some antibiotics, the bacteria are inhibited from growing at a certain concentration but only killed at a higher concentration
How are enzyme tests conducted?
- beta lactamases destroy the beta lactam ring of the penicillin molecule
- Filter paper diss impregnated with a chromogenic substrate are commonly used to detect beta lactamase enzymes
- Paper discs will change colour in the presence of beta lactamase.
How may combinations of antibiotics act together?
- in unpredictable ways:
The effect can either be synergistic, antagonist ic or indifference
What is synergism?
The combined effect of antibiotics is greater than expected and thus FAVOURABLE
What is antagonism?
The overall effectiveness of the antibiotics is reduced
What is indifference?
Each antibiotic may merely ignore the other
What is flucloxacillin?
beta lactamase resistant penicillin derivative
What is flucloxacillin?
beta lactamase resistant penicillin derivative
Why is it necessary to test bacteria resistance to antibiotics?
So the patient can be treated with an antibiotic which the infecting organism is sensitive to for the therapy to be effective.
What are the four basic methods used to test bacteria for antibiotic resistance
- Disc diffusion (Kirby Bauer) test
- E test
- Dilution method
- Enzyme tests
How is a disc diffusion test conducted?
- Either Mueller Hintin or Isosensitest agar plate is seeded with bacteria via LAWN streak
- Paper disc impregnated with antibiotic is applied to the surface
- during incubation the antibiotic will diffuse through the agar forming a concentration gradient
- A zone of inhibition will form, which reflects the:
1) susceptibility of bacteria,
2) The ability of the antibiotic to diffuse
3) the concentration of antibiotic in the disc - Tables are used to assess whether diameter of zone of inhibition indicates susceptibility or resistance to that particular antibiotic.
How is an E test conducted?
- Same principal as disc diffusion EXCEPT
- The strip that is used is impregnated with a concentration gradient of antibiotic.
- E test measures the minimum inhibitory concentration of the antibiotic
- MIC is shown by the point where the zone of inhibition bisects the graduated strip
What kind of test is the E test?
A quantitative test
How is the Dilution Method conducted?
- Doubling dilutions (e.g. 0.5, 1, 2, 4, 8, 16) of the antibiotic are made in tubes of Mueller Hintin broth
- Each dilution is inoculated with a STANDARD QUANTITY of bacteria
- After incubation, tubes are examined by growth which is indicated by TURBIDITY
- MIC = HIGHEST dilution showing NO GROWTH
- MBC is determined by inoculating agar plates with tubes showing no growth. MBC is the HIGHEST dilution factor with NO GROWTH on the plate
- Test shouled always include a known, sensitive control.
What are the clinical importance of MIC and MBC?
- MIC = minimum inhibitory concentration, the minimal concentration of antibiotic required to inhibit the growth of bacteria,
- does not kill bacteria, only inhibits their growth
-MBC = minimum bactericidal concentration, the minimal concentration of antibiotic required to kill the bacteria.
- In practice, MIC and MBC are often the same
- With some antibiotics, the bacteria are inhibited from growing at a certain concentration but only killed at a higher concentration
How are enzyme tests conducted?
- beta lactamases destroy the beta lactam ring of the penicillin molecule
- Filter paper diss impregnated with a chromogenic substrate are commonly used to detect beta lactamase enzymes
- Paper discs will change colour in the presence of beta lactamase.
How may combinations of antibiotics act together?
- in unpredictable ways:
The effect can either be synergistic, antagonist ic or indifference
What is synergism?
The combined effect of antibiotics is greater than expected and thus FAVOURABLE
What is antagonism?
The overall effectiveness of the antibiotics is reduced
What is indifference?
Each antibiotic may merely ignore the other
Why may antibiotic concentrations in blood, serum, urine or other body fluids be assayed?
1) To monitor the levels of antibiotics as antibiotics are toxic, they may accumulate in the serum if excretion via kidneys is impaired. e.g. gentamicin.
2) If there is doubt that the treatment is achieveing adequate levels. Partiularly in CSF or bone. For osteomyelitis, it may be necessary to monitor serum levels of flucloxallin if given orally.
What is flucloxacillin?
beta lactamase resistant penicillin derivative
What is the SBT?
Serum bactericidal titre
Dilutions of patient’s serum is mixed with the infecting organism to establish that the concentration of the antibiotic in the serum is greater than that required for bactericidal activity
How are microbact strips used?
To identify gram negative rod isolated from a patient.
Wells contain dehydrated culture media containing different substrates The 3 types of test in the strip are: 1) tests for enzmes 2) Tests for endproducts 3) Tests for sbstrate utilisation
Strips are analysed by comparing to a reference sheet.
There are 12 tests in total ( + oxidase )
If positive for a test the reaction index number is added.
This gives a four digit index number which can be looked up to determine the organism
What are bacteriophages?
Viruses which attack bacteria
What happens if the bacteriophage is lytic?
It will multiply in the host bacterial cell and eventually burst to release mature virus praticles
Are these bacteria-phage interactions specific?
Yes
What does phage typing depend on?
the variation in sensitivity of bacteria to phages which is shown by different bacterial strains of the same species
How is phage typing conducted?
- A lawn plate for each bacterial isolate (e.g. S. aureus) was prepared and left to dry for 5min
- A grid of 9 squares is marked on the back of each plate
- The square are labelled 1-8 with the last square C = control
- Each square (apart from C) is spot inoculated with a drop of the corresponding phage preparation. This is then left for 10 min to dry
- Plates are incubated inverted overnight
What is a positive result (of phage typing) indicated by?
a zone of no growth.
How is the phage type recorded?
If phages in squares 3, 4 and 8 have clear plaques, the phage type is 3/4/8
(Referring to clinical problem 1 page 50 of lab book) What conclusions can you draw from the results of this experiment?
Wards 1 and 6 have the same strain because they have the same phage type
Referring to clinical problem 3 in page 52 of lab manual:
Which antibiotic would you recommend as a prophylactic treatment against all three bacteria?
CIP as it is the lowest dose required to kill the bacteria
Referring to clinical problem 3 in page 52 of lab manual:
Which antibiotics would you not recommend?
Penicillin and CN as they require high doses.
CN causes kidney problems
However sometimes bacteria are sensitive to CN only so we give CN + a MIC
