Measurement of antimicrobial activity 1

Question 1

Why do we need to test antibiotic susceptibility?

Answer 1

• to guide the clinician in the choice of appropriate agents for therapy
• to measure the in vitro activity of new antimicrobial agents
• to monitor the occurrence of antibiotic-resistant microorganisms

Question 2

According to what relevant factors are threats associated with resistant infections assessed?

Answer 2

• clinical impact
• economical impact
• barriers to prevention
• incidence
• 10 year projection of incidence
• transmissibility
• availability of effective antibiotics

Question 3

How can we report antibiotic susceptibility?

Answer 3

• Sensitive (S); the standard dose of antibiotic should be enough to treat the patient infected with the strain tested.
• Resistant (R); the strand tested is unlikely to respond to the treatment with that antibiotic
• Intermediate (I); the strains are moderately resistant or moderately susceptible; indicate that the strain may be inhibited by larger doses of the antibiotic or may be inhibited in sites where the antibiotic may achieve higher concentrations e.g. urine

Question 4

What does MIC stand for?

Answer 4

Minimum Inhibitory Concentration: the lowest antibiotic concentration required to prevent microbial population from growing

Question 5

What is the MIC expressed in? (units) and what is it applied to?

Answer 5

• The Minimum Inhibitory Concentration is expressed in mg/l, mcg, ml or %w/v.
• It is applied to antibiotics and other antimicrobial agents (e.g antiseptics, disinfectants and preservatives)
• Measure only on pure chemicals (not the formulated medicine)
• At the infection site, the antibiotic must achieve a concentration greater than the MIC for the infecting organism.

Question 6

What does MBC stand for?

Answer 6

Minimum Bactericidal Concentration: the lowest concentration of the antibiotic that kills 99% of the microbial population.

Question 7

What are the antibiotic susceptibility tests?

Answer 7

• diffusion method
• MIC/ MBC determination
• breakpoint method

Question 8

In clinical laboratories, MIC methods are used to:

Answer 8

• test the susceptibility of organisms that give equivalent results in disk diffusion tests
• test organisms where disc diffusion tests may be unreliable (e.g with slow growing fastidious organisms)
• when a more accurate result is required for clinical management.
• Accepted as the standard against which other methods are assessed and for testing new agents
• MICs may also be derived from the size of the zone of inhibition in diffusion tests (e.g. using a regression curve produced by plotting the diameter of the zone of inhibition against the MIC of a range of strains of a given species)

Question 9

What are a few factors that affect susceptibility testing?

Answer 9

1. composition of the culture media
2. pH of the culture media
3. depth of agar medium
4. size and physiological state of inoculum
5. pre-incubation and pre-diffusion conditions
6. antimicrobial agents and antibiotic discs
7. incubation conditions
8. reading of the results

Question 10

What are the composition of the culture media conditions?

Answer 10

• media should be specially formulated for antibiotic susceptibility testing
• media should not contain antagonist of antimicrobial agents or substances (e.g. albumin can bind to and inactivate antimicrobial agents; gives rise to false results of resistance)

Question 11

What are the pH of the culture media conditions?

Answer 11

• Microbial growth rate is influenced by pH
• Activity of some antibiotics increases with increasing pH (amino-glycosides, macrolides etc)
• Activity of tetracycline increases with decreasing pH

Question 12

What are the depth of agar medium conditions?

Answer 12

• size of inhibition zone varies with depth of agar medium (agar in plate should be a consistent depth; 4mm)

Question 13

What are the size and physiological state of the inoculum conditions?

Answer 13

• Increasing inoculum size reduces susceptibility to antibiotics (in both diffusion and dilution tests)
• for agar diffusion methods: use size that gives semi-confluent growth

Question 14

Pre diffusion and pre incubation can decrease and increase the size of inhibition inhibition zones, respectively. How can this avoided?

Answer 14

Apply antibiotic discs soon after plates are inoculated and incubate plates soon after discs are applied

Question 15

Concept of agar diffusion methods:
The methods of control of variation and the basis of interpretation of the results fallen into two groups.
Which is more reliable?

Answer 15

Comparative methods - (Inhibition zone size of test strain and control strain are compared and set up at the same time. Lack of standardisation = less reliable)
Standardised methods - (technical details of the tests are normalised in the report of international collaborative studies)