In Vitro Determination of Susceptibility

Question 1

Empirical treatment

Answer 1

Try one agent after another until response is favorable or use combinations of agents

• undesirable due to risk of toxic or allergic effects
• undesirable alterations of normal flora or resistance can occur
• generally no sensitivity testing is done

Question 2

Rational treatment

Answer 2

Choice of the agent that is most likely to act against the pathogen and as few other cells as possible
- based on in vitro tests, clinical experience or knowledge of the organism

Question 3

Quantitation of susceptibility

Answer 3

Series of decreasing concentrations of the antimicrobial agent (drug) is prepared in a suitable medium (broth or agar) and a suspension of the infecting organism is added to each concentration

Question 4

Minimum inhibitory concentration

Answer 4

The lowest concentration capable of preventing growth

• organism is sensitive or susceptible to this concentration
• true MIC may be less than the measured MIC due to use of decreasing 2-fold concentrations of the drug

Question 5

Minimum bactericidal concentration

Answer 5

Used on cultures containing drug concentrations which show no growth

• subcultured to media without antibiotics
• if MBC is the same as the MIC or within 1-2 dilutions of the MIC, the agent is bactericidal
• if higher concentrations than the MIC are required to kill the organism, the agent is bacteriostatic

Question 6

If no growth occurs ____ of the cells are sensitive

Answer 6

100%

Question 7

It only takes _______ resistant cell to resist inhibition

Answer 7

1

- MIC or MBC determines the concentration needed to inhibit (kill) the most resistant cell in the population

Question 8

Is knowing the MIC/MBC enough to treat an infection?

Answer 8

No, you must also know whether these concentrations are likely to occur in infected tissue

Question 9

Sensitive

Answer 9

The MIC is less than the blood, urine, or tissue concentrations of the drug given to a patient at a specified dose
- for a favorable response, the specimen concentration of a drug given at a dose must be greater than the MIC

Question 10

Resistant

Answer 10

The MIC of the organism is greater than the in vivo concentrations of the drug attainable at specified doses
- concentrations of the antimicrobial drug attainable at specified doses is less than the MIC of the organism

Question 11

Intermediate or moderately susceptible

Answer 11

The MIC and the in vivo concentrations are approximately equal

• borderline between sensitive and resistant
• implies success if the dose is increased, but must consider toxicity and side effects

Question 12

Are S, I, and R categories qualitative or quantitative?

Answer 12

Qualitative

- no absolutes!!

Question 13

Disk agar diffusion

Answer 13

Standardized for rapidly growing aerobic or facultative pathogens

• utilizes Mueller-Hinton agar to determine antimicrobial resistance
• bacteria swabbed onto plate in 2 different directions
• 6 mm paper disks containing specified drug amounts are applied
• incubate in air at 37 C for 24 hrs
• zones of growth inhibition are measured to convert into S, R, or I

Question 14

Is zone size correlated to sensitivity, resistance, or inhibition?

Answer 14

No

Question 15

Mueller-Hinton agar

Answer 15

Low nutrient (protein) and cation content and pH
- blood (5%) could be added for fastidious or slow growing bacteria, but will cause problems with false positive resistance due to protein-binding of drugs or the presence of inhibitory susbstances such as thymidine and PABA

Question 16

Theory of DAD

Answer 16

As the organism grows, it is exposed to a continuous gradient of decreasing concentrations of the drug at increasing distances from the center of the disk

• points close to the disk where the drug concentration exceeds the MIC for the organism, no growth occurs
• when you measure a zone of diameter of no growth, you are indirectly measuring MIC

Question 17

Limitations, problems, and disadvantages

Answer 17

• DAD is currently based on human blood levels, is not always applicable to animals
• false resistance occurs with specific organisms and blood MH agar
• disks are not favorable for all vet med drugs
• beta lactamase produces may not be detected, leading to false sensitive interpretations
• is not useful for anaerobes or slow growing organisms
• no standard tests available for fungi, mycobacteria
• MIC methods are recommended due to above problems

Question 18

Tube sensitivity test for MIC

Answer 18

• series of tubes containing decreasing concentrations of antimicrobic (control tube with fluid only)
• addition of an invisible inoculum of bacteria doubles the volume, incubate overnight
• read MIC, subculture tubes without growth (turbidity) to determine MBC

Question 19

Do topical antibiotics have a peak concentration?

Answer 19

No

Question 20

What concentration do you start at with IV antibiotics?

Answer 20

Peak concentration