antimicrobial susceptibility testing (AST)

What is the purpose of antimicrobial susceptibility testing?

to help predict if an antimicrobial agent can eradicate a pathogen from a site

What is the aim of AST?

To lower the number of pathogens so that the immune system may overcome the infection

How do we determine if a specimen should be tested for susceptibility?

The body site

Normal flora present

Quality of the specimen

Host immune status

Which susceptibility tests are qualitative?

Disk diffusions

Which susceptibility tests are quantitative?

Dilution tests

What is the minimal inhibitory concentration (MIC)?

The minimum concentration of an antimicrobial that inhibits growth

How do we choose which antimicrobials to test?

Through consultation with drug prescribers, microbiologists, infectious disease, and therapeutics committees

Describe the general characteristics of a disk diffusion test.

MH agar is used

a lawn is formed

• inoculum conc. is 1.5 × 10⁸ organisms/mL

Incubate at 35 C for 16-18 hr

measure the zones of inhibition against CLSI guidelines for S, I or R

How do you measure the zones of inhibition on a plate?

Edge to edge across the center of the disk

What is a broth macrodilution? What is the final inoculum conc. and what is the observed result?

a quantitative dilution done in tubes in 2-fold dilutions

• final inoculum is 5 × 10⁵ CFU/mL

• the observed result is the first tube with no turbidity

What is a broth microdilution? What is the final inoculum conc. and what is the observed result?

a quantitative dilution done in multi well trays

• final inoculum is 5 × 10⁵ CFU/mL

• the observed result is the first well with no turbidity or pellet

What should be considered in a microdilution result?

• if wells were skipped (contamination)

• trailing, or heavy growth followed by sudden inhibition

• performance of a beta-lactamase test for penicillin MIC

What is the microdilution breakpoint?

the concentration of an antimicrobial agent that can be achieved in a body, or in vivo

What is a microdilution breakpoint panel?

A panel used to qualitatively report susceptibility based on the breakpoint and plus/minus one dilution

What is an agar dilution test?

antibiotics incorporated into agar and streaked for bacterial growth

What is the E test?

A test strip incorporated with antibiotics that is placed on a plate of bacteria

• the MIC is where the growth ellipse intersects with the strip

What is the mechanism of action behind MRSA?

resistance to oxacillin using the mecA gene

What is the mechanism of action behind VRSA?

acquired vanA resistance via the VRE plasmid

Why is the combination of aminoglycosides and ampicillin not synergistic against Enterococcus?

Enterococcus typically has a resistance to aminoglycosides (mRNA translation)

What are variables that must be controlled for susceptibility testing?

standardized inoculum, utilizing CLSI guidelines, quality control using ATCC specimens

What are the qualitative interpretations for susceptibility?

S = susceptible

• the organism should respond to this antibiotic

• conc. of the antibiotic at the infection site should exceed MIC

I = intermediate

• the organism may not respond to a dose that can be achieved in vivo

• questionable clinical response

R = resistant

• the organism will not be inhibited based on testing

• the conc. of the antibiotic at the infection site will be lower than the MIC

What is the D test? What does it test for?

The D test is to differentiate S. aureus genes that can efflux out certain antibiotics

Tested with erythromycin and clindamycin

• if a “D” shape shows up around the clindamycin antibiotic, the bacteria can induce resistance in vivo