LAB - Antimicrobial Susceptibility Testing Flashcards
Methods for determining susceptibility fall into three general classes:
- diffusion methods: Kirby-Bauer + mods
- dilution techniques: broth + agar
- detection of resistance mechanisms: B-lactamases
In all of the antimicrobial susceptibility test methods, this is required for testing
pure culture
- several colonies are testing in order to ensure that test results are representative of the bacterial population
this is critical when doing antimicrobial susceptibility testing (inoculum)
inoculum size
- particularly for testing susceptibility to penicillin and sulfonamides
constituents of culture media can either ______ or ______ antibiotic activity
enhance or antagonize
examples of culture media antagonists
- thymine/thymidine = end-products of folate metabolism; antagonistic to activity of trimethoprim and sulfonamides
- para-aminobenzoic acid (PABA) = structurally similar to the sulfonamides and will act as an antagonist to their activity
- divalent cations = antagonize cell entry of the aminoglycosides, cell binding of polymyxins or my chelate with tetracyclines
- increased phosphate concentrations = may reduce activity of aminoglycosides
alkaline pH
enhances activity of:
- aminoglycosides
- erythromycin
- other macrolides
- lincomycins
agar concentration and depth for antibiotic susceptibility testing
- affect diffusibility of antibiotics into agar
- must be controlled in diffusion tests
- recommended depth of agar for disc diffusions tests = 4 mm
susceptibility test media widely used in NA
Mueller-Hinton Agar
Mueller-Hinton agar
- low concentrations of PABA, thymine, thymidine
- may be used for testing sulfonamides and trimethoprim along with other antimicrobials
- broth formula = Ca++ and Mg++ to approximate physiological levels
> IF NOT, organisms tested may appear more susceptible to the aminoglycosides - agar = higher levels of cations and phosphates
- does NOT support the growth of fastidious bacteria
temperature for antimicrobial susceptibility testing
- temperature = changes will alter rates of bacterial growth and antibiotic diffusion
- accepted std = 35C
- methicillin resistance staph may not be detectable at higher temps
atmosphere for antimicrobial susceptibility testing
- incubate in room air
- 5-10% CO2 may reduce pH of culture media and thereby alter antibiotic activity
- anaerobic = prevent uptake of aminoglycosides
antimicrobial agents incubation conditions for antimicrobial susceptibility testing
- storage at -20C
- current use = refrigerated
- desiccant used to keep discs and powders dry
disc concentration for antimicrobial susceptibility testing
- total antibiotic per disc
- content is standardized and controlled by the FDA
- performance monitored daily; testing control strains of known susceptibility
diffusion rates depend on…
solubility molecular size polarity fo the antibiotic the growth medium the temperature chosen for incubation
diffusion rates depend on…
solubility molecular size polarity fo the antibiotic the growth medium the temperature chosen for incubation
growth rates depend on…
organism
nature of growth medium
temperature and atmosphere of incubation
Kirby-Bauer Method
- standardized for rapid growth bacteria
- MH agar
- standard inoculum, 10^8 CFU/mL
- inoculum density achieved by growing bacteria in TSB for several hours; adjusting turbidity to 0.5 McFarland standard!!
- adjusted broth - cotton swab - surface of MHA
- std antibiotic conctns in paper discs are applied to surface
- plate incubated within 15 mins (to avoid prediffusion)
- 35C
who establishes ZOI sizes
National Committee for Clinical laboratory standards (NCCLS)
Advantages of Kirby-Bauer
- flexible (antibiotics)
- techinically simple
deficiencies of the Kirby-Bauer Method
- inapplicability to slower-growing bacteria, anaerobes
- non-quantitative interpretation
- inaccuracy in detecting slightly elevated levels of resistance
Kirby-Bauer system requires supplementation with a … for situations where quantitative results are needed
dilution system
thick plate (how does this affect antimicrobial susceptibility testing)
smaller ZOI b/c won’t diffuse too far -> invalid!
thin plate (how does this affect antimicrobial susceptibility testing)
larger ZOI b/c antibiotic will diffuse faster!