Antimicrobial susceptibility testing

- an overview of definitions and procedures -

Definition and Purpose

• In vitro tests that measure the growth of an isolated microbe in the presence of particular drug or drugs (antibiotics) in order to predict the in vivo success or failure of antibiotic therapy

• The results - guide the choice of antibiotics (+ clinical information and experience)

Bacterial Resistance to Antimicrobial Agents

• Natural: – genetically determined for all members of a bacterial

species

• Acquired:– Present only in certain strains of a naturally sensitive

species

Natural spectrum = list of microbial species naturally sensitive to an antibiotic

Methods for antimicrobial susceptibility testing (1)

• Disk difusion (Kirby Bauer): – swab bacterial suspension on agar plate– place small filter paper disks impregnated with

a standard amount of antibiotic – incubate overnight– zone of inhibition of bacterial growth =

measure of susceptibility (predefined cutoffs):• large zone = susceptible • small / no zone = resistant • zone between the above = intermediate

Disc difusion antimicrobial susceptibility testing (Kirby Bauer)

• Pure culture of the germ to be tested: 4-6 colonies raised from culture plate with sterile bacteriological loop and suspended in sterile saline solution

• 0.5 McFarland units turbidity (adjusted by adding more colonies if required)

Disc difusion antimicrobial susceptibility testing (Kirby Bauer)

• Bacterial suspension evenly

swabed on Müller Hinton agar, blood agar, complex media with swab or L-shaped bacteriological loop

Disc difusion antimicrobial susceptibility testing (Kirby Bauer)

• Place antibiotic disks depending on known natural spectrum of tested microorganism

Disc difusion antimicrobial suscept bility Ʉtesting (Kirby Bauer)

• Antibiotics sets (e.g.):

– Current use: Penicillin G (P), Oxacillin (O), Ampicillin (A), Streptomycin (S), Kanamycin (K), Chloramphenicol (C), Tetracyclin (T), Erythromycin (E)

– Additional set for digestive infections: Bacitracin (B), Furazolidone (Fu), Neomycin (Ne), Polymyxin B (Po)

– Additional set for urinary infections: Nalidixic Acid (Nx), Colistin (Co), ...

Disc difusion antimicrobial susceptibility test – Kirby Bauer -

Various sizes of growth inhibition around different antibiotics

Interpretation: zone size diametercompared to cutoffsfor each antibiotic-microbe pair

Methods for antimicrobial susceptibility testing (2)

• MIC (minimum inhibitory concentration) = minimum concentration of antibiotic that will inhibit the growth of a microorganism

Measurement methods:- Broth dilution - E-test (Epsilometer test)

MIC (continued): Measurement methods

Broth dilution: - Bacteria inoculated in culture broth + antibiotic (various

concentrations) and incubated - MIC = the lowest concentration of antibiotic which

inhibited bacterial growth

MIC (continued): Measurement methods

E-test: - plastic strips impregnated with

decreasing antibiotic concentrations (log scale)

- strips applied on agar microbial culture and incubated

- eliptic inhibition zone intersects the MIC value scale (µg/ml) at the level of MIC

- shape resembles Greek letter ”Epsilon” (ε)

Methods for antimicrobial susceptibility testing (3)

• MBC (minimum bactericidal concentration) = minimum concentration of antibiotic that will kill 99.9-100% of the microbes of a tested bacterial strain

- Less used than MIC; more time consuming- Indicated in:

- severe infections in immunocompromised patients, - infections in anatomic sites hard to reach with antibiotics e.g.

endocarditis, osteomyelitis

Standardization of antimicrobial susceptibility testing (AST)

• Methodology for performing the tests:

– Standard operational procedures (SOPs)

– Interpretation of results (cutoff diameters S / R)

– Recommended antibiotic sets for various pathogens– .....– .....

Standardization of antimicrobial susceptibility testing

http://www.eucast.org/antimicrobial_susceptibility_testing/

Standardization of antimicrobial susceptibility testing

http://www.clsi.org

International standards, including antimicrobial susceptibility testing (AST)