Antimicrobial Susceptibility Testing (AST)

Reasons and Indications for Antimicrobial Susceptibility Testing (AST) Offer guidance to physician in selecting effective antibacterial therapy for a pathogen Performed on bacteria isolated from clinical specimens if the bacteria’s susceptibility to particular antimicrobial agents is uncertain Susceptibilities NOT performed on bacteria that are predictably susceptible to antimicrobials Ex. Group A Strep

Why Need continues for testing for Antibiotic Sensitivity Bacteria have the ability to develop resistance following repeated or subclinical (insufficient) doses, so more advanced antibiotics and synthetic antimicrobials are continually required to overcome them.

Selection of Test Batteries Generally, labs choose 10-15 antibiotics to test susceptibility for Gram Positive organisms and another 10-15 for Gram Negative organisms Too many choices can confuse physicians and be too expensive Primary objective Use the least toxic, most cost-effective, and most clinically appropriate agents Refrain from more costly, broader-spectrum agents

Antibiotic sensitivity test: the in vitro testing of bacterial cultures with antibiotics to determine susceptibility of bacteria to antibiotic therapy.

Important terms Minimum inhibitory concentration(MIC) Lowest concentration of an antimicrobial agent that visibly inhibits the growth of the organism. Minimum bactericidal concentration (MBC) Lowest concentration of the antimicrobial agent that require to kill the organism.

Susceptible ”S” Interpretive category that indicates an organism is inhibited by the recommended dose, at the infection site, of an antimicrobial agent Intermediate “I” Interpretive category that represents an organism that may require a higher dose of antibiotic for a longer period of time to be inhibited Resistant “R” Interpretive category that indicates an organism is not inhibited by the recommended dose, at the infection site, of an antimicrobial agent.

Methods of Performing AST Disk diffusion method Kirby- Bauer Gradient diffusion method (E-Test)

Disk Diffusion/ Kirby- Bauer Procedure Use a well-isolated, 18-24 hour old organism Transfer organism to a broth Either tryptic soy/sterile saline Ensure a turbidity of 0.5 McFarland Inoculate MH agar by swabbing in three different directions “Lawn of growth” Place filter paper disks impregnated with antimicrobial agents on the agar Invert and incubate for 16-18 hours at35 oC in non-CO2 McFarland standards. No. 0.5, 1 and 2.

Disk Diffusion/ Kirby-Bauer (cont’d) During incubation, drug diffuses into agar Depending on the organism and drug, areas of no growth form a zone of inhibition Zones are measured to determine whether the organism is susceptible, intermediate, or resistant to the drug

E- test/ Gradient Diffusion Method “MIC on a stick” Plastic strips impregnated with antimicrobial on one side MIC scale on the other side Read MIC where zone of inhibition intersects E strip scale

Common interpretation problems Results depends on the technique used Many factors influence results Lack of standardization of the inoculums Thickness and quality of the culture media Quality and conservation of the disks Quality control with standardized strains Condition and duration of incubation

Common interpretation problems An agar gel that is too thick leads to smaller zones

Common interpretation problems Problem with the size of the inoculums Solution: Use McFarland 0.5 photometer Scale -> same tubes

Common interpretation problems Contamination with another organism

Common interpretation problems Bad manipulation Inoculation of the Muller Hinton Swabbing Not by flooding

Common interpretation problems Problems with E-test reading

Kirby-Bauer disc testing Antibiotic-impregnated discs placed on an agar plate at the interface between test organism and susceptible control organism Resulting zones of inhibition compared, use of controls Susceptibility is inferred (standard tables)

Table of zone diameter breakpoint for Staphylococcus aureus Q: Zone of inhibition of Staphylococcus aureus was 20 mm when bacteria treated with Methicillin. Comment Comment: S.aureus is Sensitive to Penicillin

E-test Plastic strips with a predefined gradient of One antibiotic One antifungal Only one manufacturer One strip per antibiotic Wide range of antibiotics Easy to use Storage at -20°C Short shelf life, expensive

Reading E-tests Ciprofloxacin for Yersinia pestis Resistant > 4 ug/ml Intermediate 1-4 ug/ml Susceptible < 1 Upper reading

Dilution in liquid broth Tubes containing increasing antibiotic concentrations Incubation during 18 hr at 37°C Tedious MIC Bacterial growth Inhibition 0 (Control) 0,25 0,50 1 2 4 8 mg/l

Turbid .25 .5 1 2 4 8 Clean Bacterial growth Inhibition The MIC is 2 mg/l ( The first visible clean tube with lowest concentration of the Antibiotic) The MBC is 4 mg/l ( The lowest concentration of the Antibiotic without growth of bacteria on agar media)