Antibiotic Susceptibility Testing and

Data Interpretation

Dr Shabbir Simjee

Microbiologist

Co-Chair CLSI VAST

Basingstoke

England

Bangkok, 7-8 October 2014

“For clarity, these are solely my personal views/opinions and do not necessarily represent those of any organization to which I am affiliated.”

House keeping

If I’m going too fast ……….. tell me to slow down

If you need me to explain it again ………….. just ask

If you have questions ……… don’t wait till the end

Agenda

1. Antibiotic Susceptibility Testing Methodology

2. Quality Control and Interpretive Criteria - Does it Matter?

3. Q&A

Agenda

1. Antibiotic Susceptibility Testing Methodology

2. Quality Control and Interpretive Criteria - Does it Matter?

3. Q&A

What is AST Data Used For?

• Reporting S I R for the intention of treatment

• Will my antibiotic work or not?

• Looking for resistance trends over time

• AST data is used as part of the Risk Assessment process

• Continued AST is needed to ensure implemented Risk Management processes are

adequate

AST Methods

British Society of Antimicrobial Chemotherapy (BSAC)

- UK Only

European Committee on Antimicrobial Susceptibility Testing

(EuCAST)

- Human Only

Clinical and Laboratory Standards Institute (CLSI)

- Human (AST Sub-Committee)

- Fungal (AFST Sub-Committee)

- Veterinary (VAST Sub-Committee)

CLSI Approved AST Methods

1. Agar Dilution

2. Broth micro/macro dilution

3. Disk diffusion (Not going to cover today)

What Are You Measuring?

Minimal Inhibitory Concentration (MIC)

The lowest concentration of an antimicrobial

agent that prevents visible growth (to the naked

eye) of a microorganism in an agar or broth

dilution susceptibility test.

The basic for each method

Isolate bacterium

in pure culture Inoculate

broth

Standardize

turbidity

0.5 McFarland

Preparing a standard Turbidity of

0.5 McFarland

You can make the ‘Wickerham card’ in any way you like – with a pattern, or text, for

instance.

Remember that the number of the McFarland standard unit does not correspond to the

OD600 value. A McFarland standard of 0.5 should have an OD600 between about 0.08

and 0.1 using a spectrophotometer.

0.5 McFarland Water Bacteria

OD600 0.05

0.5 McFarland Water Bacteria

OD600 0.05

Preparing Antibiotic Stock Solutions

Either of the following formulas below may be used to determine

the amount of powder of diluent needed for a standard solution:

Weight (mg) = Volume (mL) x Concentration (g/mL)

Potency (g/mg)

or

Volume (mL) = Weight (mg) x Potency (g/mg)

Concentration (g/mL)

Example:

To prepare a stock solution containing 1280 g/mL of antimicrobial agent

with antimicrobial powder that has a potency of 750g/mg then 170 to 200

mg of the antimicrobial powder should be accurately weighed.

If the actual weight is 182.6 mg, the volume of solvent needed is then as

follows:

182.6 mg x 750 g/mg

Volume (mL) = (Actual Weight) x (Potency) = 107.0 mL

1280 g/mL

(Desired Concentration)

Therefore, dissolve the 182.6 mg of antimicrobial powder in 107.0 mL of

appropriate solvent.

Agar Dilution Susceptibility Test

An in vitro antimicrobial susceptibility test method conducted

using serial concentration of an antimicrobial agent incorporated

into an agar growth medium in separate Petri dishes that are

inoculated with a bacterial suspension to determine the minimal

inhibitory concentration.

The method described is as specified by CLSI. Agar is poured to

give an approximate depth of 4mm. This corresponds to

approximately 60-70ml of medium for a 150mm plate and

approximately 25-30ml for a 100mm plate

Solution Method of Preparation Antibiotic Concentration (µg/ml)

A 2.0 ml stock solution + 6.0 ml SDW 1280

B 3.0 ml Solution A + 3.0 ml SDW 640

C 3.0 ml Solution B + 3.0 ml SDW 320

D 3.0 ml Solution C + 3.0 ml SDW 160

E 3.0 ml Solution D + 3.0 ml SDW 80

F 3.0 ml Solution E + 3.0 ml SDW 40

G 3.0 ml Solution F + 3.0 ml SDW 20

H 3.0 ml Solution G + 3.0 ml SDW 10

I 3.0 ml Solution H + 3.0 ml SDW 5.0

J 3.0 ml Solution I + 3.0 ml SDW 2.5

K 3.0 ml Solution J + 3.0 ml SDW 1.25

L 3.0 ml Solution K + 3.0 ml SDW 0.62

Assuming a stock solution of 5120µg/ml

128 µg/ml 8 µg/ml 16 µg/ml 32 µg/ml 64 µg/ml

2ml soln. A

(1280µg/ml)

+

2ml soln. B

(640µg/ml)

+

2ml soln. C

(320µg/ml)

+

2ml soln. D

(160µg/ml)

+

2ml soln. E

(80µg/ml)

+

18ml agar 18ml agar 18ml agar 18ml agar 18ml agar

Check the pH of each batch of agar when the medium is prepared. The exact method used will depend largely on the type of equipment available in the laboratory. The agar medium should have a pH between 7.2 and 7.4 at room temperature after gelling. If the pH is less than 7.2, certain drugs will appear to lose potency (e.g., aminoglycosides and quinolones), while other agents may appear to have excessive activity (e.g., tetracyclines). If the pH is greater than 7.4, the opposite effects can be expected. Check the pH by one of the following means: • Macerate enough agar to submerge the tip of a pH electrode. • Allow a small amount of agar to solidify around the tip of a pH electrode in a beaker or cup. • Use a surface electrode.

pH Check

Moisture Check

• If, just before use, excess surface moisture is present on the plates, place them in an incubator (35 °C) or a laminar flow hood at room temperature with lids ajar until excess surface moisture is lost by evaporation (usually ten to 30 minutes).

• The surface of the plate should be moist, but no droplets of moisture should be apparent on the surface of the medium or on the petri dish covers when the plates are inoculated.

Incubate and record MIC

Inoculate a control agar plate (no antimicro­bial agent) first

Then, starting with the lowest concentration, inoculate the plates

con­taining the different anti­micro­bial concentra­tions

Inocu­late a second control agar plate last to ensure there was no

contam­ination or antimi­crobial agent carryover during the

inocula­tion

Let the inoculated agar plates remain at room tem­perature until the

moisture in the inoculum spots is absorbed into the agar; that is,

until the spots are dry. Invert the plates and incu­bate them at 35°C

for 16 to 20 hours.

2 µg/ml 4 µg/ml 8 µg/ml

An in vitro antimicrobial susceptibility test conducted using

serial concentrations of an antimicrobial agent incorporated in

liquid nutrient media that are inoculated with a bacterial

suspension to determine the minimal inhibitory concentration of

an antimicrobial agent.

NOTE: When this procedure is carried out in test tubes, it is

referred to as broth macrodilution; when performed in

microdilution plates, it is called broth microdilution.

Broth Dilution Susceptibility Test

Solution Method of Preparation Antibiotic Concentration (µg/ml)

A 2.0 ml stock solution + 6.0 ml SDW 1280

B 3.0 ml Solution A + 3.0 ml SDW 640

C 3.0 ml Solution B + 3.0 ml SDW 320

D 3.0 ml Solution C + 3.0 ml SDW 160

E 3.0 ml Solution D + 3.0 ml SDW 80

F 3.0 ml Solution E + 3.0 ml SDW 40

G 3.0 ml Solution F + 3.0 ml SDW 20

H 3.0 ml Solution G + 3.0 ml SDW 10

I 3.0 ml Solution H + 3.0 ml SDW 5.0

J 3.0 ml Solution I + 3.0 ml SDW 2.5

K 3.0 ml Solution J + 3.0 ml SDW 1.25

L 3.0 ml Solution K + 3.0 ml SDW 0.62

Assuming a stock solution of 5120µg/ml

0.1ml soln. A

(1280µg/ml)

0.1ml soln. B

(640µg/ml)

0.1ml soln. C

(320µg/ml)

0.1ml soln. D

(160µg/ml)

0.1ml soln. E

(80µg/ml)

0.1ml soln. F

(40µg/ml)

0.1ml soln. G

(20µg/ml)

0.1ml soln. H

(10µg/ml)

0.4ml broth

+ + + + + + + + 0.4ml broth 0.4ml broth 0.4ml broth 0.4ml broth 0.4ml broth 0.4ml broth 0.4ml broth

128µg/ml 64µg/ml 32µg/ml 16µg/ml 8µg/ml 4µg/ml 2µg/ml 1µg/ml

Commercial: - Frozen - Freeze dried

Broth Dilution

autoinoculator

Broth Dilution

MIC Drug Concentration

A

0.25 0.5 1 2 4 8 16 32 64 0.03 0.06 0.12

B

C

D

E

F

G

H

2

Repeat (?)

Repeat

.

Controls

8

≤0.03

>64

Repeat

0.06

Broth Dilution

Agenda

1. Antibiotic Susceptibility Testing Methodology

2. Quality Control and Interpretive Criteria - Does it Matter?

3. Q&A

Why use QC strains?

• QC = Quality Control strains, these can be considered ‘positive controls’

• QC are bacterial isolates that have undergone rigorous testing to ensure that under a standard test system they will always give the same MIC range with a given antibiotic

• If a QC is out of range it invalidates the AST and indicates there are problems in the method e.g. pH, ion concentrations, temperature etc

What does QC tell us…….

• As long as our QC strains are in range we have

a valid test system

• It does NOT tell us if test bacteria are

susceptible or resistant

What are interpretive criteria? • These are commonly known as breakpoints; S, I, R (Susceptible, Intermediate,

Resistant)

• Susceptible

This category implies an infection due to the isolate may be appropriately treated

with the dosage regimen of an antimicrobial agent recommended for that type of

infection and infecting species, unless otherwise indicated.

• Intermediate

This category implies an infection due to the isolate may be appropriately treated in

body sites where the drug are physiologically concentrated or when a high dosage

of drug can be used; also indicates a ‘buffer zone’ that should prevent small,

uncontrolled, technical factors from causing major discrepancies in interpretation.

• Resistant

Resistant isolates are not inhibited by the usually achievable concentrations of the

agent with normal dosage schedules and/or fall in the range where specific

microbial resistance mechanisms are likely, and clinical efficacy has not been

reliable in treatment studies.

Questions?