antimicrobial susceptibility testing overview

Amanda T. Harrington, PhD, D(ABMM)

Associate Professor, Pathology

Director, Clinical Microbiology Laboratory

Loyola University Medical Center

ANTIMICROBIAL

SUSCEPTIBILITY TESTING

OVERVIEW

Determination of antimicrobial susceptibilities of significant

bacterial isolates is one of the principal functions of the

clinical microbiology laboratory

Provide technically accurate information

Report in easily interpreted format

Results help guide clinician in selection of most appropriate

agent

Trend is to direct clinician toward most narrow spectrum, least

expensive agent to which pathogen should respond

Purpose is to ‘predict outcome of treatment’ with

antimicrobial agents tested= in vitro testing

ANTIMICROBIAL SUSCEPTIBILITY

TESTING

When should a laboratory perform susceptibility testing?

With pathogens identified from a clinically relevant infection

With pathogens for which standardized methods are available

With pathogens where resistance is known to be a clinical problem


TESTING

When should a laboratory not routinely perform susceptibility

testing?

For organisms that are not typically considered pathogens (i.e.

normal flora)

For organisms that are not clinically relevant or in relevant quantity

from the source of recovery/specimen type

“Work up everything”


TESTING

Methods currently available

Disk diffusion

Broth Microdilution

E-test

Automated instruments


TESTING

DISK DIFFUSION

DISK DIFFUSION METHOD

Also known as the Kirby-Bauer Method

Medium

Mueller Hinton agar for most bacteria

Batch to batch reproducibility

Low in sulfonamide and tetracycline inhibitors

Years of clinical experience

Supplemented with 5% sheep blood for streptococci

Other media for fastidious bacteria

Haemophilus Test Media

Supplemented GC agar base


Inoculum

Diluted at a standard concentration=Adjust to 0.5 McFarland

standard (1x108 CFU/ml)

Evenly seeded throughout the plate with the isolate of interest

Commercially prepared disks, each of which are pre-impregnated

with a standard concentration of a particular antibiotic, are then

evenly dispensed and lightly pressed onto the agar surface.

The test antibiotic immediately begins to diffuse outward from the

disks, creating a gradient of antibiotic concentration in the agar

such that the highest concentration is found close to the disk with

decreasing concentrations further away from the disk.

After an overnight incubation, the bacterial growth around each

disc is observed.


Incubation

35o C.

Ambient air with exceptions

Streptococci: CO2

Haemophilus: CO2

16-18 hours with exceptions

Staphylococci and enterococci: 24 hours

S.pneumoniae: 20-24 hours


Measure diameter of zone with caliper

Reflected light with exceptions

Staphylococci and enterococci examined by transmitted light for inner

colonies

HOW TO READ DISK DIFFUSION METHOD

Reflected Light vs. Transmitted Light

The zone around an antibiotic disk that has no growth is

referred to as “the zone of inhibition”

approximates the minimum antibiotic concentration sufficient to

prevent growth of the test isolate.

This zone is then measured in mm and compared to a

standard interpretation chart used to categorize the isolate as

susceptible, intermediately susceptible, or resistant.

INTERPRETATION OF DISK DIFFUSION

Inner colonies

Resistant subpopulation

Mixed culture

High-frequency mutants

Ignore swarming of Proteus

80% endpoint with

sulfonamides

CONSIDERATIONS

Advantages

Flexibility in antibiotic selection

Low cost

Simple to perform

No special equipment necessary

Most established and best proven method

Continues to be updated through CLSI

Qualitative results easily interpreted by clinician


Disadvantages

Not standardized for all organisms

Qualitative results only (S/I/R)

Not rapid (results available in 16-24 hours)

Time consuming since read and entered manually

Is often media dependent


BROTH MICRODILUTION

AND ETEST

Minimum inhibitory concentration (MIC) is defined as the

lowest concentration of antimicrobial agent required to inhibit

growth of the organism.

It cannot be determined from this testing whether the

concentration is bacteriocidal (i.e. the organism has been

killed).

MIC

Microtitre trays with two-fold

dilutions of antibiotics are

inoculated with a

standardized inoculum of the

bacteria and incubated under

standardized conditions.

After incubation, the MIC is

recorded as the lowest

concentration of

antimicrobial agent with no

visible growth.

BROTH MICRODILUTION TESTING

Use the record sheet for orientation of the plates

Check growth in the positive control wells

The MIC is read as the lowest concentration without visible growth

HOW TO READ BROTH MICRODILUTION

HOW TO READ MICROBROTH DILUTION

HOW TO READ BROTH MICRODILUTION

a. Control wells

b. Antibiotic 1

c. Antibiotic 2

d. Antibiotic 3

= MIC well

Etest® consists of a

predefined gradient of

antibiotic concentrations on

a plastic strip and is used to

determine the Minimum

Inhibitory Concentration

(MIC) of antibiotics

ANTIMICROBIAL GRADIENT DIFFUSION

Read MIC at the point where el l ipse intersects the scale.

I f a MIC value between two two -fold di lutions is seen, always round up to the highest value.

Remember to read the MIC value at complete inhibition of al l growth including isolated colonies.

I f the intersect dif fers on either side of the str ip, read the MIC as the greater value.

HOW TO READ E-TEST

Advantages

Generate quantitative results (MICs)

Allow testing of anaerobic and fastidious species of bacteria

Method of choice when no CLSI guidelines

Disadvantages

Increased cost over disk

Largely manual process

Quantitative data may be “challenging” to interpret for some providers

There are some systematic biases toward higher or lower MICs determined by the Etest when testing certain organism-antimicrobial agent combinations

BROTH MICRODILUTION AND E-TEST

AUTOMATED

INSTRUMENTS

BD Phoenix

Beckman Coulter (used to

be Siemens) MicroScan

WalkAway

bioMeriuex Vitek2

Thermo Scientific

Sensititer ARIS

AUTOMATED COMMERCIAL METHODS

Jorgensen, JH and Ferraro, MJ. Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices.

Clin Infect Dis. (2009) 49 (11): 1749-1755.

Cartridges or broth

microdilution plates

Manual or semi-automated

inoculation

True or extrapolated MIC

Sensitive optical detection

systems allow detection of

subtle changes in bacterial

growth

Results in 3.5-24 hours

depending on system and

organism

TESTING DEVICES

Example of bacterial growth curves generated with the phenotypic VITEK2 system.

Differences in the slope and plateau levels of the curves generated in the presence or

absence of antibiotics define whether or not a strains will be deemed resistant,

intermediary or susceptible to certain antibiotics. The layout of all current automated

systems allow for the simultaneous analysis of multiple drugs for a single bacterial

species in a single diagnostic tool. Alex van Belkum; Wm. Michael Dunne, Jr. Pathogens: Wanted—Dead or Alive ASM Microbe Dec 2015

Advantages

Generate more rapid results

Include computerized data management system

Can set interpretation rules using software

Interface with LIS

Disadvantages

More expensive

Limited to commercially available panels

Failure to detect some inducible, subtle, or emerging resistance

mechanisms

Instrument interpretation

AUTOMATED INSTRUMENTS

Threshold values established for each pathogen -antibiotic (i .e., bug-drug) combination indicating at what level of antibiotic the isolate should be considered to be sensitive, intermediate or resistant. = Clinical Breakpoints

Guidelines and recommendations for these are continuously updated by organizations worldwide, such as CLSI, EUCAST.

The CLSI zone size and MIC interpretive criteria are established by analysis of 3 kinds of data: (1) microbiologic data, including a comparison of MICs and zone sizes

on a large number of bacterial strains, including those with known mechanisms of resistance that have been defined either phenotypically or genotypically

(2) pharmacokinetic and pharmacodynamic data

(3) clinical studies results (including comparisons of MIC and zone diameter with microbiological eradication and clinical efficacy) obtained during studies prior to FDA approval and marketing of an antibiotic

FDA guidelines are for newer antibiotics

Strict quality control guidelines must be followed

INTERPRETATION OF RESULTS FROM AST

A “susceptible” result indicates that the patient's organism should respond to therapy with that antibiotic using the dosage recommended normally for that type of infection and species

A “resistant” result indicates that the organism should not be inhibited by the concentrations of the antibiotic achieved with the dosages normally used with that drug

An “intermediate” result indicates that a microorganism fal ls into a range of susceptibility in which the MIC approaches or exceeds the level of antibiotic that can ordinarily be achieved and for which cl inical response is l ikely to be less than with a susceptible strain.

Categorical reporting typically provides the cl inician with the information necessary to select appropriate therapy.

Exceptions

if the antibiotic is highly concentrated in a body fluid such as urine, or if higher than normal dosages of the antibiotic can be safely administered

Reporting of MICs could aid a physician is selecting from among a group of similar drugs for therapy

S/I/R

Jorgensen, JH and Ferraro, MJ. Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices.

Clin Infect Dis. (2009) 49 (11): 1749-1755.

Standardized bacterial inoculum size

Culture conditions (growth medium, pH, cation concentration

Blood and serum supplements and thymidine content)

Incubation conditions (atmosphere, temperature, duration)

Concentration of antimicrobials for testing

FACTORS THAT IMPACT RESULTS

antimicrobial susceptibility testing overview

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