ANTIMICROBIAL SUSCEPTIBILITY
TESTING
SHIERA MARIE APRIL G. DUGHON, RMT
Clinical Instructor
PROCEDURES IN ANTIMICROBIAL SUSCEPTIBILITY
TESTING
Antimicrobial susceptibility testing is Methods
performed on bacteria and fungi clinical ✓Disk diffusion
specimens to determine which ✓Dilution/MIC method
antimicrobial agents might be effective ***Minimal Inhibitory Concentration
in treating infections caused by theses
organism. CLSI/ Clinical and Laboratory Standards
Only organisms that are likely to be Institute - the standard that describes
contributing to an infection should be these methods , published and frequently
tested. updated
REASONS AND INDICATIONS FOR PERFORMING
AST
Factors to consider when determining whether
testing is warranted
▪Body site from which the bacterium is isolated
▪Presence of other organisms and quality of the specimen
from which the organism was grown
▪Host’s status
SELECTING ANTIMICROBIAL AGENTS TO TESTING
AND REPORTING
▪Each laboratory must determine which agents are appropriate for routine testing
against various organisms in its setting.
▪FDA must be consulted for information concerning the doses and indications for which
antimicrobial was approved and the performance of the antimicrobial agent during
initial clinical trial.
▪It is important that the drugs tested by the laboratory match the institution formulary
as close as possible.
▪Patient population must be considered in the choice of antimicrobial agents to be
tested
SELECTING ANTIMICROBIAL AGENTS TO TESTING
AND REPORTING
Selection of Test batteries
▪A laboratory will define a battery of 10-15 antimicrobial agents for routine testing
against:
✓Enterobacterales
✓Pseudomonas spp.
✓Nonfastidious gram-negative bacilli (Acinetobacter, Stenotrophomonas maltophilia, Burkholderia
cepacia)
✓Staphylococci
✓Enterococci
▪Sometimes a separate battery is performed for urine isolates for treating UTI
▪Supplemental battery for significant number of bacteria resistant to more
commonly used antimicrobials
SELECTING ANTIMICROBIAL AGENTS TO TESTING
AND REPORTING
Reporting of susceptibility test results
▪Use the least toxic, most cost effective, and most clinically effective
agents to refrain from use of costly, broader-spectrum agents
▪CLSI provides guidance for development of such selective or
cascade reporting protocol
TRADITIONAL ANTIMICROBIAL SUSCEPTIBILITY
TEST METHODS
1. Inoculation preparation and Use McFarland Standards
❖Inoculum preparation – one of the most critical steps in susceptibility
❖McFarland Turbidity Standards – specific volumes of barium sulfate solution
(1% sulfuric acid and 1.175% barium chloride)
w/ a specific optical density
❖Inoculum Standardization
TRADITIONAL ANTIMICROBIAL SUSCEPTIBILITY
TEST METHODS
2. Dilution Susceptibility Testing Methods
-use to determine MIC/ Minimal Inhibitory Concentration
❖Antimicrobial Stock Solutions- must be prepared from reference standard microbial powders
❖Broth Macrodilution (Tube Dilution) Tests- Mueller-Hinton broth is recommended for non-
fastidious organism. The MIC is determined visually as the lowest concentration that inhibits
growth or absence of turbidity. MBC can also be detected.
❖Broth Microdilution Tests – miniaturized and adapted to multiwell microdilution trays (80-
100wells)
❖Agar Dilution Tests- specific volume of antimicrobial solutions dispensed into pre measured
volumes of molten and cooled agar. Mueller-Hinton is recommended for aerobic isolates. Use
in research. Reference AST method for anaerobes and Neisseria gonorrhoeae.
TRADITIONAL ANTIMICROBIAL SUSCEPTIBILITY
TEST METHODS
3. Disk Diffusion Test (Kirby-Bauer test)
❖Establishing Zone Diameter Interpretative Breakpoints – “zone of inhibition”
❖Test performance
✓Disk Storage - -20C to maintain potency; working supply 2-8C for 1 week
✓Inoculation and Incubation
✓Reading Plates and test interpretation- use calipers or ruler to measure the
inhibition zone
MODIFIED METHODS FOR TESTING FASTIDIOUS BACTERIA
FASTIDIOUS ORGANISM BROTH DILUTION DISK DIFFUSION CONDITION
Streptococcus pneumoniae and MH BROTH + 2%-5% lysed horse MHA + 5% sheep blood 5% CO2 for 20-24hrs
blood
Streptococcus spp.
Haemophilus influenzae and HTM (MH + X and V factors) HTM (MH + X and V factors) 5% -7% CO2 for 20-24hrs
Haemophilus parainfluenzae
Neisseria gonorrhoeae and *Agar dilution Gonococcal agar 5% -7% CO2 for 20-24hrs
Gonococcal agar base
Neisseria meningitidis
Helicobacter pylori and *Agar dilution MHA + 5% aged sheep blood Microaerobic, 3days incubation
MHA + 5% aged sheep blood
Campylobacter spp.
Anaerobes *microdilution
Brucella laked sheep blood
Bacteroides fragilis Brucella broth + lysed horse blood
ADDITIONAL ORGANISM AND ANTIMICROBIAL
AGENT TESTING CONCERNS
Staphylococcus and Streptococcus Enterobacterales
▪MRSA ▪ESBL “key hole”
▪VRSA ▪AmpC
▪ICR “D-zone” ▪CPE
Enterococcus
▪High-Level Aminoglycoside resistance
▪Vancomycin Resistance
AUTOMATED AST METHODS
▪BD PHOENIX SYSTEM
▪MICROSCAN WLAKAWAY SI
▪TREK SENSITIVE
▪VITEK COMPACT
NON AUTOMATED AST METHOD:ETEST
Prinicple: An antimicrobial gradient in a
an agar medium is established as a
means of determining antimicrobial
susceptibility.
A thin plastic test strips impregnated on
the upper surface with a antimicrobial
concentration index or scale.
Interpretation: MIC is determined where
the growth ellipse intersects the Etest strip
INTERPRETATION OF IN VITRO AST RESULTS
➢Zone diameter
➢Susceptible
➢Intermediate – “buffer house”
➢Resistant
➢Nonsusceptible
METHODS OF DETECTING ANTIMICROBIAL-
INACTIVATING ENZYMES
Β-LACTAMASE TESTS
The β-lactamase are enzymes that chemically inactivate β-lactam
molecules by disrupting the β-lactam ring component of the molecule.
▪Chromogenic cephalosporin nitrocefin- commonly used
Cefinase disk commercially available (filter paper disk with nitrofecin)
QUALITY CONTROL OF AST
QC involves testing standard reference strains that have defined antimicrobial
susceptibility to the drugs tested.
The CLSI has identified ATCC/American Type Culture Collection strains useful for QC
testing.
Antibiograms –most widely used supplemental QC
END
