Antimicrobial Susceptibility Testing

Factors to Consider When

Determining Whether Testing Is
Warranted

• Body site from which the

bacterium was isolated
• Presence of other organisms and
quality of the specimen from
which the organism was grown
• Host’s status
Antimicrobial susceptibility testing (AST)
- The procedures used to produce antimicrobial susceptibility profiles and
detect resistance to therapeutic agents
vStandardization - Clinical and Laboratory Standards Institute (CLSI)
The standardized components of antimicrobial susceptibility testing include:
• Bacterial inoculum size
• Growth medium (typically a Mueller-Hinton base)
• pH
• Cation concentration
• Blood and serum supplements
• Thymidine content
• Incubation atmosphere
• Incubation temperature
• Incubation duration
• Antimicrobial concentrations
Testing Methods

1. Methods That Directly Measure Antimicrobial Activity

- bringing the antimicrobial agents of interest and the infecting
bacterium together in the same in vitro environment to determine the
effect of the drug’s presence on bacterial growth or viability.
a. Conventional
b. Commercial
c. Special screens and indicator tests
- Inoculum size is as important as culture purity and is accomplished by
comparing the turbidity of the organism suspension with a turbidity
standard.
• Antimicrobial Susceptibility Testing of the Clinical and Laboratory
Standards Institute (CLSI)
• 0.5 McFarland Standard
- 1% sulfuric acid & 1.175% barium chloride
- Comparable to 1.5 x 10 CFU/mL bacterial suspension
• Antimicrobial battery or panel.
- The antimicrobial agents chosen for testing against a particular
bacterial isolate
a. Conventional Testing Methods: Broth
Dilution
- liquid environment
- expressed as micrograms (ug) of active
drug per milliliter (mL) of broth (i.e.,
ug/mL).
- the test concentration for a drug may vary
depending on the organism and its
associated resistances.

vMcFarland Turbidity Standards

The most commonly used is the
McFarland 0.5 standard, which
contains 99.5 mL of 1% sulfuric
acid and 0.5 mL of 1.175%
barium chloride.
vMinimal Inhibitory Concentration (MIC)
- the lowest antimicrobial concentration that completely inhibits visible
bacterial growth, as detected visually or with an automated or semiautomated
method.
- Various concentrations of an antimicrobial agent are added to broth
Broth dilution testing is divided into two general
categories:
Microdilution Macrodilution
0.05 to 0.1 mL 1ml
Commonly used
*Most laboratories that perform broth microdilution use commercially
supplied microdilution panels in which the broth is already supplemented
with appropriate antimicrobial concentrations.
Inoculation and Incubation
- Standardized bacterial suspensions that match the turbidity of the 0.5
McFarland standard (i.e., 1.5 x 10. CFU/mL) usually serve as the starting
point for dilutions.
- prolonged incubation times beyond recommended limits should be
avoided, because antimicrobial deterioration may result in false or elevated
resistance patterns.
 Reading and Interpretation of Results
• bacterial growth, manifested as light to heavy turbidity or a button of growth on
the well bottom.
• well containing the lowest drug concentration that completely inhibits visible
bacterial growth is recorded as the MIC
vSusceptible (S) - Indicates that the antimicrobial agent in question may be an
ap- propriate choice for treating the infection caused by the organism.
vIntermediate (I)
vResistant (R ) - Indicates that the antimicrobial agent in question may not be an
appropriate choice for treatment
Conventional Testing
Methods: Agar Dilution

• the antimicrobial concentrations and organisms to be

tested are brought together on an agar- based medium
rather than in liquid broth.
• positive growth control plate without antibiotic
• the MIC is the lowest concentration of an antimicrobial
agent in agar that completely inhibits visible growth.
• The preparation of agar dilution plates is sufficiently labor
intensive
• It provides a means for determining MICs for N.
gonorrhoeae, which does not grow sufficiently in broth to
be tested by broth dilution methods.
preparing a series of agar plates containing the
antimicrobial agent to be tested in increasing
concentrations, usually in doubling dilutions
Conventional Testing Methods: Disk Diffusion

• detects antimicrobial resistance by challenging bacterial isolates with

antibiotic disks placed on the surface of an agar plate seeded with a
lawn of the bacterial isolate being investigated
• use of antibiotic-impregnated filter paper disks
• Incubation – bacteria grow
• the diameter of the zone of inhibition around each disk is measured
in millimeters
Disk Diffusion
• a McFarland 0.5 standardized suspension of bacteria in Mueller-Hinton
broth is swabbed over the surface of a standardized Mueller-Hinton agar
plate,
• paper disks containing specific concentrations of antimicrobial agent are
placed onto the inoculated surface. After incubation of 16 to 18 hours
• After incubation, the diameter of the zone of inhibition around each disk is
measured in millimeters
vResistant
vIntermediate
vSusceptible
• The breakpoint of an antimicrobial drug
- refers to an antimicrobial concentration in the serum associated with
optimal therapy using the customary dosing schedule. An organism is
susceptible if the MIC is at or below the break point.
Medium and Antimicrobial Agents.

• Mueller Hinton preparation - the standard agar-base medium used for

testing most bacterial organisms
• Factors
- pH and cation content, depth of the agar medium
*Most laboratories that perform disk diffusion testing purchase properly prepared and controlled Mueller-Hinton plates from
reliable commercial vendors.

Inoculation and Incubation

- the plate surface is inoculated using a swab that has been sub-
merged in a bacterial suspension standardized to match the 0.5
McFarland turbidity standard
• plates are inverted for incubation
• Most organisms are incubated at 35°C in ambient (i.e., room) air
• > 16 hours enhance resistance patterns
- methicillin resistance – staphylococci
- vancomycin resistance - enterococci
• Incubation beyond the allotted time should be avoided
Reading and Interpretation of Results

• examined to confirm that a confluent lawn of growth

• a particular isolate may have undergone mutation - blood and/or
incubation in CO2
sulfonamides and trimethoprim – hazes of growth that occur in more
obvious inhibition zones should not be ignored.
Interpretive criteria for antimicrobial
agent/organism using the CLSI interpretive
standards
• Susceptible – 17 mm
• intermediate - 14 to 16 mm
• resistant - 13 mm or less
E. coli isolate – ampicillin
Advantages and Disadvantages.

• Advantages
- convenience and user friendliness
vMost commonly used methods
• Disadvantages
- lack of interpretive criteria for organisms not included
- inability to provide more precise data
Commercial Susceptibility
Testing Systems

• The commercial methods are

variations of the conventional
dilution or disk diffusion
methods, and their accuracies
have been evaluated by
comparison of results with those
obtained by conventional
methods.
Diffusion in Agar Derivations
Automated Antimicrobial Susceptibility Test
Systems

• Vitek Legacy
• Vitek 2 systems
Alternative Approaches for Enhancing Resistance
Detection
vdetecting resistance by measuring the effect of antimicrobial presence on
bacterial growth
• certain strains of staphylococci
- oxacillin agar screen – presence of growth – susceptible
- Resistant to beta-lactam antibiotics – vancomycin
- Commercially prepared e.g Crystal MRSA ID System
- 30-mg cefoxitin disks - less than or equal to 21 mm resistant
• enterococcal resistance to vancomycin - broth microdilution method
should be performed
• oxacillin disk screen - penicillin resistance in S. pneumoniae
D-test
• indicates the presence of macrolide-inducible
resistance to clindamycin produced by an inducible
methylase that alters the common ribosomal binding
site for macrolides, clindamycin and the group B
streptogrammins
vAs complicated resistance mechanisms requiring laboratory detection
continue to emerge, screening and supplemental testing methods will con-
tinue to be developed
Predictor Antimicrobial Agents
- Staphylococcal resistance to oxacillin is used to determine and report
resistance to all currently available beta-lactams, including penicillins,
cephalosporins, and carbapenems.
- Enterococcal high-level gentamicin resistance predicts resistance to nearly all
other currently available amino- glycosides, including amikacin, tobramycin,
netilmicin, and kanamycin.
- Enterococcal resistance to ampicillin predicts resistance to all penicillin
derivatives.
 Methods that Directly Detect Specific Resistance
Mechanisms
vassaying for the presence of a particular mechanism
vPhenotypic and genotypic methods
1. Phenotypic Methods
a. Beta-Lactamase Detection
- chromogenic cephalosporin (e.g., nitrocefin) is used as the substrate
- A positive test indicates resistance to penicillin, ampicillin, amoxicillin,
carbenicillin, mezlocillin, and piperacillin.
Useful applications include detection of:
• Enterococcus resistance to ampicillin
• N. gonorrhoeae resistance to penicillin
• H. influenzae resistance to ampicillin
• Bacteroides spp. and other gram negative anaerobes resistance to penicillin
and ampicillin
• Staphylococci resistance to penicillin, if negative do zone edge test (disk
diffusion with penicillin) to detect penicillin resistance caused by other
mechanisms
Limitation - limited to organisms producing enzymes whose spectrum of
activity is known.
b. Chloramphenicol Acetyltransferase Detection (CAT)
• CAT – bacterial enzyme that detoxifies the antibiotic chloramphenicol and is
responsible for chloramphenicol resistance in bacteria
• Positive – chloramphinicol resistant
2. Genotypic Methods
• matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF MS) mass
spectrometry
- can be used to detect antimicrobial resistance by beta-lactamase, and
carbapenemases can be detected by mixing the bacterial isolate with the beta-
lactam or carbapenem substrates and then assaying the supernatant looking for
a shift in the peptide mass fingerprint (PMF) from nonhydrolyzed to hydrolyzed
products.
Tests for Activity of Antimicrobial Combinations

• Synergy: the activity of the antimicrobial combination is substantially

greater than the activity of the single most active drug alone
• Indifference: the activity of the combination is no better or worse
than the single most active drug alone
• Antagonism: the activity of the combination is substantially less than
the activity of the single most active drug alone (an interaction to be
avoided)
 Test added in severe and life threatening
infections
• Minimal Bactericidal Concentration
- The requirement of 99.9% killing defines the minimum bactericidal
concentration (MBC) of an antimicrobial agent. The MBC test is an additional
quantitative assessment of the killing effect of a drug on a specific patient isolate. This
test, done to evaluate a drug's activity, is sometimes requested in cases of life-
threatening infections.
• Time kill studies
- exposing a bacterial isolate to a concentration of antibiotic in a broth medium
and measuring the rate of killing over a specified time period.
- 1000-fold decrease in the number of viable bacteria in the antibiotic-
containing broth after a 24-hour period, compared with the number of bacte- ria in
the original inoculum, is interpreted as bactericidal activity.
Laboratory Strategies for Antimicrobial
Susceptibility Testing
• Serum Bactericidal Test (Schlichter Test)
- used to determine the bactericidal level of antibiotic in a patient's
serum against the organism isolated from that patient.
Peak and through serum specimens are drawn, serial dilutions of the
specimen are made, and a standardized inoculum of organism is added.
Susceptibility Testing of Agents of Bioterrorism

• Bacillus anthracis
• Yersinia pestis
• Burkholderia mallei
• Burkholderia pseudomallei
• Francisella tularensis,
• and Brucella spp.
üThese organisms are an imminent danger when being tested in the
laboratory, and work with these organisms should be conducted only in a
biosafety level 2 (BSL2) or higher facility by trained individuals.