Antimicrobial Susceptibility Testing:

A Primer for Clinicians

Kristi M. Kuper, Pharm.D., Deborah M. Boles, M.S., John F. Mohr, Pharm.D., and Audrey Wanger, Ph.D.

Appropriate use of antimicrobials in health care continues to be a challenge.

Reliable and reproducible antimicrobial susceptibility testing methods are
necessary to provide the clinician with valuable information that can be
translated into positive clinical outcomes at the bedside. However, there are
nuances with these testing methods that, if unrecognized, could lead to
misinterpretation of results and inappropriate antibiotic selection. This
primer describes the common antimicrobial susceptibility tests used in the
clinical microbiology laboratory and reviews how subtle differences in testing
methods and technique can influence reported results. Clinicians who have a
thorough understanding of qualitative and quantitative methods, automated
susceptibility testing systems, and commonly used screening and confirma-
tory tests for antibiotic-resistant organisms can strengthen institutional
antibiotic stewardship programs and improve patient outcomes.
Key Words: infectious disease, antimicrobial, bacterial resistance, antibiotics,
computer technology, pharmacokinetics, susceptibility, microbiology testing
methods.
(Pharmacotherapy 2009;29(11):1326–1343)

OUTLINE
Basic Principles of Antimicrobial Susceptibility Testing
Qualitative Testing
Disk Diffusion
Quantitative Testing
Minimum Inhibitory Concentration
Etest
Automated Systems
Vitek and Vitek 2
MicroScan WalkAway
Phoenix
Sensititre
Advantages and Disadvantages
Panel and Card Selection
Special Considerations in Antimicrobial Susceptibility
From the Department of Clinical Affairs, Cardinal Health,
Houston, Texas (Dr. Kuper); the Department of Pharmacy,
Lowell General Hospital, Lowell, Massachusetts (Dr. Boles);
Cubist Pharmaceuticals, Lexington, Massachusetts (Dr.
Mohr); and the Department of Pathology, University of
Texas Medical School, Houston, Texas (Dr. Wanger).
For reprints, visit http://www.atypon-link.com/PPI/loi/phco.
For questions or comments, contact Kristi M. Kuper,
Pharm.D., BCPS, Department of Clinical Affairs, Cardinal
Health, 1330 Enclave Parkway, Houston, TX 77077; e-mail:
kristine.kuper@cardinalhealth.com.
Testing
Fastidious Organisms
Anaerobes
Mycobacteria
Specialized Screening and Confirmatory Tests for
Resistant Organisms
Interpretation
Agreement Among Methods
Clinical Practice Application
Conclusion
In January 2007, the Infectious Diseases
Society of America, in conjunction with the
Society for Healthcare Epidemiology, released
guidelines for developing an institutional
program to enhance antimicrobial stewardship.1
These guidelines underscore the need for
accurate and reproducible identification of
microorganisms and antibiotic susceptibilities.
This is integral to the care of patients with
infectious diseases and plays a critical role in
antimicrobial stewardship and epidemiologic
investigations.
The clinician who has an advanced knowledge
of antimicrobials, coupled with an understanding
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
of commonly used microbiology testing methods,
can be effective in improving antimicrobial
utilization and optimizing patient care. These
skills are particularly relevant today and can help
combat increased antimicrobial resistance in the
hospital and community settings. The purposes
of this review are to help pharmacists gain a basic
understanding of common antimicrobial suscep-
tibility tests used in the clinical microbiology
laboratory, and to demonstrate how differences in
testing can influence therapeutic choices for
treating infectious diseases.
Basic Principles of Antimicrobial Susceptibility
Testing
The purpose of performing antimicrobial
susceptibility testing is to assist clinicians with
the selection of appropriate targeted antibiotic
therapy in order to optimize clinical outcomes.
Infection-related and overall mortality is reduced
when patients are treated expeditiously with an
antibiotic to which the organism is susceptible.2
Two basic methods of antimicrobial suscepti-
bility testing are available to laboratories: quali-
tative and quantitative. Disk diffusion (also
known as the Kirby-Bauer method) is a quali-
tative method of susceptibility testing that may
be prone to some degree of error depending on
the drug and organism being tested.3–7 However,
it is an acceptable option for testing among
patients with uncomplicated urinary tract
infections or other less severe infections where
eradication is augmented by the patient’s immune
system. Because of the high spontaneous cure
rates for some mild infections, an organism that
is truly resistant yet reported as susceptible by
the microbiology laboratory may not always be
clinically relevant.8 Broth microdilution and agar
dilution methodologies are considered quanti-
tative because they can measure the minimum
inhibitory concentration (MIC). The MIC is
defined as the lowest concentration of an anti-
biotic that inhibits visible growth of a micro-
organism. Both quantitative methods are considered
the reference methods for susceptibility testing
because of their high levels of reproducibility.
For convenience, most clinical laboratories use
an automated system supplemented with one or
more manual methods of susceptibility testing.
Automated systems must provide susceptibility
results that are consistent with a reference
method. Regardless of the testing methodology
selected, the results are correlated with a set of
standardized interpretations known as break-
1327
points. The term “breakpoint” can be confusing,
as it may refer to one derived from microbiologic,
clinical, or pharmacokinetic-pharmacodynamic
data. Microbiologic breakpoints refer to the MIC
for an antibiotic that distinguishes wild-type
bacterial populations from those that have either
selected or acquired resistance mechanisms.
These breakpoints are derived from moderate-to-
large numbers of in vitro MIC tests. A clinical
breakpoint is derived from the antimicrobial
MICs for the infecting organisms isolated in
prospective clinical trials. Susceptibility in this
context correlates with a high likelihood of
clinical success. Finally, a pharmacokinetic-
pharmacodynamic breakpoint predictive of
microbiologic effects is derived from human or
animal data that are modeled by using statistical
or mathematic techniques such as a Monte Carlo
simulation.9
Breakpoints for new antibiotics are approved
by the United States Food and Drug Adminis-
tration (FDA) based on composite data from the
breakpoint methodologies described previously
and are not frequently altered after the product is
released to market. A recent inventory of
antibacterial drug labels indicated that among
more than 100 currently approved drug labels,
over 70 contained breakpoints that were
outdated.10 Breakpoints may not be available for
older drugs (e.g., polymyxin or colistin for
Enterobacteriaceae) or organisms that rarely
cause human disease such as Flavobacterium
species.
Ongoing surveillance and laboratory process
standardization is governed by a nonprofit global
standards developing organization known as the
Clinical and Laboratory Standards Institute
(CLSI), previously known as the National
Committee for Clinical Laboratory Standards
(NCCLS). The CLSI promotes the development
and use of voluntary consensus standards and
guidelines within the health care community and
publishes a series of reference manuals that serve
as useful guides for antimicrobial susceptibility
testing.11, 12 These manuals are referenced by a
number-letter combination that represents the
subject matter and the version. Two manuals of
interest to the pharmacist include the M-39 and
the M-100. The M-39 is updated every 3 years
(current version is M-39A3) and provides
recommendations for antibiogram preparation.
The M-100 is updated yearly (current version is
S19) and contains organism-specific breakpoint
reference tables for disk diffusion and MIC
testing. The breakpoints published in the M-100
1328 PHARMACOTHERAPY Volume 29, Number 11, 2009
Table 1. Examples of Discrepancies Between United States Food and Drug Administration and Clinical and Laboratory
Standards Institute Minimum Inhibitory Concentration Breakpoints12–18
MIC Breakpoints (µg/ml)
FDA Approved CLSI Approved
Organism Drug Susceptible Intermediate Resistant Susceptible Intermediate Resistant
Streptococcus
pneumoniae Ceftriaxone
Nonmeningitis ≤ 0.5 1 ≥ 2a ≤1 2 ≥4
Meningitis ≤ 0.5 1 ≥ 2a ≤ 0.5 1 ≥2
Enterococcus sp Daptomycin ≤ 4b —b —b ≤ 4b —b —b
Enterobacteriaceae Tigecycline ≤2 4 ≥8 None
Doripenem ≤ 0.5 —c —c None
Acinetobacter sp Colistin or None ≤2 — ≥4
polymixin
FDA = U.S. Food and Drug Administration; CLSI = Clinical and Laboratory Standards Institute; MIC = minimum inhibitory concentration.
a
The package insert for ceftriaxone does not make a distinction between nonmeningeal and meningeal isolates of S. pneumoniae.
b
The FDA-approved breakpoint in the daptomycin package insert is for vancomycin-susceptible Enterococcus faecalis strains only, but there is
no FDA-approved breakpoint for vancomycin-susceptible Enterococcus faecium strains. The CLSI-approved breakpoint can be applied to all
Enterococcus species.
c
The FDA approved only a susceptible classification because of the absence of resistant isolates in clinical trials. Drugs with an MIC (or disk
diffusion test) that suggests nonsusceptibility of isolates should undergo additional testing.

by the CLSI are for both branded and generic
antibiotics against different microorganisms and
incorporate in vitro microbiologic information,
human and animal pharmacokinetic-pharmaco-
dynamic data, and clinical and bacteriologic
outcomes from clinical studies.9 These break-
points may be discordant with the FDA-approved
values (Table 1),12–18 as well as the values set by
the European Union Committee on Antimicrobial
Susceptibility Testing (known as EUCAST).
Qualitative Testing
Disk Diffusion
The principle behind the disk diffusion method
is that antibiotic molecules diffuse out from a
disk into the agar, creating a dynamically
changing gradient of antibiotic concentrations
while the organism being tested starts to divide
and growth progresses toward the critical mass.19
The zone edge is where the concentration of
antibiotic begins to inhibit the organism reaching
an overwhelming cell mass. At this point, the
density of cells is high enough to absorb
antibiotic in the immediate vicinity, thus
maintaining concentrations at subinhibitory
levels, and enabling the test organism to grow.
For most rapidly growing aerobic and facultative
anaerobic bacteria, the critical time it takes for
the organism to absorb the antibiotic varies from
3–6 hours, but interpretation by the microbiologist
generally occurs between 18 and 24 hours. For
some slow-growing organisms, such as Bacteroides
fragilis, the gradient is formed before the
organism reaches its critical mass, resulting in
false susceptibility.20
The standardized testing process described
here allows reproducible and consistent results.
Before testing, the microbiologist will prepare a
fresh agar plate by using an inoculum that
contains a predefined organism density. The
inoculum is prepared by suspending isolated
bacterial colonies (taken from an 18–24-hr agar
plate) in broth or saline to a turbidity matching a
0.5 McFarland standard (1 x 108 colony-forming
units/ml). This standard is used to measure the
turbidity of the bacteria and ensure a certain
number of colony-forming units in the inoculum.12
The standard concentration of the solution is
important because an inoculum that is too high
in colony-forming units can result in falsely
smaller zone sizes (i.e., false resistance), whereas
too low of an inoculum can result in falsely larger
zone sizes (i.e., false susceptibility).21, 22
The inoculum suspension should be used
within 15 minutes of preparation. This is
particularly important for fastidious organisms
(e.g., Neisseria species, Haemophilus influenzae,
and ␤-hemolytic streptococci) that lose their
viability rapidly, resulting in a low inoculum. A
sterile cotton swab is dipped into the suspension
and pressed firmly on the inside of the tube to
remove excess liquid. The entire dried surface of
the agar plate is inoculated by streaking the
surface in three directions. After each streak, the
plate is rotated 60 degrees to obtain an even
distribution of the inoculum. After streaking, the
plate should be dried for no more than 15
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
minutes. Once the agar plate is completely dry,
antibiotic disks are applied either manually or
with a dispensing apparatus. For most organisms,
no more than 12 disks should be placed on a
150-mm agar plate or 5 disks on a 90-mm plate.
It is best to place disks that have predictably
small zone sizes (e.g., gentamicin) next to disks
that give predictably larger zones of inhibition
(e.g., cephalosporins) so that zone overlap can be
prevented.23
Optimally, disks should be positioned at a
distance of 30 mm apart, and no closer than 24
mm apart when measured center to center, to
minimize inhibition zone overlap.23 Most dispenser
devices are self-tamping (disks are tapped or
pressed onto the agar surface) but may need to be
pressed down to make immediate and complete
contact with the agar surface. Once in contact
with the agar, the disk cannot be moved because
the antibiotics diffuse rapidly once the disk comes
into contact with the agar. The microbiologist’s
technique is important because movement can
disrupt the process integrity and lead to inaccurate
results.
Agar plates are incubated in an inverted position
(agar side up) under conditions appropriate for
the test organism. The incubation period is
16–18 hours for rapidly growing aerobic bacteria,
and longer for fastidious organisms or special
resistance conditions. When detecting vancomycin
or oxacillin resistance in Staphylococcus aureus,
plates need to be kept for a minimum of 24 hours
per CLSI testing procedures.23 After incubation,
the agar plate is examined to determine if a
semiconfluent and even “lawn” of growth has
been obtained before reading the plate. If
individual colonies are seen, the inoculum is too
light and the test should be repeated since the
large zone sizes are inaccurate. The same holds
true for an excessively heavy inoculum causing
overly small zones with very hazy edges.22 Either
outcome could result in false readings.
If the lawn of growth is satisfactory, the zone
diameter is read to the nearest millimeter by
using a ruler or sliding calipers. The zone
margin is identified as the area where no obvious
visible growth is seen by the naked eye, unless
otherwise specified. Zone diameters on Mueller-
Hinton agar (without blood supplements) are
read from the back of the plate, whereas blood-
containing agars are read from the surface to
ensure that the zone of inhibition is read
accurately. The line of demarcation for this zone
should be clear; faint growth or microcolonies
detectable only with a magnifying glass or by
1329
tilting the plate should be ignored. Again,
inappropriate technique could lead to erroneous
susceptibility interpretation.
The main advantages of disk diffusion testing
are simplicity, inexpensive equipment, and the
cost-effective and flexible choice of antibiotics for
testing. Disadvantages include the inability to
obtain an actual MIC and the additional time
required by the technologist for test preparation
and providing an interpretation of manually
determined zone sizes based on standards.
Commercial zone readers (e.g., BioMICV3; Giles
Scientific, Inc., Santa Barbara, CA) are available
to simplify this process.
Certain antibiotics can be problematic to test
with the disk diffusion method because of the
specific physiochemical properties of the
molecules. Vancomycin, colistin, and macrolides
have higher molecular weights and therefore
diffuse very slowly in agar. 22 The limited
diffusion and poorly resolved concentration
gradient around these disks result in only a few
millimeters of difference in zone sizes between
susceptible and resistant strains, which could
result in a potentially ambiguous reading.
Results can also be influenced by the positioning
of the light source on the plate. Most plates are
read with use of reflected light, with the
exception of linezolid, oxacillin, and vancomycin
for both S. aureus and Enterococcus species. In
these cases, the zones should be measured by
using transmitted light in order to ensure an
accurate measurement of zone diameter. If
results are unexpected or borderline, another
method of testing may be required or the test
repeated for confirmation.
Quantitative Testing
Minimum Inhibitory Concentration
In critical infections such as bacteremia and
endocarditis, accurate quantitative determination
of the exact MIC value may be useful for therapy
guidance.24, 25 In these situations, an organism
with an MIC of 0.016 µg/ml to an antimicrobial
may have a significantly different therapeutic
implication from that of the same organism-
antibiotic combination that is susceptible with an
MIC of 1 µg/ml. The pharmacodynamics of the
drug and the infection site factor into this assess-
ment. For example, a vancomycin MIC of 1.5
µg/ml or greater was found to be independently
associated with treatment failure in patients with
methicillin-resistant S. aureus (MRSA) bacteremia
(adjusted risk ratio 2.6, 95% confidence interval
1330 PHARMACOTHERAPY Volume 29, Number 11, 2009
1.3–5.4, p=0.01).26 Interpreted, this means that
patients with bacterial infections caused by
MRSA strains that have an antibiotic MIC of 1.5
or 2 µg/ml may fail therapy even though the
isolate is considered to be susceptible based on
current breakpoints. The clinician who uses the
reported antibiotic MIC for the organism coupled
with pharmacokinetic-pharmacodynamic data of
the antibiotic can customize therapy to improve
clinical outcomes and reduce mortality.27–30
Although all of these processes may seem
straightforward, MICs can vary based on the end
point, type of media, and conditions of the
incubation period. For example, end points for
bacteriostatic drugs should be read at 80%
inhibition (significant reduction in growth) and
bactericidal drugs at 100% inhibition (complete
inhibition). If a bacteriostatic drug is evaluated
at 100% inhibition (as opposed to 80% inhibition),
the MIC reported will be falsely elevated and may
result in the organism being categorized as
resistant, instead of susceptible. In addition,
capnophilic (carbon dioxide “loving”) organisms
such as pneumococci, streptococci, gonococci,
and Haemophilus species are incubated in 5%
carbon dioxide and MICs will vary based on this
carbon dioxide level.31, 32
Most testing methods used in the hospital
laboratory are correlated with either broth micro-
dilution, broth macrodilution, or agar dilution
methods. Both broth methods and agar dilution
are quantitative tests that are predominantly
performed in reference or research laboratories.
These tests are considered reference methods
because they are used to gauge the accuracy of
other testing methods. These tests are not easily
automated and require the laboratory to prepare
stock solutions of antimicrobial agents that are
then dispensed into test tubes (macrodilution),
multiple well plates (microdilution; Figure 1), or
an agar medium. Each tube, well, or agar plate
contains a differing standard concentration of
antibiotic that is inoculated with the specimen.
The MIC can then be determined based on the
level of visible growth. The broth dilution and
agar dilution methods can be used to verify the
accuracy of an automated instrument should a
discrepancy arise.
Agar dilution should not be confused with
newer colorimetric agar tests. Colorimetric agars
contain certain chemicals that cause a color
change when select organisms are present. These
tests are also referred to as agar screening
methodologies. Such agar-based testing is
outside the scope of this review.
Etest
Etest (bioMérieux, Durham, NC) was approved
by the FDA in 1991. It is a manual method of
exact MIC testing that uses a gradient technique
combining the principles of both disk diffusion
and agar dilution. However, Etest differs from
disk diffusion and is applicable for use with a
wide range of fastidious and nonfastidious
aerobic and anaerobic organisms, with varying
growth rates, critical times for antibiotic
absorption, and testing procedures. Etest offers
convenience for hospital laboratories and is
commonly used to complement other routine
automated laboratory antimicrobial susceptibility
testing methods.
Etest uses a preformed and predefined gradient
of varying antibiotic concentrations immobilized
in a dry format onto the surface of a plastic strip.
The concentration of the gradient is calibrated
across a continuous MIC range covering 15 2-fold
dilutions. When applied to the surface of an
inoculated agar plate, the antibiotic on the plastic
strip is transferred to the agar in the form of a
stable continuous gradient directly beneath and in
the immediate vicinity of the strip. The stability
of this antibiotic gradient is maintained for up to
20 hours. After incubation, a parabolic-shaped
inhibition zone centered alongside the test strip
is visible. The MIC is read at the point where the
growth or inhibition margin of the organism
Figure 1. Broth microdilution plate contains standard
dilutions of eight bacterial organisms in each row (denoted by
letters A–H). Each column of wells (denoted by numbers
1–11; 12 is a sterile control) contains a standard antibiotic
concentration that doubles when moving from right to left
(e.g., row 11 = 0.06 µg/ml, row 10 = 0.12 µg/ml, row 9 = 0.25
µg/ml, etc.). The minimum inhibitory concentration (MIC)
is determined by the first well where there is no visible
growth. For example, the MIC of this antibiotic for organism
A is 4 µg/ml, and the MIC for organism B is 8 µg/ml.
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
intersects the edge of the calibrated strip (Figure
2).33 This point can be easily identified for most
organisms. Using a magnifying glass and/or
tilting the plate can be helpful to visualize
microcolonies and hazes or other colonies within
the ellipse of inhibition.
The key advantages of Etest are that it uses a
stable gradient, it is a convenient agar-based
method, and higher inocula can be used to detect
antibiotic resistance. Furthermore, molecular
properties of the antibiotic do not affect the
performance of Etest since the gradient is
preformed and not dependent on diffusion.
Thus, the Etest gradient can accommodate large
Figure 2. This Etest strip contains graduated concen-
trations of ampicillin ranging from 0.016 µg/ml (not shown)
to 256 µg/ml placed on an agar plate growing Escherichia
coli. Since the intersection of the growth-inhibition margin
lies between two minimum inhibitory concentrations
(MICs)—0.38 and 0.5 µg/ml—the test is interpreted at the
highest value (0.5 µg/ml). This organism is defined as
susceptible since the MIC lies below the breakpoint of 8
µg/ml or lower.
1331
molecules, such as vancomycin, and lipophilic
compounds, such as amphotericin, that would
otherwise be a limitation for disk diffusion. 33
Although the Etest complements many of the
laboratory’s functions, it is not used as the sole
method of testing. An Etest is not available for
all antimicrobials. Test setup can be labor
intensive, and there is a propensity for error in
interpretation if the point of intersection with the
strip does not appear to be well marginated or
microcolonies growing in the inhibition area are
missed on visual inspection.
Etest is currently FDA approved for a variety of
antibiotics for clinical testing of aerobes, anaerobes,
pneumococci, streptococci, Haemophilus species,
and gonococci.34 Laboratories may choose to use
an Etest to confirm equivocal results with their
routine systems or test organisms with distinctive
resistance mechanisms that can be difficult to
detect with standard methods. Etest may be
helpful in detecting resistance in organisms that
are highly inoculum dependent, as they can be
falsely reported as susceptible in some automated
systems.35
Automated Systems
Before the 1970s, labor-intensive manual
susceptibility testing was the dominant method.
In 1974, the first automated system known as the
Autobac I disk elution system was introduced by
Pfizer Diagnostics.36 Now, approximately 83% of
clinical laboratories report using an automated
instrument for primary susceptibility testing.37
Several automated systems are available in the
United States. They include Phoenix (Becton
Dickinson, Franklin Lakes, NJ), Vitek (bioMérieux),
MicroScan WalkAway (Siemens Healthcare
Diagnostics, Tarrytown, NY), and Sensititre (Trek
Diagnostics, Cleveland, OH). Most systems
contain a computerized algorithm for inter-
preting results and identifying inconsistencies
between organism identification and suscepti-
bility. These automated systems use commercially
available panels that contain added growth
factors to speed organism growth, thereby
providing more rapid results compared with
traditional methods. They also use sophisticated
software to analyze the growth rates and
determine the antibiotic MIC for the organism by
using specialized decision technology. These
systems do not read the actual MIC, but rather
they use a sophisticated rules system to
determine whether to adjust to a higher or lower
MIC such that the results fit into a doubling
1332 PHARMACOTHERAPY Volume 29, Number 11, 2009
dilution result (e.g., 1, 2, 4, 8 µg/ml, etc.).
Although the general process of identification is
similar, there are differences among each system.
Vitek and Vitek 2
The Vitek was first developed in the 1960s. It
uses small plastic cards containing multiple wells
filled with either biochemical substrates or
antibiotic dilutions. Cards can be used for both
identification and susceptibility testing for most
aerobic gram-positive and gram-negative
organisms. The system uses a manual filler sealer
module to inoculate the cards with the organism.
After inoculation, a reader incubator module uses
a single wavelength of light to measure turbidity
and color changes in the card wells. Robotics
technology is used to move the cards every 15
minutes to different reading areas. The Vitek
then uses regression analysis to calculate the
MIC.38
The Vitek 2 system is the second generation of
Vitek and offers a more sophisticated model of
data analysis as well as a fully automated process
for card identification, organism suspension
dilution, and card filling. Once these steps are
complete, the Vitek 2 seals the cards into a
chamber to prevent contamination during
processing. The cards are then loaded into the
reader incubator, which ejects them at the end of
testing. The Vitek 2 uses colorimetric technology
with use of three wavelengths of light to provide
broad profiles for the most clinically significant
organisms. The data analysis is performed by
using algorithms to look at a variety of parameters
and test conditions to ensure accurate results and
early detection of resistance mechanisms through
the use of proprietary software.38 The Vitek 2
Compact offers a condensed version of the Vitek
2; although it requires more technician involve-
ment before reading the cards, the compact
system is desirable for smaller hospitals or clinics
with limited space. Species identification with
these systems is complete in an average of 3
hours with rapid methods, but may take up to
5.7 hours for slow-growing organisms that
require colorimetric testing methodology.
Susceptibility results may take up to 15 hours,
with a mean of about 9 hours.39, 40
MicroScan WalkAway
The MicroScan offers a choice of an overnight
panel or the more rapid identification-suscep-
tibility panel that uses fluorescent technology. In
contrast to the Vitek system, the MicroScan
panels are the size of conventional microdilution
trays. Depending on the laboratory’s preference,
either a combination identification-susceptibility
panel or separate identification and susceptibility
panels may be used. The WalkAway system
consists of an incubator-reading unit that can
read either as a conventional panel or a fluorescent
rapid-read panel. Panels are manually inoculated
by using a proprietary device known as the
RENOK (rehydrator-inoculator device). Once
the panels are placed into the incubator-reader
unit, 41 the remainder of the process is fully
automated. A bar code reader identifies each
panel, incubates it for the appropriate amount of
time, and moves the panels to the reading position.
Organism identification can be determined within
an average of 2.5 hours by rapid methods but
may take 6–18 hours with use of conventional
testing methodologies. Final results for
susceptibilities take an average of 20 hours, with
a range of 16.8–27.8 hours depending on type of
organism.40, 41
Phoenix
This system uses an optimized colorimetric
oxidation-reduction indicator for susceptibility
testing and a variety of fluorometric and colori-
metric indicators for bacterial identification. The
panels are inoculated manually and require a
specific organism dilution for accuracy. If the
inoculation is incorrect, the system will auto-
matically reject the panel from the incubation
and reading phase. After inoculation, the panels
are loaded into the incubator-reader module, and
all subsequent steps are fully automated. The
Phoenix database then analyzes the kinetic
measurements of bioreactivity within individual
wells. The average time to identifi-cation with
the Phoenix system is 4.3 hours. The time to
final susceptibility results ranges from 7.5–16
hours, with a mean of 10.5 hours.40, 42 Although
all the systems offer some basic epidemiologic
software and data for antibiogram production,
the Phoenix database allows for greater flexibility
and data analysis.42
Sensititre
The Sensititre susceptibility system is a version
of the broth dilution method and can provide
both in vitro qualitative and quantitative
susceptibility results in a dried plate format.
Isolated colonies for testing are diluted according
to a predefined standard and then used to
inoculate a 96-well microdilution plate. A
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
fluorescent precursor may be added at the same
time as the test organism. Inoculated panels are
sealed with adhesive film to prevent evaporation
and then incubated at 34–36°C for 18–24 hours.
During incubation, bacterial surface enzymes will
cleave key bonds on the precursor and trigger the
release of fluorophores, which emit fluorescence.
Unlike the Vitek and Phoenix systems, the
contents of the wells can then be examined
manually for bacterial growth. However,
Sensititre can also be read with an automated
reading system that is able to detect the fluores-
cence. The amount of fluorescence detected is
directly related to the activity of bacterial surface
enzymes and, therefore, to the presence of
bacterial growth and resistance to the antibiotic.
Additional interpretations of susceptibility results
are performed by the software using rules derived
from FDA standards.43
Advantages and Disadvantages
One major advantage of automated suscepti-
bility methodologies is a reduction in labor.44–46
Another advantage is that these systems can
provide faster reporting of susceptibility results,
potentially leading to the earlier initiation of
appropriate antibiotic therapy. Although the
reduction in labor requirements and faster
reporting are significant advantages to using
automation in the microbiology laboratory,
definite disadvantages exist.
First, all automated systems require the manual
step of preparing an inoculum. This step is
critical to the accuracy of the results because
over- or underinoculating or mixing cultures can
result in false or misleading results. Second, all
automated systems can test only a limited range
of organisms. Slow-growing or fastidious
organisms must still be tested by alternative
methods. Mucoid Pseudomonas aeruginosa
isolated from patients with cystic fibrosis also
presents a challenge for automated systems
because of the thick cell wall caused by
exopolysaccharide-alginate and its slow growth.
In one study, antibiotic susceptibilities for 498
strains from patients with cystic fibrosis (one
third of which were mucoid) were performed by
using Vitek and MicroScan WalkAway systems,
and results were compared with those of a
reference broth microdilution method. “Very
major” error rates (i.e., erroneously reported as
susceptible, yet determined to be resistant by a
reference method) were 17% and 10.4–12.4%,
respectively. (See Agreement Among Methods
1333
section for a detailed description of types of
errors associated with testing.) The authors
concluded that these commercial systems
performed poorly for cystic fibrosis isolates in
contrast to high result correlations that had been
reported previously with reference methods and
Etest.47
Panel and Card Selection
All of the automated systems offer several
panel choices for the susceptibility portion of the
testing. Panels and cards are separated into
gram-positive, gram-negative, and fungal panels
(for select systems).48 Each manufacturer’s Web
site provides examples of the potential panels
and cards. The choice of panel should be aligned
with the hospital’s antimicrobial formulary.
Pharmacy personnel play an important role in
panel selection by ensuring that formulary
antibiotics are reported on the culture and
sensitivity reports.
Depending on the system, separate cards may
need to be purchased for organism identification,
as well as gram-positive and gram-negative
susceptibilities. The identification panel may be
used alone when susceptibilities are not clinically
significant. For example, Lactobacillus and
Pasteurella species are not tested because approved
susceptibilities are lacking, and Staphylococcus
saprophyticus will only be identified because
susceptibilities will not change the course of
therapy.
Interchangeability between panels is limited by
several factors. Whenever a new panel or card is
used, a series of quality assurance tests must be
performed before starting the new panel; this
may take an average of 20–30 days and limits the
number of times that panels can be changed.
Most microbiology laboratories purchase supplies
in bulk to obtain better pricing. In an effort to
reduce cost, the laboratory may need to finish the
existing panel supply before proceeding to a new
one. This can be a challenge when changes are
made to the antibiotic formulary. If the new
antibiotic is not on the susceptibility panel, then
manual tests must be conducted to verify
susceptibilities to the new agents and the results
for nonformulary antibiotics need to be
suppressed. Customized testing panels can be
developed for a specific institution based on their
specific formulary or to increase the number of
dilutions tested to provide a better range of
MICs. However, this may not be a cost-effective
strategy, especially for institutions with dynamic
1334 PHARMACOTHERAPY Volume 29, Number 11, 2009
formularies. Finally, testing space on the antibiotic
susceptibility cards is not infinite, and therefore
not all MICs can be tested. Generally, the range
of MICs tested are doubling dilutions (e.g., 0.25,
0.5, 1, 2 µg/ml). In some cases, only the break-
point is reported (e.g., MIC ≤ 2 µg/ml).
Special Considerations in Antimicrobial
Susceptibility Testing
Fastidious Organisms
As their category implies, most fastidious
organisms do not grow well enough in automated
antimicrobial testing systems and require some
type of supplementation (e.g., blood). Disk
diffusion was initially developed for susceptibility
testing of rapidly growing aerobic bacteria,
including enterococci, staphylococci, Enterobacte-
riaceae, and P. aeruginosa. Modifications for
performing disk diffusion testing with certain
fastidious organisms such as H. influenzae,
Neisseria gonorrhea, Streptococcus pneumoniae,
and other Streptococcus species have been
described by CLSI and include using agar that is
enriched with glucose, yeast extract, and other
substances. Modifications for broth microdilution
can also be used for testing fastidious organisms,
although results must be read manually and not
by automated instruments.49 Automated testing
methodologies can be developed to correlate
results that are obtained with CLSI recommen-
dations for testing of fastidious organisms.
Because of the stability of the gradient, Etest
can also be used for testing very slow-growing
fastidious organisms, as previously mentioned.
Clinical situations often warrant susceptibility
testing of rarely encountered, unusual, and/or
opportunistic pathogens for which no CLSI
guidelines are available. In these cases, scientific
references can be used to guide the choice of
appropriate media, incubation conditions, and
antibiotics to test. Because of the lack of
guidelines, accompanying interpretations may be
difficult to find, so the MIC value is reported
without interpretation. These situations should
be handled on an individual basis in consultation
with an infectious diseases physician. Factors
that may influence the course of action include
the organism(s), site of infection, pharmacokinetics
of the antibiotic, and previous experience with a
similar antibiotic for treating the same condition.
Anaerobes
Most hospital laboratories do not routinely
perform anaerobic susceptibilities and must send
the sample out to a reference laboratory if
requested. Indications for anaerobic susceptibility
testing include the management of patients with
serious infections requiring long-term therapy
and selection of appropriate therapy for
organisms that are not predictably susceptible.
The CLSI recommends that agar dilution (known
as the Wadsworth method) be used as the
reference method for anaerobic susceptibility
testing.50 A broth microdilution method is also
approved but is limited to rapidly growing
anaerobes (Bacteroides species). Another
disadvantage is that the method is only described
for a limited number of antimicrobials, and the
panels are not commercially available. Because of
these limitations, performing susceptibility
testing on obligate anaerobes is not a standard
procedure. 51 However, due to the increasing
resistance rates reported among some anaerobes
in recent years, the CLSI recommends suscep-
tibility testing for clinically significant anaerobes
(e.g., B. fragilis, Prevotella species, and Fusobac-
terium species) and creating an antibiogram.52–55
The Etest can be used for testing individual isolates
as well as batch-testing for an antibiogram.
Mycobacteria
Because of minimal equipment requirements
for acid-fast bacilli smears and their ability to
produce rapid results, almost all laboratories
have the capacity to conduct these tests. Further
identification and susceptibility testing of
mycobacteria are generally performed in larger
institutions or reference laboratories with the
equipment necessary to further process these
organisms. A negative pressure room and a
sterile hood are required, along with a specialized
set of materials and techniques specific to the
selection and testing of mycobacteria. Clinical
laboratories that test mycobacteria use an
automated or semiautomated broth-based system
for growth as well as susceptibility of Mycobac-
terium tuberculosis. Susceptibility testing of
rapidly growing mycobacteria can be performed
by either broth microdilution or by Etest.56, 57
Specialized Screening and Confirmatory Tests
for Resistant Organisms
Microbiologists can use certain antibiotics to
screen for key resistance patterns among common
bacteria. Screening tests may differ from
traditional susceptibility methods in some cases
because the antibiotic used to detect the resis-
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
tance may be incongruous with the antibiotic
selected for treatment. Most screening tests used
in the clinical laboratory are designed to detect
methicillin-, oxacillin-, or vancomycin-resistant
gram-positive organisms (most commonly MRSA,
vancomycin-resistant Enterococcus, and ␤-
lactamase–producing gram-negative organisms).
There are several methods that can be used to
detect oxacillin resistance among S. aureus. In
addition to disk diffusion or broth microdilution,
other methodologies used include the oxacillin
salt agar screen and the cefoxitin disk screen.12 A
latex agglutination test for penicillin-binding
protein 2a is also available, but this is not
commonly used in the traditional hospital
microbiology laboratory because of the ease of
other methods and cost. All methods vary in
terms of their specificity and sensitivity, and the
results can vary depending on strain type.58, 59
The oxacillin salt agar screen test uses Mueller-
Hinton agar supplemented with 4% sodium
chloride and oxacillin 6 µg/ml inoculated with a
0.5 McFarland suspension of the organism. The
plates are incubated for 24 hours at a maximum
temperature of 35 o C. Testing at higher
temperatures may interfere with the detection of
methicillin resistance.23 The appearance of more
than one colony indicates resistance. This screen
has a high sensitivity for detecting resistant
strains, but sensitivity decreases when very
heteroresistant strains are tested or when the
MIC is borderline.59, 60 Recently, the cefoxitin
disk screen has been recognized as a better
predictor of methicillin resistance than many
other classic methods because cefoxitin is a more
potent inducer of mecA than are penicillins.61
The substitution of a cefoxitin disk for an
oxacillin disk results in an easier test for the
microbiologist to read and has similar sensitivity
and specificity to detect oxacillin resistance in
Staphylococcus species. The incubation time
required for testing S. aureus and Staphylococcus
lugdunensis is only 16 hours,23 but some studies
have shown that results can be achieved even
earlier.62 In addition, the cefoxitin disk screen
has equal sensitivity but improved specificity in
coagulase-negative Staphylococcus species. The
disadvantage of this test is that it may falsely
detect resistance in some mecA-negative strains
and may fail to detect resistance in some strains
of mecA-positive Staphylococcus simulans.63
Brain-heart infusion agar supplemented with
vancomycin 6 µg/ml can be used to screen for
vancomycin resistance in S. aureus isolates with a
vancomycin MIC of 4 µg/ml or more.64 Organisms
1335
that grow on the agar should be suspected as
possible vancomycin-intermediate or vancomycin-
resistant S. aureus and confirmed by using a
validated MIC method such as broth dilution,
agar dilution, or Etest, or through approved
antimicrobial susceptibility testing.65 Screening
and detection of heterogeneous vancomycin-
intermediate S. aureus presents a significant
challenge in the clinical laboratory. Although
methods exist in research, there is no standard-
ized method for identifying heterogeneous
vancomycin-intermediate S. aureus consistently
in the clinical microbiology laboratory.66
Brain-heart infusion agar supplemented with
vancomycin 6 µg/ml can also be used as a screen
for vancomycin resistance in enterococci. 67
Plates are inoculated, incubated, and interpreted
in the same manner as the MRSA screen agar
plates described previously. Additional screening
agars are also available for testing for high-level
aminoglycoside resistance in enterococci by
using brain-heart infusion agar supplemented
with gentamicin 500 µg/ml and streptomycin
2000 µg/ml. The presence of high-level amino-
glycoside resistance indicates a lack of synergistic
effect when an aminoglycoside is combined with
a cell-wall inhibitor. Gentamicin high-level
resistance is associated with high-level resistance
to other aminoglycosides, such as tobramycin,
netilmicin, amikacin, and kanamycin. Streptomycin
resistance occurs by means of a separate
mechanism. This is the justification for testing
both gentamicin and streptomycin for high-level
resistance in enterococci.68
Some strains of macrolide-resistant S. aureus
and coagulase-negative Staphylococcus species
have a transferable resistance mechanism known
as macrolide-lincosamide-streptogramin B
(MLSB) resistance. This inducible resistance can
result in clindamycin treatment failures,69, 70 but
it can be detected by using a disk approximation
test known as the double disk diffusion, which is
frequently referred to as the D-test. A 2-µg
clindamycin disk is placed 15–26 mm away from
the edge of a 15-µg erythromycin disk on
Mueller-Hinton agar. After incubation, when
inducible resistance is not present, the zone of
inhibition around the clindamycin disk will
appear as a concentric circle. However, if the
edge of the clindamycin zone closest to the
erythromycin disk is flattened (thus causing the
D shape), then the isolate is reported as
clindamycin resistant (Figure 3).12 Although the
CLSI recommends a distance of up to 26 mm,
one recent study found that using a 22-mm
1336 PHARMACOTHERAPY Volume 29, Number 11, 2009
distance was associated with inaccuracies, but
placing the disks 15 mm apart resulted in 100%
sensitivity and specificity.71
One of the most frequently used screening tests
detects ␤-lactamases, of which the most common
among gram-negative bacteria is the enzyme
known as TEM-1. This enzyme causes ampicillin
and penicillin resistance in H. influenzae and N.
gonorrhea.72 Most hospitals will perform a ␤-
lactamase screening test for these two organisms
because these ␤-lactamases may produce low-
level resistance that may go undetected. There
are three categories of ␤-lactamase tests:
colorimetric, acidimetric, and iodometric. In the
hospital microbiology laboratory, the colorimetric
method is the most commonly used and involves
sticks or disks impregnated with certain
compounds such as nitrocefin or cephalosporin-
pyridinium-2-azo-p-dimethylaniline that produce
a color change when the ␤-lactam is hydrolyzed.
Acidometric and iodometric tests are referred to
as linked detection systems because more than
one chemical reaction must occur to produce an
interpretive result. 73 These tests are typically
performed by using penicillin as a substrate and
therefore may only detect enzymes that hydrolyze
penicillins.74 These ␤-lactamase tests also may be
performed on Moraxella catarrhalis. However,
because ␤-lactamase–positive M. catarrhalis is so
Figure 3. Positive double disk diffusion test (D-Test) shows
induction of clindamycin (CC) resistance by erythromycin
(E) in this methicillin-resistant Staphylococcus aureus
isolate. This is indicated by the blunting of the clindamycin
zone of inhibition, which appears as a D shape.
frequent, it is safe to assume resistance will be
present, and treatment with a ␤-lactam–enzyme
inhibitor combination will be necessary.
Hospital microbiology laboratories also have
the ability to detect extended-spectrum ␤-
lactamases (ESBLs). Although ESBLs have been
identified in many organisms,75 CLSI procedures
for screening and confirmation of ESBLs exist
only for Klebsiella pneumoniae, Klebsiella oxytoca,
Escherichia coli, and clinically relevant Proteus
mirabilis strains (e.g., a bacteremic isolate). 12
Screening for ESBLs in E. coli, K. pneumoniae, or
K. oxytoca involves performing a preliminary
susceptibility test against cefpodoxime, ceftazidime,
aztreonam, cefotaxime, or ceftriaxone. The process
is similar for P. mirabilis but does not involve the
use of aztreonam or ceftriaxone. If the results
suggest the presence of an ESBL (based on CLSI
procedure), a phenotypic confirmatory test is
performed by using either disk diffusion or broth
microdilution. The disk diffusion confirmatory
test involves placing a 30-µg ceftazidime disk and
a 30-µg cefotaxime disk, both alone and in
conjunction with clavulanic acid 10 µg, on a
Mueller-Hinton agar plate. If the difference
between the zone diameter for the single antibiotic
versus the combination with clavulanic acid is 5
mm or greater, then the organism is classified as
ESBL-positive. For broth microdilution, the
ESBL test is positive if a three or more 2-fold
concentration decrease in the MIC occurs between
the original single antibiotic (e.g., ceftazidime or
cefotaxime) and the combination of the reference
antibiotic with clavulanic acid (e.g., going from 8
µg/ml with a single antibiotic to 1 µg/ml or less
with the inhibitor present).12 Etest strips are also
available that contain cefotaxime or ceftazidime
on one end of the strip and cefotaxime or
ceftazidime plus clavulanic acid on the other end
of the strip (Figure 4).33 The interpretation for
the Etest ESBL test is the same as that for broth
microdilution. Several automated systems can
also be used to detect ESBLs. Early and appro-
priate detection of ESBL-producing organisms
allows for better customization of therapy, which
can lead to improvements in morbidity and
mortality.76
In addition to ESBLs, there are several ␤-
lactamase–producing enzymes that may also
affect interpretation and antibiotic selection.
One of the most common is the AmpC type ␤-
lactamases. The AmpC ␤-lactamases are
commonly isolated from extended-spectrum,
cephalosporin-resistant, gram-negative bacteria
such as Serratia, Enterobacter, and Citrobacter
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
species. 77 The AmpC enzymes are inhibitor-
resistant ␤-lactamases. They are resistant to
clavulanic acid and other ␤-lactamase inhibitors
but susceptible to cloxacillin. High level of
expression of AmpC may prevent recognition of
an ESBL because of this resistance to clavulanic
acid, which is used to detect the presence of
ESBLs.77 The presence of AmpC ultimately could
lead to a false-negative ESBL screen. One
possible solution is to include cefepime as an
ESBL screening agent because high-level AmpC
expression is minimally affected with this drug.
There is also a commercially available Etest for
the detection of AmpC, but it is not FDA
approved. The Etest contains a double-sided
Figure 4. Confirmatory extended-spectrum ␤-lactamase
(ESBL) with use of the Etest. The strip on the left contains
cefotaxime + clavulanic acid (CTL) and cefotaxime (CT).
The strip on the right contains ceftazidime + clavulanic acid
(TZL) and ceftazidime (TZ). The organism is ESBL-positive
based on the deformation of the CT ellipse, but may also be
determined by the CT and TZ minimum inhibitory
concentrations (MICs) and the CT:CTL MIC ratio or the
TZ:TZL MIC ratio.33
1337
strip whereby one side contains a cephamycin
(e.g., cefotetan or cefoxitin) alone and the other
side contains a cephamycin with cloxacillin. A
positive test for AmpC is noted if the cephamycin
MIC decreases by three or more 2-fold dilutions
in the presence of cloxacillin or a deformation of
the inhibition ellipses around the Etest.78, 79
Organisms with increased carbapenem MICs
are of great concern. The metallo-␤-lactamases
are carbapenem-hydrolyzing ␤-lactamases that
emerged in Japan in the 1990s.80 These enzymes
effectively hydrolyze both ␤-lactam antibiotics
and carbapenems except aztreonam. Although
the clinical prevalence of these enzymes is low,
they have been reported in Enterobacter and
Pseudomonas species. Because of difficulty of
detection, a screening method for metallo-␤-
lactamases can be used when an increase in the
MIC of carbapenems is observed. 81, 82 The
metallo-␤-lactamase enzymes are zinc mediated
and can be repressed by using ethylene-
diaminetetraacetic acid (EDTA). A commercially
available Etest may be used for metallo-␤-
lactamase detection, but it is not FDA approved.
One half of the Etest strip is impregnated with
imipenem and the other half contains imipenem
plus EDTA. A reduction in the MIC of imipenem
of three or more 2-fold dilutions in the presence
of EDTA represents a positive metallo-␤-
lactamase organism.82, 83
Carbapenemase-producing Enterobacteriaceae
(CPE), such as K. pneumoniae, are endemic in the
northeastern part of the United States 84 and
present a new challenge in antimicrobial
susceptibility testing and breakpoint reporting.
They can be missed by automated testing
systems85 and may require manual testing. Only
recently have formalized screening and
confirmatory standards been developed by the
CLSI. 12 For screening purposes, a zone of
inhibition of 19–21 mm for an ertapenem 10-µg
disk or a zone of 16–21 mm for a meropenem 10-
µg disk suggests a possible CPE. No inter-
pretative standards exist for imipenem since it
performs poorly as a screen for carbapenemases.
Broth microdilution using imipenem, meropenem,
and ertapenem 1 µg/ml can also be used as a
screen. An MIC of 2–4 µg/ml for imipenem and
meropenem or an MIC of 2 µg/ml for ertapenem
may indicate the presence of a carbapenemase
despite being considered susceptible based on
standard interpretations. To confirm the presence
of a CPE, a modified Hodge test must be
performed. This test involves streaking CPE-
positive and -negative reference strains along
1338 PHARMACOTHERAPY Volume 29, Number 11, 2009
with the clinical isolate onto a Mueller-Hinton
agar plate that has already been inoculated with a
reference strain of E. coli. An ertapenem disk is
placed in the center. If the test is read as positive,
the CLSI suggests that the actual carbapenem
MIC be reported along with a comment indicating
that the isolate demonstrates carbapenemase
production and that the clinical efficacy of
treating these organisms with carbapenems has
not been established. If the test is negative, the
CLSI recommends interpreting the carbapenem
MICs by using current interpretative breakpoints.
Interpretation
On completion of antimicrobial susceptibility
testing and determination of the MIC or zone
diameter, the organism may be classified into one
of three interpretative categories: susceptible,
intermediate, or resistant. There is an inverse
relationship between results and the inter-
pretative categories when conducting MIC
testing versus disk diffusion testing. Organisms
with an MIC less than (or equal to) a certain
concentration (µg/ml) or a zone diameter greater
than (or equal to) a specific millimeter measure-
ment are considered to be susceptible and will
likely respond to treatment with a standard
antibiotic dosage as indicated. Organisms are
considered to have intermediate susceptibility to
an antibiotic when the MICs approach the top
end of the usually achievable serum concentrations
for a standard dose or when the zone diameter
falls within a certain range. The intermediate
category also serves as a buffer zone to help
prevent major categoric errors caused by slight
changes in the zone sizes due to the influence of
technical variables. Antibiotics used for the
treatment of intermediately susceptible organisms
may achieve clinical success when the antibiotic
being used concentrates at the site of infection
(e.g., fluoroquinolones for urinary tract infec-
tions) or when a higher than normal dosage is
used (e.g., ␤-lactams). If the MIC is greater than
(or equal to) or the zone diameter is less than (or
equal to) the resistant breakpoint, this implies
that the infection is not likely to respond to
therapy because the physiologic concentrations
required to overcome resistance would cause
toxicity in humans.
There are some caveats to these interpretative
categories. For some organisms, such as P.
aeruginosa, there are no interpretative categories
for disk diffusion tests, only MIC tests. For S.
pneumoniae, breakpoints vary depending on the
source of the isolate. For example, S. pneumoniae
with a penicillin MIC of 1 µg/ml in a patient with
meningitis would be defined as resistant since it
is above the defined resistant breakpoint of 0.12
µg/ml or greater. The same isolate from a
nonmeningeal source, such as sputum, would be
considered susceptible because the MIC fell
below the susceptible breakpoint of 2 µg/ml or
lower for nonmeningitis isolates. This
differentiation by site of infection also exists with
␤-lactam–␤-lactamase inhibitors and intravenous
cephalosporins. Some antibiotic-organism
combinations only have a susceptible and
nonsusceptible or susceptible and resistant
categorization. For example, the susceptibility
breakpoint for doripenem tested against
Enterobacteriaceae is 0.5 µg/ml or lower, whereas
any MIC above this is only granted a non-
susceptible classification. For piperacillin-
tazobactam and P. aeruginosa, there is no
intermediate interpretative standard, only
susceptible (MIC ≤ 64/4 µg/ml) or resistant (MIC
≥ 128/4 µg/ml).17, 86
Agreement Among Methods
Because of the impracticality of reference
methods for routine susceptibility testing in the
clinical microbiology laboratory, automated
susceptibility testing methods and the Etest have
become the microbiologist’s workhorse tools.
However, these nonreference method testing
systems must show that the results obtained with
the test system agree with the results obtained
from a reference method. The FDA sets
standards to ensure new devices and new drugs
to be added to the panels of the automated
systems are substantially equivalent to a reference
method for determining susceptibility testing.
The reference methods for susceptibility testing
are broth dilution and agar dilution.87
When conducting these studies (frequently
referred to as a 510[k]), the testing device is
being evaluated to determine if the results are the
same as those of a proven reference method. A
testing system is said to have “essential agree-
ment” when the device under evaluation has an
MIC within ± one 2-fold dilution compared with
the reference method MIC determination.
“Categorical agreement” is when the interpretive
results (susceptible, intermediate, or resistant)
between a new device under evaluation and a
standard reference method are the same, by using
FDA interpretive criteria as presented in the
FDA-approved pharmaceutical antimicrobial
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
agent package insert. It is possible for there to be
essential agreement with categorical disagree-
ment. When this occurs, it is considered a “minor
error” and frequently results in an intermediate
susceptibility with one method and susceptible
or resistant with another method. For drugs
without an intermediate category, there cannot be
minor errors. A “major error” occurs when the
testing method determines that the organism is
resistant, but the reference method determines
that the organism is susceptible. Finally, a “very
major error” occurs when the converse is true—
the testing method indicates the organism is
susceptible but the reference method indicates
resistant (Table 2).
The FDA has issued guidance that outlines
acceptable performance when susceptibility
testing is conducted by methods other than a
reference method. 87 First, the percentage
categorical and essential agreement must be
greater than 89.9%. Categorical agreement less
than 90% may be acceptable if there is very good
essential agreement, with the majority of the
discrepancies as minor discrepancies. In
addition, the major error rate must be less than
3% of all the susceptible organisms tested.
Finally, the very major error rate based on the
number of resistant organisms tested must
include an upper 95% confidence limit for the
true very major error rate of 7.5% or less, and the
lower 95% confidence limit for the true very
major error rate of 1.5% or less. If 100 resistant
organisms were tested, the very major error rate
should not be greater than 2/100 (95%
confidence interval 0.24–7.04).
Two studies demonstrate these concepts for
gram-negative and gram-positive organisms. In
the first study, the investigators tested 101
bacteremic isolates of MRSA recovered from
2002–2006.88 Each isolate was tested by using
both the broth microdilution and agar dilution
methods, and the results were compared with
those of Etests performed on two different brands
of Mueller-Hinton agar plates. Vancomycin MIC
results from the Etests were consistently 1–2-fold
higher than the MICs determined with the
reference methods. In the second study, the
investigators evaluated the accuracy of three
automated testing systems and tested the
susceptibility of five broad-spectrum ␤-lactams
against 100 strains of P. aeruginosa.89 Although
the rate of major errors was minimal, the
investigators noted that two of the systems
produced minor error rates of 8–32% for
cefepime and that all three systems had minor
1339
Table 2. Agreement Categories According to Reference
Method versus Test Method Results
Reference Test Method Agreement
Method Result Result Category
Resistant Resistant Agreement
Intermediate Minor error
Susceptible Very major error
Intermediate Resistant Minor error
Intermediate Agreement
Susceptible Minor error
Susceptible Resistant Major error
Intermediate Minor error
Susceptible Agreement
error rates when testing aztreonam. Clinically,
these discrepancies could manifest as lower-than-
expected susceptibility rates on a hospital
antibiogram. If there are sudden sharp declines
in susceptibility of P. aeruginosa to cefepime,
confirmatory testing of a subset of isolates using
another testing method may be prudent.
Clinical Practice Application
Patients infected with an organism that is
considered susceptible to an antibiotic do not
respond 100% of the time; conversely, patients
who are infected with an organism that is
resistant to an antibiotic do not universally fail
treatment. Infections caused by susceptible
isolates respond to appropriate therapy
approximately 90% of the time, whereas
infections caused by resistant isolates actually
respond to the inappropriate antibiotic about
60% of the time—the “90-60” rule.8 Either the
susceptibility methodologies or patient factors
likely explain the variability seen in patient
outcomes based solely on susceptibility results.
Thus, the interpretation should be used as part of
the antibiotic therapy selection process. An
understanding of the antibiotic’s pharmacokinetic
properties, the antibiotic’s MIC for the infecting
organism, and the pharmacodynamic relationship
between the two can help boost the patient’s
outcome to the 90% clinical success rate.
Although there are hundreds of in vitro
microbiology tests that can be performed in the
hospital microbiology laboratory, maintaining a
working knowledge of common clinical
laboratory tests enables pharmacists to become
valuable resources in helping to translate bench
results into bedside application. For example,
many practitioners interpret the results of culture
and susceptibility tests at “face value” and do not
understand the discordance between laboratory
1340 PHARMACOTHERAPY Volume 29, Number 11, 2009
tests and clinical interpretation. Some practi-
tioners associate a lower MIC with increased
efficacy. However, this is erroneous because each
organism-drug combination varies and the MIC
does not reflect pharmacodynamics or patient-
specific factors such as renal function, site of
infection, or volume of distribution. Pharmacists
can translate these objective measurements into
meaningful decisions for clinicians who deliver
patient care.
Collective susceptibility testing results, compiled
as an antibiogram, allow hospitals to use local
susceptibility data to guide empiric therapy
before culture and susceptibility results. This
enables the clinician to preliminarily “predict”
the susceptibility of the infecting organism and
ensure the early, appropriate therapy associated
with improved patient outcomes.90–92 The ability
to recognize erroneous results in an antibiogram
is important. As previously mentioned, there are
certain known limitations with both automated
and manual systems that can influence results.
Under-standing these discrepancies can help
institutions distinguish between true resistance
issues and laboratory testing errors. Advances in
rapid diagnostic testing will undoubtedly
influence antibiotic utilization and ultimately
patient outcomes. The higher cost of the tests
and laboratory resources may be offset by
reductions in unnecessary drug therapies.
The benefits of applied microbiology principles
in the clinical pharmacy setting are numerous
and are summarized as follows:
Results interpretation
• Detecting antibiogram inaccuracies
• Translating how changes in CLSI or FDA
breakpoints affect antibiotic susceptibility
trending
• Recognizing the influence of human factors
on test results
• Recognizing organism–antibiotic testing
mismatches
• Assisting in the selection of antimicrobial
susceptibility testing panels based on
formulary
Clinical therapeutics
• Pharmacodynamic dosing based on patient-
level MIC data
• Antimicrobial stewardship
• Translating susceptibilities into empiric
therapy guidelines
• Educating prescribers to optimize antimicrobial
selection
• Reducing patient morbidity and mortality
Financial savings
• Researching alternative sources for manual
susceptibility testing from pharmaceutical
manufacturers
• Reducing total health care resource con-
sumption through early and appropriate
antimicrobial therapy
Conclusion
Maintaining an active knowledge base of the
various antimicrobial susceptibility testing
methods and their limitations can be challenging.
Qualitative testing methods are helpful and easily
reproduced in the clinical laboratory setting with
minimal investment in equipment and personnel.
However, errors in specimen preparation, disk
placement, and zone interpretation can lead to
inaccurate results. Quantitative methods are
more accurate and can provide specific MICs that
can be used to optimize the pharmacodynamics
of antibiotic therapy. Agar and broth dilution
methods are the most accurate, but are difficult
to perform in a busy hospital microbiology
laboratory. The development of Etests and the
deployment of automated testing systems in most
hospital laboratories have allowed for faster
result interpretation and earlier initiation of
appropriate therapy. However, clinicians must be
aware of the potential for major and minor errors
that can occur between these systems.
Numerous screening tests exist and can help
the clinician identify key resistance issues
secondary to the presence of certain enzymes.
These tests are increasingly valuable as resistance
patterns continue to emerge. The development
of molecular-based technologies will allow for
faster identification of select resistant organisms,
thus shortening the duration of time to appro-
priate, targeted therapy. Clinicians who have a
thorough understanding of the strengths and
weaknesses of these various testing methodologies
can successfully apply this information to benefit
both individual patient care and global antibiotic
stewardship within their institution.
Acknowledgments
The authors would like to thank Sheila Maness,
M.T., for assisting with content development and
Allison Krug, M.P.H., of Artemis Biomedical
Communications, LLC, for assistance with editing the
final manuscript.
References
1. Dellit TH, Owens RC, McGowan JE Jr, et al. Infectious
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
Diseases Society of America and the Society for Healthcare
Epidemiology of America guidelines for developing an
institutional program to enhance antimicrobial stewardship.
Clin Infect Dis 2007;44:159–77.
2. Kollef MH. Inadequate antimicrobial treatment: an important
determinant of outcome for hospitalized patients. Clin Infect
Dis 2000;31(suppl 4):S131–8.
3. Biedenbach DJ, Jones RN, Erwin ME. Interpretive accuracy of
the disk diffusion method for testing newer orally administered
cephalosporins against Morganella morganii. J Clin Microbiol
1993;31:2828–30.
4. Jevitt LA, Thorne GM, Traczewski MM, et al. Multicenter
evaluation of the Etest and disk diffusion methods for
differentiating daptomycin-susceptible from non–daptomycin-
susceptible Staphylococcus aureus isolates. J Clin Microbiol
2006;44:3098–104.
5. Lo-Ten-Foe JR, de Smet AM, Diederen BM, Kluytmans JA, van
Keulen PH. Comparative evaluation of the VITEK 2, disk
diffusion, Etest, broth microdilution, and agar dilution
susceptibility testing methods for colistin in clinical isolates,
including heteroresistant Enterobacter cloacae and Acinetobacter
baumannii strains. Antimicrob Agents Chemother
2007;51:3726–30.
6. Tenover FC, Williams PP, Stocker S, et al. Accuracy of six
antimicrobial susceptibility methods for testing linezolid
against staphylococci and enterococci. J Clin Microbiol
2007;45:2917–22.
7. Wiegand I, Geiss HK, Mack D, Sturenburg E, Seifert H.
Detection of extended-spectrum ␤-lactamases among
Enterobacteriaceae by use of semiautomated microbiology
systems and manual detection procedures. J Clin Microbiol
2007;45:1167–74.
8. Rex JH, Pfaller MA. Has antifungal susceptibility testing come
of age? Clin Infect Dis 2002;35:982–9.
9. Turnidge J, Paterson DL. Setting and revising antibacterial
susceptibility breakpoints. Clin Microbiol Rev 2007;20:
391–408.
10. Infectious Diseases Society of America. Statement of the
Infectious Diseases Society of America before the Food and
Drug Administration part 15 hearing panel on antimicrobial
resistance. April 28,2008. Arlington, VA: Infectious Diseases
Society of America, 2008.
11. Clinical and Laboratory Standards Institute. Our history.
Available from http://www.clsi.org. Accessed April 13, 2009.
12. Clinical and Laboratory Standards Institute. Performance
standards for antimicrobial susceptibility testing; 19th
informational supplement. Approved standard M100-S19.
Wayne, PA: Clinical and Laboratory Standards Institute, 2009.
13. JHP Pharmaceuticals. Coly-Mycin M parenteral (colistemethate
sodium) package insert. Rochester, MI; 2007.
14. Roche Laboratories, Inc. Rocephin (ceftriaxone sodium)
package insert. Nutley, NJ; 2009.
15. Cubist Pharmaceuticals, Inc. Cubicin (daptomycin) package
insert. Lexington, MA; 2008.
16. Wyeth Pharmaceuticals, Inc. Tygacil (tigecycline) package
insert. Philadelphia, PA; 2009.
17. Ortho-McNeil Pharmaceutical, Inc. Doribax (doripenem)
package insert. Raritan, NJ; 2009.
18. Abraxis Pharmaceutical Products. Polymixin B package insert.
Schaumburg, IL; 2008.
19. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic
susceptibility testing by a standardized single disk method. Am
J Clin Pathol 1966;45:493–6.
20. Sutter VL, Kwok Y, Finegold SM. Susceptibility of Bacteroides
fragilis to six antibiotics determined by standardized
antimicrobial disc susceptibility testing. Antimicrob Agents
Chemother 1973;3:188–93.
21. Laverdiere M, Welter D, Sabath LD. Use of a heavy inoculum
in the in vitro evaluation of the anti-staphylococcal activity of
19 cephalosporins. Antimicrob Agents Chemother
1978;13:669–75.
22. Ericsson H. The paper disc method for determination of
bacterial sensitivity to antibiotics: studies on the accuracy of
1341
the technique. Scand J Clin Lab Invest 1960;12:408–13.
23. Clinical and Laboratory Standards Institute. Performance
standards for antimicrobial disk susceptibility tests; approved
standard—tenth edition, M02-A10. Wayne, PA: Clinical and
Laboratory Standards Institute, 2009.
24. Baddour LM, Wilson WR, Bayer AS, et al. Infective
endocarditis: diagnosis, antimicrobial therapy, and management
of complications: a statement for healthcare professionals from
the committee on rheumatic fever, endocarditis, and Kawasaki
disease, council on cardiovascular disease in the young, and the
councils on clinical cardiology, stroke, and cardiovascular
surgery and anesthesia, American Heart Association, endorsed
by the Infectious Diseases Society of America. Circulation
2005;111:e394–434.
25. Soriano A, Marco F, Martínez JA, et al. Influence of
vancomycin minimum inhibitory concentration on the
treatment of methicillin-resistant Staphylococcus aureus
bacteremia. Clin Infect Dis 2008;46:193–200.
26. Lodise TP, Graves J, Graffunder E, et al. Relationship between
vancomycin MIC and failure among patients with methicillin-
resistant Staphylococcus aureus bacteremia treated with
vancomycin. Antimicrob Agents Chemother 2008;52:3315–20.
27. Forrest A, Nix DE, Ballow CH, Goss TF, Birmingham MC,
Schentag JJ. Pharmacodynamics of intravenous ciprofloxacin in
seriously ill patients. Antimicrob Agents Chemother
1993;37:1073–81.
28. Mohr JF, Wanger A, Rex JH. Pharmacokinetic/pharmacodynamic
modeling can help guide targeted antimicrobial therapy for
nosocomial gram-negative infections in critically ill patients. Diagn
Microbiol Infect Dis 2004;48:125–30.
29. Rafati MR, Rouini MR, Mojtahedzadeh M, et al. Clinical
efficacy of continuous infusion of piperacillin compared with
intermittent dosing in septic critically ill patients. Int J
Antimicrob Agents 2006;28:122–7.
30. Tam VH, McKinnon PS, Akins RL, Rybak MJ, Drusano GL.
Pharmacodynamics of cefepime in patients with gram-negative
infections. J Antimicrob Chemother 2002;50:425–8.
31. Hamilton-Miller JM, Shah S, Loebenberg D. Susceptibility of
pneumococci to evernimicin: effect of CO 2 and different
methodologies. Clin Microbiol Infect 2001;7:339–40.
32. Johnson J, Bouchillon S, Pontani D. The effect of carbon
dioxide on susceptibility testing of azithromycin,
clarithromycin and roxithromycin against clinical isolates of
Streptococcus pneumoniae and Streptococcus pyogenes by broth
microdilution and the Etest: Artemis project—first-phase study.
Clin Microbiol Infect 1999;5:327–30.
33. AB Biodisk. Etest technical manual. Available from
http://www.abbiodisk.com/pdf/etm_html/01_etm.htm. Accessed
April 13, 2009.
34. AB Biodisk. Product list for the USA 2008. Available from
http://www.abbiodisk.com/pdf/pricelist/M0000549.pdf.
Accessed April 13, 2009.
35. Jones RN, Biedenbach DJ, Marshall SA, Pfaller MA, Doern
GV. Evaluation of the Vitek system to accurately test the
susceptibility of Pseudomonas aeruginosa clinical isolates against
cefepime. Diagn Microbiol Infect Dis 1998;32:107–10.
36. Felmingham D, Brown DF. Instrumentation in antimicrobial
susceptibility testing. J Antimicrob Chemother 2001;48(suppl
1):81–5.
37. Baselski V, Buchanan K, Carey R, Clarridge J, Weissfeld A.
Survey of clinical microbiology laboratory workloads,
productivity rates, and staffing vacancies. Washington, DC:
American Society for Microbiology Office of Public and
Scientific Affairs, 2005.
38. Sanders CC, Peyret M, Moland ES, et al. Potential impact of
the VITEK 2 system and the advanced expert system on the
clinical laboratory of a university-based hospital. J Clin
Microbiol 2001;39:2379–85.
39. Eigner U, Schmid A, Wild U, Bertsch D, Fahr AM. Analysis of
the comparative workflow and performance characteristics of
the VITEK 2 and Phoenix systems. J Clin Microbiol
2005;43:3829–34.
40. Sellenriek P, Holmes J, Ferrett R, Drury R, Storch GA.
1342 PHARMACOTHERAPY Volume 29, Number 11, 2009
Comparison of MicroScan Walk-Away, Phoenix, and VITEK 2
microbiology systems used in the identification and
susceptibility testing of bacteria. Presented at the 105th
meeting of the American Society of Microbiology, Atlanta,
Georgia, June 5–9, 2005. Available from http://www.bd.com/ds/
technicalCenter/whitepapers/lr900.pdf. Accessed April 13,
2009.
41. Chapin KC, Musgnug MC. Validation of the automated reading
and incubation system with Sensititre plates for antimicrobial
susceptibility testing. J Clin Microbiol 2003;41:1951–6.
42. Donay JL, Mathieu D, Fernandes P, et al. Evaluation of the
automated Phoenix system for potential routine use in the
clinical microbiology laboratory. J Clin Microbiol 2004;42:
1542–6.
43. Trek Diagnostic Systems, Inc. Sensititre 510(k) substantial
equivalence determination decision summary; assay only
template. K052091. Available from http://www.fda.gov/cdrh/
reviews/K052091.pdf. Accessed April 13, 2009.
44. Camporese A. The impact of automation on organizational
changes in a community hospital clinical microbiology
laboratory. Infect Med 2004;12:118–25.
45. Cartwright RY, Davies JR, Dulake C, Hart RJ, Morris CA,
Wilkinson PJ. A study of workload units in five microbiology
laboratories. J Clin Pathol 1985;38:208–14.
46. Freeman R. Microbiology workload [editorial]. J Clin Pathol
2002;55:734.
47. Burns JL, Saiman L, Whittier S, et al. Comparison of two
commercial systems (Vitek and MicroScan-WalkAway) for
antimicrobial susceptibility testing of Pseudomonas aeruginosa
isolates from cystic fibrosis patients. Diagn Microbiol Infect Dis
2001;39:257–60.
48. Pfaller MA, Diekema DJ, Procop GW, Rinaldi MG. Multicenter
comparison of the VITEK 2 antifungal susceptibility test with
the CLSI broth microdilution reference method for testing
amphotericin B, flucytosine, and voriconazole against Candida
spp. J Clin Microbiol 2007;45:3522–8.
49. Jorgensen JH, Hindler JF. New consensus guidelines from the
Clinical and Laboratory Standards Institute for antimicrobial
susceptibility testing of infrequently isolated or fastidious
bacteria. Clin Infect Dis 2007;44:280–6.
50. Clinical and Laboratory Standards Institute. Methods for
antimicrobial susceptibility testing of anaerobic bacteria;
approved standard M11-A7. Wayne, PA: Clinical and
Laboratory Standards Institute, 2007.
51. Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing:
special needs for fastidious organisms and difficult-to-detect
resistance mechanisms. Clin Infect Dis 2000;30:799–808.
52. Aldridge KE, Ashcraft D, Cambre K, Pierson CL, Jenkins SG,
Rosenblatt JE. Multicenter survey of the changing in vitro
antimicrobial susceptibilities of clinical isolates of Bacteroides
fragilis group, Prevotella, Fusobacterium, Porphyromonas, and
Peptostreptococcus species. Antimicrob Agents Chemother
2001;45:1238–43.
53. Lassmann B, Gustafson DR, Wood CM, Rosenblatt JE.
Reemergence of anaerobic bacteremia. Clin Infect Dis
2007;44:895–900.
54. Snydman DR, Jacobus NV, McDermott LA, et al. National
survey on the susceptibility of Bacteroides fragilis group: report
and analysis of trends in the United States from 1997 to 2004.
Antimicrob Agents Chemother 2007;51:1649–55.
55. Clinical and Laboratory Standards Institute. Analysis and
presentation of cumulative antimicrobial susceptibility test
data; approved guideline—third edition: approved standard
M39-A3. Wayne, PA: Clinical and Laboratory Standards
Institute, 2009.
56. Clinical and Laboratory Standards Institute. Laboratory
detection and identification of mycobacteria: approved standard
M48A. Wayne, PA: Clinical and Laboratory Standards Institute,
2007.
57. Pfyffer GE. Mycobacterium: general characteristics, laboratory
detection, and staining procedure. In: Murray PR, Baron EJ,
Jorgensen JH, Landry ML, Pfaller MA, eds. Manual of clinical
microbiology, 9th ed. Washington, DC: American Society of
Microbiology; 2007:543–72.
58. Swenson JM, Lonsway D, McAllister S, et al. Detection of
mecA-mediated resistance using reference and commercial
testing methods in a collection of Staphylococcus aureus
expressing borderline oxacillin MICs. Diagn Microbiol Infect
Dis 2007;58:33–9.
59. Swenson JM, Williams PP, Killgore G, O’Hara CM, Tenover
FC. Performance of eight methods, including two new rapid
methods, for detection of oxacillin resistance in a challenge set
of Staphylococcus aureus organisms. J Clin Microbiol 2001;39:
3785–8.
60. Louie L, Matsumura SO, Choi E, Louie M, Simor AE.
Evaluation of three rapid methods for detection of methicillin
resistance in Staphylococcus aureus. J Clin Microbiol 2000;38:
2170–3.
61. McKinney TK, Shar ma VK, Craig WA, Archer GL.
Transcription of the gene mediating methicillin resistance in
Staphylococcus aureus (mecA) is corepressed but not coinduced
by cognate mecA and ␤-lactamase regulators. J Bacteriol
2001;183:6862–8.
62. Bennett K, Sharp SE. Rapid differentiation of methicillin-
resistant Staphylococcus aureus and methicillin-susceptible
Staphylococcus aureus from blood cultures by use of a direct
cefoxitin disk diffusion test. J Clin Microbiol 2008;46:3836–8.
63. Swenson JM, Tenover FC. Results of disk diffusion testing with
cefoxitin correlate with presence of mecA in Staphylococcus spp.
J Clin Microbiol 2005;43:3818–23.
64. Hageman JC, Patel JB, Carey RC, Tenover FC, McDonald LC.
Investigation and control of vancomycin-intermediate and -
resistant Staphylococcus aureus: a guide for health departments and
infection control personnel. Atlanta, GA, 2006. Available from
http://www.cdc.gov/ncidod/dhqp/pdf/ar/visa_vrsa_guide.pdf.
Accessed April 10, 2009.
65. Centers for Disease Control and Prevention. Algorithm for
testing S. aureus with vancomycin. Available from http://www.
cdc.gov/ncidod/dhqp/pdf/ar/VRSA_testing_algo09v4.pdf.
Accessed April 10, 2009.
66. Liu C, Chambers HF. Staphylococcus aureus with hetero-
geneous resistance to vancomycin: epidemiology, clinical
significance, and critical assessment of diagnostic methods.
Antimicrob Agents Chemother 2003;47:3040–5.
67. Swenson JM, Clark NC, Ferraro MJ, et al. Development of a
standardized screening method for detection of vancomycin-
resistant enterococci. J Clin Microbiol 1994;32:1700–4.
68. Cetinkaya Y, Falk P, Mayhall CG. Vancomycin-resistant
enterococci. Clin Microbiol Rev 2000;13:686–707.
69. Sidberr y GK, Tekle T, Carroll K, Dick J. Failure of
clindamycin treatment of methicillin-resistant Staphylococcus
aureus expressing inducible clindamycin resistance in vitro.
Clin Infect Dis 2003;37:1257–60.
70. Drinkovic D, Fuller ER, Shore KP, Holland DJ, Ellis-Pegler R.
Clindamycin treatment of Staphylococcus aureus expressing
inducible clindamycin resistance. J Antimicrob Chemother.
Aug 2001;48:315–16.
71. O’Sullivan MV, Cai Y, Kong F, Zeng X, Gilbert GL. Influence of
disk separation distance on accuracy of the disk approximation
test for detection of inducible clindamycin resistance in
Staphylococcus spp. J Clin Microbiol 2006;44:4072–6.
72. Bradford PA. Extended-spectrum ␤-lactamases in the 21st
century: characterization, epidemiology, and detection of this
important resistance threat. Clin Microbiol Rev 2001;14:
933–51.
73. Livermore DM. ␤-Lactamases in laboratory and clinical
resistance. Clin Microbiol Rev 1995;8:557–84.
74. BD Diagnostic Systems. BBL paper discs for the detection of ␤-
lactamase enzymes: Cefinase discs package insert. Franklin
Lakes, NJ; 2004. Available from http://www.bd.com/ds/
productCenter/231650.asp. Accessed July 22, 2008.
75. Schwaber MJ, Raney PM, Rasheed JK, et al. Utility of National
Committee for Clinical Laboratory Standards guidelines for
identifying extended-spectrum ␤-lactamases in non–Escherichia
coli and non–Klebsiella spp. of Enterobacteriaceae. J Clin
Microbiol 2004;42:294–8.
 ANTIMICROBIAL SUSCEPTIBILITY TESTING PRIMER Kuper et al
76. Hyle EP, Lipworth AD, Zaoutis TE, Nachamkin I, Bilker WB,
Lautenbach E. Impact of inadequate initial antimicrobial
therapy on mortality in infections due to extended-spectrum ␤-
lactamase–producing Enterobacteriaceae. Arch Intern Med 85.
2005;165:1375–80.
77. Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-
type ␤-lactamases. Antimicrob Agents Chemother 2002;46: 86.
1–11.
78. Bolmström A, Engelhardt A, Bylund L, Ho T, Walsh TR. 87.
Evaluation of two new Etest strips for AmpC detection
[abstract]. Abstr Intersci Conf Antimicrob Agents Chemother
2006;46:145.
79. Engelhardt A, Yusof A, Ho P, Johansson C, Sjöström K. 88.
Evaluation of a new Etest strip for detection of plasmidic
AmpC. Presented at the 18th European congress of clinical
microbiology and infectious diseases, Barcelona, Spain, April
19–22, 2008. 89.
80. Rasmussen BA, Bush K. Carbapenem-hydrolyzing ␤-
lactamases. Antimicrob Agents Chemother 1997;41:223–32.
81. Picão RC, Andrade SS, Nicoletti AG, et al. Metallo-␤-
lactamase detection: comparative evaluation of double-disk
synergy versus combined disk tests for IMP, GIM, SIM, SPM, or 90.
VIM producing organisms. Clin Microbiol 2008;46:2028–37.
82. Galani I, Rekatsina PD, Hatzaki D, Plachouras D, Souli M,
Giamarellou H. Evaluation of different laboratory tests for the
detection of metallo-␤-lactamase production in Enterobac- 91.
teriaceae. J Antimicrob Chemother 2008;61:548–53.
83. Bolmström A, Qwarnstrom A, Gales A, Walsh TR. Evaluation
of new Etest to detect metallo-␤-lactamases (MBL) in routine
clinical testing [abstract]. Abstr Intersci Conf Antimicrob 92.
Agents Chemother 2000;40:154.
84. Moland ES, Black JA, Ourada J, Reisbig MD, Hanson ND,
1343
Thomson KS. Occurrence of newer ␤-lactamases in Klebsiella
pneumoniae isolates from 24 U.S. hospitals. Antimicrob Agents
Chemother 2002 46:3837–42.
Tenover FC, Kalsi RK, Williams PP, et al. Carbapenem
resistance in Klebsiella pneumoniae not detected by automated
susceptibility testing. Emerg Inf Dis 2006:12:1209–13.
Wyeth Pharmaceuticals Inc. Zosyn (piperacillin-tazobactam)
package insert. Philadelphia, PA; 2007.
U.S. Food and Drug Administration. Class II special controls
guidance document: antimicrobial susceptibility test (AST)
systems; guidance for industry and FDA. Issued March 5, 2007.
Rockville, MD: U.S. Food and Drug Administration, 2007.
Prakash V, Lewis JS, Jorgensen JH. Vancomycin MICs for
methicillin-resistant Staphylococcus aureus isolates differ based
on the susceptibility test method used [letter]. Antimicrob
Agents Chemother 2008;52:4528.
Sader HS, Fritsche TR, Jones RN. Accuracy of three automated
systems (MicroScan WalkAway, VITEK, and VITEK 2) for
susceptibility testing of Pseudomonas aeruginosa against five
broad-spectrum ␤-lactam agents. J Clin Microbiol 2006;44:
1101–4.
Ibrahim EH, Sherman G, Ward S, Fraser VJ, Kollef MH. The
influence of inadequate antimicrobial treatment of bloodstream
infections on patient outcomes in the ICU setting. Chest
2000;118:146–55.
Lodise TP, McKinnon PS, Swiderski L, Rybak MJ. Outcomes
analysis of delayed antibiotic treatment for hospital-acquired
Staphylococcus aureus bacteremia. Clin Infect Dis 2003;36:
1418–23.
Luna CM, Vujacich P, Niederman MS, et al. Impact of BAL
data on the therapy and outcome of ventilator-associated
pneumonia. Chest 1997;111:676–85.