Overview of Antibacterial Susceptibility Testing

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Overview of antibacterial susceptibility testing

AUTHOR: Virginia M Pierce, MD
SECTION EDITOR: Stephen B Calderwood, MD
DEPUTY EDITOR: Keri K Hall, MD, MS
All topics are updated as new evidence becomes available and our peer review process is complete.
Literature review current through: May 2024.

This topic last updated: Apr 06, 2023.
INTRODUCTION
The clinical microbiology laboratory serves as a valuable ally to clinicians in the diagnosis and
treatment of infectious diseases. In particular, the isolation of bacteria from clinical samples
yields information that can be used to guide the selection of appropriate antibiotic regimens
based on knowledge of the most likely susceptibility profile of certain bacterial species.
Through the use of in vitro antimicrobial susceptibility testing, the laboratory can specifically
determine which antibiotics effectively inhibit the growth of a given bacterial isolate,
allowing for targeted therapy. Antimicrobial resistance is a growing concern in both
community and health care settings; as such, decisions surrounding empirical antibiotic
treatment are becoming more complicated, and the importance of routine antimicrobial
susceptibility testing to guide therapeutic decisions has increased.
Multiple different methods for antimicrobial susceptibility testing exist, including

conventional methods, automated systems, and newer molecular techniques.
Understanding these methods allows clinicians to correctly interpret susceptibility testing
results reported by the clinical microbiology laboratory. In general, antimicrobial
susceptibility testing methods used in clinical laboratories should:
● Provide rapid and accurate information to the clinician

● Be relatively inexpensive
● Be relatively easy to perform
This topic will provide an overview of antimicrobial susceptibility testing with a focus on
bacterial isolates. Routine susceptibility testing of viruses and parasites is not performed in
most clinical laboratories and is beyond the scope of this discussion. An overview of
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antimicrobial susceptibility testing in fungi is presented elsewhere. (See "Antifungal

susceptibility testing".)
BASIC CONCEPTS OF ANTIMICROBIAL RESISTANCE
Intrinsic versus acquired resistance — Bacteria can have either intrinsic or acquired
resistance to antimicrobials.
Intrinsic resistance is the inherent resistance to an antimicrobial that all or almost all
members of a species display, rendering susceptibility testing unnecessary [1]. As an
example, Klebsiella pneumoniae is intrinsically resistant to the antimicrobial ampicillin.
In contrast, acquired resistance is the development of resistance to an antimicrobial to which

members of the wild-type bacterial population are susceptible. Bacteria can acquire
resistance through chromosomal mutations; through the horizontal transfer of genes via
plasmids, integrons, transposons, or transformation; or through a combination of these
mechanisms [2]. Unlike intrinsic resistance, acquired resistance in a specific bacterial isolate
is not reliably predictable. The goal of antimicrobial susceptibility testing is to determine the
degree of acquired resistance to antibiotics that might be employed therapeutically.
Constitutive versus inducible resistance mechanisms — The expression of some bacterial

resistance mechanisms is variable, potentially complicating their detection in the
microbiology laboratory and requiring special consideration.
Constitutively expressed resistance mechanisms are expressed continuously, while inducible

expression occurs following exposure to a particular inciting agent [3]. For example, use of
third generation cephalosporins for infections caused by certain Enterobacterales can induce
production of a chromosomally encoded AmpC beta-lactamase, which results in resistance to
this subgroup of beta-lactam antimicrobials [4]. (See "Beta-lactam antibiotics: Mechanisms of
action and resistance and adverse effects", section on 'Chromosomal beta-lactamases'.)
Heteroresistance — The phenotypic expression of an antimicrobial resistance mechanism

within a bacterial population can be homogeneous or heterogeneous [3].
Heterogeneous expression, or heteroresistance, can lead to bacterial subpopulations within

a microbiologic sample that have varying degrees of phenotypic resistance, making the in
vitro identification of resistance more difficult. Some conventional antimicrobial susceptibility
testing methods can be insufficiently sensitive for the identification of heteroresistance,
leading to the misclassification of certain bacterial strains as susceptible. Antimicrobial
susceptibility testing methods that use higher inocula can overcome this barrier, facilitating
the detection of small subpopulations with intermediate or resistant minimum inhibitory
concentrations. Heteroresistant vancomycin-intermediate Staphylococcus aureus is an

example of an organism with the capability of heterogenous expression [5].
INDICATIONS FOR SUSCEPTIBILITY TESTING
Antimicrobial susceptibility testing should be performed when clinically significant bacteria

are isolated from patient specimens and the resulting information can be used to guide
treatment. The microbiology laboratory is primarily responsible for making this
determination [6].
Susceptibility testing may not provide clinically useful information and thus may not be
routinely performed in the following circumstances:
● When the antimicrobial susceptibility pattern of a particular organism is predictable. As

an example, testing Streptococcus pyogenes for susceptibility to penicillin is not routinely
performed because isolates that are not susceptible to penicillin have not been
reported [1].
● When the isolated organism is likely to represent the normal flora of the body site from
which the specimen was collected (rather than a pathogen). As an example,
Lactobacillus spp are considered part of the normal bacterial flora of the female genital
tract, so their isolation in a vaginal culture would not trigger susceptibility testing.
● When insufficient numbers of bacterial colonies are grown in cultures of certain

specimen types. As an example, organisms that grow in rare amounts in cultures of
noninvasively collected urine specimens are likely to represent contamination and be of
little clinical significance.
● When the results of in vitro antimicrobial susceptibility testing do not reliably predict in
vivo effectiveness or a clinical therapeutic response. As an example, susceptibility
testing of first- and second-generation cephalosporins against Salmonella and Shigella
spp is not recommended due to the poor correlation between in vitro susceptibility
testing results and clinical outcomes [1].
● When guidance regarding the performance of standardized susceptibility testing for a

particular organism is not available from professional groups such as the Clinical and
Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial
Susceptibility Testing (EUCAST), or from regulatory authorities such as the United States
Food and Drug Administration (FDA). For certain infrequently isolated organisms
(Capnocytophaga spp, for example), such guidance does not exist, making routine
susceptibility testing and interpretation of these results difficult.
Host factors also inform the decision to perform susceptibility testing. In particular, clinicians
should notify the microbiology laboratory if a patient is immunosuppressed, so that it can
modify the approach to selecting appropriate bacterial isolates for susceptibility testing.
Some bacteria thought to be nonpathogenic in immunocompetent hosts can cause serious
infections in immunocompromised individuals, and their isolation (particularly from a
normally sterile site) may warrant antimicrobial susceptibility testing.
CONVENTIONAL METHODS
Conventional antimicrobial susceptibility testing methods are phenotypic in vitro tests that
provide a direct measurement of antimicrobial activity. These techniques measure the
activity of a specific antimicrobial agent against a clinical bacterial isolate by assessing
bacterial growth in the presence of that agent. Much of the antimicrobial susceptibility
testing performed in clinical laboratories relies on these conventional methods because they
provide accurate and reproducible results. However, these methods have several limitations:
● They rely on bacterial growth. This requirement is often the rate-limiting step and can
make susceptibility testing of more fastidious bacteria, such as Granulicatella spp
(formerly known as nutritionally variant streptococci), more difficult to perform. The
Clinical and Laboratory Standards Institute (CLSI) offers some guidance to microbiology
laboratories on the testing of organisms that are fastidious or infrequently
encountered in clinical practice ( table 1) [7].
● Standardization of the testing methods is necessary to ensure accuracy as well as intra-

and inter-laboratory reproducibility. This includes the selection of isolated colonies of
the organism (taking care to avoid testing mixtures of different types of
microorganisms), preparation of a standardized inoculum, and adherence to validated
testing procedures. Performance standards for antimicrobial susceptibility testing are
available from CLSI and the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) [1,7-11].
● There are instances in which certain conventional testing methods are not
recommended given their poor accuracy and reproducibility. As an example, disk
diffusion is insensitive for the detection of vancomycin-intermediate S. aureus, and so
this method should not be used when testing S. aureus isolates for susceptibility to
vancomycin [1,12].
Conventional antimicrobial susceptibility testing methods can be categorized into those that
provide qualitative results and those that provide quantitative results, as below.
Qualitative methods (disk diffusion/Kirby-Bauer method) — Overall, qualitative

conventional antimicrobial susceptibility testing methods tend to be easier to perform and
interpret compared with quantitative methods. They classify the activity of an antimicrobial
agent against a specific organism into one of four interpretive categories (susceptible,
susceptible-dose dependent, intermediate, or resistant (see 'Interpretation of results' below))
[1,8]. The qualitative conventional testing method most commonly used by clinical
microbiology laboratories is the disk diffusion, or Kirby-Bauer, method, due to its simplicity,
reliability, and high degree of standardization [13,14].
Performance — In brief, the procedure involves swabbing a standardized inoculum of

bacteria (approximately 1 to 2 x 108 colony-forming units [CFU]/mL) onto a Mueller-Hinton
(or other medium appropriate for the bacterial species) agar plate [8]. Commercially
prepared paper disks, each embedded with a fixed concentration of a specific antimicrobial,
are placed on the agar surface. During overnight incubation, the antimicrobials diffuse into
the agar medium, creating a concentration gradient as each antimicrobial diffuses further
away from the disk. Bacterial growth is inhibited at sites where the concentration of an
antimicrobial is high enough to prevent proliferation of the organism. After incubation, the
diameters of the complete growth inhibition zones around each disk are measured
( picture 1). Prespecified breakpoints are used to interpret the zone sizes and classify them
as susceptible, susceptible-dose dependent, intermediate, or resistant. (See 'Interpretation
of results' below.)
The diameter of the zone of inhibition is affected by both the susceptibility of the organism
to a specific antibiotic and the rate at which the drug diffuses into the agar medium. Overall,
there is a correlation between the size of the zone of inhibition and the minimum inhibitory
concentration (MIC), but this relationship is not always linear [15]. As a result, simply
measuring the zone of inhibition without knowledge of the breakpoints should not be
performed [1,7,8].
Advantages — The disk diffusion method is a highly standardized method, with

breakpoints established as correlates to reference broth microdilution MIC breakpoints for
many organism-antimicrobial combinations [16]. In addition to its low cost and simplicity, an
advantage of disk diffusion is the flexibility in the choice of tested antimicrobials; this choice
can be customized in accordance with institutional formularies and local resistance patterns
and can be modified easily when indicated. The qualitative results of disk diffusion testing
(ie, susceptible, susceptible-dose dependent, intermediate, or resistant) are also readily
interpreted by clinicians.
Limitations — Despite the numerous advantages of the disk diffusion method, there are
also some important limitations. The lack of automation of the procedure makes its use in
high-volume microbiology laboratories more difficult.
Furthermore, though this method is validated for most commonly encountered bacteria, its
use with some more fastidious or slow-growing bacteria has not been as well studied or
standardized. As an example, disk diffusion testing of HACEK group bacteria, such as
Aggregatibacter spp, Cardiobacterium spp, Eikenella corrodens, and Kingella spp, is not
recommended by CLSI for this reason [7].
Finally, there are some circumstances in which the qualitative nature of disk diffusion
susceptibility results is a limitation; for certain infections, a quantitative MIC is required to
determine the best therapeutic approach. For example, recommended treatment regimens
for endocarditis caused by viridans group streptococci are stratified by the penicillin MIC,
necessitating the use of a quantitative antimicrobial susceptibility testing method [1,17].
Quantitative methods — Quantitative methods for antimicrobial susceptibility testing allow

determination of the MIC, which is the lowest concentration of a specific agent that is
needed to inhibit visible growth of an organism. Breakpoints published by the CLSI, EUCAST,
or, in the United States, the Food and Drug Administration (FDA) [18], are used to classify
MICs into susceptible, intermediate, or resistant interpretive categories.
Quantitative techniques are considered to be the most precise for the comparison of in vitro
efficacy of different antibiotics against a specific organism. Furthermore, as noted above,
there are certain infections for which a quantitative result is required in order to optimize the
antimicrobial treatment regimen [17].
Several quantitative antimicrobial susceptibility methods are currently available, including

agar dilution, broth dilution (macrodilution and microdilution), and gradient methods, as
detailed below.
Agar dilution — The agar dilution method is a well-studied and reproducible technique
that is considered a reference standard for the susceptibility testing of many organism-
antimicrobial combinations [1,7,9,10]. However, because of the high cost and labor-intensive
nature of the method, the majority of clinical microbiology laboratories restrict its use to
certain specific applications, such as screening for high-level gentamicin resistance in
enterococci [1]. This technique is also recommended for use with fastidious bacteria that
require special growth conditions, such as Helicobacter pylori and Neisseria gonorrhoeae [7].
With this method, a fixed concentration of a specific antimicrobial is added into molten agar
and allowed to solidify [9]. Agar plates containing serial twofold dilutions of the antimicrobial
are created, with concentrations spanning the relevant range for testing the organism of
interest. As many as 36 different bacterial isolates can be tested on a single agar plate in
locations called spots, with a standardized inoculum (approximately 104 CFU) of an isolate
inoculated onto a particular spot. Plates are incubated for 16 to 20 hours and subsequently
examined for growth. The plate with the lowest antimicrobial concentration that visibly
inhibits bacterial growth is designated as the MIC. (See 'Interpretation of results' below.)
A principle advantage of the agar dilution method is that MICs can be generated without
using any special laboratory instrumentation.
As above, the main limitations of the agar dilution method are the high cost of reagents and
the labor required to prepare the agar plates, which are generally not commercially available
and cannot be batch-produced and stored for future use.
Broth dilution — Broth dilution is performed by creating a series of twofold dilutions of a

specific antimicrobial, including concentrations that cover the breakpoints for the organism
of interest, and determining the lowest concentration of antimicrobial that inhibits growth
(under standardized incubation conditions) of the bacterial isolate [9]. This concentration is
the MIC. The principles of the broth dilution technique apply to both the broth macrodilution
and broth microdilution methods.
Broth (tube) macrodilution — The broth macrodilution method is mainly reserved for
research settings, such as the evaluation of new antimicrobial compounds.
In the broth macrodilution procedure, serial antimicrobial dilutions are manually prepared in
tubes containing 1 to 2 mL of broth medium [9,14]. Each tube is inoculated with an equal
volume of a standardized bacterial suspension (containing approximately 5 x 105 CFU/mL).
After overnight incubation, the tubes are visually inspected for the presence of turbidity,
which is an indicator of bacterial growth. The tube with the lowest concentration of the
specific antimicrobial in which turbidity is absent is the MIC (see 'Interpretation of results'
below). The precision of the broth macrodilution method is considered to be plus or minus
one twofold dilution.
Broth macrodilution is a time-honored technique that has been well studied and
standardized, making it a reliable tool for the detection of antimicrobial resistance. However,
limitations include its lack of automation, making it particularly labor intensive and subject to
errors introduced during the manual preparation of the antibiotic dilutions. As a result, this
method is not used for routine antimicrobial susceptibility testing in clinical microbiology
laboratories.
Broth microdilution — The broth microdilution method is a popular method for

antimicrobial susceptibility testing in clinical microbiology laboratories.
It is considered a miniaturized and more automated version of the macrodilution method

(see 'Broth (tube) macrodilution' above). It employs the same principles as the macrodilution
procedure, but the antibiotic dilutions are prepared in small volumes of broth medium
(usually 0.1 mL per well) in 96-well plates ( picture 2) [9,13]. Panels containing dilutions of
commonly used antibiotics are commercially available (frozen or lyophilized), which saves
labor and reduces the risk of error associated with preparing dilutions within the clinical
laboratory. A standardized inoculum of bacteria (approximately 5 x 105 CFU/mL) is added into
each well and the plates are incubated for 16 to 20 hours. After incubation, the wells can be
examined either manually or through an automated tray reader for the presence of turbidity.
As with the macrodilution method, the concentration of antibiotic that inhibits bacterial
growth is defined as the MIC. (See 'Interpretation of results' below.)
Like agar dilution, broth microdilution testing is considered a reference method [1]. The
results of broth microdilution testing are generally reproducible, and the 96-well plate format
facilitates the concurrent testing of multiple antimicrobials within a relatively small footprint.
Test panels are commercially available, and some such panels allow for simultaneous
organism identification. As an additional advantage, broth microdilution can also be used for
the susceptibility testing of many fastidious bacteria [7].
Disadvantages include its high cost relative to some other methods, such as disk diffusion.
Additionally, when using commercial test panels, the included antibiotics may not be well
matched to an institution's formulary. Furthermore, because of the miniaturization of the
microdilution method, one logarithm fewer bacteria are analyzed than with the
macrodilution method; therefore, resistance mechanisms present in only a small subset of a
bacterial population might not be represented within the tested inoculum, and the organism
might falsely appear to be uniformly susceptible to a drug. (See 'Heteroresistance' above.)
Antimicrobial gradient method — The antimicrobial gradient method is an agar-based

technique that relies on the creation of a concentration gradient of an antimicrobial to
determine the susceptibility of a bacterial isolate to the antimicrobial ( picture 3).
As with the disk diffusion method, the gradient procedure involves the preparation of a
standardized bacterial suspension containing approximately 1 to 2 x 108 CFU/mL organisms
and streaking it onto a Mueller-Hinton (or other medium appropriate for the bacterial
species) agar plate. Commercially produced plastic strips, each impregnated with a graded
concentration of a specific antimicrobial, are then placed on the inoculated plate. After
overnight incubation, the MIC of each antimicrobial is determined by identifying the
intersection of the elliptical zone of growth inhibition with the antimicrobial gradient on the
strip.
Multiple reports have confirmed that results of gradient method testing correlate well with
those of agar dilution and broth dilution for certain organism-antimicrobial combinations
[19-23]. However, the gradient method should only be used for testing organism-
antimicrobial combinations approved by the FDA or demonstrated in well-controlled studies
to be equivalent in performance to CLSI reference methods, since the method is not robust
for all applications; for example, the method may overstate the vancomycin MIC for S. aureus
by one or more doubling dilutions.
One strength of the gradient technique is that it can be used to test fastidious organisms
with special growth requirements [13,14]. Because each antimicrobial is tested individually
(and not as part of a predetermined panel), the gradient method also allows for flexibility in
the selection of which antimicrobials to test for a specific isolate.
Important limitations of the gradient method are its cost and lack of automation, both of
which preclude its use for testing numerous antimicrobials against routine isolates.
Interpretation of results — Conventional qualitative disk diffusion and quantitative

susceptibility testing methods yield an inhibitory zone diameter and an MIC, respectively, for
the given isolate-antibiotic pair. Breakpoints published by the CLSI, EUCAST, or, in the United
States, the FDA, are used to interpret the zone sizes or MIC values and classify them into
interpretive categories [24,25]. The categories used by CLSI are:
● Susceptible – Indicates that the antibiotic concentration that inhibits the isolate's
growth is usually achieved with administration of the dose recommended for the type
of infection and infecting organism. Clinical efficacy is expected.
● Susceptible-dose dependent – Indicates that, to achieve the antibiotic concentration

that inhibits the isolate’s growth, it is necessary to use a dosing regimen that results in
higher drug exposure (by means of higher doses, more frequent doses, or both) than
that achieved with the regimen used to establish the susceptible breakpoint. Like the
intermediate category below, the susceptible-dose-dependent category includes a
buffer zone to avoid major discrepancies in interpretation based upon small,
uncontrolled technical factors, especially for drugs with a narrow margin between
effective and toxic doses.
● Intermediate – Indicates that the MIC of the antimicrobial approaches usually

attainable blood and tissue levels and/or that response rates may be reduced
compared with susceptible isolates. Clinical efficacy may be achieved when the
antimicrobial is physiologically concentrated at the site of infection (eg, beta-lactams,
aminoglycosides, and fluoroquinolones in the urine). The CLSI also defines the
intermediate category to include a buffer zone to avoid major discrepancies in
interpretation based upon small, uncontrolled technical factors, especially for drugs
with a narrow margin between effective and toxic doses.
● Resistant – Indicates that the concentrations usually achieved when the antibiotic is
administered at conventional doses do not inhibit the isolate's growth, that the MIC or
disk diffusion zone diameter falls into a range where specific microbial resistance
mechanisms are likely, or that reliable clinical efficacy of the antimicrobial against the
organism has not been established in clinical studies.
For commonly encountered bacteria, establishment of breakpoints involves extensive review

and consideration of microbiologic, clinical, and pharmacodynamic data. For uncommonly
encountered bacteria, breakpoints are largely informed by available wild-type MIC
distributions. For each organism or group of organisms, CLSI also provides supplemental
information regarding known resistance mechanisms or patterns, indications for
antimicrobial susceptibility testing, how the interpretive criteria were derived, and special
testing notes.
AUTOMATED METHODS
Most automated systems depend on the optical detection of bacterial growth in the
presence of a specific antimicrobial. Because these optical systems can detect more subtle
changes in growth than would be visible to the human eye, they can determine antimicrobial
susceptibility patterns more rapidly than conventional methods [13].
Multiple such automated systems are available; all use similar techniques and involve
inoculation of antimicrobial microdilution trays or small cards with known quantities of
bacteria, followed by incubation in an instrument that monitors growth in each well using
photometric, fluorometric, or turbidimetric measurements ( picture 4) [26]. Using these
automated systems, multiple antimicrobials can be tested against a specific isolate at a time,
and many samples can be processed simultaneously.
The software of automated systems also offers certain advantages. It can include
interpretive rules (which can be customized in some cases) to facilitate detection of unusual
or important susceptibility patterns that warrant particular action, such as confirmation
before reporting or expedited notification of clinicians or infection control personnel.
Additionally, these automated systems can interface with the electronic medical record to
quickly make results available to clinicians, which has been associated with improvements in
clinical outcomes [27,28].
The principal limitation of automated antimicrobial susceptibility testing systems is cost,

which is prohibitive for some clinical microbiology laboratories. Earlier automated systems
also had difficulty detecting inducible antimicrobial resistance patterns, such as those seen
with certain beta-lactamases; newer versions, however, have improved the ability to detect
these specific resistance mechanisms, making them reliable tools for routine antimicrobial
susceptibility testing of many commonly isolated bacteria [13]. There can be a lag between
the publication of new interpretive criteria by the Clinical and Laboratory Standards Institute
(CLSI) or the Food and Drug Administration (FDA) and the incorporation of the new
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breakpoints into the database of an automated system. In this setting, clinical microbiology
laboratories must be aware of these discrepancies and perform validation studies to assure
adequate performance of the automated system with the revised breakpoints before
adopting them.
In the United States, FDA clearance of an automated system indicates that it provides results
that are substantially equivalent to those generated using appropriate reference methods
(eg, broth microdilution, agar dilution) for the organisms and antimicrobial agents claimed in
the manufacturer's package insert [1].
METHODS TO DETECT SPECIFIC TYPES OF RESISTANCE
Treatment regimens can often be devised on the basis of qualitative or quantitative

antimicrobial susceptibility testing results for a given clinical isolate without knowledge of
the specific acquired resistance mechanism or mechanisms present. Determination of
specific resistance mechanisms (eg, extended-spectrum beta-lactamase production among
Enterobacterales) is more commonly useful for infection control and epidemiologic
purposes. However, there are some situations in which knowing that an isolate expresses a
specific resistance mechanism can be clinically valuable.
Beta-lactamase testing — The beta-lactamases produced by many commonly encountered

bacteria, including Staphylococcus spp, Haemophilus influenzae, N. gonorrhoeae, and
Enterococcus spp, can be detected in the clinical microbiology laboratory. These enzymes
confer resistance to penicillin and ampicillin through hydrolysis of the beta-lactam ring. In
many cases, this enzymatic hydrolysis can be ascertained within minutes from colony growth
(in contrast to the overnight incubation required by growth-dependent disk diffusion or
minimum inhibitory concentration [MIC] tests), providing rapid information that can guide
antimicrobial selection. In other cases, supplemental beta-lactamase testing should be
performed to confirm the results of routine susceptibility testing before reporting results; for
example, Staphylococcus spp that initially test susceptible to penicillin should only be
reported as penicillin-susceptible if the results of beta-lactamase testing are negative [1].
The chromogenic cephalosporin beta-lactamase method can be used to detect beta-

lactamases in staphylococci, H. influenzae, N. gonorrhoeae, Enterococcus spp, and other
bacteria. It is performed by placing a small amount of test organism onto a commercially
prepared paper disk embedded with nitrocefin, a chromogenic cephalosporin. If the test
organism produces a beta-lactamase that hydrolyzes nitrocefin's beta-lactam ring, a pink
color is produced ( picture 5) [1]. Some organisms, such as H. influenzae and N.
gonorrhoeae, produce beta-lactamase constitutively. In contrast, staphylococci may produce
detectable levels of beta-lactamase only after exposure to an inducing agent; therefore, the
inoculum of staphylococci used for the chromogenic cephalosporin beta-lactamase test

should be taken from growth in the zone margin surrounding a penicillin or cefoxitin disk.
For S. aureus, the penicillin zone edge test, or the "beach and cliff" test, can also be used to
detect beta-lactamases [1]. In this procedure, a standard inoculum of the organism is plated
onto a Mueller-Hinton agar plate. A commercially available penicillin disk is added to the
agar plate, which is then incubated for 16 to 18 hours. After incubation, the zone edge of
inhibition of growth around the penicillin disk is examined. A sharp zone edge (a "cliff")
indicates the presence of a beta-lactamase, while a fuzzy zone edge (a "beach") indicates the
absence of a beta-lactamase ( picture 6). The penicillin zone edge test is more sensitive
than the nitrocefin-based method for the detection of beta-lactamase production by S.
aureus [29,30]; as such, a negative nitrocefin-based beta-lactamase test must be confirmed
with a penicillin zone edge test [1].
Inducible clindamycin resistance testing — The Clinical and Laboratory Standards

Institute (CLSI) recommends that testing for inducible clindamycin resistance be performed
on all Staphylococcus spp and Streptococcus pneumoniae isolates and for beta-hemolytic
streptococci whenever antimicrobial susceptibility testing is clinically indicated (eg, group B
streptococci isolated from recto-vaginal specimens obtained from pregnant women allergic
to penicillin).
The genetic underpinning of this resistance mechanism is the erm gene, which mediates the
methylation of 23S ribosomal ribonucleic acid (RNA), thereby modifying the binding target
site for macrolides and clindamycin [31]. Clindamycin alone is a poor inducer of the
methylase; as such, bacterial isolates can appear falsely susceptible to clindamycin when
conventional antimicrobial susceptibility testing methods are used. The addition of
erythromycin, however, allows for the induction of this resistance mechanism and accurate
identification of the presence of the erm gene in Staphylococcus spp, S. pneumoniae, and beta-
hemolytic streptococci [1,31-34].
Standardized inducible clindamycin resistance testing can be performed using either the D-
zone test (a disk diffusion method) or broth microdilution [1]. For the D-zone test,
commercially available erythromycin- and clindamycin-impregnated disks are placed on an
agar plate inoculated with a standard inoculum of the organism of interest; the distance
between the disks is specified according to the organism. The plates are incubated for 16 to
24 hours and then visually inspected for the presence of a flattening of the clindamycin zone
of inhibition adjacent to the erythromycin disk (D-zone) ( picture 7). An isolate with a
positive D-zone should be reported as clindamycin-resistant.
To test for inducible clindamycin resistance using the broth microdilution method, specific
concentrations of erythromycin and clindamycin are included in the same well, which is
inoculated with a bacterial suspension in the usual manner for broth microdilution testing.
After overnight incubation, the well is examined for bacterial growth; any visible growth
indicates inducible clindamycin resistance. Manufacturers of automated antimicrobial
susceptibility testing systems have integrated this test into their systems such that inducible
clindamycin resistance can be determined concurrently with other antimicrobial
susceptibility testing results.
High-level aminoglycoside resistance screening — CLSI recommends screening for high-

level aminoglycoside resistance for severe enterococcal infections, such as endocarditis [1].
Antimicrobial treatment of enterococcal endocarditis usually consists of a combination of a
cell wall-active agent and an aminoglycoside, if the isolate is susceptible [17]. (See
"Antimicrobial therapy of left-sided native valve endocarditis", section on 'Enterococci' and
"Antimicrobial therapy of prosthetic valve endocarditis", section on 'Enterococci'.)
Disk diffusion, broth microdilution, and agar dilution methods can be used for this purpose
[1]. All three methods have been shown to have good sensitivity and specificity for the
detection of high-level aminoglycoside resistance when standard methods are applied [35].
Inconclusive results, however, can sometimes be seen with the disk diffusion method,
requiring resolution with either broth microdilution or agar dilution [1,35].
● The disk diffusion test is performed on Mueller-Hinton agar and follows the standard
procedure (see 'Performance' above), but the disks are impregnated with high
concentrations of gentamicin or streptomycin (120 mcg of gentamicin or 300 mcg of
streptomycin). The plates are manually examined after 16 to 18 hours of incubation;
zones of inhibition around each disk are measured and interpreted according to
qualitative breakpoints.
● For the broth microdilution procedure, a standardized inoculum of the enterococcal

isolate is added to a well containing brain-heart infusion broth and a high
concentration of either gentamicin (500 mcg/mL) or streptomycin (1000 mcg/mL). The
well is examined after 24 hours of incubation, and any visible growth is indicative of
high-level resistance to the aminoglycoside. For streptomycin, if no growth is observed
after 24 hours, a second read is performed after an additional 24 hours of incubation.
● The agar dilution technique involves spotting a standardized inoculum of the bacterial
isolate onto a brain-heart infusion agar plate containing a high concentration of either
gentamicin (500 mcg/mL) or streptomycin (2000 mcg/mL); as with the broth
microdilution method, growth (>1 colony) after 24 hours of incubation (or 48 hours for
streptomycin) indicates resistance.
GENOTYPIC METHODS
Although phenotypic methods remain the cornerstone of antimicrobial susceptibility testing

in clinical microbiology laboratories, molecular assays that test for specific resistance genes
have been increasingly incorporated into routine use. Techniques commonly used to detect
bacterial nucleic acid sequences conferring antibiotic resistance include polymerase chain
reaction (PCR) and deoxyribonucleic acid (DNA) hybridization. A number of such genotypic
assays have been cleared by the United States Food and Drug Administration (FDA) [36], in
some cases as screening tools for the identification of multidrug-resistant bacteria in
hospital settings, and in others for diagnostic purposes [37-40]. A list of assays can be found
on the FDA website.
Genotypic antimicrobial susceptibility testing methods have several important strengths.

Some molecular assays can be performed directly on clinical specimens or primary cultures,
in contrast to conventional antimicrobial susceptibility testing methods that require initial
bacterial growth, subculture, and pure colony isolation, resulting in substantially faster
turnaround times. Furthermore, some molecular assays are more automated, objective, and
simple to perform than conventional methods, which can reduce both labor and errors.
Additionally, when a given phenotypic resistance pattern can occur as a consequence of
more than one genetic determinant, molecular tests can provide more specific information
about the presence of particular resistance genes that are of institutional epidemiological or
infection control concern. In other cases, a genotypic test may provide potentially useful
information about resistance difficult to detect using standard phenotypic approaches (eg, in
the case of poor growth masking higher minimum inhibitory concentrations, low in vitro
expression, or heteroresistance).
Molecular antimicrobial susceptibility testing methods also have important limitations that
have thus far restricted their clinical application:
● Available FDA-cleared assays are designed to identify one or a few specific genetic
resistance targets in a given bacterial species. This can be sufficient when antimicrobial
resistance is mediated by one or two specific genes, as with the mecA gene, which
regulates the vast majority of methicillin resistance in human isolates of S. aureus. For
many other bacteria, however, the genetic underpinnings of antimicrobial resistance
are much more complex, and reliance on molecular assays that target only a few known
resistance genes to predict antimicrobial susceptibility would be misleading. This caveat
is especially relevant among members of the family Enterobacterales, in which
hundreds of resistance mechanisms have been described. In addition, emergence of
genetic variants may interfere with detection of specific genetic resistance targets or
lead to discrepancies between genotypic and phenotypic susceptibility results [41].
● Genotypic results do not obviate the need for phenotypic antimicrobial susceptibility
testing. While FDA-cleared molecular assays have good sensitivity and specificity for the
detection of specific genetic resistance elements, phenotypic testing is still required to

confirm those results and to provide information about other possible therapeutic
options. As an example, a clinician treating a patient with an infection caused by
methicillin-resistant S. aureus may not only need to know that the isolate is resistant to
methicillin, but also whether the isolate is susceptible to other antimicrobials. Because
molecular testing is performed in addition to (rather than in replacement of)
phenotypic antimicrobial susceptibility testing, its use has the potential to increase both
costs and labor in clinical microbiology laboratories.
● The detection of a specific genetic resistance target is not always associated with the
expression of this gene in vivo. In some circumstances, characterizing bacteria as
resistant based on unexpressed genetic potential may result in over-reporting of
resistance.
● The majority of genotypic tests are significantly more expensive than phenotypic
antimicrobial susceptibility tests and do not allow for high-throughput testing, both of
which are important considerations in laboratories responsible for a large volume of
clinical testing.
The Clinical and Laboratory Standards Institute (CLSI) provides a practical approach for how
laboratories can use and report the results of molecular tests for detection of antibiotic
resistance, including guidance for how to proceed when phenotypic and genotypic results
are discordant [1].
REPORTING SUSCEPTIBILITY RESULTS
The selection of antimicrobials to use for susceptibility testing on a given isolate depends on
several factors, including the type of organism, the site of infection, and the institutional
formulary.
The Clinical and Laboratory Standards Institute provides guidance regarding the selection of
appropriate antimicrobial agents for testing commonly encountered bacteria, as well as
many infrequently isolated organisms [1,7]. These recommendations are based on clinical
efficacy data when available, United States Food and Drug Administration clinical indications
for use, consensus recommendations, prevalence of resistance, minimizing emergence of
resistance, and cost [1].
Once antimicrobial susceptibility testing is complete, the clinical microbiology laboratory also
must decide which results should be released into the clinical information system. An
institutional committee comprised of microbiologists, infectious diseases physicians, and
pharmacists is often responsible for selecting the number and types of antimicrobials that
should be reported for an individual organism under specific circumstances. The rationale
for selective reporting is to help guide antimicrobial prescribing and reduce the
inappropriate use of broad-spectrum antimicrobials when more targeted agents would
suffice. As an example, a laboratory might test S. pyogenes isolates for susceptibility to
carbapenems, but it would be inappropriate to routinely prescribe such broad-spectrum
agents to treat infections caused by this pathogen, and so the carbapenem susceptibility
results should generally be suppressed from reporting into the clinical information system.
Results that are not routinely reported should be released to the clinicians under specific
circumstances, such as in cases of unexpected resistance or multidrug resistance (in which
case, the susceptibility testing results for broader spectrum or second-line agents may be
needed to guide the regimen selection). Direct communication between clinicians and
clinical microbiology laboratory personnel is frequently helpful in such scenarios to establish
whether additional testing should be undertaken. The clinical microbiology laboratory
should save isolates from cultures for at least one week after the culture is reported as final,
in the event that additional testing is required.
SUMMARY
● Antimicrobial susceptibility testing should be performed when clinically significant

bacteria are isolated from patient specimens and the resulting information can be used
to guide treatment. Testing is most useful when the antimicrobial susceptibility of an
isolate is not predictable based on the genus and species, the organism is not part of
the normal flora of the specimen site, the results of in vitro susceptibility testing predict
clinical effectiveness, and guidance for the standardized performance of susceptibility
testing is available. (See 'Indications for susceptibility testing' above.)
● The disk diffusion test, or Kirby-Bauer method, is simple, reliable, and the most
commonly used qualitative testing method. A standardized inoculum of bacteria is
grown on a Mueller-Hinton agar plate, upon which commercially prepared paper disks,
each containing a fixed concentration of an antimicrobial, are placed. The diameters of
the zones of growth inhibition around each of the disks are measured and are
qualitatively interpreted based on published breakpoints (susceptible, susceptible-dose
dependent, intermediate, or resistant) ( picture 1). This method cannot be used for
fastidious or slow-growing bacteria. (See 'Qualitative methods (disk diffusion/Kirby-
Bauer method)' above and 'Interpretation of results' above.)
● Agar dilution tests, in which standardized inocula of bacteria are grown on agar
prepared with a fixed concentration of an antimicrobial, allow for multiple samples to
be tested on a single set of plates. This technique is recommended for use with
fastidious bacteria that require special growth conditions, such as Helicobacter pylori
and Neisseria gonorrhoeae. Because the agar dilution test is costly and labor-intensive,
the majority of clinical microbiology laboratories restrict its use to certain specific
applications, such as screening for high-level gentamicin resistance in enterococci. (See
'Agar dilution' above.)
● The broth microdilution method is commonly used in clinical microbiology laboratories.

A standardized inoculum of bacteria is incubated in dilutions of antimicrobials
commercially prepared in 96-well plates, and the lowest concentration of antimicrobial
that inhibits visible growth of bacteria is the minimum inhibitory concentration (MIC)
( picture 2). (See 'Broth microdilution' above.)
● The antimicrobial gradient method can be useful for testing fastidious organisms that
require special growing conditions. Commercially produced plastic strips with graded
antimicrobial concentrations are placed upon medium inoculated with a standardized
inoculum of bacteria, and MICs are determined by the intersection of the elliptically
shaped zone of growth inhibition and the strip ( picture 3). (See 'Antimicrobial
gradient method' above.)
● Automated susceptibility testing provides results more rapidly than traditional

laboratory methods that require overnight incubation. Clinical microbiology
laboratories must be aware that there can be a lag between the publication of new
interpretive criteria and the incorporation of the new breakpoints into the database of
an automated system. (See 'Automated methods' above.)
● Treatment regimens can often be devised on the basis of antimicrobial susceptibility

testing results without knowledge of the specific acquired resistance mechanisms
present. However, such specific identification of beta-lactamase production in
staphylococci and some other organisms, and inducible clindamycin resistance in
staphylococci, pneumococci, and beta-hemolytic streptococci, can be clinically valuable.
(See 'Methods to detect specific types of resistance' above.)
● The incorporation of genotypic antimicrobial susceptibility testing into routine use in

clinical laboratories has thus far been limited, in large part because of the high
associated cost of genotypic testing, the fact that genotypic testing does not obviate
the need for phenotypic testing, and the current limitation of US Food and Drug
Administration (FDA)-cleared assays to the detection of only one or a few specific
genetic resistance targets. (See 'Genotypic methods' above.)
● Multiple factors, including the type of organism, the site of infection, and the
institutional formulary, should inform the selection of antimicrobials for susceptibility
testing and the selective reporting of susceptibility results. (See 'Reporting susceptibility
results' above.)
ACKNOWLEDGMENTS
The UpToDate editorial staff acknowledges Mary Jane Ferraro, PhD, MPH, Jatin M Vyas, MD,
PhD, and Sarah E Turbett, MD, who contributed to an earlier version of this topic review.
Use of UpToDate is subject to the Terms of Use.
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Topic 467 Version 27.0
GRAPHICS
Methods to determine antimicrobial susceptibility for infrequently isolated

or fastidious bacteria
Organism Method
Abiotrophia species and Granulicatella species (formerly known as Broth microdilution

nutritionally deficient or nutritionally variant streptococci)
Aerococcus species Broth microdilution
Aeromonas species (includes members of the Aeromonas caviae complex, Broth microdilution
Aeromonas hydrophilia complex, and Aeromonas veronii complex)
Disk diffusion
Bacillus species (not Bacillus anthracis) and related genera Broth microdilution
Campylobacter jejuni/coli Broth microdilution
Disk diffusion
Corynebacterium species (including Corynebacterium diphtheriae) and related Broth microdilution

coryneform genera
Erysipelothrix rhusiopathiae Broth microdilution
Gemella species Broth microdilution
HACEK group: Aggregatibacter species, Cardiobacterium species, Eikenella Broth microdilution

corrodens, Kingella species
Helicobacter pylori Agar dilution
Lactobacillus species Broth microdilution
Lactococcus species Broth microdilution
Leuconostoc species Broth microdilution
Listeria monocytogenes Broth microdilution
Micrococcus species Broth microdilution
Moraxella catarrhalis Broth microdilution
Disk diffusion
Pasteurella species Broth microdilution
Disk diffusion
Pediococcus species Broth microdilution
Rothia mucilaginosa Broth microdilution
Vibrio species (including Vibrio cholerae) Broth microdilution
Disk diffusion
Potential agents of bioterrorism: Bacillus anthracis, Yersinia pestis, Broth microdilution

Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella
species
Adapted from: Clinical and Laboratory Standards Institute (CLSI). Methods for antimicrobial dilution and disk susceptibility
testing of infrequently isolated or fastidious bacteria, 3rd ed. CLSI Guideline M45, Clinical and Laboratory Standards Institute,
Wayne, Pennsylvania 2015.
Graphic 114733 Version 2.0
Disk diffusion antimicrobial susceptibility testing (Kirby-Bauer procedure)
The susceptibility of a bacterial isolate to antimicrobials is determined by measuring the diameter of

the zone of growth inhibition (in millimeters) around each of the antibiotic-impregnated disks. The
zone sizes are then classified as susceptible, intermediate, or resistant based on breakpoint tables
published by the Clinical and Laboratory Standards Institute (CLSI), the European Committee on
Antimicrobial Susceptibility Testing (EUCAST), or the United States Food and Drug
Administration (FDA).
Courtesy of Mary Jane Ferraro, PhD, MPH.
Broth microdilution antimicrobial susceptibility testing
Commercially prepared serial dilutions of commonly used antimicrobials are added to a 96-well plate.
After incubation with a standardized inoculum of a bacterial isolate, minimum inhibitory
concentrations are determined and classified as susceptible, intermediate, or resistant based on
the Clinical and Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial
Susceptibility Testing (EUCAST), or United States Food and Drug Administration (FDA) breakpoints.
Antimicrobial gradient method
A commercially prepared strip containing a graded concentration of a specific antimicrobial is placed

on a lawn of a standardized inoculum of bacteria. After incubation, the minimum inhibitory
concentration is determined by identifying the intersection of the elliptical zone of growth inhibition
with the strip.
Miniaturized minimum inhibitory concentration test card for automated

susceptibility instrument
Automated systems use microdilution trays or cards inoculated with known quantities of bacteria to
determine minimum inhibitory concentrations. Growth is monitored using photometric, fluorometric,
or turbidimetric systems.
Nitrocefin beta-lactamase test
The organism of interest is placed onto a commercially available nitrocefin disk. Nitrocefin is a
chromogenic cephalosporin; in the presence of a bacterial beta-lactamase that hydrolyzes the beta-
lactam ring, the disk changes color from yellow to pink.
Penicillin zone edge or "beach and cliff" test
A standard inoculum of the organism of interest is inoculated onto a Mueller-Hinton agar plate and
incubated in the presence of a penicillin disk. After incubation, the zone edge of inhibition of growth
around the penicillin disk is examined.
(A) A fuzzy zone edge, or "beach," indicates the absence of a beta-lactamase.
(B) A sharp zone edge, or "cliff," indicates the presence of a beta-lactamase.
Detection of inducible clindamycin resistance using the D test
Commercially available erythromycin and clindamycin disks are placed in a standardized fashion on
an agar plate inoculated with a standardized inoculum of the test organism. The plates are incubated
for 16 to 24 hours and then visually inspected.
(A) Flattening of the clindamycin zone of inhibition (D-zone) adjacent to the erythromycin disk,
indicating inducible clindamycin resistance.
(B) An isolate that is erythromycin-resistant but clindamycin-susceptible.
(C) An isolate that is susceptible to both clindamycin and erythromycin.
(D) An isolate with constitutive resistance to both clindamycin and erythromycin.
C: clindamycin disk; E: erythromycin disk.
Contributor Disclosures
Virginia M Pierce, MD No relevant financial relationship(s) with ineligible companies to
disclose. Stephen B Calderwood, MD Consultant/Advisory Boards: Day Zero Diagnostics [Whole
genome sequencing for microbial identification and determination of antimicrobial susceptibility]. All of
the relevant financial relationships listed have been mitigated. Keri K Hall, MD, MS No relevant
financial relationship(s) with ineligible companies to disclose.
Contributor disclosures are reviewed for conflicts of interest by the editorial group. When found, these
are addressed by vetting through a multi-level review process, and through requirements for
references to be provided to support the content. Appropriately referenced content is required of all
authors and must conform to UpToDate standards of evidence.
Conflict of interest policy

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21/06/2024, 00:22 Overview of antibacterial susceptibility testing - UpToDate

Official reprint from UpToDate®

Literature review current through: May 2024.

Multiple different methods for antimicrobial susceptibility testing exist, including

● Provide rapid and accurate information to the clinician

antimicrobial susceptibility testing in fungi is presented elsewhere. (See "Antifungal

BASIC CONCEPTS OF ANTIMICROBIAL RESISTANCE

In contrast, acquired resistance is the development of resistance to an antimicrobial to which

Constitutive versus inducible resistance mechanisms — The expression of some bacterial

Constitutively expressed resistance mechanisms are expressed continuously, while inducible

Heteroresistance — The phenotypic expression of an antimicrobial resistance mechanism

Heterogeneous expression, or heteroresistance, can lead to bacterial subpopulations within

concentrations. Heteroresistant vancomycin-intermediate Staphylococcus aureus is an

INDICATIONS FOR SUSCEPTIBILITY TESTING

Antimicrobial susceptibility testing should be performed when clinically significant bacteria

● When the antimicrobial susceptibility pattern of a particular organism is predictable. As

● When insufficient numbers of bacterial colonies are grown in cultures of certain

● When guidance regarding the performance of standardized susceptibility testing for a

● Standardization of the testing methods is necessary to ensure accuracy as well as intra-

Qualitative methods (disk diffusion/Kirby-Bauer method) — Overall, qualitative

Performance — In brief, the procedure involves swabbing a standardized inoculum of

Advantages — The disk diffusion method is a highly standardized method, with

Quantitative methods — Quantitative methods for antimicrobial susceptibility testing allow

Several quantitative antimicrobial susceptibility methods are currently available, including

Broth dilution — Broth dilution is performed by creating a series of twofold dilutions of a

Broth microdilution — The broth microdilution method is a popular method for

It is considered a miniaturized and more automated version of the macrodilution method

Antimicrobial gradient method — The antimicrobial gradient method is an agar-based

Interpretation of results — Conventional qualitative disk diffusion and quantitative

● Susceptible-dose dependent – Indicates that, to achieve the antibiotic concentration

● Intermediate – Indicates that the MIC of the antimicrobial approaches usually

For commonly encountered bacteria, establishment of breakpoints involves extensive review

The principal limitation of automated antimicrobial susceptibility testing systems is cost,

METHODS TO DETECT SPECIFIC TYPES OF RESISTANCE

Treatment regimens can often be devised on the basis of qualitative or quantitative

Beta-lactamase testing — The beta-lactamases produced by many commonly encountered

The chromogenic cephalosporin beta-lactamase method can be used to detect beta-

inoculum of staphylococci used for the chromogenic cephalosporin beta-lactamase test

Inducible clindamycin resistance testing — The Clinical and Laboratory Standards

High-level aminoglycoside resistance screening — CLSI recommends screening for high-

● For the broth microdilution procedure, a standardized inoculum of the enterococcal

Although phenotypic methods remain the cornerstone of antimicrobial susceptibility testing

Genotypic antimicrobial susceptibility testing methods have several important strengths.

detection of specific genetic resistance elements, phenotypic testing is still required to

REPORTING SUSCEPTIBILITY RESULTS

● Antimicrobial susceptibility testing should be performed when clinically significant

● The broth microdilution method is commonly used in clinical microbiology laboratories.

● Automated susceptibility testing provides results more rapidly than traditional

● Treatment regimens can often be devised on the basis of antimicrobial susceptibility

● The incorporation of genotypic antimicrobial susceptibility testing into routine use in

Use of UpToDate is subject to the Terms of Use.

11. European Committee on Antimicrobial Susceptibility Testing (EUCAST). www.eucast.org

Microbiol 2011; 49:S20.

40. Sullivan KV, Diekema DJ. Overview of molecular diagnostics in multiple-drug-resistant or

Methods to determine antimicrobial susceptibility for infrequently isolated

Abiotrophia species and Granulicatella species (formerly known as Broth microdilution

Aerococcus species Broth microdilution

Campylobacter jejuni/coli Broth microdilution