Note: For testing the response of staphylococci to benzylpenicillin, only the categories “susceptible”

and “resistant” (corresponding to the production of β-lactamase) are recognized.

Other categories
1. Susceptible-Dose Dependent (SDD):
“susceptible-dose dependent” is a new category for antibacterial susceptibility testing. If a particular
isolate falls under this category, we have to remember that the susceptibility of that isolate depends on
the dosing regimen used. Higher doses or more frequent doses or both is used to achieve concentration
levels that is likely to be clinically effective against the isolate. While prescribing that antibiotics
clinicians should give propoer consideration to the maximum approved dosage regimen.
2. Nonsusceptible (NS)
The “nonsusceptible” category is used for isolates for which only a susceptible interpretive criterion has
been designated because of the absence or rare occurrence of resistant strains.
Isolates for which the antimicrobial agent MICs are above or zone diameters below the value indicated
for the susceptible breakpoint should be reported as nonsusceptible.

References and further reading:

 CLSI: Performance Standards for Antimicrobial Susceptibility Testing.

Abstract

An important task of the clinical microbiology laboratory is the performance of antimicrobial

susceptibility testing of significant bacterial isolates. The goals of testing are to detect possible
drug resistance in common pathogens and to assure susceptibility to drugs of choice for
particular infections. The most widely used testing methods include broth microdilution or rapid
automated instrument methods that use commercially marketed materials and devices. Manual
methods that provide flexibility and possible cost savings include the disk diffusion and gradient
diffusion methods. Each method has strengths and weaknesses, including organisms that may be
accurately tested by the method. Some methods provide quantitative results (eg, minimum
inhibitory concentration), and all provide qualitative assessments using the categories
susceptible, intermediate, or resistant. In general, current testing methods provide accurate
detection of common antimicrobial resistance mechanisms. However, newer or emerging
mechanisms of resistance require constant vigilance regarding the ability of each test method to
accurately detect resistance.

Emergence of Antimicrobial Resistance and the Rationale for Performing Susceptibility

Testing

The performance of antimicrobial susceptibility testing by the clinical microbiology

laboratory is important to confirm susceptibility to chosen empirical antimicrobial
agents, or to detect resistance in individual bacterial isolates. Empirical therapy
continues to be effective for some bacterial pathogens because resistance mechanisms
have not been observed e.g., continued penicillin susceptibility of Streptococcus
pyogenes . Susceptibility testing of individual isolates is important with species that
may possess acquired resistance mechanisms (eg, members of the
Enterobacteriaceae, Pseudomonas species, Staphylococcus species, Enterococcus spec
ies, and Streptococcus pneumoniae ).

Overview of Commonly Used Susceptibility Testing Methods

Broth dilution tests. One of the earliest antimicrobial susceptibility testing methods
was the macrobroth or tube-dilution method [1]. This procedure involved preparing
two-fold dilutions of antibiotics (eg, 1, 2, 4, 8, and 16 µg/mL) in a liquid growth
medium dispensed in test tubes [1, 2]. The antibiotic-containing tubes were inoculated
with a standardized bacterial suspension of 1–5×105CFU/mL. Following overnight
incubation at 35°C, the tubes were examined for visible bacterial growth as evidenced
by turbidity. The lowest concentration of antibiotic that prevented growth represented
the minimal inhibitory concentration (MIC). The precision of this method was
considered to be plus or minus 1 two-fold concentration, due in large part to the
practice of manually preparing serial dilutions of the antibiotics [3]. The advantage of
this technique was the generation of a quantitative result (ie, the MIC). The principal
disadvantages of the macrodilution method were the tedious, manual task of preparing
the antibiotic solutions for each test, the possibility of errors in preparation of the
antibiotic solutions, and the relatively large amount of reagents and space required for
each test.

The miniaturization and mechanization of the test by use of small, disposable, plastic
“microdilution” trays (Figure 1) has made broth dilution testing practical and popular.
Standard trays contain 96 wells, each containing a volume of 0.1 mL that allows
approximately 12 antibiotics to be tested in a range of 8 two-fold dilutions in a single
tray [2, 4]. Microdilution panels are typically prepared using dispensing instruments
that aliquot precise volumes of preweighed and diluted antibiotics in broth into the
individual wells of trays from large volume vessels. Hundreds of identical trays can be
prepared from a single master set of dilutions in a relatively brief period. Few clinical
microbiology laboratories prepare their own panels; instead frozen or dried
microdilution panels are purchased from one of several commercial suppliers. The
cost of the preprepared panels range from approximately $10 to $22 each. Inoculation
of panels with the standard 5×105CFU/mL is accomplished using a disposable device
that transfers 0.01 to 0.05 mL of standardized bacterial suspension into each well of
the microdilution tray or by use of a mechanized dispenser. Following incubation,
MICs are determined using a manual or automated viewing device for inspection of
each of the panel wells for growth [2].
Figure 1

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A broth microdilution susceptibility panel containing 98 reagent wells and a

disposable tray inoculator

Figure 1

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A broth microdilution susceptibility panel containing 98 reagent wells and a

disposable tray inoculator

The advantages of the microdilution procedure include the generation of MICs, the
reproducibility and convenience of having preprepared panels, and the economy of
reagents and space that occurs due to the miniaturization of the test. There is also
assistance in generating computerized reports if an automated panel reader is used.
The main disadvantage of the microdilution method is some inflexibility of drug
selections available in standard commercial panels.

Antimicrobial gradient method. The antimicrobial gradient diffusion method uses the
principle of establishment of an antimicrobial concentration gradient in an agar
medium as a means of determining susceptibility. The Etest (bioMérieux AB
BIODISK) (Figure 2) is a commercial version available in the United States. It
employs thin plastic test strips that are impregnated on the underside with a dried
antibiotic concentration gradient and are marked on the upper surface with a
concentration scale. As many as 5 or 6 strips may be placed in a radial fashion on the
surface of an appropriate 150-mm agar plate that has been inoculated with a
standardized organism suspension like that used for a disk diffusion test. After
overnight incubation, the tests are read by viewing the strips from the top of the plate.
The MIC is determined by the intersection of the lower part of the ellipse shaped
growth inhibition area with the test strip.

Figure 2

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A Staphylococcus aureus isolate tested by the Etest gradient diffusion method with
vancomycin (VA), daptomycin (DM), and linezolid (LZ) on Mueller-Hinton agar. The
minimum inhibitory concentration of each agent is determined by the intersection of
the organism growth with the strip as measured using the scale inscribed on the strip.

Figure 2

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A Staphylococcus aureus isolate tested by the Etest gradient diffusion method with
vancomycin (VA), daptomycin (DM), and linezolid (LZ) on Mueller-Hinton agar. The
minimum inhibitory concentration of each agent is determined by the intersection of
the organism growth with the strip as measured using the scale inscribed on the strip.

The gradient diffusion method has intrinsic flexibility by being able to test the drugs
the laboratory chooses. Etest strips cost approximately $2-$3 each and can represent
an expensive approach if more than a few drugs are tested. This method is best suited
to situations in which an MIC for only 1 or 2 drugs is needed or when a fastidious
organism requiring enriched medium or special incubation atmosphere is to be tested
(eg, penicillin and ceftriaxone with pneumococci) [5-7]. Generally, Etest results have
correlated well with MICs generated by broth or agar dilution methods [5-9].
However, there are some systematic biases toward higher or lower MICs determined
by the Etest when testing certain organism-antimicrobial agent combinations [6, 10].
This can represent a potential shortcoming when standard MIC interpretive criteria
derived from broth dilution testing [10] are applied to Etest MICs that may not be
identical.

Disk diffusion test. The disk diffusion susceptibility method [2, 11, 12] is simple and
practical and has been well-standardized. The test is performed by applying a bacterial
inoculum of approximately 1–2×108CFU/mL to the surface of a large (150 mm
diameter) Mueller-Hinton agar plate. Up to 12 commercially-prepared, fixed
concentration, paper antibiotic disks are placed on the inoculated agar surface (Figure
3). Plates are incubated for 16–24 h at 35°C prior to determination of results. The
zones of growth inhibition around each of the antibiotic disks are measured to the
nearest millimeter. The diameter of the zone is related to the susceptibility of the
isolate and to the diffusion rate of the drug through the agar medium. The zone
diameters of each drug are interpreted using the criteria published by the Clinical and
Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical
Laboratory Standards or NCCLS) [13] or those included in the US Food and Drug
Administration (FDA)-approved product inserts for the disks. The results of the disk
diffusion test are “qualitative,” in that a category of susceptibility (ie, susceptible,
intermediate, or resistant) is derived from the test rather than an MIC. However, some
commercially-available zone reader systems claim to calculate an approximate MIC
with some organisms and antibiotics by comparing zone sizes with standard curves of
that species and drug stored in an algorithm [14, 15].

Figure 3

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A disk diffusion test with an isolate of Escherichia coli from a urine culture. The
diameters of all zones of inhibition are measured and those values translated to
categories of susceptible, intermediate, or resistant using the latest tables published by
the CLSI.

Figure 3

View largeDownload slide

A disk diffusion test with an isolate of Escherichia coli from a urine culture. The
diameters of all zones of inhibition are measured and those values translated to
categories of susceptible, intermediate, or resistant using the latest tables published by
the CLSI.

The advantages of the disk method are the test simplicity that does not require any
special equipment, the provision of categorical results easily interpreted by all
clinicians, and flexibility in selection of disks for testing. It is the least costly of all
susceptibility methods (approximately $2.50-$5 per test for materials). The
disadvantages of the disk test are the lack of mechanization or automation of the test.
Although not all fastidious or slow growing bacteria can be accurately tested by this
method, the disk test has been standardized for testing streptococci, Haemophilus
influenzae, and N. meningitidis through use of specialized media, incubation
conditions, and specific zone size interpretive criteria [12].

Automated instrument systems. Use of instrumentation can standardize the reading of

end points and often produce susceptibility test results in a shorter period than manual
readings because sensitive optical detection systems allow detection of subtle changes
in bacterial growth. There are 4 automated instruments presently cleared by the FDA
for use in the United States. Three of these can generate rapid (3.5–16 h) susceptibility
test results, while the fourth is an overnight system [16]. The MicroScan WalkAway
(Siemens Healthcare Diagnostics) is a large self-contained incubator/reader device
that can incubate and analyze 40–96 microdilution trays. The WalkAway utilizes
standard size microdilution trays that are hydrated and inoculated manually and then
placed in one of the incubator slots in the instrument. The instrument incubates the
trays for the appropriate period, examining them periodically with either a photometer
or fluorometer to determine growth development. Gram-negative susceptibility test
panels containing fluorogenic substrates can be read in 3.5–7 h. Separate gram-
positive and gram-negative panels read using turbidimetric end points are ready in
4.5–18 hours.

The BD Phoenix Automated Microbiology System (BD Diagnostics) has a large

incubator reader with a capacity to process 99 test panels that contain 84 wells
devoted to antibiotic doubling dilutions and are inoculated manually. The Phoenix
monitors each panel every 20 min using both turbidometric and colorimetric
(oxidation-reduction indicator) growth detection. Test panels for gram-negative,
gram-positive, S. pneumoniae, β-hemolytic, and viridans group streptococci are
available. MIC results are generated in 6–16 h.

The Vitek 2 System (bioMérieux) is highly automated and uses very compact plastic
reagent cards (credit card size) that contain microliter quantities of antibiotics and test
media in a 64-well format. The Vitek 2 employs repetitive turbidimetric monitoring of
bacterial growth during an abbreviated incubation period. The instrument can be
configured to accommodate 30–240 simultaneous tests. The susceptibility cards allow
testing of common, rapidly growing gram-positive, and gram-negative aerobic
bacteria, and S. pneumoniae in a period of 4–10 h. An older, less automated, Vitek 1
System is still used in some laboratories. The system is more limited with a 45-well
card and does not include S. pneumoniae .

The Sensititre ARIS 2X (Trek Diagnostic Systems) is an automated, overnight,

incubation and reading system with a 64-panel capacity. The test panels are standard
96-well microdilution plates that can be inoculated with a Sensititre Autoinculator.
Growth is determined by fluorescence measurement after 18–24 h of incubation. Test
panels are available for gram-positive and gram-negative bacteria, S.
pneumoniae, Haemophilus species, and nonfermentative gram-negative bacilli.

The Phoenix, Sensititre ARIS 2X, Vitek 1 and 2, and WalkAway instruments have
enhanced computer software used to interpret susceptibility results including “expert
systems” for analyzing test results for atypical patterns and unusual resistance
phenotypes [16]. Two studies [17, 18] have shown that providing rapid susceptibility
test results can lead to more timely changes to appropriate antimicrobial therapy,
substantial direct cost savings attributable to ordering of fewer additional laboratory
tests, performance of fewer invasive procedures, and a shortened length of stay. These
benefits are best realized when coupled with extended laboratory staffing schedules,
and real-time, electronic transmission of verified results. One of the early
shortcomings of rapid susceptibility testing methods was a lessened ability to detect
some types of antimicrobial resistance including inducible β-lactamases and
vancomycin resistance. However, the recently FDA-cleared instruments have made
significant improvements in large part through modifications of the instruments'
computer software to either provide extended incubation for problematic organism-
drug combinations, or by editing of susceptibility results using expert software to
prevent unlikely results from being reported. In some cases these modifications result
in prolonged incubation (ie, >10 h) of test panels to assure accurate results, thus
rendering them less “rapid.”

Selection of Drugs for Routine Testing

The laboratory must test and report the antimicrobial agents that are most appropriate
for the organism isolated, for the site of the infection, and the institution's formulary
[13, 19]. The CLSI provides tables that list the antimicrobial agents appropriate for
testing members of the Enterobacteriaceae, Pseudomonas, and other gram-negative
glucose nonfermenters, staphylococci, enterococci,
streptococci, Haemophilus species, etc. [13]. The listings include recommendations
for agents that are important to test routinely, and those that may be tested or reported
selectively based on the institution's formulary.

The availability of antimicrobial agents for testing by the laboratory's routine testing
methodology must next be determined. The disk diffusion and gradient diffusion
procedures offer the greatest flexibility including testing of newly available drugs.
Most broth microdilution or automated test panels contain ⩽96 wells, effectively
limiting the number of agents tested or the range of dilutions of each drug that can be
included. Manufacturers of commercially prepared panels have attempted to deal with
this problem by offering a number of different standard panel configurations, or by
including fewer dilutions of each drug in a single panel [19]. Another solution to this
problem is testing antimicrobial agents that have activities that are essentially the
same as the desired formulary drugs. The CLSI susceptibility testing document [13]
lists groups of some antimicrobial agents with nearly identical activities that can
provide practical alternatives for testing.

Interpretation of Susceptibility Test Results

The results of a susceptibility test must be interpreted by the laboratory prior to

communicating a report to a patient's physician. Optimal interpretation of MICs
requires knowledge of the pharmacokinetics of the drug in humans, and information
on the likely success of a particular drug in eradicating bacteria at various body sites
[20]. This is best accomplished by referring to an expert source such as the CLSI,
which publishes interpretive criteria for MICs of all relevant antibiotics for most
bacterial genera [13]. Indeed, both MIC values and disk diffusion zone diameters must
be interpreted using a table of values that relate to proven clinical efficacy of each
antibiotic and for various bacterial species [12]. The CLSI zone size and MIC
interpretive criteria are established by analysis of 3 kinds of data: (1.) microbiologic
data, including a comparison of MICs and zone sizes on a large number of bacterial
strains, including those with known mechanisms of resistance that have been defined
either phenotypically or genotypically; (2) pharmacokinetic and pharmacodynamic
data; and (3) clinical studies results (including comparisons of MIC and zone diameter
with microbiological eradication and clinical efficacy) obtained during studies prior to
FDA approval and marketing of an antibiotic [20].

A “susceptible” result indicates that the patient's organism should respond to therapy
with that antibiotic using the dosage recommended normally for that type of infection
and species [13, 20]. Conversely, an organism with a MIC or zone size interpreted as
“resistant” should not be inhibited by the concentrations of the antibiotic achieved
with the dosages normally used with that drug [13, 20]. An “intermediate” result
indicates that a microorganism falls into a range of susceptibility in which the MIC
approaches or exceeds the level of antibiotic that can ordinarily be achieved and for
which clinical response is likely to be less than with a susceptible strain. Exceptions
can occur if the antibiotic is highly concentrated in a body fluid such as urine, or if
higher than normal dosages of the antibiotic can be safely administered (eg, some
penicillins and cephalosporins). At times, the “intermediate” result can also mean that
certain variables in the susceptibility test may not have been properly controlled, and
that the values have fallen into a “buffer zone” separating susceptible from resistant
strains [13, 20]. Generally, reporting of a category result of susceptible, intermediate,
or resistant provides the clinician with the information necessary to select appropriate
therapy. Reporting of MICs could aid a physician is selecting from among a group of
similar drugs for therapy of infective endocarditis or osteomyelitis, in which therapy
is likely to be protracted.

It is important that the tables used for susceptibility test interpretations represent the
most current criteria. Indeed, the CLSI documents are reviewed and updated
frequently, usually once per year. Use of old or outdated information from the original
editions of FDA-approved drug labels or older CLSI tables could represent a serious
shortcoming in the reporting of patients' results.
What Is the Acceptable Accuracy of a Susceptibility Test Method?

When assessing the accuracy of various susceptibility testing methods as compared to

standard reference methods, the terms very major and major errors have been used to
describe false-susceptible or false-resistant results, respectively. In evaluations of new
susceptibility testing methods it is important to examine a representative number of
strains that are resistant to various drugs to verify the ability of the new test to detect
resistance and to test a number of susceptible strains to determine the rate of major
errors that might be expected in a typical clinical laboratory setting [16, 21]. To be
cleared for marketing in the United States, the FDA requires that very major errors
attributable to a test device should be <1.5% for individual species/drug comparisons,
major errors should not exceed 3%, and an overall essential MIC agreement of >90%
of device MICs within one doubling dilution of a CLSI reference MIC [22]. A recent,
international standard on susceptibility test device evaluation proposes similar but not
identical criteria for acceptable accuracy [23]. The emergence of new antimicrobial
resistance mechanisms, including some that may be difficult to detect (eg,
vancomycin intermediate susceptibility in S. aureus and carbapenemase production in
some gram-negative organisms) requires that the performance of susceptibility
devices be constantly reassessed and updated when needed. In some cases, it has been
necessary to employ special ancillary testing methods (eg, single concentration
screening agars, modified Hodge test for carbapenemase production) [13] to
supplement routine testing by a commercial instrument system.

Current Test Methods and Future Directions

The antimicrobial susceptibility testing methods described in this article provide

reliable results when used according to the procedures defined by the CLSI or by the
manufacturers of the commercial products. However, there is considerable
opportunity for improvement in the area of rapid and accurate recognition of bacterial
resistance to antibiotics. There is a need for development of new automated
instruments that could provide faster results and also save money by virtue of lower
reagent costs and reduced labor requirements. To accomplish this, it will likely be
necessary to explore different methodologic approaches for detection of bacterial
growth. The direct detection of resistance genes by polymerase chain reaction or
similar techniques has limited utility, because only a few resistance genes are firmly
associated with phenotypic resistance (eg, mecA, vanA, and vanB ) [24]. There are
hundreds of β-lactamases, and numerous mutations, acquisitions, and expression
mechanisms that result in fluoroquinolone, aminoglycoside, and macrolide resistance
[25]; too many to be easily detected by current molecular techniques. Thus, it seems
likely that phenotypic measures of the level of susceptibility of bacterial isolates to
antimicrobial agents will continue to be clinically relevant for years to come.