1898 SECTION 16 INFECTIOUS DISEASES

of cells present contained in the total population of bacteria at the infection

site that are more resistant to the antimicrobial than the MIC
would indicate. This is particularly true for most -lactam antibiotics
and gram-negative bacilli, where a 100-fold increase in the size of an
inoculum can increase the -lactams MIC so as to make a susceptible
organism tested at a lower inoculum resistant at a higher inoculum.
Use of the antimicrobial in vivo (where a higher inoculum of bacteria
is often present) could select these more resistant subpopulations,
resulting in poor clinical response. A specific example occurs with
infections owing to strains of Enterobacter and Citrobacter species.
These bacteria overproduce chromosomal -lactamases that are not
detected in vitro.25 Treatment of a patient with a -lactam susceptible
to degradation by the -lactamase then can result in an unacceptably
high frequency of resistance development and treatment failure.
8 Many other factors also can influence the in vitro MIC value
obtained and its subsequent application to the in vivo situation.
The bacterial growth medium used and cation content can affect
the activity of many drugs significantly. For example, aminoglycosides
are less active against P. aeruginosa in a medium supplemented
with physiologic concentrations of magnesium and calcium (NCCLS
standardized method) than in a medium without these cations. MIC
values of antibiotics that are highly bound to plasma proteins are
significantly higher when the test medium contains human serum.
Since testing of these drugs in a serum-supplemented medium has
not gained widespread acceptance, their in vivo activity may be overestimated
by in vitro MIC test results. Fortunately, the standardized
guidelines for testing and quality assurance procedures proposed by
the NCCLS attempt to minimize the impact of these problems and
are followed by most clinical and research laboratories. 26 However,
when a patient infected with an apparently susceptible organism fails
therapy, it is important for the clinician to consider these potential confounding
factors as possibly being related to the observed failure. In
such situations, consideration of antimicrobial pharmacokinetics and
pharmacodynamics also often can help to better predict therapeutic
response as compared with organism susceptibility alone.

QUALITATIVE ANTIMICROBIAL SUSCEPTIBILITY

TEST METHODS
DISK DIFFUSION ASSAY

The disk diffusion assay method for susceptibility testing (BauerKirby method) was developed in the 1960s by Bauer and coworkers
as a way to reduce the labor needed for tube dilution susceptibility
testing.27 It still remains one of the more common susceptibility test
methods used in the clinical microbiology laboratory owing to its
high degree of standardization, reliability, flexibility, low cost, and
simplicity of test interpretation. 25 Up to 12 user-selected antibioticimpregnated
disks are placed on an agar plate previously streaked
with a standard suspension of bacteria (12 108 CFU/mL). The
drug contained in the disk diffuses in a concentration gradient out
into the agar. The plate is incubated (18 to 24 hours at 35 C [95F]),
and visual bacterial growth occurs only in areas in which the drug
concentrations are below those required for growth inhibition. The
diameters of the zones of inhibition are measured via calipers or automated
scanners and are compared with standard zone size ranges
that determine susceptibility, intermediate susceptibility, or resistance
to the antimicrobials that were tested (Fig. 1035). Although factors
such as agar composition, incubation temperature, bacterial inoculum,
and antibiotic paper disk composition can influence results, the
standards for testing conditions and interpretive zone sizes are well
defined by the NCCLS.

FIGURE 1035. Disk diffusion susceptibility test. Antibiotic-impregnated disks

are placed on the surface of a plate previously inoculated with the test organism.
The plate is incubated for 18 hours, and the subsequent zones of inhibition
are measured. The zone size correlates with the sensitivity of the organism. The
larger the zone, the more sensitive is the organism to the specific antibiotic.
On the basis of predetermined zone breakpoints, organisms may be classified
as susceptible, resistant, or intermediately susceptible to the antibiotic. (Photograph
courtesy of the Anti-Infective Research Laboratory, Wayne State
University, Detroit, Michigan.)

Although a survey that was conducted of clinical microbiology

laboratories in the 1990s indicated that only approximately onequarter
of laboratories performed disk diffusion as the primary susceptibility
test (the remainder used primarily microdilution methods/
automated testing systems), the lack of realization of substantial cost
savings from automated susceptibility testing systems (discussed in
more detail below), along with the emergence of more difficult-todetect
resistance mechanisms, may result in an increased use of this
time-proven susceptibility testing method, especially for smaller clinical
laborotories.23,28

QUALITATIVE VERSUS QUANTITATIVE

SUSCEPTIBILITY TESTING OF MICROORGANISMS
MIC data often are reported qualitatively by deeming