For bacteria, the other way to ascertain the drug
susceptibilities of an organism is by laboratory
testing. Current techniques of laboratory suscepti-
bility testing involve growth of the organism, so the
rapidity with which the results are available depends
upon the rate of
in vitro
growth of the organism.
The results from fast-growing organisms, such as
most bacteria, can be known within days. Infection
with one of these pathogens can be treated empiri-
cally until the susceptibility results are available
then, if the results indicate that there is a more
appropriate antibiotic, the patient can be switched to
that drug. Susceptibility testing of slow-growing
pathogens, such as mycobacteria, yields results only
after weeks.
The most common laboratory methods for
testing antimicrobial susceptibility are the disk diffu-
sion susceptibility test and the microdilution broth
susceptibility test (Jorgensen and Ferraro 1998). In
the disk diffusion susceptibility test (also called the
Bauer-Kirby test) antibiotic impregnated disks of
filter paper are placed on the surface of an agar
culture plate with defined medium that has previ-
ously been uniformly inoculated with a suspension
containing a standard number of the organism to be
tested. The antibiotics that are studied are those that
have an established clinical role in treatment of
infections caused by the species of organism being
tested. The plate is incubated under ambient air at
35şC. During the incubation, antibiotic diffuses out
of the disk and into the agar, establishing a logarith-
mic concentration gradient. Bacterial growth is
inhibited close to the disk where the concentration of
antibiotic exceeds the minimal inhibitory concentra-
tion (MIC) for the organism while bacterial growth
proceeds at a distance from the disk where the
antibiotic concentration is lower. Over time, con-
tinuing diffusion of the antibiotic causes the MIC to
be located farther and farther from the disk resulting
in a larger and larger zone of growth inhibition. At
some point, called the critical time, the density of
the growing bacteria beyond the MIC is great
enough that subsequent exposure to higher concen-
trations of the antibiotic does not inhibit further
growth. Consequently, a discrete interface develops
at what was the location of the MIC at the critical
time: bacterial growth is present outside, clear agar
is present inside. This interface demarcates the
definitive zone of inhibition, the width of which is
proportional to the MIC for the organism. Because
a standard amount of antibiotic is present in the disks
used in the test, there is a defined inverse relation-
ship between zone width and MIC. Tables provide
critical values for zone widths based on the MICs
that correspond to the widths. The critical values
define three susceptibility categories: susceptible,
resistant, or intermediate. Susceptible organisms
have an MIC lower than the antibiotic concentrations
that are ordinarily obtained by usual doses of the
drug, resistant organisms have an MIC higher than
the drug concentrations that are usually achievable
clinically, and intermediate organisms have an MIC
in the range of drug concentrations usually achieved
only by maximal doses of the drug. Note that, for a
variety of reasons, the most important of which is
that
in vitro
testing is not a perfect model of
in vivo
drug action,
in vitro
susceptibility of an organism
does not assure
in vivo
effectiveness of the drug.
The clinician must be knowledgeable concerning the
drugs, the species of pathogen, and the sites of
infection for which there is clinical experience of a
disparity between
in vitro
and
in vivo
susceptibility
results.
The microdilution broth susceptibility test (or
microtube dilution test) provides a direct estimate of
the MIC of the organism being tested. A range of
clinically meaningful concentrations of antibiotic are
added to defined broth culture medium in the wells
of a microtiter plate. The wells are then inoculated
with a suspension containing a standard number of
the organism to be tested. The plate is incubated at
35şC for 16 to 18 h and then evaluated either
visually or by instrumental means for turbidity due
to bacterial growth. The MIC is measured as the
lowest antibiotic concentration studied that inhibits
growth of the organism. The actual MIC is some-
where between that concentration and the next lower
concentration studied but exactly where in that range
cannot be ascertained without further testing. The
measured MIC is used to determine the susceptibility
category of the organism. The susceptibility cate-
gory is reported along with or in place of the MIC.
REFERENCES
Ardron MJ, Westengard JC, and Dutcher TF. 1994. Band
neutrophil counts are unnecessary for the diagnosis of
infection in patients with normal total leukocyte
counts. Am J Clin Pathol 102:646.
Bentley SA, Pegram MD, and Ross DW. 1987. Diagnosis
of infective and inflammatory disorders by flow
cytometric analysis of blood neutrophils. Am J Clin
Pathol 88:177.
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