ANTIBIOTIC SENSITIVITY TEST
INTRODUCTION
Once we have identified the bacterium which is causing the
infection we need to find out the antibiotics that would be
effective against it. This is done by antibiotic sensitivity testing.
there are various methods which can be employed for this
purpose.
OBJECTIVES
After reading this chapter, you will be able to:
Various terminologies related to Antibiotic susceptibility
testing
Principle for Antibiotic susceptibility testing.
The procedure for performing Antibiotic susceptibility
testing
Different methods used for Antibiotic susceptibility
testing
Categories of Antibiotics
Antibiotics are categorized as bactericidal, if they kill
the susceptible bacteria or bacteriostatic, if they
reversibly inhibit the growth of bacteria. In general the
use of bactericidal antibiotics is preferred but many
factors may dictate the use of a bacteriostatic antibiotic.
When a bacteriostatic antibiotic is used the duration of
therapy must be sufficient to allow cellular and humoral
defense mechanisms to eradicate the bacteria. If
possible, bactericidal antibiotics should be used to treat
infections of the endocardium or the meninges. Host
defenses are relatively ineffective at these sites and the
dangers imposed by such infections require prompt
eradication of the organisms.
In vitro sensitivity tests
Bacterial pathogens are tested for their susceptibility to
antibiotics to guide antibiotic treatment. Sensitivity tests are
generally performed from single pure bacterial colonies on an
agar plate. Direct sensitivity tests are set up directly from
specimens or liquid cultures, producing quicker, but less
standardized results. In order to guide the appropriate antibiotic
treatment of bacterial infections, bacterial pathogens isolated
from clinical specimens are usually tested against a selection of
antibiotics to assess their degree of susceptibility. This is
usually done with bacteria that have been grown on solid media.
Sensitivity tests are performed from single pure colonies and
require a further 18–24 hrs of incubation. Thus while culture
results may be available within 24 hrs of receipt of a specimen,
sensitivity results usually take an additional day.
In some situations, direct sensitivity tests are performed, either
from the specimen itself (e.g. Urine) or from a liquid broth with
bacterial growth (e.g. Blood culture bottle). In this case,
sensitivity tests are setup at the same time as the specimen is
subcultured to agar plates. Although this speeds up the process,
there are several disadvantages:
(i) it is difficult to ensure the correct inoculum (the number
of bacteria spread onto the agar surface)
(ii) the inoculum may be mixed (more than one type of
bacteria), making the results difficult to interpret and requiring
the test to be repeated
(iii) the selection of antibiotics tested may be inappropriate for
the bacterium subsequently grown.
Disk sensitivity tests
Disk sensitivity tests are performed on agar plates. A small disk
of filter paper, preimpregnated with a defined quantity of
antibiotic, is placed on the surface of an agar plate that has
already been inoculated with a suspension of bacteria. The
antibiotic diffuses out of the disk into the agar, along a
concentration gradient, as the plates are incubated (for 18–24
h). If the bacterial strain is sensitive to the antibiotic, then a
zone of inhibition (no growth) occurs around the disk.
The diameter of the zone depends on a number of factors
including,
(i) the quantity of antibiotic within the disk
(ii) the degree of susceptibility of the bacteria to the antibiotic
(iii) the physicochemical properties of the antibiotic;
(iv) the depth (in mm) of the agar plate;
(v) the concentration of bacteria in the inoculum (semiconfluent
growth is required).
There are two methods employed to determine the sensitivity
pattern. The comparative disk test (stokes’ method) uses both
a test organism and a control organism on the same plate. The
control organism is of defined sensitivity to the antibiotics
being tested, and this method allows a direct comparison of the
diameter of the zones of inhibition between the test and control
organisms.
Standardized disk testing
This uses carefully standardized agar plates and inocula. A
standardized inoculum of the test organism is plated out across
the whole surface of the agar plate (control organisms are tested
on a separate plate). The diameter of the zones of inhibition are
measured in mm, and the organism.
Reported as sensitive or resistant based on defined cut-off
points (for example <18 mm=resistant).
One disadvantage of disk testing is that it is usually only
possible to have a maximum of six different antibiotic disks on
a standard agar plate.
Minimum inhibitory concentration (MIC)
The MIC is the minimum (lowest) concentration of an
antibiotic that will inhibit the growth of a bacterial strain.
Conventionally, this is determined using a series of doubling
dilutions of the antibiotic in liquid culture medium, to produce
a range of concentrations in test tubes (macrodilution) or in a
microtiter tray (microdilution). After inoculation of the test
strain into each antibiotic concentration, bacterial growth is
determined by visible turbidity after 18–24 h of incubation (Fig.
12.4). The MIC is the lowest concentration of antibiotic with no
visible bacterial growth.
MIC tests can also be done by extended breakpoint sensitivity
tests. These methods are technically time-consuming and
relatively expensive. An alternative method is by use of
commercially available E-test strips. These are specialized
antibiotic-impregnated strips which, like disk testing, are
placed on the surface of inoculated agar plates. During
incubation, antibiotic diffuses into the agar forming a zone of
inhibition. There is a manufactured concentration gradient
within the strip, and numerical gradations are marked along the
edge of the strip to reflect this. The MIC is determined by
measuring the point at which the edge of the zone of inhibition
crosses the e-test.
Antibiotic MIC tests are usually performed only in certain
situations in a clinical bacteriology laboratory. They are most
commonly used when a very precise assessment of the in vitro
susceptibility of a bacterial strain is required, for instance in the
treatment of pneumococcal meningitis (topic f3) or
Streptococcal endocarditis. MIC tests are also used to assess the
overall degree of activity of antibiotics against different strains
of the same bacterial species, particularly when evaluating or
developing new antimicrobial agents. A simple way of
describing the relative activity of an antibiotic against a group
of organisms, is by using the terms mic50 and mic90. These are
the lowest concentrations of the antibiotic that inhibit 50 and
90% of the bacterial strains tested, respectively.
Minimum bactericidal concentration (MBC)
The MBC is the lowest concentration of the antibiotic that
will ‘kill’ a bacterial strain. The definition of ‘killing’ is a
99.9% (3 Iog10) reduction in viable bacteria. The MBC test
is an extension of an MIC test.
The simplest method for determining the MBC is to perform a
subculture from antibiotic concentrations with no visible
growth in the MIC test on to antibiotic free agar. This will
determine whether the bacteria have been inhibited from
growing but are still viable, or whether they have been killed.
Some antibiotics are highly bactericidal. In this case the
MIC and MBC are usually very similar. Bacteristatic
antibiotics on the other hand have much higher MBC than
MIC.
Occasionally a bacterial strain may have a high MBC
but low MIC with a normally bactericidal antibiotic (e.g.
Penicillin). This is described as bacterial ‘tolerance’ to
the antibiotic.
MBC tests are very difficult to standardize and are
often not entirely reproducible. The clinical relevance
of MBC tests and the demonstration of tolerance is
less clear than with MIC determinations, but they are
occasionally performed to guide antibiotic therapy in
some difficult cases of infection.
Automated sensitivity tests
There are a variety of commercially available automated
systems available to help reduce the technical time required to
perform and record routine sensitivity tests. For example, the
results of disk sensitivity tests and breakpoint sensitivity
tests can be read using a camera interfaced to a computer
system. Other systems utilize liquid cultures, and detect the
effect of antibiotics on the rate of bacterial growth through
measurement of turbidity (nephelometry) or the production of
co2. These automated systems can significantly shorten the
necessary incubation time, with the possibility of some
results being available within the same working day. They
can also significantly reduce the time taken to produce
sensitivity results for slow-growing organisms, notably
mycobacterium tuberculosis.
Clinical relevance of in vitro antibiotic sensitivity tests
It must be remembered that in vitro sensitivity tests are only a
guide to the appropriate antibiotic treatment. A laboratory
report indicating that organism A,
is resistant to antibiotic B does not necessarily mean that
antibiotic B will not work, and vice versa. Whilst in vitro tests
are designed to try and reflect the in
vivo situation (e.g. through utilization of appropriate
breakpoints to reflect antibiotic pharmacokinetic
parameters), they can never take account of all the
human and bacterial biological variables.
There are a number of factors to consider when interpreting
laboratory reports that include sensitivity test results:
(i) many infections will resolve spontaneously (assuming a
normal immune system). If a patient has already responded
clinically to a certain antibiotic treatment, then it is not
always necessary to change the antibiotic if the laboratory
report indicates that the organism isolated is ‘resistant’.
(ii) the organism identified on the laboratory report may not be
the primary pathogen.
(iii) an organism reported with sensitivity results does not
always require treatment. A good example of this is with
catheter-specimens of urine. Treatment is generally required
only if the patient is symptomatic (i.e. Treat the patient not
the result!).
(iv)an antibiotic with apparent in vitro activity may not work
clinically, as there are many pharmacokinetic and other
factors to consider in choosing the most appropriate
antibiotic therapy.
Conclusion
Summarize findings and emphasize the critical role of AST in
combating antibiotic resistance and improving patient care
outcomes. Discuss implications for public health policy and
antibiotic stewardship practices, underscoring the importance
of prudent antibiotic use based on AST results.
References
Cite comprehensive sources including textbooks, research
articles, and authoritative guidelines (e.g., WHO, CDC) on
AST methodologies and antibiotic resistance.
