In Vitro Testing of
Antimicrobial Agents
Maryam Monajemzadeh , Pathologist
Children Medical Center Hospital
Tehran University Of Medical Sciences
History
 Paul Ehrlich
1910
Upon making the observation that some dyes stained
bacterial cells, but not animal cells, Ehrlich
determined that there was a fundamental difference
between the 2 types of cells. He began the search for
the “magic bullet”- a drug that would kill a
microbial pathogen without harming the human
host.
History
After 605 tests to find a cure for syphilis,
Ehrlich was successful in 1910. He proved
that arsphenamine, a compound of arsenic,
was effective in treating lab animals. The
ability of the new drug, named Salvarson, to
cure syphilis, proved that chemicals could be
used to selectively kill microorganisms
without harming the human host
permanently.
Paul Ehrlich
History
 Gerhard Domagk 1932

accidentally discovered the first sulfa drug,
sulfanilamide, while testing a red dye called
Prontosil on streptococci
Gerhard
Domagk
History
 Fleming 1928

noticed that colonies of Staphylococcus were
inhibited by mold


Fleming identified the mold as a species of
Penicillium
with further testing, it was shown that Penicillium was
a bacteria-killing substance
Alexander
Fleming
History
 Ernst Chain & Howard Florey


successfully purified penicillin
1941 - 1st test on an ill human



the patient improved dramatically,
but died when the penicillin ran out
mass development of penicillin
was spurred on by WW2
first antibiotic developed for the
general public
Sir Howard Florey
Dr. Ernst Chain
Importance:
More important than pathogen
identification
Uses of Antibiotic Sensitivity
Testing
 Antibiotic sensitivity test: A laboratory test
which determines how effective antibiotic
therapy is against a bacterial infections.
 Antibiotic sensitivity testing will control the
use of Antibiotics in clinical practice
 Testing will assist the clinicians in the choice
of drugs for the treatment of infections.
Components of Antibiotic
Sensitivity Testing
 1.The identification of relevant pathogens in
exudates and body fluids collected from
patients
 2. Sensitivity tests done to determine the
degree of sensitivity or resistance of
pathogens isolated from patient to an
appropriate range of antimicrobial drugs
 3. Assay of the concentration of an
administered drug in the blood or body fluid of
patient required to control the schedule of
dosage.
Antibiotic Sensitivity Testing Is
Essential of selection of
Antibiotics
The main objective:
 To predict the outcome of treatmentwith the
antimicrobial agents
Susceptible:
Resistant:
 There is a high probablity
 Tx with that antibiotic is
that the patient will
respond to tx with
appropriate dosage
regimen for that antibiotic.
likely to fail
90-60 rule
 For many infections we can expect tx success
about 90% of the time when the organism
tests as susceptible to that tx
 Success will occur in around 60% of cases
when the organism tests as resistant to that tx
 60% response= the natural response to many
bacterial infections in the immunogically nl
host
Intermediate:
 Higher doses
 Normally concentrated
 effective
Intermediate:
 Higher doses is dangerous
 Limited penetration
 ineffective
Buffer zone:
 Prevents strains with borderline
susceptibilities from incorrectly categorized
as resistant
Patient has been given the
empirical therapy before report of
lab
 Confirm the susceptibility:
•
Alternatives
o
o
o
adverse reactions
Price
Narrow spectrum
 Indicate resistant
Susceptibility tests:
Only with pathogens:
 for
which standard methods are
available
 Resist or suscep is a major concern, not
normal flora or contaminations
Uses of Antibiotic Sensitivity
Testing
 Helps to guide the Physician in choosing
Antibiotics
 The accumulated results on different
pathogens their sensitivity will guide the
physician in choosing empirical treatment in
serious patients before the individual’s
laboratory results are analyzed in the
Microbiology laboratory.
 Reveals the changing trends in the local
isolates.
 Helps the local pattern of antibiotic
prescribing.
Why Need continues for testing for
Antibiotic Sensitivity
 Bacteria have the ability
to develop resistance
following repeated or
subclinical (insufficient)
doses
 so more advanced
antibiotics and synthetic
antimicrobials are
continually required to
overcome them.

Testing Antibiotic
Susceptibility
 Antibiotic sensitivity test: A laboratory test
which determines how effective antibiotic
therapy is against a bacterial infections.
 Antibiotic sensitivity test: the in vitro
testing of bacterial cultures with antibiotics to
determine susceptibility of bacteria to
antibiotic therapy.
Definitions:
o Susceptible : an infection due to a specific
isolate can be appropriately treated with the
recommended dosage of antibiotic.
o Resistant : isolate will not or is unlikely to
respond to achievable concentrations of the
antibiotic using normal doses.
o Intermediate: infection caused by the specific
isolate can be treated with an antibiotic if treated
with high doses
 or if theinfection is in an anatomic site where
the antibiotic is concentrated, for example, blactam antibiotics in the urine.
Host factors affecting treatment
 Dosage
 route of administration
 immune status
 distribution space of the antibiotic
 pharmokinetic characteristics of the antibiotic
 and the hepatic and/or renal functional status of the




patient
Serum protein binding
Drug interactions
Multiple simultaneous infections
Virulence of organism
 Site and severity of infection
The pharmacological concept for
breakpoints
The concentration range tested for a drug and the
interpretative criteria for various categories are based
on extensive studies that correlate with:



Serum achievable levels for each antimicrobial agent
Particular resistance mechanisms
Successful therapeutic outcome
From breakpoints to interpretation
Sensitive strain
Intermediate strain
Resistant strain
Measuring antimicrobial sensitivity of a strain
isolated from a patient, to determine its status
as S, I or R is an individual problem
Defining the status of a bacterial species or genus
is an epidemiological problem distributed
across time and space that requires monitoring
 In order to have intra- and interlaboratory
reproducibility, and have different
laboratories obtain the same results, these
variables must be standardized.
Inhibitory Methods for Susceptibility
Testing
1. Dilution Testing
2. Epsilometer
3. Disk Diffusion
4. Direct Tests for β-lactamase
Recent years:
 Trend to commercial broth microdilution
 Automated instrument methods
Disk diffusion:
 Flexibility in drug selection
 Respond quickly to changes in breakpoints
 Low cost
 Standard
 Readily understood by clinicians
Microdilution and agar gradient
 Quantitative results
 Fastidious or anarobic
 Automated
 There is no clear evident that MICs are more
relevant than susceptiblity category results