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In Vitro Testing of Antimicrobial Agents


Title: Antimicrobial Susceptibility Test and Assay Author: Usanee Anukool Last modified by: vahid Created Date: 1/14/2005 1:35:25 AM Document presentation format – PowerPoint PPT presentation

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Title: In Vitro Testing of Antimicrobial Agents

In Vitro Testing of Antimicrobial Agents
  • Maryam Monajemzadeh , Pathologist
  • Children Medical Center Hospital
  • Tehran University Of Medical Sciences

  • 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.

  • 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
  • Gerhard Domagk 1932
  • accidentally discovered the first sulfa drug,
    sulfanilamide, while testing a red dye called
    Prontosil on streptococci

Gerhard Domagk
  • Fleming 1928
  • noticed that colonies of Staphylococcus were
    inhibited by mold
  • Fleming identified the mold as a species of
  • with further testing, it was shown that
    Penicillium was a bacteria-killing substance

Alexander Fleming
  • 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
  • first antibiotic developed for the general public

Sir Howard Florey Dr. Ernst Chain
  • 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 that the patient will
    respond to tx with appropriate dosage regimen for
    that antibiotic.
  • Tx with that antibiotic is 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

  • Higher doses
  • Normally concentrated
  • effective

  • 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
  • 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
  • The accumulated results on different pathogens
    their sensitivity will guide the physician in
    choosing empirical treatment in serious patients
    before the individuals laboratory results are
    analyzed in the Microbiology laboratory.
  • Reveals the changing trends in the local
  • Helps the local pattern of antibiotic prescribing.

Why Need continues for testing for Antibiotic
  • Bacteria have the ability to develop resistance
    following repeated or subclinical (insufficient)
  • 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.

  • Susceptible an infection due to a specific
    isolate can be appropriately treated with the
    recommended dosage of antibiotic.
  • Resistant isolate will not or is unlikely to
    respond to achievable concentrations of the
    antibiotic using normal doses.
  • 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,
    b-lactam 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
  • Serum achievable levels for each antimicrobial
  • 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

Inhibitory Methods for Susceptibility Testing
  1. Dilution Testing
  2. Epsilometer
  3. Disk Diffusion
  4. Direct Tests for ß-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