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.

HistoryAfter 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 FloreyDr. 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: There is a high probablity

that the patient will respond to tx with appropriate dosage regimen for that antibiotic.

Resistant: 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% response60% 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 adverse reactionso Priceo 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, 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 theinterpretative 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

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

Sensitive strain

Intermediate strain

Resistant strain

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