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:

 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% 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 reactions o Price o 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

isolates.

in the local  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 o o 

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 the

infection 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 infecti on

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.

2.

3.

4.

Dilution Testing Epsilometer Disk Diffusion 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