Antibiotic Antibiotic Sensitivity TestingSensitivity Testing

Modern era in Antibiotics begins Modern era in Antibiotics begins with Fleming.with Fleming.

History of Antibiotic DiscoveryHistory of Antibiotic Discovery

Uses of Antibiotic Sensitivity Uses of Antibiotic Sensitivity TestingTesting

Antibiotic sensitivity test:Antibiotic sensitivity test: A laboratory A laboratory test which determines how effective test which determines how effective antibiotic therapy is against a bacterial antibiotic therapy is against a bacterial infections. infections.

Antibiotic sensitivity testing will control the Antibiotic sensitivity testing will control the use of use of AntibioticsAntibiotics in clinical practice in clinical practice

Testing will assist the clinicians in the Testing will assist the clinicians in the choice of drugs for the treatment of choice of drugs for the treatment of infections.infections.

Components of Antibiotic Components of Antibiotic Sensitivity TestingSensitivity Testing

1.The identification of relevant pathogens in 1.The identification of relevant pathogens in exudates and body fluids collected from exudates and body fluids collected from patientspatients

2. Sensitivity tests done to determine the 2. Sensitivity tests done to determine the degree of sensitivity or resistance of degree of sensitivity or resistance of pathogens isolated from patient to an pathogens isolated from patient to an appropriate range of antimicrobial drugsappropriate range of antimicrobial drugs

3. Assay of the concentration of an 3. Assay of the concentration of an administered drug in the blood or body fluid administered drug in the blood or body fluid of patient required to control the schedule of of patient required to control the schedule of dosage.dosage.

Antibiotic Sensitivity Testing Is Antibiotic Sensitivity Testing Is Essential of selection of Essential of selection of

AntibioticsAntibiotics

Isolation and Identification of Isolation and Identification of Bacteria precedes the selection of Bacteria precedes the selection of

Antibiotic Testing MethodsAntibiotic Testing Methods

Uses of Antibiotic Sensitivity Uses of Antibiotic Sensitivity TestingTesting

Helps to guide the Veterinarian in choosing Helps to guide the Veterinarian in choosing AntibioticsAntibiotics

The accumulated results on different The accumulated results on different pathogens their sensitivity will guide the pathogens their sensitivity will guide the veterinarian in choosing empirical treatment in veterinarian in choosing empirical treatment in serious patients before the individual’s serious patients before the individual’s laboratory results are analyzed in the laboratory results are analyzed in the Microbiology laboratory.Microbiology laboratory.

Reveals the changing trends in the local Reveals the changing trends in the local isolates.isolates.

Helps the local pattern of antibiotic prescribing.Helps the local pattern of antibiotic prescribing.

Why Need continues for testing Why Need continues for testing for Antibiotic Sensitivityfor Antibiotic Sensitivity

Bacteria have the Bacteria have the ability to develop ability to develop resistance following resistance following repeated or subclinical repeated or subclinical (insufficient) doses, so (insufficient) doses, so more advanced more advanced antibiotics and antibiotics and synthetic antimicrobials synthetic antimicrobials are continually required are continually required to overcome them.to overcome them.

Testing for Antibiotic sensitivityTesting for Antibiotic sensitivity

The method includes several steps The method includes several steps including obtaining a bacterial including obtaining a bacterial sample; identifying the type of sample; identifying the type of bacteria in the bacterial sample; bacteria in the bacterial sample; selecting a set of antibiotics based selecting a set of antibiotics based on the identity of the bacteria in the on the identity of the bacteria in the bacterial sample; obtaining a control bacterial sample; obtaining a control sample from the bacterial sample; sample from the bacterial sample;

Testing Antibiotic SusceptibilityTesting Antibiotic Susceptibility

Antibiotic sensitivity test:Antibiotic sensitivity test: A A laboratory test which determines how laboratory test which determines how effective antibiotic therapy is against a effective antibiotic therapy is against a bacterial infections. bacterial infections.

Antibiotic sensitivity test:Antibiotic sensitivity test: the in the in vitro testing of bacterial cultures with vitro testing of bacterial cultures with antibiotics to determine susceptibility antibiotics to determine susceptibility of bacteria to antibiotic therapy. of bacteria to antibiotic therapy. BauerBauer-Kirby test.-Kirby test.

How results to be Reported How results to be Reported after Sensitivity Testingafter Sensitivity Testing

One should follow guidelines in reporting the results One should follow guidelines in reporting the results and make matters simple with clear words asand make matters simple with clear words as

Organism A isolated and sensitive ( or Organism A isolated and sensitive ( or resistant ) to Antibiotic Bresistant ) to Antibiotic B

Such a report is relevant to present clinical condition Such a report is relevant to present clinical condition “ “ that the minimum inhibitory that the minimum inhibitory concentration( MIC ) of the antibiotic for it has concentration( MIC ) of the antibiotic for it has been measured in some way and that, if the been measured in some way and that, if the organism is reported as sensitive the MIC is organism is reported as sensitive the MIC is less than a half or quarter of the concentration less than a half or quarter of the concentration of antibiotics likely to be found in the infected of antibiotics likely to be found in the infected tissues of a patient given the usual schedule tissues of a patient given the usual schedule of doses i.e. that the infection is treatable”of doses i.e. that the infection is treatable”

What is Resistance in Antibiotic What is Resistance in Antibiotic Sensitivity TestingSensitivity Testing

Resistance implies that the infection is not Resistance implies that the infection is not treatable with the tested Antibiotic treatable with the tested Antibiotic because its MIC exceeds achievable safe because its MIC exceeds achievable safe tissue or urine levels.tissue or urine levels.

Intermediate sensitivity means they show Intermediate sensitivity means they show aa unimodal distribution, but they can unimodal distribution, but they can often be reclassified as sensitive or often be reclassified as sensitive or resistant if retested. resistant if retested.

Kirby-Bauer methods Kirby-Bauer methods A commonly used method in basic A commonly used method in basic

laboratorieslaboratories Kirby-Bauer antibiotic testingKirby-Bauer antibiotic testing

((KB testingKB testing or or disk diffusion disk diffusion antibiotic sensitivity testingantibiotic sensitivity testing) is a ) is a test which uses antibiotic-test which uses antibiotic-impregnated wafers to test whether impregnated wafers to test whether particular bacteria are susceptible to particular bacteria are susceptible to specific antibiotics specific antibiotics

How to perform Kirby- Bauer How to perform Kirby- Bauer testingtesting

The basics are easy:  The bacterium is The basics are easy:  The bacterium is swabbed on the agar and the antibiotic swabbed on the agar and the antibiotic discs are placed on top.  The antibiotic discs are placed on top.  The antibiotic diffuses from the disc into the agar in diffuses from the disc into the agar in decreasing amounts the further it is away decreasing amounts the further it is away from the disc.  If the organism is killed or from the disc.  If the organism is killed or inhibited by the concentration of the inhibited by the concentration of the antibiotic, there will be antibiotic, there will be NO growth NO growth in the in the immediate area around the disc: This is immediate area around the disc: This is called the called the zone of inhibition zone of inhibition . . 

Steps in Antibiotic sensitivity Steps in Antibiotic sensitivity testingtesting

Streaking the InoculumStreaking the Inoculum Kirby-Bauer (also Kirby-Bauer (also

Bauer-Kirby) disk Bauer-Kirby) disk diffusion antibiotic diffusion antibiotic susceptibility testing susceptibility testing applies a defined applies a defined inoculum (compared to inoculum (compared to McFarland 0.5 OD McFarland 0.5 OD standard streaked as a standard streaked as a lawn onto a large lawn onto a large Mueller-Hinton agar or Mueller-Hinton agar or Blood agar plate (in 3 Blood agar plate (in 3 directions to ensure directions to ensure confluence).confluence).

Making proper inoculumMaking proper inoculum

Swab a Mueller-Hinton Swab a Mueller-Hinton plate with each of the plate with each of the bacteria.  Dip a sterile bacteria.  Dip a sterile swab into the broth swab into the broth and express any and express any excess moisture by excess moisture by pressing the swab pressing the swab against the side of the against the side of the tube. tube.

Bacteria are inoculated as Bacteria are inoculated as lawn culturelawn culture

Method of Method of inoculation- Good inoculation- Good results are results are obtained by obtained by placing a standard placing a standard loopful of inoculum loopful of inoculum suspension on the suspension on the plate and then plate and then spreading it with a spreading it with a dry sterile swab.dry sterile swab.

Disk Diffusion MethodDisk Diffusion Method

After completely swabbing the plate, After completely swabbing the plate, turn it 90 degrees and repeat the turn it 90 degrees and repeat the swabbing process. (It is not swabbing process. (It is not necessary to re-moisten the swab.)  necessary to re-moisten the swab.)  Run the swab around the Run the swab around the circumference of the plate before circumference of the plate before discarding it in the discard bag. discarding it in the discard bag.

Placing the Antibiotic disksPlacing the Antibiotic disks

Then, using a Then, using a dispenser such as dispenser such as the one pictured, the one pictured, antibiotic-antibiotic-impregnated disks impregnated disks are placed onto the are placed onto the agar surface. As the agar surface. As the bacteria on the lawn bacteria on the lawn grow, they are grow, they are inhibited to varying inhibited to varying degrees by the degrees by the antibiotic diffusing antibiotic diffusing from the diskfrom the disk

Zone sizes differ on sensitivity Zone sizes differ on sensitivity patternpattern

It has been It has been determined that determined that zones of inhibition of zones of inhibition of a certain diameter a certain diameter (varies for antibiotic (varies for antibiotic and to a lesser and to a lesser extent, bacterial extent, bacterial species) correlate species) correlate with sensitivity or with sensitivity or resistance to the resistance to the antibiotic tested antibiotic tested

Look at the Charts for Look at the Charts for establishing the zones of establishing the zones of

SensitivitySensitivity The zone sizes are looked up on a The zone sizes are looked up on a

standardized chart to give a result of standardized chart to give a result of sensitivie, resistant, or intermediate. sensitivie, resistant, or intermediate. Many charts have a corresponding Many charts have a corresponding column that also gives the column that also gives the MIC MIC (minimal inhibitory concentration) for (minimal inhibitory concentration) for that drug. that drug.

InterpretationInterpretation Place the metric ruler across the zone of Place the metric ruler across the zone of

inhibition, at the widest diameter, and measure inhibition, at the widest diameter, and measure from one edge of the zone to the other edge.  from one edge of the zone to the other edge.  HOLDING THE PLATE UP TO THE LIGHT MIGHT HOLDING THE PLATE UP TO THE LIGHT MIGHT HELP.    HELP.   

The disc diameter will actually be part of that The disc diameter will actually be part of that number.   If there is NO zone at all, report it as number.   If there is NO zone at all, report it as 0---even though the disc itself is around 7 mm. 0---even though the disc itself is around 7 mm.      

Zone diameter is reported in millimetres, Zone diameter is reported in millimetres, looked up on the looked up on the chart, chart, and result reported as and result reported as S (sensitive), R (resistant), or I (intermediate).    S (sensitive), R (resistant), or I (intermediate).   

The area of Inhibition is The area of Inhibition is measured with a Scalemeasured with a Scale

Record the Record the results for results for everyone on everyone on your table in your table in the table the table below.   below.  

The disk diffusion methods are The disk diffusion methods are commonly used for routine testingcommonly used for routine testing

Read the plates in Read the plates in traanmitted lighttraanmitted light

The zone of inhibition The zone of inhibition guides the right choice of guides the right choice of

AntibioticAntibiotic

How results to be Reported How results to be Reported after Sensitivity Testingafter Sensitivity Testing

One should follow guidelines in reporting the results One should follow guidelines in reporting the results and make matters simple with clear words asand make matters simple with clear words as

Organism A isolated and sensitive ( or Organism A isolated and sensitive ( or resistant ) to Antibiotic Bresistant ) to Antibiotic B

Such a report is relevant to present clinical condition Such a report is relevant to present clinical condition “ “ that the minimum inhibitory that the minimum inhibitory concentration( MIC ) of the antibiotic for it has concentration( MIC ) of the antibiotic for it has been measured in some way and that, if the been measured in some way and that, if the organism is reported as sensitive the MIC is organism is reported as sensitive the MIC is less than a half or quarter of the concentration less than a half or quarter of the concentration of antibiotics likely to be found in the infected of antibiotics likely to be found in the infected tissues of a patient given the usual schedule tissues of a patient given the usual schedule of doses i.e. that the infection is treatable”of doses i.e. that the infection is treatable”

The strips with multiple The strips with multiple Antibiotics can be tested in one Antibiotics can be tested in one

gogo

Other methods of Antibiotic Other methods of Antibiotic susceptibility testingsusceptibility testing

Other methods to test antimicrobial Other methods to test antimicrobial susceptibility include the Stokes susceptibility include the Stokes method, E-test (also based on method, E-test (also based on antibiotic diffusion). Agar and Broth antibiotic diffusion). Agar and Broth dilution methods for Minimum dilution methods for Minimum Inhibitory Concentration Inhibitory Concentration determination. determination.

Limitation of Disk Diffusion Limitation of Disk Diffusion MethodsMethods

Disk diffusion methods Disk diffusion methods are not suitable for are not suitable for slow grwoing bacterial slow grwoing bacterial pathogenspathogens

The great limitation The great limitation being for testing on being for testing on Mycobacterium Mycobacterium tuberculosistuberculosis which which needs varied, needs varied, technically demanding technically demanding methodsmethods

Antibiotic Sensitivity testing can Antibiotic Sensitivity testing can be done with automationbe done with automation

There is a growing need for There is a growing need for Automation in Antibiotic sensitivity Automation in Antibiotic sensitivity

testingtesting

Limitation of Antibiotic Limitation of Antibiotic Sensitivity usageSensitivity usage

Both Microbiologists and Clinician Both Microbiologists and Clinician should however bear in mind that the should however bear in mind that the

response therapy in vivo may not response therapy in vivo may not always reflect the results of testing always reflect the results of testing the sensitivity of patient's pathogen the sensitivity of patient's pathogen

in vitro.in vitro.