Antibiotic Sensitivity Testing

Antibiotic Antibiotic Sensitivity TestingSensitivity Testing

Dr.T.V.Rao MDDr.T.V.Rao MD

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 choice Testing will assist the clinicians in the choice of drugs for the treatment of infections.of drugs for the treatment of 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 Physician in choosing Helps to guide the Physician in choosing AntibioticsAntibiotics

The accumulated results on different pathogens The accumulated results on different pathogens their sensitivity will guide the physician in their sensitivity will guide the physician in choosing empirical treatment in serious patients choosing empirical treatment in serious patients before the individual’s laboratory results are before the individual’s laboratory results are analyzed in the Microbiology laboratory.analyzed in the 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. Dr.T.V.Rao MDDr.T.V.Rao MD

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 of tissues of a patient given the usual schedule of doses i.e. that the infection is treatable”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 test which uses antibioticantibiotic--impregnated wafers to test whether impregnated wafers to test whether particular particular bacteriabacteria are susceptible to 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 inhibitionzone 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 stanMcFarland 0.5 OD standarddard streaked as a lawn streaked as a lawn onto a large Mueller-onto a large Mueller-Hinton agar or Blood Hinton agar or Blood agar plate (in 3 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 necessary swabbing process. (It is not necessary to re-moisten the swab.) Run the swab to re-moisten the swab.) Run the swab around the circumference of the plate around the circumference of the plate before discarding it in the discard bag. before discarding it in the discard bag.


Placing the Antibiotic disksPlacing the Antibiotic disks

Then, using a Then, using a dispenser such as the dispenser such as the one pictured, one pictured, antibiotic-antibiotic-impregnated disks are impregnated disks are placed onto the agar placed onto the agar surface. As the 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 S and result reported as S (sensitive), R (resistant), or I (intermediate). (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 of tissues of a patient given the usual schedule of doses i.e. that the infection is treatable”doses i.e. that the infection is treatable”

Stokes’ MethodStokes’ Method In original Stokes’ In original Stokes’

method the inoculum method the inoculum of the control strain is of the control strain is evenly spread over evenly spread over the upper and lower the upper and lower thirds of a plate and thirds of a plate and that of the test strain that of the test strain over the central over the central third;uninoculated third;uninoculated gaps 2 – 3 mm wide gaps 2 – 3 mm wide are left to test from are left to test from the control areas.the control areas.


Control strainsControl strainsin Stokes’ Methodin Stokes’ Method

The Microbiology laboratories should The Microbiology laboratories should always practice to test the quality control always practice to test the quality control of their work with the use of standard of their work with the use of standard strains in Stokes method with following strains in Stokes method with following standard strains and results are standard strains and results are comparedcompared

1 1 Escherichia coli NCTC 10418Escherichia coli NCTC 10418

2 Pseudomonas aeruginosa NCTC 106622 Pseudomonas aeruginosa NCTC 10662

3 Staphylococcus aureus NCTC 65713 Staphylococcus aureus NCTC 6571

Stokes Method for Antibiotic Stokes Method for Antibiotic Sensitivity testingSensitivity testing

In the In the Stokes controlled Stokes controlled sensitivity testsensitivity test, a control , a control organism is inoculated on organism is inoculated on part of a plate and the test part of a plate and the test organism is plated on the organism is plated on the remainder. Disks are remainder. Disks are placed at the interface and placed at the interface and the zones of inhibition are the zones of inhibition are compared. The use of a compared. The use of a sensitive control shows sensitive control shows that the antibiotic is active, that the antibiotic is active, so that if the test organism so that if the test organism grows up to the disk it may grows up to the disk it may safely be assumed that the safely be assumed that the test organism is resistant test organism is resistant to that drug. to that drug.


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, method, E-testE-test (also based on (also based on antibiotic diffusion). Agar and Broth antibiotic diffusion). Agar and Broth dilution methods for dilution methods for Minimum Inhibitory ConcentrationMinimum Inhibitory Concentration determination. determination.

Testing Minimum Inhibitory Testing Minimum Inhibitory ConcentrationConcentration

In alternative measure of susceptibility is In alternative measure of susceptibility is to determine the to determine the Minimum Inhibitory Minimum Inhibitory Concentration (MIC)Concentration (MIC) and the and the Minimum Minimum Bactericidal Concentration (MBC)Bactericidal Concentration (MBC) of a of a drug. A series of broths are mixed with drug. A series of broths are mixed with serially diluted antibiotic solutions and a serially diluted antibiotic solutions and a standard inoculum is applied. After standard inoculum is applied. After incubation, the MIC is the first broth in incubation, the MIC is the first broth in which growth of the organism has been which growth of the organism has been


What is Minimum What is Minimum Inhibitory Inhibitory

concentrationconcentration Minimum inhibitory concentrationMinimum inhibitory concentration ((MICMIC), in ), in microbiologymicrobiology, is the lowest , is the lowest concentrationconcentration of an of an antimicrobialantimicrobial that that will inhibit the visible will inhibit the visible growthgrowth of a micro of a micro organism after overnight incubation. organism after overnight incubation. Minimum inhibitory concentrations are Minimum inhibitory concentrations are important in diagnostic laboratories to important in diagnostic laboratories to confirm resistance of micro organisms confirm resistance of micro organisms to an antimicrobial agent and also to to an antimicrobial agent and also to monitor the activity of new monitor the activity of new antimicrobial agents.antimicrobial agents.[[

The Antibiotics are diluted to various The Antibiotics are diluted to various dilution to test the minimum dilution to test the minimum

inhibitory concentrationinhibitory concentration

MIC test results graphedMIC test results graphed

What is E TestWhat is E Test Etest is an antimicrobial gradient Etest is an antimicrobial gradient

technique in which 15 reference MIC technique in which 15 reference MIC dilutions of an antibiotic have been dilutions of an antibiotic have been repackaged with innovative dry chemistry repackaged with innovative dry chemistry technology onto a plastic strip. The technology onto a plastic strip. The predefined gradient provides precise and predefined gradient provides precise and accurate assessment of antimicrobial accurate assessment of antimicrobial activity against both fastidious and non-activity against both fastidious and non-fastidious microorganisms. fastidious microorganisms.


The strips are impregnated The strips are impregnated with various concentration with various concentration

of Antibioticsof Antibiotics

E = testing on E = testing on various isolatesvarious isolates

Multiple drug resistant Multiple drug resistant organismsorganisms

Multiple drug resistant organisms are Multiple drug resistant organisms are resistant to treatment with several, often resistant to treatment with several, often unrelated, antimicrobial agents as unrelated, antimicrobial agents as described above in described above in ShigellaShigella. Some of the . Some of the most important types of multiple drug most important types of multiple drug resistant organisms that have been resistant organisms that have been encountered.encountered.


MRSA - methicillin/oxacillin-resistant MRSA - methicillin/oxacillin-resistant Staphylococcus aureusStaphylococcus aureusVRE - vancomycin-resistant VRE - vancomycin-resistant enterococcienterococciESBLs - extended-spectrum beta-ESBLs - extended-spectrum beta-lactamases (which are resistant to lactamases (which are resistant to cephalosporins and monobactams)cephalosporins and monobactams)PRSP - penicillin-resistantPRSP - penicillin-resistant Streptococcus pneumoniaeStreptococcus pneumoniae


Examples of Bacteria of Clinical Examples of Bacteria of Clinical ImportanceImportance

Methicillin resistance in Methicillin resistance in Staphylococcu aureusStaphylococcu aureus

Detection of MRSA made simple Detection of MRSA made simple with Disk Diffusion Methodswith Disk Diffusion Methods

The MRSA among the Staphylococcus The MRSA among the Staphylococcus aureus needs detection in special aureus needs detection in special condtions either by carrying out the condtions either by carrying out the test on medium containing 5 % Nacl test on medium containing 5 % Nacl with Methicillin disk with 5 or 10 with Methicillin disk with 5 or 10 microgramsmicrograms

Recently the detection was made Recently the detection was made simple with testing on Oxacillin or simple with testing on Oxacillin or Cefotixin disksCefotixin disks

Newer Methods in MRSA Newer Methods in MRSA detectiondetection

Recently developed chromogenic Recently developed chromogenic media combine primary growth and media combine primary growth and selectivity with differentiation from selectivity with differentiation from coagulase negative staphylococci. coagulase negative staphylococci. These media show improved These media show improved specificity when compared with specificity when compared with traditional media. Sensitivity is also traditional media. Sensitivity is also improved but requires 48hrs improved but requires 48hrs incubation to achieve >85%. incubation to achieve >85%.

ChromChromagaragar for detection of MRSA for detection of MRSA

BBL™ CHROMagar™BBL™ CHROMagar™ MRSA MRSA is a selective and differential is a selective and differential medium for the qualitative medium for the qualitative direct detection of nasal direct detection of nasal colonization by Methicillin colonization by Methicillin resistant resistant Staphylococcus Staphylococcus aureusaureus (MRSA) to aid in the (MRSA) to aid in the prevention and control of prevention and control of MRSA infections in MRSA infections in healthcare settings. The test healthcare settings. The test is performed on anterior is performed on anterior nares swab specimens from nares swab specimens from patients and healthcare patients and healthcare workers to screen for MRSA workers to screen for MRSA colonization. colonization. BBL BBL CHROMagarCHROMagar MRSA is not MRSA is not intended to diagnose MRSA intended to diagnose MRSA infection nor to guide or infection nor to guide or monitor treatment for monitor treatment for infections infections

Detection of ESBL strains in Detection of ESBL strains in EnterobacteriaceaeEnterobacteriaceae

Methods to detect ESBL Methods to detect ESBL producersproducers

Various Various combination of combination of Antibiotic disks Antibiotic disks are used for the are used for the detection of detection of ESBL producers ESBL producers in Gram in Gram negative bacillinegative bacilli..

Antibiotic disks helps in Antibiotic disks helps in detection of ESBL producersdetection of ESBL producers

Various Antibiotic Various Antibiotic disks kept at disks kept at specified place specified place helps in detection helps in detection of ESBL producersof ESBL producers

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 AssaysAntibiotic Assays The amount of antibiotic in the Blood or other The amount of antibiotic in the Blood or other

body fluids of patients measured for following body fluids of patients measured for following reasonsreasons

1 To ensure adequate theraputic concentrations 1 To ensure adequate theraputic concentrations are reached – in serious infections with parental are reached – in serious infections with parental administrationadministration

2 To avoid toxic concentration2 To avoid toxic concentration 3 To study pharmacokinetics of Antibiotic3 To study pharmacokinetics of Antibiotic 4 To identify the clinical response with identified 4 To identify the clinical response with identified

concentrationconcentration At present Automated methods are At present Automated methods are

popularly used as the testing protocols are popularly used as the testing protocols are technically demandingtechnically demanding


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.

Various Methods of Antibiotic Various Methods of Antibiotic sensitivity testing are used for sensitivity testing are used for

quality control studiesquality control studies

Created for awareness to Medical Created for awareness to Medical and Paramedical staff in the and Paramedical staff in the

Developing worldDeveloping world

Dr.T.V.Rao MD ‘s Basic learning Dr.T.V.Rao MD ‘s Basic learning seriesseries

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Antibiotic Sensitivity Testing