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Antibiotic sensitivity testing skill based learning l.jpg

ANTIBIOTIC SENSITIVITY TESTINGSKILL BASED LEARNING

Dr.T.V.Rao MD

Dr.T.V.Rao MD


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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.

Dr.T.V.Rao MD


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What is the goal of Antibiotic Sensitivity testing?

  • The goal of antimicrobial susceptibility testing is to predict the in vivo success or failure of antibiotic therapy. Tests are performed in vitro, and measure the growth response of an isolated organism to a particular drug or drugs. The tests are performed under standardized conditions so that the results are reproducible. The test results should be used to guide antibiotic choice. The results of antimicrobial susceptibility testing should be combined with clinical information and experience when selecting the most appropriate antibiotic for our patients.

Dr.T.V.Rao MD


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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.

Dr.T.V.Rao MD


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Why Need Continues for Testing 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.

  • Antibiotic sensitivity testing is essential part of Medical Care

Dr.T.V.Rao MD


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Introduction

  • Susceptibility test, main purposes:

    • As a guide for treatment

      • Sensitivity of a given m.o. to known conc. of drugs

      • Its concentration in body fluids or tissues

    • As an epidemiological tool

      • The emergence of resistant strains of major pathogens (e. g. Shigella, Salmonella typhi)

      • Continued surveillance of the susceptibility pattern of the prevalent strains (e. g. Staphylococci, Gram-negative bacilli)

Dr.T.V.Rao MD


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Introduction

  • Methods for antimicrobial susceptibility testing

    • Indirect method

      • cultured plate from pure culture

    • Direct method

      • Pathological specimen

      • e.g. urine, a positive blood culture, or a swab of pus

Dr.T.V.Rao MD


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What Does the Laboratory Need to Knowabout Antimicrobial Susceptibility Testing (AST) ?

  • Which organisms to test?

  • What methods to use?

  • What antibiotics to test?

  • How to report results?

Dr.T.V.Rao MD


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Routine Susceptibility Tests

  • Disk diffusion (Kirby Bauer)

  • Broth micro-dilution MIC

    • NCCLS reference method

  • Etest

Dr.T.V.Rao MD


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Preparing for Testing

  • Inoculum preparation

    - Number of test organisms can be determined using different methods:

    • Direct count (Microscopic examination)

    • The optical density (OD) at 600 nm (Spectrophotometry)

    • Plate count: making dilution first

    • Turbidity standard (McFarland) routinely performed.

Dr.T.V.Rao MD


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Choosing the Appropriate Antibiotic

  • Drugs for routine susceptibility tests:

    • Set 1:the drugs that are availablein most hospitals and for which routine testing should be carried out for every strain

    • Set 2:the drugs that are tested only:

      • at the special request of the physician

      • or when the causative organism isresistantto the first-choice drugs

      • or when other reasons (allergy to a drug, or its unavailability) make further testing justified

Dr.T.V.Rao MD


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Table 1: Basic sets of drugs for routine susceptibility tests(http://w3.whosea.org/)

Dr.T.V.Rao MD


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Antimicrobial Susceptibility Testing

  • Diffusion method

    • Put a filter disc, or a porous cup/a bottomless cylinder containing measured quantity of drugs on the a solid medium that has been seeded with test bacteria

  • Dilution method

    • vary amount of antimicrobial substances incorporated into liquid or solid media

    • followed by inoculation of test bacteria

Dr.T.V.Rao MD


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Susceptibility Testing Methods

Incubate plate

18-24 hr, 35 C

Measure and record zone of inhibition around each disk

Inoculate

MH plate

Place disks

on agar plate


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Diffusion Method

  • Disc diffusion method : The Kirby-Bauer test

    • Antibiotic-impregnated filter disc*

    • Susceptibility test against more than one antibiotics by measuring size of “inhibition zone ”

    • 1949: Bondi and colleagues paper disks

    • 1966: Kirby, Bauer, Sherris, and Tuck  filter paper disks

      • Demonstrated that the qualitative results of filter disk diffusion assay correlated well with quantitative results from MIC tests

Dr.T.V.Rao MD


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Disc Diffusion Method

  • Procedure(Modified Kirby-Bauer method: National Committee for Clinical Laboratory Standards. NCCLS)

    • Prepareapproximately.108 CFU/ml bacterial inoculum in a saline or tryptic soy broth tube(TSB) or Mueller-Hinton broth (5ml)

      • Pick 3-5 isolated colonies from plate

      • Adjust the turbidity tothesame as the McFarland No. 0.5 standard.*

    • Streak the swabon the surface of the Mueller-Hinton agar(3 times in 3 quadrants)

    • Leave 5-10 min to dry the surface of agar

Dr.T.V.Rao MD


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Examining purity of plateSelect the Colonies from Pure Isolates

Transmitted light

Reflected light

Dr.T.V.Rao MD


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Disk Diffusion Test

Prepare inoculum

suspension

Prepare inoculum

suspension

Select colonies

Dr.T.V.Rao MD


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Prepare the Material for Inoculation

Standardize inoculum

Suspension as per Mac farland standard

Mix well

Dr.T.V.Rao MD


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Swab the plate with optimal sample

Remove sample

Swab plate

Dr.T.V.Rao MD


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Select the Disks and Apply

Select disks

Dr.T.V.Rao MD


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Incubate Overnight

Dr.T.V.Rao MD


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Disc Diffusion Method

  • Place the appropriate drug-impregnated disc on the surface of the inoculated agar plate

  • Invert the plates and incubate them at35oC, o/n (18-24 h)

  • Measure the diameters of inhibition zone in mm

Dr.T.V.Rao MD


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Read the Results with Precision

Transmitted

Light

Dr.T.V.Rao MD


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Disc Diffusion Method

  • Measurement of the diameters of inhibition zone

    • Measure from the edge where the growth stats, BUT there are three exceptions

      • With sulfonamides and co-trimoxazole, ignore slight growth within the zone

      • Certain Proteus spp. may swarm into the area of inhibition

      • When beta-lactamase producing Streptococci are tested, zone of inhibition are produced with a heaped-up, clearly defined edge, regardless of the size of the inhibition zone, they should be reported as resistant

Dr.T.V.Rao MD


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Look at the Charts for establishing the zones of Sensitivity

  • The zone sizes are looked up on a standardized chart to give a result of sensitive, resistant, or intermediate. Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug.

Dr.T.V.Rao MD


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Disc Diffusion Method

Reporting the Results

  • Interpretation of results

    • By comparing with the diameters with “standard tables”

    • Susceptible

    • Intermediate susceptible

      • Low toxic antibiotics: Moderate susceptible

      • High toxic antibiotics: buffer zone btw resistant and susceptible

    • Resistant

Dr.T.V.Rao MD


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Factors Affecting Size of Zone of Inhibition

  • Larger zones with light inoculum and vice versa

  • If after application of disc, the plate is kept for longer time at room temperature, small zones may form

  • Larger zones are seen with temperatures < 35oC

  • Ideal 16-18 hours; less time does not give reliable results

  • Inoculum density

  • Timing of disc application

  • Temperature of incubation

  • Incubation time

Dr.T.V.Rao MD


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Factors Affecting Size of Zone of Inhibition

  • Size of the plate

  • Depth of the agar medium(4 mm)

  • Proper spacing of the discs (2.5 cm)

  • Smaller plates accommodate less number of discs

  • Thin media yield excessively large inhibition zones and vice versa

  • Avoids overlapping of zones

Dr.T.V.Rao MD


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Factors Affecting Size of Zone of Inhibition

  • Deterioration in contents leads to reduced size

  • Affects rate of growth, diffusion of antibiotics and activity of antibiotics

  • Tetracycline, novobiocin, methicillin zones are larger

  • Aminoglycosides, erythromycin zones are larger

  • Subjective errors in determining the clear edge

  • Potency of antibiotic discs

  • Composition of medium

  • Acidic pH of medium

  • Alkaline pH of medium

  • Reading of zones

Dr.T.V.Rao MD


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Quality Assurance in Antibiotic Susceptibility Testing

  • Visit - WHO-Regional Office for South East Asia website

    • Medium:Mueller-Hinton agar plates

      • Enterococcus faecalis (ATCC 29212 or 33l86) and a disc of co-trimoxazole 20 mm in diameter of the inhibition zone

    • Procedure: Modified Kirby-Bauer method recommended by National Committee on Clinical Laboratory Services (NCCLS)

    • Susceptibility test with quality control strains

Dr.T.V.Rao MD


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Quality Assurance in Antibiotic Susceptibility Testing with Control strains

  • Susceptibility test with quality control strains

    for every new batch of Mueller-Hinton agar

    • Staphylococcus aureus (ATCC 25923)

    • Escherichia coli (ATCC 25922)

    • Pseudomonas aeruginosa (ATCC 2785 )

Dr.T.V.Rao MD


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Quality Assurance in Antibiotic Susceptibility Test

  • Salient features of quality control

    • Use antibiotic discs of 6 mm diameter

    • Use correct content of antimicrobial agent per disc

    • Store supply of antimicrobial discs at -20oC

    • Use Mueller-Hinton medium for antibiotic sensitivity determination

    • Use appropriate control cultures

    • Use standard methodology for the test

Dr.T.V.Rao MD


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Need for Modified Methods

  • Modified Methods in Disc diffusion for Antibiotic sensitivity testing to be used for detections of following bacterial isolates

  • 1 MRSA

  • 2 ESBL

  • 3 Enterobacteriaceae and Gram negative bacteria and Carbapenems resistant using Modified Hodge test

Dr.T.V.Rao MD


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Dilution Method

  • Minimum Inhibition Concentration (MIC)

    • The lowest concentration of antimicrobial agent thatinhibitsbacterial growth/ multiplication

  • Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC)

    • The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive


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Antimicrobial susceptibilitytesting using micro-broth dilutions

ug/ml

64 32 16 8 4 2

96 well microtiter plate


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Broth Dilution Method

  • Procedure Making dilutions (2-fold) of antibiotic in broth Mueller-Hinton, Tryptic Soy Broth

    • Inoculation of bacterial inoculum, incubation, overnight

      • Controls: no inoculum, no antibiotic

    • Turbidity visualization  MIC

    • Sub culturing of non-turbid tubes, overnight

    • Growth (bacterial count)  MBC


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Creating Dilutions

Dr.T.V.Rao MD


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128 64 32 16 8 4 2 C1 C2

64 32 16 8 4 2 1 C1 C2

Broth Dilution Method

Day 1

Add 1 ml of test bacteria (1*106 CFU/ml) to tubes containing 1 ml broth and concentration of antibiotic (mg/l)

Controls:

C1 = No antibiotic, check viability on agar plates immediately

C2 = No test bacteria

Bacterial conc.= 5*105 CFU/ml

Incubate 35 oC, o/n


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64 32 16 8 4 2 1 C1 C2

64 32 16

Broth Dilution Method

Day 2

Record visual turbidity

Subculture non-turbid tubes to agar plates (use 0.01 ml standard loop)

MIC = 16 mg/l

0.01 ml (spread plate), Incubate 35 oC, o/n

Day 3

Determine CFU on plates:

At 16 mg/ = 700 CFU/ml > 0.1% of 5*105 CFU/ml

MBC = 32 mg/l


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Broth Dilution Method

  • 100% of original bacterial conc.

    • = 5*105 CFU/ml

  • 0.1%

    • = [(5*105)*0.1]/100 CFU/ml

    • = 500 CFU/ml

  • The bacteria count should be less than 5 CFU on agar plate subcultured with 0.01 ml

    • 500*0.01 = 5 CFU


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Broth Dilution Method are Technically Difficult

  • Disadvantages :

    • Only one antibiotic & one organism can be tested each time

    • Time-consuming

  • Solutions??

    • Agar dilution method

    • Disc diffusion method

    • Micro broth dilution method

Dr.T.V.Rao MD


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Micro broth Dilution Method

  • Micro dilution plates:

    • “Micro dilution/ Micro broth dilutions”

    • 96 wells/ plate: simultaneously performed with many tests organisms/ specimens, less reagent required

  • Manually prepared

  • Commercially prepared

    • Frozen or Dried/ lyophilized

    • Consistent performance but high cost

    • May suffer from degradation of antibiotic during shipping and storage


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Agar Dilution Method

  • Procedure

    • Making dilutions of antimicrobial agent in melted media and pouring plates

      • One concentration of antibiotic/ plate

      • Possible for several different strains/plate

64 uGu/ml 32 ug/ml 16 ug/ml


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Agar Dilution Method

  • Procedure

    • Inoculation of bacterial inoculum (McFarland No. 0.5)

      • Using a replicating inoculator device called “A Steers-Foltz replicator”

      • Delivers 0.001 ml of bacterial inoculum

    • Incubation

    • Spot of growth

MIC

32 ug/ml


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Minimal inhibitory concentration

  • The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium

  • Interpret:

    • Susceptible

    • Intermediate

    • Resistant

Dr.T.V.Rao MD


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Clinical Conditions when MICs are Useful

  • Endocarditis

  • Meningitis

  • Septicemia

  • Osteomyelitis

  • Immunosuppressed patients (HIV, cancer, etc.)

  • Prosthetic devices

  • Patients not responding despite “S” Reports

Dr.T.V.Rao MD


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Inoculum PreparationMIC Testing (NCCLS Reference Method)

  • Standardize inoculum suspension

  • Final inoculum concentration

    3 – 5 x 105 CFU/ml

    (3 – 5 x 104 CFU/well)

Dr.T.V.Rao MD


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Select Micro titration plate and prepare optimal inoculum

Prepare inoculum

suspension

Micro dilution MIC tray

Dr.T.V.Rao MD


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Dilute & mix inoculumsuspension

Dr.T.V.Rao MD


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Pour inoculum into reservoir and inoculate MIC tray

Dr.T.V.Rao MD


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Incubate overnightDo not forget to check the purity of Inoculum

Inoculate

purity plate

Dr.T.V.Rao MD


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Optimal Use of Purity Plates

  • Sub final test suspension to non-selective medium (after inoculating MIC test)

  • Streak for isolation (avoid several specimens per plate - may not reveal contaminants if no isolated colonies)

  • Examine before reading MIC (usually at 16-20 h)

  • Re-incubate if Antibiograms questionable


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Read MICs

-

+

0.5

1

2

4

8

16

32

64

>64

>64


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The gradient technique, Etest®

  • Etest is a well established AST method in microbiology laboratories around the world. The Etest technique comprises a predefined gradient of antibiotic concentrations on a plastic strip, and can be used to determine the Minimum Inhibitory Concentration (MIC) of antibiotics, antifungal agents and antimycobacterial agents.

Dr.T.V.Rao MD


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E test – MIC Reports are helpful in Critical management decisions

  • Quantitative MIC data is a prerequisite for the management of critical infections, including sepsis, especially among critical care patients. Etest is particularly valuable in such situations, when on-scale MICs are needed for treatment decisions.

Dr.T.V.Rao MD


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Antimicrobial Gradient TestingE-test®

Read plates

after

recommended

Incubation

Read MIC

where elipse

intersects

scale


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MIC of the Bacteria can be read Directly

Dr.T.V.Rao MD


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MIC on a strip

abbiodisk.com


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Serum Susceptibility Tests

  • To determine drug concentration in the patient’s serum = MIC*SIT

    • The Serum Inhibitory Titer (SIT)

      • The highest dilution of patient’s serum that inhibit bacteria

  • To determine the ability of drug in the patient’s serum to kill bacteria

    • The Serum Bactericidal Level (SBL)

      • The lowest dilution of patient’s serum that kills bacteria

      • Technically Demanding

Chiang Mai University


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Antibiotic Sensitivity testing can be done with automation

Dr.T.V.Rao MD


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VITEK 2 Automates Reporting of Resistance

  • Integrated in the VITEK 2 system is the Advanced Expert System (AES™), a software which validates and interprets susceptibility test results, and detects antibiotic resistance mechanisms. The AES Expert System is the most developed software system in this field, and is capable of identifying even emerging and low-level resistance.

Dr.T.V.Rao MD


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What is the Role of Microbiology Departments

  • Each laboratory should have a staff member with the time, interest, and expertise to provide leadership in antibiotic testing and resistance. This person would read relevant publications, network with other laboratories, and evaluate potentially useful tests to detect new forms of resistance before new CLSI-recommended tests become available”

  • - Ken Thomson, Emerging Infect. Dis., 2001

Dr.T.V.Rao MD


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References

1Usanee Anukool (Ph.D.) Clinical Microbiology,AMS,

Chiang Mai University

2National Committee For Clinical Laboratory Standards. 1998. NCCLS document M100 - S8 . Performance Standards for Antimicrobial Susceptibility Testing. 8th edition, NCCLS, Waynae, Pa.

Dr.T.V.Rao MD


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For Articles of Interest on Antibiotics follow me on

Dr.T.V.Rao MD


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  • Created by Dr.T.V.Rao MD for ‘e’ learning resources for Microbiologists in Developing World

  • Email

  • doctortvrao@gmail.com

Dr.T.V.Rao MD


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