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Dr.T.V.

Rao MD

Dr.T.V.Rao MD

1

 Antibiotic sensitivity test: A laboratory

Uses of Antibiotic Sensitivity Testing

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 2

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 3

Routine Susceptibility Tests
Disk diffusion (Kirby Bauer)
Broth microdilution MIC
 NCCLS reference method

Etest
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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.
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V.Rao MD 6 .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.

Susceptibility Testing Methods  Inoculate MH plate Place disks on agar plate Incubate plate 18-24 hr. 35 C Measure and record zone of inhibition around each disk .

and Tuck  filter paper disks  Demonstrated that the qualitative results of filter disk diffusion assay correlated well with quantitative results from MIC tests Dr.V. Bauer.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. Sherris.Rao MD 8  .T.

Rao MD 9  .V.5 standard. 108 CFU/ml bacterial inoculum in a saline or tryptic soy broth tube (TSB) or Mueller-Hinton broth (5 ml)  Pick 3-5 isolated colonies from plate  Adjust the turbidity to the same as the McFarland No.T. NCCLS)  Prepare approximately. 0.*  Streak the swab on the surface of the Mueller-Hinton agar (3 times in 3 quadrants)  Leave 5-10 min to dry the surface of agar Dr.Disc Diffusion Method Procedure (Modified Kirby-Bauer method: National Committee for Clinical Laboratory Standards.

T.Rao MD 10 .V.Examining purity of plate Select the Colonies from Pure Isolates  Reflect ed light Transmitted light Dr.

V.Rao MD 11 .T.Disk Diffusion Test  Prepare inoculum suspension Prepare inoculum Select colonies suspension Dr.

V.Prepare the Material for Inoculation  Standardize inoculum Suspension as per Mac farland standard Mix well Dr.Rao MD 12 .T.

Swab the plate with optimal sample  Remove sample Swab plate Dr.Rao MD 13 .T.V.

Rao MD 14 .Select the Disks and Apply  Select disks Dr.T.V.

Rao MD 15 .V.Incubate Overnight  Dr.T.

Disc Diffusion Method  Place the appropriate drugimpregnated disc on the surface of the inoculated agar plate Invert the plates and incubate them at 35 oC.V. o/n (18-24 h)    Measure the diameters of inhibition zone in mm Dr.Rao MD 16 .T.

Read the Results with Precision  Transmitted Light Dr.V.T.Rao MD 17 .

Disc Diffusion Method  Measurement of the diameters of inhibition zone  Measure from the edge where the growth stats. clearly defined edge. ignore slight growth within the zone Certain Proteus spp. may swarm into the area of inhibition When beta-lactamase producing Streptococci are tested.T. zone of inhibition are produced with a heaped-up.Rao MD 18 . they should be reported as resistant Dr. BUT there are three exceptions    With sulfonamides and co-trimoxazole.V. regardless of the size of the inhibition zone.

resistant.V.T.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. Dr. Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug.Rao MD 19 . or intermediate.

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.V.T.Rao MD 20 .

V.T. less time does not give reliable results 21 .Rao MD  Larger zones are seen with temperatures < 35 oC  Ideal 16-18 hours. the  Temperature of incubation  Incubation time Dr. small zones may form  If after application of disc.Factors Affecting Size of Zone of Inhibition   Inoculum density  Timing of disc application  Larger zones with light inoculum and vice versa plate is kept for longer time at room temperature.

Rao MD 22 .5 cm)  Avoids overlapping of Dr.V.Factors Affecting Size of Zone of Inhibition  Size of the plate   Smaller plates accommodate less number of discs excessively large inhibition zones and vice versa zones  Depth of the agar medium (4 mm)  Thin media yield  Proper spacing of the discs (2.T.

novobiocin.  Alkaline pH of  Aminoglycosides.V.Factors Affecting Size of Zone of Inhibition  Potency of antibiotic discs  Deterioration in contents leads to reduced size  Composition of medium  Affects rate of growth. medium  Reading of zones  Subjective errors in Dr.Rao MD .T. diffusion of antibiotics and activity of antibiotics methicillin zones are larger erythromycin zones are larger determining the clear edge 23  Acidic pH of medium  Tetracycline.

T.V.Rao MD 24 .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.

V.T.  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 ) Quality Assurance in Antibiotic Susceptibility Testing with Control strains Dr.Rao MD 25 .

V.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 -20 oC    Use Mueller-Hinton medium for antibiotic sensitivity determination Use appropriate control cultures Use standard methodology for the test Dr.T.Rao MD 26 .

V.Rao MD 27 .T.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.

1% of the original inoculum to survive 28 .Dilution Method  Minimum Inhibition Concentration (MIC)  The lowest concentration of antimicrobial agent that inhibits bacterial growth/ multiplication  Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC)  The lowest concentration of antimicrobial agent that allows less than 0.

Antimicrobial susceptibility testing using micro-broth dilutions  ug/ml 64 32 16 8 4 2 • • • • • • • • 96 well microtiter plate • • • • • .

incubation. Tryptic Soy Broth  Inoculation of bacterial inoculum. no antibiotic  Turbidity visualization  MIC  Sub culturing of non-turbid tubes. overnight  Controls: no inoculum.Broth Dilution Method Procedure  Making dilutions (2-fold) of antibiotic in broth Mueller-Hinton. overnight  Growth (bacterial count)  MBC 30 .

T.Rao MD 31 .Creating Dilutions  Dr.V.

= 5*105 CFU/ml Incubate 35 oC. o/n .Broth Dilution Method  128 64 32 16 8 4 2 C1 C2 Day 1 Add 1 ml of test bacteria (1*106 CFU/ml) to tubes containing 1 ml broth and concentration of antibiotic (mg/l) 64 32 16 8 4 2 1 C1 C2 Controls: C1 = No antibiotic. check viability on agar plates immediately C2 = No test bacteria 32 Bacterial conc.

01 ml (spread plate).01 ml standard loop) 0. o/n MIC = 16 mg/l Day 3 Determine CFU on plates: At 16 mg/ = 700 CFU/ml > 0.1% of 5*105 CFU/ml MBC = 32 mg/l 33 64 32 16 .Broth Dilution Method Day 2 64 32 16 8 4 2 1 C1 C2 Record visual turbidity Subculture non-turbid tubes to agar plates (use 0. Incubate 35 oC.

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.  = 5*105 CFU/ml  0.01 = 5 CFU 34 .1%  = [(5*105)*0.Broth Dilution Method   100% of original bacterial conc.

Rao MD 35 .T.V.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.

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 36 .Micro broth Dilution Method  Micro dilution plates:  “Micro dilution/ Micro broth dilutions”  96 wells/ plate: simultaneously performed with many tests organisms/ specimens.

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 37 .

5)    Using a replicating inoculator device called “A SteersFoltz replicator” Delivers 0. 0.001 ml of bacterial inoculum   Incubation Spot of growth MIC 32 ug/ml 38 .Agar Dilution Method  Procedure  Inoculation of bacterial inoculum (McFarland No.

Minimal inhibitory concentration   The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium Interpret:  Susceptible  Intermediate  Resistant Dr.Rao MD 39 .V.T.

) Prosthetic devices  Patients not responding despite “S” Reports Dr. cancer.Rao MD 40 . etc.T.Clinical Conditions when MICs are Useful Endocarditis  Meningitis  Septicemia  Osteomyelitis  Immunosuppressed patients (HIV.V.

Rao MD 41  .T.Inoculum Preparation MIC Testing (NCCLS Reference Method)  Standardize inoculum suspension  Final inoculum concentration 3 – 5 x 105 CFU/ml (3 – 5 x 104 CFU/well) Dr.V.

T.Rao MD 42 .V.Select Micro titration plate and prepare optimal inoculum  Micro dilution MIC tray Prepare inoculum suspension Dr.

Rao MD 43 .V.T.Dilute & mix inoculum suspension  Dr.

V.Rao MD 44 .Pour inoculum into reservoir and inoculate MIC tray  Dr.T.

Rao MD 45 .T.Incubate overnight Do not forget to check the purity of Inoculum  Inoculate purity plate Dr.V.

may not reveal contaminants if no isolated colonies)  Examine before reading MIC (usually at 16-20 h)  Re-incubate if Antibiograms questionable .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 .

+ 8 16 32 64 >6 4 >6 4 .5 1 2 4 .Read MICs 0.

antifungal agents and antimycobacterial agents.V. Etest®  AST method in microbiology laboratories around the world.Rao MD 48 . 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. Dr.T. Etest is a well established The gradient technique.

Rao MD decisions.T.V. 49 .E test – MIC Reports are helpful in Critical management decisions   Quantitative MIC data is a prerequisite for the management of critical infections. especially among critical care patients. Etest is particularly valuable in such situations. when on-scale MICs are needed for treatment Dr. including sepsis.

Antimicrobial Gradient Testing E-test®  Read plates after recommended Incubation Read MIC where elipse intersects scale .

Rao MD 51 .V.T.MIC of the Bacteria can be read Directly  Dr.

MIC on a strip abbiodisk.com .

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 5-Jan-06 Chiang Mai University 53 .

V.Antibiotic Sensitivity testing can be done with automation  Dr.T.Rao MD 54 .

Rao MD 55 . The AES Expert System is the most developed software system in this field.V. and detects antibiotic resistance mechanisms. and is capable of identifying even emerging and low-level resistance.T.VITEK 2 Automates Reporting of Resistance  Integrated in the VITEK 2 system is the Advanced Expert System (AES™).  Dr. a software which validates and interprets susceptibility test results.

and expertise to provide leadership in antibiotic testing and resistance. 2001 Dr.. and evaluate potentially useful tests to detect new forms of resistance before new CLSI-recommended tests become available”  .T. network with other laboratories.V.Ken Thomson.Rao MD 56 . This person would read relevant publications. Dis. Emerging Infect.  Each laboratory should have a staff member What is the Role of Microbiology Departments with the time. interest.

References  Dr. 8th edition. 1998.1Usanee Anukool (Ph.S8 . Chiang Mai University 2National Committee For Clinical Laboratory Standards.) Clinical Microbiology. NCCLS document M100 .V.AMS. Waynae. Pa. NCCLS.Rao MD 57 .T.D. Performance Standards for Antimicrobial Susceptibility Testing.

T.For Articles of Interest on Antibiotics follow me on  Dr.V.Rao MD 58 .

V.com Dr. Created by Dr.Rao MD 59 .T.Rao MD for „e‟ learning resources for Microbiologists in Developing World  Email  doctortvrao@gmail.V.T.