h. sensitivitas
TRANSCRIPT
Dr.T.V.Rao MD 1
ANTIBIOTIC SENSITIVITY TESTINGSKILL BASED LEARNING
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.
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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
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Routine Susceptibility Tests
Disk diffusion (Kirby Bauer)
Broth micro-dilution 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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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
Antimicrobial Susceptibility Testing
Susceptibility Testing Methods
InoculateMH plate
Place diskson agar plate
Incubate plate18-24 hr, 35 CMeasure and record zone of inhibition around each disk
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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
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Disc Diffusion Method
Procedure (Modified Kirby-Bauer method: National Committee for Clinical Laboratory Standards. NCCLS) Prepare approximately. 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. 0.5 standard.* 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
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Examining purity of plateSelect the Colonies from Pure
Isolates
Reflected light
Transmitted light
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Disk Diffusion Test
Select colonies
Prepare inoculumsuspension
Prepare inoculumsuspension
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Prepare the Material for Inoculation
Standardize inoculumSuspension as per Mac farland standard
Mix well
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Swab the plate with
optimal sample
Remove sample
Swab plate
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Select the Disks and Apply
Select disks
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Incubate Overnight
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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 at 35 oC, o/n (18-24 h)
Measure the diameters of inhibition zone in mm
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Read the Results with Precision
TransmittedLight
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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
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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.
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Disc Diffusion MethodReporting 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
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Factors Affecting Size of Zone of Inhibition
Inoculum density
Timing of disc application
Temperature of incubation
Incubation time
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 < 35 oC
Ideal 16-18 hours; less time does not give reliable results
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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
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Factors Affecting Size of Zone of Inhibition
Potency of antibiotic discs
Composition of medium
Acidic pH of medium
Alkaline pH of medium
Reading of zones
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
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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
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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 )
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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 -20 oC Use Mueller-Hinton medium for
antibiotic sensitivity determination Use appropriate control cultures Use standard methodology for the
test
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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
Need for Modified Methods
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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 0.1% of the original inoculum to survive
Antimicrobial susceptibilitytesting using micro-broth
dilutions
•
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•• •
•
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96 well microtiter plate
ug/ml64 32 16 8 4 2
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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
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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
128 64 32 16 8 4 2 C1 C2
64 32 16 8 4 2 1 C1 C2
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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
64 32 16 8 4 2 1 C1 C2
0.01 ml (spread plate), Incubate 35 oC, o/n
64 32 16
Day 3Determine 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
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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 MethodProcedure
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
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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
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Inoculum Preparation MIC Testing
(NCCLS Reference Method)
Standardize inoculum suspension
Final inoculum concentration3 – 5 x 105
CFU/ml(3 – 5 x 104
CFU/well)
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Select Micro titration plate and prepare optimal
inoculum
Micro dilution MIC tray
Prepare inoculumsuspension
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Dilute & mix inoculumsuspension
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Pour inoculum into reservoir and
inoculate MIC tray
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Incubate overnightDo not forget to check the purity of
Inoculum
Inoculate purity plate
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
- +
64
32
16
8
4
2
1
>64
0.5
Read MICs
>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.
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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.
Antimicrobial Gradient TestingE-test®
Read platesafter
recommendedIncubation
Read MICwhere elipse
intersectsscale
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MIC of the Bacteria can be read Directly
MIC on a stripabbiodisk.com
5-Jan-06 Chiang Mai University 53
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
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Antibiotic Sensitivity testing can be done with
automation
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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.
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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
What is the Role of Microbiology Departments
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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.
References
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