Download - MIC MBC 2012_rev
Sri Agung Fitri Kusuma, M.Si., Apt.
Pharmacokinetics/Pharmacodynamics
• General terms for any drug, not antibiotic specific
• The term pharmacokinetics is used to define the time course of drug absorption, distribution, metabolism and excretion.
• The term pharmacodynamics refers to the relationship between drug concentration at the site of action and pharmacologic response.
– However, when we apply these principles to antimicrobial therapy there are a number of factors that can alter the predicted outcome of therapy.
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Dilution methodvary amount of antimicrobial substances
incorporated into liquid or solid mediafollowed by inoculation of test bacteria
Diffusion methodPut 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
Antimicrobial Susceptibility Testing
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Dilution Method
Broth dilution/ Agar dilution methods Permit quantitative results:
Indicating amount of a given drug necessary to inhibit (bacteriostatic activity) or kill (bactericidal activity) the microorganisms tested
Minimum Inhibition Concentration (MIC) Minimum Bactericidal Concentration (MBC)
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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
How do you know if a particular drug will be effective?
Minimum inhibitory concentration (MIC)
Minimum bactericidal concentration (MBC)
Antimicrobials are usually regarded as bactericidal if the MBC is no more than four times the MIC
the agent with the lowest MIC or MBC against a bacterium becomes the preferred choice
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Broth Dilution Method
ProcedureMaking dilutions (2-fold) of antibiotic in broth
○ Mueller-Hinton, Tryptic Soy BrothInoculation of bacterial inoculum, incubation,
overnight○ Controls: no inoculum, no antibiotic
Turbidity visualization MICSubculturing of non-turbid tubes, overnightGrowth (bacterial count) MBC
http://www.medschool.lsuhsc.edu/Microbiology/Flash/MICMBC.htm
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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
p. 519
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Broth Dilution Method
Disadvantages :Only one antibiotic & one organism can be
tested each timeTime-consuming
Solutions??Agar dilution methodDisc diffusion methodMicrobroth dilution method
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Microbroth Dilution Method Microdilution plates:
“Microdilution/ Microbroth dilutions”96 wells/ plate: simultaneously performed with
many tests organisms/ specimens, less reagent required
Manually prepared Commercially prepared
Frozen or Dried/ lyophilizedConsistent performance but high cost May suffer from degradation of antibiotic during
shipping and storage
Antimicrobial Susceptibility Testing – Broth Dilution Method
Macro dilution Method- Using test tube- Media : 1-5 mL/tube
Micro dilution Method- Using 96-wells microtiter plates- Media : 0.1 – 0.2 mL/well
Antimicrobial susceptibilityAntimicrobial susceptibilitytesting using micro-broth testing using micro-broth dilutionsdilutions
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96 well microtiter plate96 well microtiter plate
ug/mlug/ml64 32 16 8 4 264 32 16 8 4 2
- +
64
32
16
8
4
2
1
>64
0.5
MICs
>64
Agar Dilution Method
Dilution tests agar also be carried out using a series of agar plates containing known antimicrobial concentration
Appropriate bacterial suspensions are inoculated onto each plate and the presence or absence of growth is recoded after suitable incubation
In case of solid media, agar plates of defined thickness (approximatelly 3 mm)
Agar Dilution Method
Diencerkan dgn pelarutnya (lihat Farmakope) dan air suling steril dalam labu ukur
1 ml
1 ml
1 ml 1 ml
Antibiotik berbentukcairan
9 ml air suling steril
kocok
1 ml 1 ml
A B C
A B C19 ml MHA bersuhu 40-50C
Goyang2kan, lalu biarkan membeku
Antibiotik berbentukpadat digerus, lalu ditimbang teliti
Agar Dilution Method
1 2 3
- Bagi permukaan dasar cawan menjadi 4 bagian- Gores setiap bagian dengan 1 ose bakteri uji berbeda yang berumur 18-24 jam ( 4 jenis bakteri : a, b, c dan d)
Buat kontrol positif dan negatifKontrol positif : MHA + 1 ose bakteri uji berbeda yang berumur 18-24 jamKontrol negatif : MHA
+ -1, 2, 3, kontrol positif dan negatif diinkubasi 37C 18-24 jam
Amati pertumbuhan koloni pada cawan petri 1, 2 dan 3 Dibandingkan cawan petri kontrol positif dan negatif.
www.themegallery.com
Agar Dilution Method
PENGAMATAN CAWAN PETRI
1 2 3
a b c d a b c d a b c d
PERTUMBUHAN KOLONI
- - + - + - + - + + + -
MIC terletak pada cawan petri terakhir yang tidak tampak pertumbuhan koloni
Contoh : - Untuk bakteri a : MIC terletak cawan petri 1 - Untuk bakteri b : MIC terletak cawan petri 2 - Untuk bakteri c : MIC terletak sebelum cawan petri 1 - Untuk bakteri d : MIC terletak pada atau sesudah cawan petri 3
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Agar Dilution Method
ProcedureMaking dilutions of antimicrobial agent in
melted media and pouring plates○ One concentration of antibiotic/ plate○ Possible for several different strains/plate
64 ug/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
Agar Dilution Method
Kelebihan Agar Dilution Method/MIC padat : Dapat digunakan untuk menentukan MIC
dari suspensi zat antibiotik yang keruh sulit untuk membedakan kekeruhan yang disebabkan zat antibiotik atau oleh pertumbuhan bakteri uji.
Dapat digunakan untuk menentukan MIC zat antibiotik terhadap beberapa bakteri uji sekaligus.
Antibiotic-impregnated discs placed on an agar plate at theinterface between test organism and susceptible control organism
Resulting zones of inhibition compared, use of controls
Susceptibility is inferred (standard tables)
MIC from antibiotic diffusion in agar
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E-TEST® agar diffusion MIC determination
Continuous scale - not just doubling dilutions.
Expensive
Reading E-tests
Susceptible < 1
Resistant > 4 ug/ml
Ciprofloxacin for Yersinia pestis
Intermediate 1-4 ug/ml
Upper reading
Common interpretation problemsProblems with E-test reading
Antimicrobial susceptibility tests
Antimicrobial susceptibility testing is expensive Antimicrobial susceptibility testing is expensive (costs include all supplies)(costs include all supplies)
Kirby-Bauer– 12 discs panel = $1.35
E-test (Performed only in certain situations)– One strip = $2.50
Kirby-Bauer is quicker and easier
p. 519; tests have been modified
Determine correlation between disc diffusion zone diameter & broth (or agar) MIC
Comparison of Kirby Bauer and MIC result
Minimum bactericidal concentration (MBC)
MBC testing is required for evaluation of novel antimicrobials
The MBC is the lowest concentration (in mg/L) of antimicrobial that results in ≥ 99.9 % killing of the bacterium under test
MBC are determined by spreading 0.1 mL volume of all clear (no growth) tubes from a dilution MIC test onto separate agar plates.
Minimum bactericidal concentration (MBC)
After incubation at 35-37oC for 18-24 hours, the numbers of colonies growing on each plate are recorded
The first concentration of drug that produced no growth or < 50 colonies after subculture is considered the MBC (the initial inoculum 5 X 105 CFU/mL)
Test for fungistatic activity
Tests for fungisatic activity have been based on the established bacterial techiques
The medium is different (SDA or RPMI plus 2% dextrose)
The inoculum density used is reduced (104 CFU/mL)
More incubation times (72 hours for filamentous fungi)
Tests for fungicidal activity
About 20 µL from MFC test are subculture onto suitable growth medium from each clear tube
This plate are incubated until growth is evident on the growth control subculture.
MFC is the lowest drug concentration showing no growth or fewer than 3 colonies per plate to obtain 99-99.5% killing activity