mic mbc 2012_rev

37
Sri Agung Fitri Kusuma, M.Si., Apt.

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Page 1: MIC MBC 2012_rev

Sri Agung Fitri Kusuma, M.Si., Apt.

Page 2: MIC MBC 2012_rev

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.

Page 3: MIC MBC 2012_rev

5-Jan-06 Chiang Mai University 3

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

Page 4: MIC MBC 2012_rev

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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)

Page 5: MIC MBC 2012_rev

5-Jan-06 Chiang Mai University 5

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

Page 6: MIC MBC 2012_rev

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

Page 7: MIC MBC 2012_rev

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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

Page 8: MIC MBC 2012_rev

http://www.medschool.lsuhsc.edu/Microbiology/Flash/MICMBC.htm

Page 9: MIC MBC 2012_rev

5-Jan-06 Chiang Mai University 9

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

Page 10: MIC MBC 2012_rev

5-Jan-06 Chiang Mai University 10

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

Page 11: MIC MBC 2012_rev

5-Jan-06 Chiang Mai University 11

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

Page 12: MIC MBC 2012_rev

p. 519

Page 13: MIC MBC 2012_rev

5-Jan-06 Chiang Mai University 13

Broth Dilution Method

Disadvantages :Only one antibiotic & one organism can be

tested each timeTime-consuming

Solutions??Agar dilution methodDisc diffusion methodMicrobroth dilution method

Page 14: MIC MBC 2012_rev

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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

Page 15: MIC MBC 2012_rev

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

Page 16: MIC MBC 2012_rev

Antimicrobial susceptibilityAntimicrobial susceptibilitytesting using micro-broth testing using micro-broth dilutionsdilutions

• •

96 well microtiter plate96 well microtiter plate

ug/mlug/ml64 32 16 8 4 264 32 16 8 4 2

Page 17: MIC MBC 2012_rev

- +

64

32

16

8

4

2

1

>64

0.5

MICs

>64

Page 18: MIC MBC 2012_rev

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)

Page 19: MIC MBC 2012_rev

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

Page 20: MIC MBC 2012_rev

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.

Page 21: MIC MBC 2012_rev

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

Page 22: MIC MBC 2012_rev

5-Jan-06 Chiang Mai University 22

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

Page 23: MIC MBC 2012_rev

5-Jan-06 Chiang Mai University 23

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

Page 24: MIC MBC 2012_rev

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.

Page 25: MIC MBC 2012_rev

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

Page 26: MIC MBC 2012_rev

26

E-TEST® agar diffusion MIC determination

Continuous scale - not just doubling dilutions.

Expensive

Page 27: MIC MBC 2012_rev

Reading E-tests

Susceptible < 1

Resistant > 4 ug/ml

Ciprofloxacin for Yersinia pestis

Intermediate 1-4 ug/ml

Upper reading

Page 28: MIC MBC 2012_rev

Common interpretation problemsProblems with E-test reading

Page 29: MIC MBC 2012_rev

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

Page 30: MIC MBC 2012_rev

Kirby-Bauer is quicker and easier

p. 519; tests have been modified

Page 31: MIC MBC 2012_rev

Determine correlation between disc diffusion zone diameter & broth (or agar) MIC

Page 32: MIC MBC 2012_rev

Comparison of Kirby Bauer and MIC result

Page 33: MIC MBC 2012_rev

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.

Page 34: MIC MBC 2012_rev
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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)

Page 36: MIC MBC 2012_rev

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)

Page 37: MIC MBC 2012_rev

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