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.

Antimicrobial Susceptibility Testing

Dilution method
vary amount of antimicrobial substances

incorporated into liquid or solid media followed by inoculation of test bacteria

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
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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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Chiang Mai University

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
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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

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

Subculturing of non-turbid tubes, overnight

Growth (bacterial count) MBC

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Chiang Mai University

http://www.medschool.lsuhsc.edu/Microbiol ogy/Flash/MICMBC.htm

Broth Dilution Method

Day 1 128 64 32 16 8 4 2 C1 C2 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

1 C1 C2

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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Chiang Mai University

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 0.01 ml standard loop) 0.01 ml (spread plate), Incubate 35 oC, 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

64

32

16

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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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p. 519

Broth Dilution Method

Disadvantages :
Only one antibiotic & one organism can be

tested each time Time-consuming

Solutions??
Agar dilution method

Disc diffusion method

Microbroth 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/ lyophilized Consistent performance but high cost May suffer from degradation of antibiotic during shipping and storage
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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 susceptibility testing using micro-broth dilutions

ug/ml 64 32 16 8 96 well microtiter plate 4 2

MICs

- +
0.5 1 2 4

8
16 32

64
>64 >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

Antibiotik berbentuk padat digerus, lalu ditimbang teliti

1 ml 1 ml

kocok

1 ml

A
Diencerkan dgn pelarutnya (lihat Farmakope) dan air suling steril dalam labu ukur 1 ml

C 9 ml
air suling steril

1 ml 1 ml Goyang2kan, lalu biarkan membeku

Antibiotik berbentuk cairan

19 ml MHA bersuhu 40-50C

Agar Dilution Method

- 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 negatif Kontrol positif : MHA + 1 ose bakteri uji berbeda yang berumur 18-24 jam Kontrol 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.

Agar Dilution Method

PENGAMATAN 1 a b c d a b CAWAN PETRI 2 c d a b 3 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
www.themegallery.com

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 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 SteersFoltz replicator Delivers 0.001 ml of bacterial inoculum

Incubation Spot of growth

MIC
32 ug/ml

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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.

MIC from antibiotic diffusion in agar

Antibiotic-impregnated discs placed on an agar plate at the interface between test organism and susceptible control organism Resulting zones of inhibition compared, use of controls Susceptibility is inferred (standard tables)

E-TEST agar diffusion MIC determination

Continuous scale - not just doubling dilutions. Expensive

26

Reading E-tests

Ciprofloxacin for Yersinia pestis

Resistant > 4 ug/ml

Intermediate 1-4 ug/ml

Susceptible < 1 Upper reading

Common interpretation problems

Problems with E-test reading

Antimicrobial susceptibility tests

Antimicrobial susceptibility testing is expensive (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 L 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