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

Farmakokinetik/Farmakodinamik

• Istilah umum untuk obat apapun, tidak spesifik hanya antibiotik

• Istilah Farmakokinetik digunakan untuk menentukan waktu
perjalanan dari proses penyerapan obat, distribusi, metabolisme
dan ekskresi.
• Istilah farmakodinamik merujuk pada hubungan antara konsentrasi
obat di lokasi aksi dan respon farmakologi.

– Namun, ketika kita menerapkan prinsip-prinsip ini untuk terapi

antimikroba ada sejumlah faktor yang dapat mengubah hasil
prediksi terapi.
Introduction
 Susceptibility test, main purposes:
 As a guide for treatment
○ Sensitivity of a given m.o. to known conc. of
drugs
○ Its concentration in body fluids or tissues

 As an epidemiological tool
○ The emergence of resistant strains of major
pathogens (e. g. Shigellae, Salmonella typhi)
○ Continued surveillance of the susceptibility
pattern of the prevalent strains (e. g.
Staphylococci, Gram-negative bacilli)

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Introduction
 Drugs for routine susceptibility tests:
 Set 1: the drugs that are available in most
hospitals and for which routine testing should be
carried out for every strain

 Set 2: the drugs that are tested only:

○ at the special request of the physician
○ or when the causative organism is resistant
to the first-choice drugs
○ or when other reasons (allergy to a drug, or
its unavailability) make further testing
justified
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Table 1: Basic sets of drugs for routine susceptibility tests
(http://w3.whosea.org/)
Set 1 Set 2
Staphylococcus Benzyl penicillin Gentamicin
Oxacillin Amikacin
Erythromycin Co-trimoxazole
Tetracycline Clindamycin
Chloramphenicol

Intestinal Ampicillin Norfloxacin

Chloramphenicol
Co-trimoxazole
Nalidixic acid
Tetracycline

Enterobacteriaceae Sulfonamide Norfloxacin

Urinary Trimethoprim Chloramphenicol
Co-trimoxazole Gentamicin
Ampicillin
Nitrofurantoin
Nalidixic acid
Tetracycline

Blood and tissues Ampicillin Cefuroxime

Chloramphenicol Ceftriaxone
Cotrimoxazole Ciprofloxacin
Tetracycline Piperacillin
Gentamicin Amikacin

Pseudomonas aeruginosa Piperacillin Amikacin

Gentamicin
Tobramycin

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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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DEFinisi MIC & MBC
 Minimum Inhibition Concentration (MIC)
 Konsentrasi terendah zat antimikroba yang masih
dapat menghambat pertumbuhan bakteri /
multiplikasinya
 Minimum Bactericidal Concentration (MBC) or
Minimum Lethal Concentration (MLC)
 Konsentrasi terendah zat antimikroba yang
memungkinkan kurang dari 0,1% dari inokulum asli
untuk bertahan hidup

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Nilai MIC -MBC vs Kerja Antimikroba
 Hasil: menunjukkan kadar obat yang perlu diberikan
untuk menghambat (aktivitas bakteriostatik) atau
membunuh (aktivitas bakterisida) mikroorganisme
diuji
 Antimikroba biasanya dianggap sebagai
bakterisida jika MBC tidak lebih dari empat kali
MIC
 Zat antimikroba dengan MIC atau MBC terendah
terhadap suatu bakteri, menjadi pilihan yang lebih
disukai

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 Pengujian Kerentanan Mikroba

 Metode Pengeceran
 zat antimikroba dengan jumlah yang
bervariasi, dimasukkan ke dalam media
yang cair atau padat
 Dilanjutkan dengan inokulasi bakteri uji
 Metode Difusi
 Letakkan cakram kertas, atau lubang
menggunakan perforator yang mengandung
obat dengan kuantitas yang terukur pada
media padat yang telah diinokulasi dengan
bakteri uji
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Metode Pengeceran Cair (MIC Cair)
 Menggunakan media cair pertumbuhan
untuk mengencerkan konsentrasi
antibiotik uji
 Membuat pengenceran (2 kali lipat) dari
antibiotik dalam kaldu 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)
5-Jan-06
 MBC
Chiang Mai University 10
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)
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 8 4 2 1 C1 C2 Controls:
C1 = No antibiotic, check
Bacterial conc.= 5*105 CFU/ml viability on agar plates
immediately
Incubate 35 oC, o/n
C2 = No test bacteria

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 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
64 32 16
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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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 4 2

•
• •
•
•
• • •
• • •
• •

96 well microtiter plate

Read MICs
MICs
- +
0.51
2
4
8
16
32
64

>64 >64
PENENTUAN MIC (Metode Mikrodilusi)

MIC
Bacterial growth Inhibition

0 (Control) 0,25 0,50 1 2 4 8 mg/l

Reporting Time: Disk Diffusion or MIC

 “Resistance may be reported any time growth is

observed after a minimum of 16 h incubation”
 After minimum of 16 h, read test:
 if “R”, report
 If “S”, re-incubate and read again at 24 h

National Committee for Clinical Laboratory Standards (NCCLS) M2-A8, M7-A6

 MIC Interpretive Criteria (g/ml)

Drug Susc Int Res

cefazolin 8 16  32

gentamicin 4 8  16
 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 B C 9 ml
air suling
steril
Diencerkan dgn 1 ml 1 ml 1 ml
pelarutnya
(lihat Farmakope)
dan air suling steril Goyang2kan, lalu
Antibiotik dalam labu ukur biarkan membeku
berbentuk
cairan
A B C
19 ml MHA bersuhu 40-50C
 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)
1 2 3
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
37C 18-24 jam

Amati pertumbuhan koloni pada cawan petri 1, 2

dan 3
Dibandingkan cawan petri kontrol positif dan
negatif.
 Agar Dilution Method : Kekeruhan suspensi bakteri

 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

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Pengamatan MIC (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
www.themegallery.com
 Kelebihan MIC padat

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.
E-TEST® agar diffusion MIC determination

Continuous scale - not

just doubling dilutions.

Expensive

32
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
Kirby Bauer vs E-test : Price

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
 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