Antibakteri 2011

Antibiotik

Cakupan Pengertian Sejarah Mekanisme antimikroba Resistensi Metode uji aktivitas anti mikroba

Pengertian Antibiotik Substansi dihasilkan oleh mikroorganisme (:

bacteria, fungi, actinomycetes) utk menekan pertumbuhan mikroorganisme lain dan membunuhnya.

Sekarang istilah antibiotik sering disamakan dg antimikroba karena banyak yg merupakan senyawa semisintetik maupun senyawa hasil sintesis metabolit mikroba.

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Antibiotik bersifat : Antibiotik bersifat :

• Toksik secara selektif pada bakteri, tetapi tidak toksik pada host • Merusak struktur yang ada pada bakteri, tetapi tidak ada pada host.

Kategori Antibiotik Bakteriostatik

Secara reversibel menghambat pertumbuhan bakteri

Bakterisidal Membunuh bakteri

SejarahSejarah In 1928, Sir Alexander Fleming, a In 1928, Sir Alexander Fleming, a

Scottish biologist, menemukan bahwa Scottish biologist, menemukan bahwa Penicillium notatum,Penicillium notatum, merusak merusak pertumbuhan bakteri staphylococcuspertumbuhan bakteri staphylococcus

Fleming’s Petri DishFleming’s Petri Dish

Mekanisme Kerja

1.Menghambat sintesis dinding sel

2.Merubah permeabilitas membran

3.Menghambat sintesis protein

4.Menghambat sintesis asam nukleat

5.Menghambat biosintesis asam folat (anti metabolit)

Antibiotik Penghambat Sintesis Dinding Sel

Umumnya bersifat bakterisidal Menghambat sintesis peptidoglikan

Komposisi peptidoglikan : Peptido-glycan Polymer

(amino acids + sugars) Gula; NAG & NAM

N-acetylglucosamine (NAG)

N-acetylmuramic acid (NAM)

Asam Amino berikatan silang dg NAG & NAM

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Cross-linking of peptidoglycanOld New

Mekanisme penghambatan sintesis peptidoglikan :

Pd sikloserin (analog D-ala) Menghambat konversi enzimatik L-ala mjd D-

ala Menghambat sintesis D-ala-D-ala

Pd basitrasin : Menghambat reaksi defosforilasi dalam

transport sub unit peptidoglikan menembus membran

TRANSPORT OF TRANSPORT OF PEPTIGOGLYCAN SUBUNIT PEPTIGOGLYCAN SUBUNIT ACROSS MEMBRANEACROSS MEMBRANE

Cell membraneCell membrane

undecaprenolundecaprenol

PPPP

Cell wallCell wall




PP

PP

Cell wallCell wall




PPPP

Cell wallCell wall




PPPP

Cell wallCell wall




PPPP

Cell wallCell wall

MINUSMINUS BACITRACINBACITRACIN

CellCell membranemembrane


PP

Cell wallCell wall


21PLUSPLUS BacitracinBacitracin



PPPP

Cell wallCell wall


basitrasin

Mekanisme penghambatan sintesis peptidoglikan (lanjutan) :

Pd vancomycin : Berikatan dg D-ala-D-ala Menghambat ikatan silang (transpeptidasi)

Pd antibiotik betalaktam (penisilin, sefalosporin, monobactam) :

Berikatan dan menghambat enzim (penicillin binding protein) yg mengkatalisis transpeptidasi

Antibiotik Perubah Permeabilitas Membran

Mekanisme pd polimixin B : Berikatan dengan lipid A (komponen dari

lipopolisakarida) Berikatan dg fosfolipid Sehingga merusak membran luar (pada gram

negatif)

Antibiotik Penghambat Sintesis Protein

Inhibitor Tahap Inisiasi 30S Ribosomal Subunit (Aminoglycosides,

Tetracyclines, Spectinomycin) 50S Ribosomal Subunit (Chloramphenicol,

Macrolides)

Inhibitor tahap Elongasi Elongation Factor G (Fusidic acid)

Review of Initiation of Protein Synthesis

30S1 32 GTP

1 2 3 GTP

Initiation Factors

mRNA

3

1

2 GTP

30S Initiation Complex

f-met-tRNA

Spectinomycin

Aminoglycosides

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GDP + Pi 50S

70S Initiation Complex

AP

X mRNA

Review of Elongation of Protein Synthesis

GTP

AP

Tu GTP Tu GDP

Ts

TsTu

+

GDPTs

Pi

P ATetracycline

AP

Erythromycin

Fusidic Acid

Chloramphenicol

G GTPG GDP + Pi

G

GDP

AP

+GTP

X ikatan peptida

X translokasi

X EF-G

X aminoasil tRNA

Aminoglikosida (bactericidal)streptomycin, kanamycin, gentamicin, tobramycin, amikacin, netilmicin, neomycin (topical)

Macrolides (bacteriostatic)erythromycin, clarithromycin, azithromycin, spiramycin

Chloramphenicol, Lincomycin, Clindamycin (bacteriostatic)

Tetracyclines (bacteriostatic)tetracycline, minocycline, doxycycline

Spectinomycin (bacteriostatic)

Inhibitor Sintesis RNA Inhibitor Sintesis DNA

Antibiotik Penghambat Sintesis Asam Nukleat

Inhibitor sintesis RNA :

Menghambat enzim RNA polimerase

Rifampin, Rifamycin, Rifampicin, Rifabutin (bactericidal)

Inhibitor sintesis DNA :

Menghambat enzim DNA girase (menghambat supercoiling)

Quinolones (bactericidal)nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, lomefloxacin, sparfloxacin

Antimetabolit (Penghambat Biosintesis Asam Folat)

Sulfonamides, Sulfones (bacteriostatic)

merupakan analog para-aminobenzoic acid (PABA) dan menghambat pembentukan asam dihidropteroat.

Trimethoprim, Methotrexate, (bacteriostatic)

These antimicrobials binds to dihydrofolate reductase and inhibit formation of tetrahydrofolic acid

p-aminobenzoic acid + Pteridine

Dihydropteroic acid

Dihydrofolic acid

Tetrahydrofolic acid

Pteridine synthetase

Dihydrofolate synthetase

Dihydrofolate reductase

Thymidine

Purines

Methionine

Trimethoprim

Sulfonamides

Kombinasi sufonamid dan trimetoprim

ResistensiResistensi (klinis) terjadi bila Kadar Hambat

Minimum (MIC) antibiotik thd strain bakteri melebihi konsentrasi aman scr in vivo.

mekanisme resistensi

Perubahan permeabilitas membran sel :

- Influx : menyebabkan obat tdk bs masuk

sel (krn mutasi transporter)

- Efflux : Menyebabkan obat dikeluarkan dr sel

(mis pd tetrasiklin) Obat diinaktivasi oleh enzim mikroba (-

lactamase, Chloramphenicol Acetyl Transferase)

mekanisme resistensi (lanjutan)

Target berubah (krn mutasi) : Penicillin binding proteins (penicillins) RNA polymerase (rifampin) 30S ribosome (streptomycin)

Terbentuknya enzim sensitif dg enzim resisten : Plasmid mediated acquisition of a resistant enzyme (sulfonamides, trimethoprim)

Figure 20.20

Antibiotic Resistance

Antimicrobial Resistance

Relative or complete lack of effect of antimicrobial against a previously susceptible microbe

Increase in MIC

Antibiotic Selection for Resistant Bacteria

What Factors Promote Antimicrobial Resistance?

Exposure to sub-optimal levels of antimicrobial

Exposure to microbes carrying resistance genes

Inappropriate Antimicrobial Use

Prescription not taken correctlyAntibiotics for viral infectionsAntibiotics sold without medical

supervisionSpread of resistant microbes in

hospitals due to lack of hygiene

Inappropriate Antimicrobial Use

Lack of quality control in manufacture or outdated antimicrobial

Inadequate surveillance or defective susceptibility assays

Poverty or war Use of antibiotics in foods

Antibiotics in Foods

Antibiotics are used in animal feeds and sprayed on plants to prevent infection and promote growth

Multi drug-resistant Salmonella typhi has been found in 4 states in 18 people who ate beef fed antibiotics

MRSA “mer-sah”

Methicillin-Resistant Staphylococcus aureus

Most frequent nosocomial (hospital-acquired) pathogen

Usually resistant to several other antibiotics

Antimicrobial peptides Broad spectrum antibiotics from plants and

animals Squalamine (sharks) Protegrin (pigs) Magainin (frogs)

The Future of Chemotherapeutic Agents

Antisense agents Complementary DNA or peptide nucleic acids that

binds to a pathogen's virulence gene(s) and prevents transcription

The Future of Chemotherapeutic Agents

Antibiotic susceptibility testing (in vitro)

Bacteriostatic Antibiotics Minimum inhibitory concentration (MIC) Lowest concentration that results in

inhibition of visible growth (colonies on a plate or turbidity of liquid culture)

Bactericidal Antibiotics Minimum bactericidal concentration (MBC) Lowest concentration that kills 99.9% of

the original inoculum

Metode Uji Aktivitas AntimikrobaMetode Dilusi cair atau dilusi padat Pada prinsipnya, antibiotik diencerkan hingga

diperoleh beberapa konsentrasi. Pada dilusi cair, masing-masing konsentrasi obat ditambah suspensi kuman dalam media. Pada dilusi padat, tiap konsentrasi obat dicampur dengan media agar, lalu ditanami kuman.

Metode Difusi Cara Kirby Bauer Cara Sumuran (Cup Plate) Cara Pour Plate

•Skema penyiapan suspensi bakteri

Diambil 1 koloni bakteri dari stok bakteri

(Staphylococcus aureus atau Escherichia coli)

Dimasukkan dalam BHI 1 ml

Diinkubasi 18-24 jam pada suhu 37ºC

Diambil 100 µl, dimasukkan dalam 1 ml BHI

Diinkubasi 4-8 jam pada suhu 37ºC

Diencerkan dengan NaCl 0,9% sampai konsentrasi bakteri 108 CFU/ml

(Kekeruhan disamakan dengan Standar Mc Farland)

Diencerkan sampai 106 CFU/ml dengan BHI DS

(diperoleh suspensi bakteri dg konsentrasi 106 CFU/ml)

CONTOH

1 2 3 4 K1 K4K3K2

Tabung uji

+ suspensi bakteri 106 CFU/ml

Tabung kontrol

K1 : Kontrol media

K2 : Kontrol pelarut

K3 : Kontrol sampel

K4 : Kontrol suspensi bakteri

Diinkubasi selama 24 jam pada suhu 37C

Diamati kejernihan dalam tabung uji (Menentukan KHM)

Digores ke media padatAgar darah (Staphylococcus aureus) atau Agar Mc.Conkey ( Escherichia coli)


Diamati tumbuh tidaknya koloni bakteri di atas media agar (Menentukan KBM)

Metode Dilusi Cair

NoTabung

Perlakuan% b/v

(kadar akhir)

HASIL PENGAMATAN

Kejernihan Koloni

1. 0,6125% K +2. 1,25% J -3. 2,5% J -4. 5 % J -5. K -6. K -7. K.I J -8. K.II J -9. K.III K -

10. K.IV K +

Contoh Hasil Uji Aktivitas

Keterangan :KK = Kekeruhan + = Ada koloni bakteriJ = Jernih - = Tidak ada koloni bakteriK = Keruh

Kadar akhir = kadar ekstrak awal dibagi 2Jadi, KHM = 1,25% b/v KBM = 1,25% b/v

Kontrol (data dan gambar belum ditampilkan)K.I (Kontrol media) = 1 ml BHI DSK.II (Kontrol pelarut) = 0,5 ml aquadest + 0,5 ml BHI DSK.III (Kontrol sampel) = 0,5 ml lart obat + 0,5 ml BHI DSK.IV (Kontrol bakteri) = 0,5 ml suspensi bakteri dlm BHI DS + 0,5 ml akuades

1 2 3 4

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KHM

KBM

8 4 02 1

Tetracycline (μg/ml)

MIC = 2 μg/ml

Determination of MIC

1 2 3 4 K1 K4K3K2

Petri uji

suspensi bakteri 106 CFU/mlDg cara spread plate

Petri kontrol

K1 : Kontrol media

K2 : Kontrol pelarut

K3 : Kontrol sampel

K4 : Kontrol suspensi bakteri


Diamati tumbuh tidaknya koloni bakteri di atas media agar (Menentukan KHM/KBM)

Metode Dilusi Padat

+ media padat + media padat

METODE METODE DIFUSIDIFUSI

a. Kirby-a. Kirby-BauerBauer

No No growthgrowth

Kirby-BauerKirby-Bauer

SusceptibleSusceptible Not susceptibleNot susceptible

BacterialBacterial lawnlawn

GrowthGrowth

Antibiotic diskAntibiotic disk

Chl Amp

Ery

Str

Tet

Disk Diffusion Test

Size of zone of inhibition depends on sensitivity, solubility, rate of diffusion. Compare results to

MIC tables generated using standards.

Disk Diffusion

Zone diameter (mm) Approx. MIC(ug/ml) for:Antimicrobial agent

(amt. per disk)and organism R I MS S R S

Ampicillin (10ug/disk)

Enerobacteriacae <11 12-13 >14 >32 <8

Haemophilus spp. <19 >20 >4 <2

Enterococci <16 >17 >16

Tetracycline (30 g) <14 15-18 >19 >16 <4

Zone Diameter Standards for Disk Diffusion Tests

Measuring Antimicrobial Sensitivity

• MIC: Minimal inhibitory concentration

E-Test method

TUgas Presentasi Power point ttg : Patogen, patogenesis, cara replikasi (perkembangbiakan), obatPenyakit :

• DHF• Malaria• Cacar• Polio• Herpes• AIDS

• Avian Influenza• SARS• Cikungunya• Kanker servix• toxoplasmosis

Antibakteri 2011

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