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.

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

Sejarah

In 1928, Sir Alexander Fleming, a Scottish biologist, menemukan bahwa Penicillium notatum, merusak pertumbuhan bakteri staphylococcus

Flemings 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

Cross-linking of peptidoglycan
Old New

12

Mekanisme penghambatan sintesis peptidoglikan : Pd sikloserin (analog D-ala) Menghambat konversi enzimatik L-ala mjd Dala Menghambat sintesis D-ala-D-ala
Pd basitrasin : Menghambat reaksi defosforilasi dalam transport sub unit peptidoglikan menembus membran

TRANSPORT OF PEPTIGOGLYCAN SUBUNIT ACROSS MEMBRANE

Cell membrane
undecaprenol

Cell wall

TRANSPORT OF PEPTIGOGLYCAN SUBUNIT ACROSS MEMBRANE

Cell membrane
undecaprenol

Cell wall

P
P

TRANSPORT OF PEPTIGOGLYCAN SUBUNIT ACROSS MEMBRANE

Cell membrane
undecaprenol

Cell wall

P P

TRANSPORT OF PEPTIGOGLYCAN SUBUNIT ACROSS MEMBRANE

Cell membrane
undecaprenol

Cell wall

P P

TRANSPORT OF PEPTIGOGLYCAN SUBUNIT ACROSS MEMBRANE

Cell membrane
undecaprenol

Cell wall

TRANSPORT OF PEPTIGOGLYCAN SUBUNIT ACROSS MEMBRANE

Cell membrane
undecaprenol

Cell wall

MINUS BACITRACIN

TRANSPORT OF PEPTIGOGLYCAN SUBUNIT ACROSS MEMBRANE

Cell membrane
undecaprenol

Cell wall

P P

PLUS Bacitracin

21

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

30S
1 2 3 GTP Initiation Factors

1 3 2 GTP

f-met-tRNA mRNA Spectinomycin

3

X mRNA

GDP + Pi
P A

50S
1 2 GTP

70S Initiation Complex

Aminoglycosides

30S Initiation Complex

Review of Elongation of Protein Synthesis

X aminoasil tRNA
P A

Tetracycline

P A

Tu GTP GTP Ts

Tu GDP + Ts

Pi

Tu Ts

GDP

Fusidic Acid X EF-G

G GDP + P A

GDP + G

Chloramphenicol X ikatan peptida

GTP

Pi

G GTP P A

Erythromycin X translokasi

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)

Antibiotik Penghambat Sintesis Asam Nukleat

Inhibitor Sintesis RNA Inhibitor Sintesis DNA

Inhibitor sintesis RNA :

Rifampin, Rifamycin, Rifampicin, Rifabutin (bactericidal)

Menghambat enzim RNA polimerase

Inhibitor sintesis DNA :

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

Menghambat enzim DNA girase (menghambat supercoiling)

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

Kombinasi sufonamid dan trimetoprim

p-aminobenzoic acid + Pteridine Sulfonamides

Pteridine synthetase

Dihydropteroic acid Dihydrofolate synthetase Dihydrofolic acid Trimethoprim Dihydrofolate reductase

Tetrahydrofolic acid Thymidine Purines Methionine

Resistensi
Resistensi (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 (blactamase, 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)

Antibiotic Resistance

Figure 20.20

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 correctly Antibiotics for viral infections Antibiotics sold without medical supervision Spread 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 (hospitalacquired) pathogen Usually resistant to several other antibiotics

The Future of Chemotherapeutic Agents

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

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 Antimikroba

Metode 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

CONTOH

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 37C Diambil 100 l, dimasukkan dalam 1 ml BHI Diinkubasi 4-8 jam pada suhu 37C 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)

Metode Dilusi Cair

1 2 3 4

K1

K2

K3

K4

Tabung uji

Tabung kontrol

+ suspensi bakteri

106

CFU/ml

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 padat Agar darah (Staphylococcus aureus) atau Agar Mc.Conkey ( Escherichia coli) Diinkubasi selama 24 jam pada suhu 37C

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

Contoh Hasil Uji Aktivitas

No Tabung Perlakuan % b/v (kadar akhir) HASIL PENGAMATAN Kejernihan Koloni

KHM

1. 0,6125% K + 2. 1,25% J 3. 2,5% J 4. 5% J 5. K Keterangan : 6.Kekeruhan K bakteri KK = + = Ada koloni J = 7.Jernih K.I - = Tidak adaJkoloni bakteri K = 8.Keruh K.II J 9. K.III K Kadar akhir = kadar ekstrak awal dibagi 2 10. K.IV K + Jadi, KHM = 1,25% b/v KBM = 1,25% b/v Kontrol (data dan gambar belum ditampilkan) K.I (Kontrol media) = 1 ml BHI DS K.II (Kontrol pelarut) = 0,5 ml aquadest + 0,5 ml BHI DS K.III (Kontrol sampel) = 0,5 ml lart obat + 0,5 ml BHI DS K.IV (Kontrol bakteri) = 0,5 ml suspensi bakteri dlm BHI DS + 0,5 ml akuades

KBM
1

Determination of MIC

Tetracycline (g/ml) MIC = 2 g/ml

Metode Dilusi Padat

+ media padat
1 2 3 4
K1 K2 K3 K4

+ media padat

Petri uji

Petri kontrol

suspensi bakteri CFU/ml Dg cara spread plate

106

K1 : Kontrol media K2 : Kontrol pelarut K3 : Kontrol sampel K4 : Kontrol suspensi bakteri

Diinkubasi selama 24 jam pada suhu 37C Diamati tumbuh tidaknya koloni bakteri di atas media agar (Menentukan KHM/KBM)

METODE DIFUSI a. KirbyBauer

Kirby-Bauer

Susceptible
Bacterial lawn No growth Growth

Not susceptible

Antibiotic disk

Disk Diffusion Test

Str

Tet

Ery

Chl

Amp

Size of zone of inhibition depends on sensitivity, solubility, rate of diffusion. Compare results to MIC tables generated using standards.

Disk Diffusion

Zone Diameter Standards for Disk Diffusion Tests

Zone d iamet er (mm) Antimicrobial ag ent (amt. per disk) and organism Ampicillin (10ug/d isk) Enerobacteriacae Haemophilus spp. Enterococci Tetracycline (30 g) <11 <19 <16 <14 15-18 >17 >19 12-13 >14 >20 >32 >4 >16 >16 <4 <8 <2 Approx. MIC (ug/ml) for:

MS

Measuring Antimicrobial Sensitivity

E-Test method

MIC: Minimal inhibitory concentration

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