ANTIBIOTICS

Abrahm Mikru (PhD) Dpt of Biology, Hawasa

Univ
 Antibiotics
2

 Low molecular weight compounds that kill or

inhibit the growth of bacteria
 Can be ingested or injected into the human body
with minimal side effects (selective toxicity).
 Many produced by soil bacteria (streptomyces) and
fungi (penicillin).

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

Characteristics of good antibiotic:

 Few or no side effects,

 broad spectrum of activity,

 good distribution in the body,

 minimal effect on normal microbiota.

 Discovery process
4

1.Produced biologically by soil microbes; screening on

hypersensitive strains.
2. Chemical synthesis or, chemical modification of biologically
produced antibiotics (semi-synthetic),
 Combinatorial chemistry: chemical group added to basic

antibiotic structure,
 New antibiotic may not get to target inside the bacterial cell.

3. Rational drug design: synthesize chemicals that will bind

target molecule based on target structure.
4. Antimicrobials that target virulence genes/ virulence factors

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Antibiotic types based on actions mechanism
5

BACTERIOSTATIC BACTERICIDAL
 Sulfonamides  Beta lactams (penicillins,
cephalosporins, imipenem)
 Drugs inhibiting  Bactrim
protein synthesis except
aminoglycosides  Vancomycin
 Macrolides,  Fluoroquinolones
chloramphenicol,
tetracyclines etc.  Aminoglycosides

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Classification based on mechanisms of actions
6

1. Inhibitors of cellwall synthesis

2. Inhibitors of protein synthesis
3. Inhibitors of nucleic acid synthesis
4. Antimetabolites
5. Interferers with membrane integrity

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Antibiotic types based on actions mechanism
7

1. Bacterial cell wall synthesis inhibitors:

A. Beta lactams such as penicillins, cephalosporins,
carbapenems, monobactams.
 Usually bactericidal.
 Inhibit last step in peptidoglycan synthesis
(trans-peptidation).
 May trigger endogenous enzymes that degrade
peptidoglycan.
 Spectrum of different betalactams varies.
Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
8 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
 O S CH3
C CH
R C N C C 3

C N C COOH
O

Penicillinase
(β-Lactamase)

O S CH3
C CH
R C N C 3

O C N C COOH
OH

Penicilloic Acid
9 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
10 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre noteswww.uccs.edu/
 Transpeptidase

NAG-NAM E
nz
y
PENICILLINS
D-ALA m
e

L-GLU X
LYS

E
D-ALA nz
y
m
D-ALA e

11 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 1.Bacterial cell wall synthesis inhibitors
12

B. Glycopeptides such as vancomycin, teichoplanin.

Glycopeptides bind D-Ala-D-Ala portion of UDP-
NAM pentapeptide.
 Prevent trans-glycosylation and trans-peptidation.

More effective against G+ve bacteria than G-ve.

 Medically important use against multidrug resistant

bacteria.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 E
nz Transglycosylase
NAG-NAM y
Enzyme
m
e
D-ALA

L-GLU

LYS

VAN
D-ALA

D-ALA

13 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 2. Protein synthesis inhibitors:
15

A. Amino-glycosides such as kanamycin,

gentamycin.
 They bind 30S ribosomal subunits; prevent protein

synthesis. bactericidal.
B. Tetra-cyclines such as tetracycline, doxycline.
 Bind 30S ribosomal subunit; prevent protein

synthesis; generally bacteriostatic. 60S

50S

Prokaryote Eukaryote
30S
Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes 40S
 2. Protein synthesis inhibitors:
16

C. Macrolids such as erythromycin and azithromycin.

D. Lincosamid such as lincomycin and clindamycin.
 Bind 50S ribosomal subunit; prevent elongation of

peptide chain.
 Bacteriostatic for most bacteria. Bactericidal for some

G+ve bacteria.
 Chloramphenicol and Linzolide also inhibit protein

synthesis by similar mechanism, i.e binding to 50S r

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Antibiotics binding to the 50S ribosomal subunit
and inhibiting protein synthesis

 Erythromycin and other macrolides

 Chloramphenicol

 Linezolid

 Streptogramins
 2. Protein synthesis inhibitors:
18

E. Strepto-gramins (keto-lides) such as synercid

(mixtures of dalfopristin and quinupristin)
 Bind 50S ribosomal subunit; prevent elongation of

peptide chain.
 Individual components bacteriostatic; mixture

bactericidal.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

19 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
 Nascent polypeptide 50S
chain A

MACROLIDES Transferase
site
aa
mRNA

template

P 30S
20 TRANSLOCATION
Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
 3. Inhibitors of nucleic acid synthesis or
replication:
21

A. Quinolones such as ciprofloxacin and norfloxacin.

 Bind to DNA gyrase; prevent DNA supercoiling.

 Broad bactericidal activity; effective against some intracellular

bacteria.
B. Metronidazole such as flagyl.
 Interferes with DNA replication; creates breaks in DNA.

 Must be activated by flavodoxin or ferredoxin of host

 Effective against anaerobic and microaerophilic bacteria; some

protozoa.
C. Rifampin such as rifadin binds to beta subunit of bacterial RNA
polymerase. Broad spectrum of action.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 4. Antimetabolite.
22

A. Trimethoprim and sulfonamides such as bactrim

and septra
 Inhibit enzymes in tetrahydrofolate biosynthetic

pathway (Vit B12)

 Broad spectrum against bacteria some fungi and

protozoa.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 PABA + Pteridine

Dihydropteroate
SULFONAMIDE
Synthetase
DIHYDROPTEROIC ACID

Dihydrofolate Synthetase

DIHYROFOLIC ACID

Dihydrofolate Reductase TRIMETHOPRIM

23 TETRAHYDROFOLIC ACID
Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
 5.ANTIBIOTICS AFFECTING CELL
MEMBRANES
24

A. POLYMYXINS:
 Surface active amphipathic agent

 Interact strongly with phospholipids and

disrupt the structure of cell membranes

B. DAPTOMYCIN: Depolarizes the cell

membrane

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Antibiotic resistance
25
mechanisms:
 1. Limiting access of the antibiotic,
 A. Outer membrane of G-ve bacteria act as barrier to entry of
antibiotics.
 Mutations can restrict entry of more than one type of antibiotic.
 B. Reduced uptake across cell membrane (not common
mechanism).
 2. Enzymatic Inactivation of antibiotics:
 A. betalactamase cleave betalactam ring, often specific for
subset of bata lactams.
 Effect countered by clavulanic acid, sulbactam.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 O S CH3
C CH
R C N C C 3

C N C COOH
O

Penicillinase
(Betalactamase)

O S CH3
C CH
R C N C 3

O C N C COOH
OH

Penicilloic Acid
26 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
 Antibiotic resistance
27
mechanisms:
2. Enzymatic Inactivation of antibiotics:
 B. Aminoglycoside modifying enzymes act by

adding groups (phosphoryl, adenyl, acetyl) to

antibiotic. Located outside cell membrane in G-ves.
C. Acetyl transferases add acetyl group to
antibiotics (chloramphenicol acetyl transferase,
streptogramin acetyl transferase.
 D. Oxidation of tetracycline-mechanism unknown.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Antibiotic resistance
28
mechanisms:
 3. Active efflux of antibiotics:
 Efflux pumps confer resistance to tetracycline,
found in G-ve and G+ve bacteria.
 ATP-dependent efflux pumps confer resistance to
some macrolides.
 Fluoroquinolones may be expelled by efflux
pumps.
 Streptogramin pump found in Staphylococci.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Antibiotic resistance
29
mechanisms:
4. Modification or protection of antibiotic target:
A. penicillin binding proteins modified;
 Betalactams no longer able to bind.

 Chaning D-Ala-D-Ala to D-Ala-D-lactate prevents binding of

glycopeptides.
B. Ribosome protection:
 Cytoplasmic protein prevents tetracycline binding to ribosome.

 High amino acid homology to elongation factors.

C. rRNA methylase methylates adenine on 23S rRNA,

 confers resistance to macrolides, streptogramins, lincosamides.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Antibiotic resistance
30
mechanisms:
4. Modification or protection of antibiotic target:
D. Point mutation that alter affinity of enzymes for the
antibiotics:
 Altered DNA gyrase B subunit prevents binding of

quinolones.
 Altered RNA polymerase reduces affinity for rifampin.

 Altered enzymes in tetrahydrofolate synthetic pathway

block activity of trimethoprim and sulfonamides.

 Failure to activate an antibiotic- decreased production of

flavodoxin by host prevents activation of metronidazole.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
 Regulation of resistance genes:
31

A. Repressors, tetracycline efflux pumps,

 Tetracycline plus repressor complex unable to bind to

promoter region of resistance gene (tetB).

 TetB protein produced while tetracycline is present.

B. Attenuation: stem loop structure formed when

erythromycin present,
 Ribosomes cannot move along mRNA.

 Different loop forms; allows translation of erm gene

(methylase),
 Ribosome methylated, bacteria able to grow.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Regulation of resistance genes:
32

C. Transcriptional activators:
 Vancomycin resistance gene in Enterococcus sp.

 Some batalactamase genes in enterobacter and

Pseudomonas spp.
D. Insertion sequence and promoter mutations:
 Can increase expression of resistance genes if located

upstream of the gene.

 Examples are ampC gene of Enterobacter sp. And ermF of

Bacteroides sp.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Cross resistance and linkage.
33

A. Multidrug efflux pumps excrete different types of

antibiotics.
B. Modification of antibiotic binding site used by different
types of antibiotics.
C. Multiple resistance genes linked on plasmid.

Antibiotic tolerance: bacterium stops growing when

antibiotic is present,
 Resume growing when level declines.

 Eg. Vancomycin tolerance in S. pneumonia.

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

34 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
35 Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes
 Disc diffusion test (Kirby-Bauer) for antibiotic
sensitivity
36

Muller Hinton agar

Cotton swab
Test bacterium
Inoculum 0.5 Mc Farland

Interpretations
R = resistant
S = Sensitive
I = Intermediate

Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes

 Factors affecting anti-microbial activity in
vitro
37

 1. PH of the environment - some drugs more active at acidic or alkaline

PH
 2. Components of medium - media composition components enhance or
inhibit bacterial growth.
 3. Stability of drug
 4. Size of inoculum: the larger the bacterial inoculum, the lower the
apparent sensitivity of the organisms.
 5. Length of incubation: short exposure of moisture to the drug inhibits
their growth but does not kill them; longer exposure of moisture to a drug
gives a chance for resistant mutants to emerge.
 6. Metabolic activity of moistures - actively and rapidly growing micro-
organisms are more susceptible to drug action than those in the resting
phase.
Abraham Mikru (PhD) Hawassa Univ Virology Lecturre notes